Diet supplemented either with dried chicory root or chicory inulin significantly influence kidney and liver mineral content and antioxidative capacity in growing pigs.

According to the Regulation No. 1831/2003 of the European Parliament and European Union Council, the use of antibiotics as a dietary supplements has been prohibited. It seems that the administration of prebiotics, instead of antibiotics, into the pig's diet, may regulate the intestinal microbiota and has a long-term health-related impact on the host. Inulin-type fructans can stimulate mineral absorption from the gut. Additionally, it may regulate energy metabolism and activate enzymatic mechanisms preventing oxidative stress. The goal of the present study was to estimate the influence of dietary supplementation with dried chicory root or native chicory inulin on 1) liver histology; 2) liver and kidney lipid metabolism indices, activity of selected enzymes, concentration of macro- and micronutrients and heavy metals; 3) blood plasma, liver and kidney oxidative stress biomarkers and 4) blood plasma water-electrolyte homeostasis indices in growing pigs. The nutritional study was conducted on 24 piglets assigned to 3 dietary groups (n = 8): control (C) fed a basal diet and two experimental groups receiving basal diet supplemented with 2% of inulin (IN) either 4% of dried chicory root (CR). The animals were fed with a group-specific diets for 40 days and then subjected to euthanasia. Subsequently, blood, liver and kidney samples were harvested for further processing. In the control and experimental groups, no apparent morphological abnormalities in the liver tissues were seen. The percent of periodic acid Schiff positive glycogen liver cells was significantly lower in the CR group as compared to C and IN groups (P < 0.001). Chicory root supplementation improved blood plasma prooxidative-antioxidative balance - PAB (P < 0.001) and liver PAB (P < 0.01) and thiobarbituric acid reactive substances - thiobarbituric acid-reactive substances (P < 0.05). Feeding the CR diet increased calcium (P < 0.001) and potassium (P < 0.05) and decreased cadmium (P ≥ 0.05) content in the liver when compared to the C group. Administration of the CR and IN diets increased selenium (Se) and sodium concentrations, whereas decreased zinc content both in the liver (P < 0.01; P < 0.05 and P < 0.05, respectively) and in the kidney (P < 0.01; P < 0.001 and P < 0.001, respectively) of pigs. Additionally, a higher concentration of lead (P < 0.05) was observed in the kidney of pigs fed the CR diet. In conclusion, both dietary supplements had a potential to significantly improve the Se status and oxidoreductive homeostasis in growing pigs.

Effect of dietary inclusion of 1% or 3% of native chicory inulin on the large intestinal mucosa proteome of growing pigs.

Native chicory inulin is one of the promising alternatives to replace antibiotic growth promoters in young animals. Several potential mechanisms of prebiotic action have been proposed, such as modification of the intestinal microbiota composition leading to improved epithelial integrity and gut mucosal immunity of the host. The current study was focused on inulin effect on the large intestinal proteome and its implications for gut barrier functions. Therefore, we used proteomic techniques to determine changes in the large intestinal mucosa proteome of growing pigs after 40-day supplementation with native chicory inulin. The experiment was performed on 24 piglets fed from the 10th day of life an unsupplemented cereal-based diet or inulin-enriched diets (1% or 3%) with an average degree of polymerisation ≥ 10. At the age of 50 days, animals were sacrificed and tissue samples were collected from the cecum, and proximal and distal colon. Feeding diets supplemented with both levels of native inulin increased cecal and colonic expression of molecular chaperones, protein foldases and antioxidant proteins, which are collectively responsible for maintaining mucosal cell integrity as well as protecting against endotoxins and reactive oxygen species. This may confirm the beneficial effect of inulin on the gut health in growing pigs.

The current proteomic landscape of the porcine liver.

The main objective of the study was to create a reproducible protein map of the liver in healthy growing piglets. The analysis was performed on liver homogenates obtained from 8 castrated male piglets (PIC x Penerlan P76) at the 50 days. Two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry allowed to determine the proteomic profile of the liver. Liver proteins were separated at pH 4 - 7, followed by 12% SDS-PAGE. As a result, 470 ± 44 spots were present on the 2-D maps, of which 265 were successfully identified, representing products of 142 unique genes. Of these, 26 gene products have not been previously observed on the protein maps of porcine liver. Gene ontology analysis showed that the most of identified gene products belonged to the known metabolic pathways: protein processing in endoplasmic reticulum, arginine and proline metabolism, microbial metabolism in diverse environments, carbon metabolism, Epstein-Barr virus infection, propionate metabolism, biosynthesis of amino acids, proteasome. These results can undoubtedly serve as a useful and prospective prerequisite for the future analysis of the liver proteome changes in different physiological and pathophysiological conditions.

Sperm mitochondrial dysfunction and oxidative stress as possible reasons for isolated asthenozoospermia.

Reduced sperm motility, defined as asthenozoospermia, is a frequent cause of male infertility, and is mainly connected with the dysfunction of sperm mitochondria. The aim of this study was to identify the proteins, and thereby the metabolic pathways, responsible for asthenozoospermia, using 2-DE and MALDI-TOF MS, and correlate the results obtained with those of two mitochondrial tests: JC-1 and MitoSox Red. The JC-1 test was performed to test sperm mitochondrial activity, and the MitoSox Red test was performed to check whether the observed sperm poor motility is associated with mitochondrial reactive oxygen species (ROS) production. To identify proteins strictly connected with reduced sperm motility, men with isolated asthenozoospermia (n = 4 versus 10 normozoospermic controls) alone were included in the study. The proteomic analyses resulted in the identification of 25 sperm proteins that are differentially expressed in asthenozoospermic individuals. Most of the identified proteins were downregulated and were involved in energy production; however, we have also identified structural sperm proteins and proteins secreted by the epididymis. The latter, together with the results from MitoSox Red assay, may provide insights into the pathophysiological basis of asthenozoospermia.

An update on medium- and low-abundant blood plasma proteome of horse.

The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation - time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a result 136 spots were successfully identified, representing the products of 46 unique genes. Of these, 22 gene products have not been previously identified in horse serum/plasma samples using proteomic tools. Gene ontology analysis showed that almost 30% of all identified gene products belong to the coagulation and complement cascades. These results can undoubtedly serve as a useful and prospective prerequisite for the future analysis of horse plasma proteome changes in different physiological and pathophysiological conditions. The use of the medium- and low-abundant protein enrichment tool increased their abundance and allowed us to identify a higher number of protein gene products. The highest depletion efficiency was observed for the most abundant plasma proteins, that is albumin, IgG heavy chains and serotransferrin.

Dietary chicory root and chicory inulin trigger changes in energetic metabolism, stress prevention and cytoskeletal proteins in the liver of growing pigs - a proteomic study.

Currently, a wide array of plant preparations exerting health-promoting properties are commonly used as feed additives. Among them, Cichorium intybus L. have gained considerable attention as a source of compounds showing prebiotic character. Large body of evidence suggests that products of prebiotic fermentation (short-chain fatty acids) may influence the expression of genes encoding liver enzymes involved in the regulation of energetic metabolism. Given the above, the present study was aimed at estimating the influence of a diet supplemented with chicory root or water extract of chicory inulin on liver proteome in growing pigs. The study was performed on 24 castrated male piglets (PIC × Penarlan P76). Animals were assigned to three equal groups (n = 8) and fed cereal-based isoenergetic diets: control and supplemented with 2% of inulin extract from chicory root or 4% of dried chicory root. Liver proteins were separated using two-dimensional electrophoresis, followed by the identification of statistically valid protein spots with the aid of MALDI-TOF mass spectrometry. Both experimental factors significantly modulated the expression of liver proteins associated with energetic metabolism, particularly those involved in cholesterol and triglyceride metabolism. Additionally, both dietary additives induced increased expression of proteins involved in hepatocyte protection against oxidative stress. In the present study, we have shown for the first time that diet supplementation with dried chicory root or inulin caused significant changes in the expression of liver cytoskeletal proteins. Close attention should be paid to the downregulation of cytokeratin 18, hepatic acute phase protein that can enhance the anti-inflammatory properties of inulin-type fructans.

Differentially expressed proteins in the blood serum of piglets in response to a diet supplemented with inulin.

In the present study we introduced a two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation time of flight mass spectrometry-based proteomic workflow to identify proteins that show altered expression as a result of the addition of 2% of water extract of inulin-type fructans to the diet of growing piglets. This analysis allowed us to detect an average of 240 spots per gel with a mass range from 10 to 250 kDa and a pH ranging from 3 to 10. Twenty protein spots were found to show statistically significant differences in their expression. Of these, 7 protein spots were up-regulated, whereas 13 showed down-regulation in response to the experimental diet. In total, 13 spots were identified, representing 8 distinct gene products. The experimental diet caused a significant change in proteins directly or indirectly involved in hemostasis and the innate immune response. Increased levels of fibrinogen along with decreased plasminogen expression may indicate that a fructan-rich diet favours the deposits of fibrin and promotes blood clotting. We also found increased expression of vitronectin and the alpha subunit of the complement component C8 which may protect the host organism against excessive cytolitic activity of the activated complement. The piglets from the experimental group had slightly increased values of IgG and IgA, whereas the IgM level tended to be decreased. The fructan-rich diet did not have any influence on plasma total cholesterol, HDL and LDL cholesterol and triglyceride levels.

Feeding milk replacer instead of whole milk affects blood plasma proteome and lipid profile in preruminant calves.

The study was undertaken to determine the effect of feeding milk or milk-replacer on the blood plasma proteome and lipid profile in calves during the second week of life. Feeding milk-replacer significantly decreased the expression of plasma apoA-I. Age of calves affected apoA-I expression, which was higher on the 8th than on the 11th and 14th day of life. A significant effect of interaction between diet and age was also observed. The expression of apoA-IV, was significantly affected by diet and was lower in calves fed milk replacer. Expression of this protein was significantly lower at the 8th day of life and was up-regulated in the calves fed milk-replacer at the second week of life. Calves fed milk-replacer had greater expression of haptoglobin, which differed significantly between days of blood sampling, being higher on the 8th than on the 11th and 14th day. The interactive effect of diet and age affected haptoglobin expression, which was successively down-regulated in calves fed milk re- placer. Diet had a significant effect on the plasma lipid profile. Animals fed milk had a greater concentration of TC, HDLC and LDLC. The composition of milk-replacer, especially fat source, is probably the main factor that affects expression of proteins involved in cholesterol metabolism and level of components of lipid profile in calves fed formula. We claim that the initially increased level of haptoglobin, followed by its decrease during the second week of life in calves fed milk-replacer may indicate the presence of short-term stress induced by changes in the feeding system.

Dietary supplementation with dried chicory root triggers changes in the blood serum proteins engaged in the clotting process and the innate immune response in growing pigs.

The aim of the study was to characterize the systemic immune and metabolic alterations in the blood serum of growing pigs in response to a dietary supplementation with 4% of dried chicory roots. This was achieved by examining the influence of the experimental diet on serum protein changes especially these related with immunology and lipid metabolism. Serum proteins with the isoelectric point ranging from pH 3.0 to 10.0 were separated using high resolution two-dimensional electrophoresis. As a result, we found that experimental diet triggered significant changes in 37 protein spots. Of these, 14 were up-regulated, whereas 23 showed down-regulation. Of 37 significantly altered protein spots, 24 were successfully identified, representing 14 distinct gene products. Implementation of the dried chicory roots into the diet of growing pigs caused a significant down-regulation of apolipoprotein C-II complement component C6, C-reactive protein, CD14 antigen, C4b binding protein α and ß chains, and fibrinogen. Piglets fed experimental diet had similar IgA, IgG and IgM concentrations, although the level of IgM tended to be lower compared to the control group. It is concluded that diet supplemented with 4% of dried chicory root may exert anti-inflammatory properties and affect lipid metabolism in growing pigs.

Proteome of spleen CD4 lymphocytes in mouse preimplantation pregnancy.

Pregnancy exerts profound impact on female immune system. The first signs of pregnancy recognition by immune system are observed even before implantation. The most visible effects are present in the local compartment, i.e. in uterine draining lymph nodes and the decidua, while peripheral changes are less obvious. In our recent paper we indicated that costimulation phenotype of APCs in spleens of female mice during the preimplantation period of pregnancy differs from mice in pseudopregnancy. However, the effect of differential costimulation in the context of the T lymphocyte function at periphery in early pregnancy is still unknown. For that reason, we decided to investigate global protein expression in splenic CD4(+) lymphocytes in order to identify and validate the most important biomarkers characteristic for the preimplantation period of pregnancy at periphery. Two-dimensional electrophoresis (2-DE) and mass spectrometry (MS) were utilized to analyze the protein expression pattern of magnetically sorted CD4(+) lymphocytes from spleens of pregnant and pseudopregnant females at 3.5 days after mating. The first goal of this study was to create a 2-DE map of the splenic CD4(+) T cells of pregnant mice. As a result, 106 protein spots from 373 were identified using MS. The comparison of lymphocyte protein patterns between pregnant and pseudopregnant mice depicted differential expression of 11 identified proteins belonging to the group of proteins involved in cytoskeletal structure, cell motility and metabolism. Profoundly diminished expression of cofilin-1, F-actin capping protein subunit alpha and malate dehydrogenase proteins in lymphocytes of pregnant mice indicates that preimplantation pregnancy could change the activation state of peripheral CD4(+) lymphocytes.

Immunohistochemical identification of aquaporin 2 in the kidneys of young beef cattle.

Aquaporin 2 (AQP2) is a small, integral tetrameric plasma membrane protein that is expressed in mammalian kidneys. The specific constitution of this protein and its selective permeability to water means that AQP2 plays an important role in hypertonic urine production. Immunolocalization of AQP2 has been studied in humans, monkeys, sheep, dogs, rabbits, rats, mice and adult cattle. We analyzed the expression of AQP2 in kidneys of 7-month-old Polish-Friesian var. black and white male calves. AQP2 was localized in the principal cells of collecting ducts in medullary rays penetrating the renal cortex and in the collecting ducts of renal medulla. AQP2 was expressed most strongly in the apical plasma membrane, but expression was observed also in the intracellular vesicles and basolateral plasma membrane. Our study provides new information concerning the immunolocalization of AQP2 in calf kidneys.

Blood plasma protein and lipid profile changes in calves during the first week of life.

The present study was undertaken to determine blood plasma protein and lipid profile changes in healthy Polish Holstein-Fresian calves of Black-and-White variety. Blood was drawn immediately after birth, before first colostrum intake and at the 3rd, 6th, 12th, 24th, 36th, 48th and 72nd hour of life. Subsequent four blood samples were collected at 24 hour intervals until the 7th day of life. Plasma proteins within the isoelectric point ranging from 3.0 to 10.0 were separated using high resolution two-dimensional electrophoresis. Among the 74 protein spots detected and analyzed, 16 were significantly altered during the first week of life. Differentially expressed spots were excised from the gels and subjected to peptide mass fingerprinting using MALDI-TOF MS. In total, 12 spots were successfully identified, which correspond to three proteins, namely: apolipoprotein A-I, apolipoprotein A-IV and fibrinogen gamma-B chain. A gradual increase in plasma triglyceride, total cholesterol, HDL and LDL cholesterol values was shown during the first seven days of calves life. The lowest concentration of these indicators were observed at birth and was followed by a rapid increase during the first week of postnatal life. These changes appear to be related to the transition in energy sources, from a maternal nutrient supply comprising mainly carbohydrates and amino acids to a diet which was rich in fat--colostrum and milk. This was reflected by the intense up-regulation of plasma proteins related with lipid transport and lipoprotein metabolism during the first week of life.

Defining the blood plasma protein repertoire of seven day old dairy calves - a preliminary study.

During the early postnatal period in calves various adaptational changes occur. These functional, morphological and also metabolic alteration are reflected by blood plasma protein changes as they are secreted and shed from many cells and tissues. Blood plasma protein pattern of an adult cattle differs in some respect when compared with neonatal calves. There exist a very few data concerning 2-D maps of neonatal calves blood plasma. The above prompted us to establish protein pattern of this biological fluid characteristic of healthy, 7 day old, Polish Black-and-White (Polish Friesian) breed calves. Blood plasma proteins of the isoelectric point ranging from 4.0 to 7.0 were analyzed by the aid of high resolution two-dimensional electrophoresis (2-DE). Subsequently, 79 excised protein spots corresponding to 23 different gene products were identified using matrix-assisted laser desorption/ionisation mass spectrometer (MALDI-TOF MS). Protein map obtained in the present study may be useful in assessing the changes in the calves blood plasma protein profiles occurring in response to different physiological and/or pathophysiological factors.

Urinary excretion of low molecular weight proteins in goats during the neonatal period.

Urinary protein excretion occurs in neonates of many animal species, as well as in human neonates. However, the incidence, dynamics, and mechanism of proteinuria have not been unambiguously explained. The aims of this study were to investigate into excretion of selected protein fractions of molecular weight less than 69 kDa (LMW), evaluation of intensity and dynamics of changes during the first month of kids' life, and an attempt to explain the causes of neonatal proteinuria. The analysis were carried out on 16 kids of White Improved goats, over the period from birth until 30 days of age, using clearance methods. Urine proteins were separated electrophoretically (SDSPAGE), and their concentration and percentage content was determined by densitometric method with the use of archiving and image analysis software. The proteins found in the urine were grouped as HMW, LMW and albumin. For six fractions of LMW proteins, excretion rates and percentage content of the urinary total LMW protein pool were calculated. It has been demonstrated that neonatal proteinuria in goat kids is associated with a high level of excretion of proteins of lower molecular weight than albumin (69 kDa). A strong dynamics of changes in excretion of particular LMW protein fractions with age was observed, which may imply not only an increased permeability of glomerular filtration barrier, especially over the first days of life, but also a selectivity of reabsorption mechanisms in the nephrons. An increased permeability of glomerular filtration barrier for proteins during the first days of life may represent the adaptive mechanism for removal of protein excess from the organism. The urinary LMW protein pool may also contain proteins resulting from the hydrolysis in the tubular cells.