Regulatory role of phosphoproteins in the development of bovine small intestine during early life.

The small intestine is the primary site of nutrient digestion and absorption, which plays a key role in the survival of neonatal calves. A comprehensive assessment of the phosphoproteomic changes in the small intestine of neonatal calves is unavailable; therefore, we used phosphopeptide enrichment coupled with liquid chromatography-tandem mass spectrometry to investigate the changes in the phosphoproteome profile in the bovine small intestine during the first 36 h of life. Twelve neonatal male calves were assigned to one of the following groups: (1) calves not fed colostrum and slaughtered approximately 2 h postpartum (n = 3), (2) calves fed colostrum at 1 to 2 h and slaughtered 8 h postpartum (n = 3), (3) calves fed 2 colostrum meals (at 1-2 and 10-12 h) and slaughtered 24 h postpartum (n = 3), (4) calves fed 3 colostrum meals (at 1-2, 10-12, and 22-24 h) and slaughtered 36 h postpartum (n = 3). Mid-duodenal, jejunal, and ileal samples of the calves were collected after slaughter. We identified 1,678 phosphoproteins with approximately 3,080 phosphosites, which were mainly Ser (89.9%), Thr (9.8%), and Tyr (0.3%) residues; they belonged to the prodirected (52.9%), basic (20.4%), acidic (16.6%), and Tyr-directed (1.7%) motif categories. The regional differentially expressed phosphoproteins included zonula occludens 2, sorting nexin 12, and protein kinase C, which are mainly associated with developmental processes, intracellular transport, vesicle-mediated transport, and immune system process. They are enriched in the endocytosis, tight junction, insulin signaling, and focal adhesion pathways. The temporal differentially expressed phosphoproteins included occludin, epsin 1, and bridging integrator 1, which were mainly associated with macromolecule metabolic process, cell adhesion, and growth. They were enriched in the spliceosomes, adherens junctions, and tight junctions. The observed changes in the phosphoproteins in the tissues of small intestine suggest the protein phosphorylation plays an important role in nutrient transport and immune response of calves during early life, which needs to be confirmed in a larger study.

Changes in bacterial community and expression of genes involved in intestinal innate immunity in the jejunum of newborn lambs during the first 24 hours of life.

The newborn gut undergoes rapid colonization by commensal microorganisms and possible exposure to pathogens. The contribution of colostrum intake to host protection is well known; however, limited research exists on the intestinal innate immunity corresponding to colostrum intake during the passive immune transfer period in newborn ruminants. The aim of this study was to investigate the changes in bacterial community and expression of genes encoding toll-like receptors (TLR), mucins (MUC), antimicrobial peptides, and tight junctions in the jejunum of lambs that were fed colostrum during the first 24 h of life. Twenty-seven newborn lambs were used in this study, of which 18 lambs were bottle-fed pooled bovine colostrum within the first 2 h after birth to obtain an intake of approximately 8% of body weight. Lambs were slaughtered at 12 (n = 9) and 24 h (n = 9) after birth. The remaining 9 lambs without any feeding were slaughtered at 30 min after birth (0 h). Tissue and ligated segment samples from the jejunum were collected immediately after the lambs were slaughtered. The bacterial profile in the ligated jejunum segment was assessed using amplicon sequencing. The gene expression in the jejunum tissue was determined using quantitative real-time PCR. The relative abundances of Escherichia-Shigella, Lactobacillus, Lactococcus, and Streptococcus increased, whereas those of Sphingomonas, Phyllobacterium, Bradyrhizobium, and Rudaea decreased during the first 24 h of life. Expression of TLR2 and ß-defensin 109-like was upregulated at 12 h after birth, but a recovery was detected at 24 h; TLR3, TLR5, LYZ, MUC1, MUC13, MUC20, and CLDN7 showed a higher expression level in samples taken at 24 h than in those taken at 0 h. In addition, expression level of CLDN1, CLDN4, and the junctional adhesion molecule-1 tended to be higher at 24 h than at 0 h after birth. Correlation analysis indicated that TLR2 expression was negatively correlated with the relative abundance of Lactobacillus and Bradyrhizobium, whereas TLR5 expression was positively correlated with the relative abundance of Escherichia-Shigella and Pelagibacterium. These results suggest that TLR, MUC, antimicrobial peptides, and CLDN act together and play an important role in intestinal defense during the passive immune transfer period. They are potentially associated with microbial colonization. The findings from this study provide novel information to elucidate the role of colostrum components in regulating the development of the intestinal mucosal immune barrier in newborn lambs during the passive immune transfer period.

Quantitative comparative phosphoproteomic analysis of the effects of colostrum and milk feeding on liver tissue of neonatal calves.

Posttranslational modifications, mostly phosphorylation, are critical for protein structure and function. However, the association between liver phosphoproteins in neonatal calves and colostrum intake is not well understood. In this study, we examined the liver phosphoproteome profile in neonatal calves after receiving colostrum or milk. Liver tissue samples were collected from control calves (CON, n = 3) 2 h after birth and from calves that received colostrum (CG, n = 3) or milk (MG, n = 3) 24 h after birth. Hepatic phosphoprotein expression profiles were analyzed using quantitative proteomics based on the liquid chromatography-tandem mass spectrometry method. In total, 1,587 phosphorylated sites were identified in 1,011 liver proteins. The most abundant phosphorylation site AA was serine (87.5%), followed by threonine (11.9%) and tyrosine (0.5%). Among the 1,011 phosphoproteins, 219, 453, and 26 displayed differential expression in the CG versus MG, CG versus CON, and MG versus CON comparisons, respectively. Differentially expressed phosphoproteins in the CG-MG comparison included 3-phosphoinositide-dependent protein kinase 1, glucose transporter member 4, protein kinase N2, and vinculin, which were mainly involved in the glycogen metabolic process, transport, growth and development, and cell adhesion process, according to Gene Ontology analysis. Pathway analysis indicated their enrichment in the insulin signaling pathway, spliceosome, and adherens junction. The CG-CON comparison identified differentially expressed phosphoproteins and their target genes that were largely involved in the cellular process, macromolecule metabolic process, developmental process, and transport. Pathway analysis indicated their association with endocytosis, mechanistic target of rapamycin, AMP-activated protein kinase, and insulin signaling pathways. These data demonstrate that changes in the phosphoproteins of liver tissues may play an important role in energy metabolism and immune response in the calves that received colostrum. These results provide novel insights into the crucial roles of protein phosphorylation during the early life of newborn calves.

Evaluation of colostrum bioactive protein transfer and blood metabolic traits in neonatal lambs in the first 24 hours of life.

Colostrum is a unique resource that contributes to the passive transfer of immunity and plays a central role in the health status of neonatal ruminants. However, digestion and absorption of colostral proteins in the gut remain incompletely understood. Therefore, this study aimed to investigate the effect of bovine colostrum feeding on blood metabolic traits and to quantify colostral bioactive proteins in the gastrointestinal digesta and blood to evaluate intestinal transfer in neonatal lambs in the first 24 h of life. Fifty-four newborn lambs were used in this study, including 27 lambs fed pooled bovine colostrum and slaughtered at 6 (C6h), 12 (C12h), or 24 h (C24h) after birth; 18 lambs not fed any colostrum or milk and slaughtered at birth (N0h) or 24 h (N24h) after birth; and 9 milk-fed lambs slaughtered at 24 h (M24h) after birth. Lambs receiving colostrum or milk were bottle-fed within the first 2 h to obtain intakes of 8% of body weight at birth. Samples of blood and digesta from the abomasum, jejunum, and ileum were collected after slaughter. Serum concentrations of glucose, insulin, total protein, and aspartate aminotransferase were higher in colostrum-fed lambs than in N0h lambs. Serum concentrations of insulin, total protein, insulin-like growth factor 1, and γ-glutamyl transpeptidase were higher in C24h lambs than in N24h or M24h lambs. Apparent efficiencies of IgG absorption in C6h, C12h, and C24h lambs were 14.4, 26.8, and 17.2%, respectively, whereas apparent efficiencies of lactoferrin (LF), α-lactalbumin (α-LA), and ß-lactoglobulin (ß-LG) absorption were very low in colostrum-fed lambs, with mean values of 0.06, 0.002, and 0.003%, respectively. Concentrations of IgG, LF, α-LA, and ß-LG in the digesta of the abomasum, jejunum, and ileum rapidly decreased from C6h to C24h lambs, and the disappearance rates of IgG, LF, α-LA, and ß-LG were higher in lambs from C6h to C12h (62.1, 75.7, 91.3, and 95.0% for IgG, LF, α-LA, and ß-LG, respectively) than from C12h to C24h (34.6, 22.5, 7.5, and 2.2% for IgG, LF, α-LA, and ß-LG, respectively). These results indicated that bovine colostrum feeding improved the metabolic and immunological status of lambs, and that ingested colostral IgG was prone to intact uptake into the blood, whereas almost all ingested LF, α-LA, and ß-LG disappeared in the lumen of the gastrointestinal tract in a time-dependent manner. The findings provide novel information for exploring selective absorption of colostral compounds in the small intestine of lambs.

Label-free quantitative proteomics analysis reveals the fate of colostrum proteins in the intestine of neonatal calves.

The contribution of intestinally absorbed colostral immunoglobulins to the transmission of passive immunity is widely reported in neonatal calves. However, changes in the colostral proteome in the gastrointestinal digesta remain unclear. Therefore, this study aimed to investigate changes in colostral proteome affected by gastrointestinal proteases in neonatal calves. Twenty-one neonatal Holstein calves were used in this study, including 18 colostrum-fed calves slaughtered at 8 (CI, n = 6), 24 (CII, n = 6), and 36 h (CIII, n = 6) postpartum and 3 milk-fed calves slaughtered 24 h postpartum (MI, n = 3). The ingested colostrum and milk samples were collected from the mid-jejunum segment, following the sacrifice. The undigested colostrum or milk along with their ingested colostrum or milk samples were investigated using a label-free proteomics approach. Hierarchical clustering and principal component analysis of the quantified proteins revealed that the ingested colostrum from the CII and CIII groups and the ingested mature milk from the MI group appeared to share similar patterns. Analysis of the intestinal digesta revealed a time-dependent decrease in caseins, lactoferrin, and osteopontin protein levels, and an increase in cationic trypsin, chymotrypsin, and carboxypeptidase. Several protease inhibitors, such as α-1-antiproteinase, α-2-antiplasmin, and early lactation protein, were identified in the colostrum and intestinal digesta. In addition, we detected identical levels in the intestinal digesta and colostrum for albumin, α-1-acid glycoprotein, and plasminogen. Pathway analysis indicated that proteins increased in the intestinal digesta belonged to the following categories: biosynthesis of antibiotics, carbon metabolism, and biosynthesis of amino acids. These results indicated that selected colostral proteins were digested by gastrointestinal proteases, contributing to their intestinal absorption in calves. These findings provide new insights into the fate of the colostral proteome in the gastrointestinal tract and may aid in the identification of factors contributing to health management in neonatal calves.

Alterations of the circular RNA profile in the jejunum of neonatal calves in response to colostrum and milk feeding.

Circular RNA (circRNA) have been suggested to contribute to regulating gene expression in various tissues and cells of eukaryotes. However, little is known regarding the expression pattern of circRNA and their potential function in the small intestine of neonatal calves that receive colostrum. In the current study, jejunum tissue samples were collected from control calves (2 h after birth; CT; n = 3) and neonatal calves that ingested colostrum (24 h after birth; CO; n = 3) or milk (24 h after birth; MK; n = 3) to compare the circRNA expression patterns using a high-throughput RNA sequencing approach. A total of 21,213, 17,861, and 21,737 circRNA were identified in the CT, CO, and MK groups, respectively. Only 13,254 of these circRNA were common to the 3 groups, suggesting high specificity of circRNA expression depending on nutrient type. In total, 243, 249, and 283 circRNA were differentially expressed in the CO versus CT, CO versus MK, and MK versus CT comparisons, respectively. Gene ontology analysis showed that the differentially expressed circRNA and their predicted or known target genes from the CO and MK groups were mainly involved in macromolecule metabolic process, response to stress, and vesicle-mediated transport. Moreover, pathway analysis showed that the Rap1 signaling pathway, focal adhesion, ubiquitin-mediated proteolysis, and extracellular matrix-receptor interaction were the most significantly enriched pathways. These data collectively indicate that circRNA are abundant and dynamically expressed when calves receive colostrum and act as microRNA sponges to regulate their target genes for jejunum function during the early development of newborn calves.

Changes in serum metabolites in response to ingested colostrum and milk in neonatal calves, measured by nuclear magnetic resonance-based metabolomics analysis.

Uptake of colostrum is of central importance for establishing a passive immunity transfer in neonatal calves. Studies of absorption and transmission of colostral immunoglobulins have been widely reported; however, changes in the serum in response to the absorption of colostral components in neonatal calves have not been completely characterized. Here, a nuclear magnetic resonance-based metabolomics approach was used to investigate the changes in metabolites in ingested colostrum, milk, and serum after neonatal calves were fed colostrum or milk. Twenty-seven neonatal male Holstein calves were assigned to 1 of the following groups: (1) calves not fed colostrum or milk and slaughtered approximately 2 h after birth (control group, n = 6), (2) calves fed colostrum at 1 to 2 h after birth and slaughtered 8 h after birth (n = 6), (3) calves fed 2 colostrum meals (at 1-2 and 10-12 h after birth) and slaughtered 24 h after birth (n = 6), (4) calves fed 3 colostrum meals (at 1-2, 10-12, and 22-24 h after birth) and slaughtered 36 h after birth (n = 6), or (5) calves fed 2 milk meals (1-2 and 10-12 h after birth) and slaughtered 24 h after birth (n = 3). Concentrations of valine, leucine, lactate, lysine, and isoleucine were higher and concentrations of lactose were lower in the groups fed colostrum and milk compared with groups not fed colostrum and milk, respectively. Metabolite changes between groups fed or not fed colostrum and milk were similar and may reflect the primary metabolic requirements of ingestion by the small intestine of neonatal calves. Concentrations of serum metabolites choline, valine, leucine, and glutamate were higher in the serum of calves that received colostrum compared with control calves. Furthermore, concentrations of serum phenylalanine, valine, and glutamate were significantly higher, whereas serum concentrations of citrate and very low density lipoproteins were lower in calves that received colostrum compared with calves fed milk. Our results indicate that concentrations of leucine, valine, and glutamate, which were higher in the calves that ingested colostrum, may transfer into the bloodstream, and that these metabolites are associated with health benefits in the neonatal calves that received colostrum. These findings provide novel information to help us understand the mechanism by which colostrum components are metabolized and absorbed in the small intestine and then transferred into bloodstream of neonatal calves.

Supplementing different ratios of short- and medium-chain fatty acids to long-chain fatty acids in dairy cows: changes of milk fat production and milk fatty acids composition.

Milk fat synthesis might be promoted by the dietary addition of long-chain fatty acids (LCFA) or short- and medium-chain fatty acids (SMCFA). This study evaluated unprotected lipid supplementation with different ratios of SMCFA to LCFA, which had equivalent fatty acid (FA) proportions (by weight) to those in milk, on milk fat production and milk FA composition. Thirty-six Holstein cows (183±46 d in milk) were divided into 3 treatments according to a randomized block design. Cows in 3 treatments received supplements of 80 g/d of SMCFA mixture and 320 g/d of LCFA mixture (ratio of SMCFA to LCFA was 20:80); 400 g/d of butterfat (ratio of SMCFA to LCFA was 40:60); or 240 g/d of SMCFA mixture and 160 g/d of LCFA mixture (ratio of SMCFA to LCFA was 60:40). The FA compositions of the SMCFA mixture and the LCFA mixture were similar to the de novo synthesized FA (except C4:0) and preformed FA (except trans FA) found in the butterfat, respectively. Fatty acid supplements and butterfat were consumed by cows daily before the morning feeding during the 8-wk experimental period. Dry matter intake and milk yield were not different among the treatments. The milk fat percentage and total SMCFA concentration in milk fat tended to increase linearly and the proportion of milk total solids increased linearly with increasing ratios of SMCFA to LCFA in the supplements, whereas milk fat yield was not changed. We suggest that increasing ratios of SMCFA to LCFA in diets has the potential to improve milk fat synthesis.

[Changes in atrial natriuretic peptide and vasopressin during the pressor response to central osmotic stimulation].

In order to study the mechanism of pressor response to central osmotic stimulation, rats were administered with hypertonic artificial cerebrospinal fluid (ACSF) intracerebroventricularly. Carotid arterial pressure and heart rate were recorded. Ten minutes after administration, blood samples, hypothalamus and hypophysis were taken for the determination of atrial natriuretic peptide (ANP) and vasopressin (AVP) by radioimmunoassay. The results showed that after central administration of hypertonic ACSF, the plasma level of AVP increased significantly with no apparent change in ANP. In hypothalamus and hypophysis, the content of ANP was increased while that of AVP decreased.

[Effects of human atrial natriuretic factor-(99-126) on plasma and brain vasopressin in stroke-prone spontaneously hypertensive rats].

In order to investigate the interaction between atrial natriuretic factor (ANF) and arginine vasopressin (AVP) in the pathogenesis of essential hypertension, the effects of intravenous (iv) or intracerebroventricular (icv) injection of human ANF-(99-126) on plasma and brain AVP, as well as mean arterial pressure (MAP), urinary volume (UV) and sodium (UNaV) excretion in stroke-prone spontaneously hypertensive rats (SHRsp) and age-matched normotensive Wistar-Kyoto rats (WKY) were studied. The results showed that ten minutes after iv injection of ANF, MAP decreased by 9.4% and 12.2% (P less than 0.05), UV increased about 9 and 20 folds (P less than 0.01), UNaV increased about 16 and 29 folds (P less than 0.01) in SHRsp and WKY rats, respectively. No such significant changes in these parameters were found in the icv group. Although iv and icv injection of ANF caused significant decrease of plasma AVP in both strains, the decrease was less marked in SHRsp than in WKY rats, while the maximum decreases were 58% (iv) and 31% (icv) in SHRsp, the corresponding values were 80% (iv) and 65% (icv) in WKY. Intravenous and intracerebroventricular injection of ANF also induced significant increase of hypothalamic AVP in both SHRsp and WKY rats, but no significant change could be found in hypophyseal AVP content. The results suggest that decreased sensitivity of AVP inhibition as well as less marked hypotensive, diuretic and natriuretic effects to ANF in SHRsp might play a role in the pathogenesis of their hypertension.

Anti-shock effects of synthetic effective compositions of fructus aurantii immaturus. Experimental study and clinical observation.

Satisfactory results have been obtained in treating infective shock with injection of natural Fructus Aurantii immaturus (nat-FAI). The results of animal experiments and clinical observations on the anti-shock effects of the synthetic effective compositions of Fructus Aurantii immaturus (syn-FAI) are reported. The cardiac output increased from 0.53 to 0.87 L/min (P less than 0.01), and the cardiac index increased from 0.99 to 1.63 L/m2/min (P less than 0.01) in the endotoxic shock dogs after the treatment with syn-FAI. At the same time the blood stream in bulbar conjunctiva became accelerated and the dilated microvessels began to get smaller in most dogs. Of fifty children with infective shock treated with syn-FAI, forty-eight showed curative effects, with a total effective rate of 96%. The anti-shock effective compositions in FAI have been proved to be synephrine and N-methyltyrosamine. Moreover, syn-FAI has shown a more stable property, less side-effects and better clinical results than nat-FAI.

[Studies on the chemical constituents of Aconitum szechenyianum Gay].

Five constituents were isolated from Aconitum szechenyianum Gay. The structures of these compounds were identified as beta-sitosterol (I), aconitine (II), 3-acetylaconitine (III), songorine (IV) and a new compound whose structure was elucidated to be szechenyine (V) on the basis of UV, IR, 1HNMR, 13CNMR and Mass spectra.