Agrimonia procera exerts antimicrobial effects, modulates the expression of defensins and cytokines in colonocytes and increases the immune response in lipopolysaccharide-challenged piglets.

BACKGROUND: Because antibiotic use in livestock is assumed to contribute to the emerging public health crisis of antibiotic resistance, alternatives are required. Phytogenic additives are extensively studied due to their antibiotic properties. Components of Agrimonia species have been reported as candidate antimicrobials that possess antioxidative and anti-inflammatory properties. We studied the impact of Agrimonia procera (AP) on the growth of selected strains of gut bacteria, the effect of AP on the mRNA abundance of genes involved in inflammation and bacterial defense in a colon carcinoma cell line, the effect of AP in piglets challenged with lipopolysaccharides, and the effect of AP on the growth performance of healthy piglets. RESULTS: The in vitro growth rate of different bacteria strains was negatively affected by AP, especially in Pediococcus pentosaceus and all tested E. coli strains. Stimulation of Caco-2 cells with TNFα resulted in elevated mRNA expression of CXCL1, IL-8 and GPX2. After pretreatment of cells with AP, stimulation of Caco-2 cells with TNFα still resulted in elevated mRNA expression of CXCL1 and IL-8 at all measured points in time. However, mRNA expression in AP-pretreated cells was lower after 6 h and 24 h. In addition, expression of DEFB1 and GPX2 was significantly elevated after TNFα stimulation. In vivo, application of lipopolysaccharides induced significantly increased animal body temperatures. Piglets pretreated with AP prior to lipopolysaccharide application showed a faster and larger increase in body temperature than controls. In addition, piglets pretreated with AP appeared to release more TNFα than controls. In healthy piglets, AP treatment had no impact on growth performance parameters. Fecal dry matter and total plasma antioxidant capacity tended to be higher in piglets treated with AP than in control piglets (P = 0.055 and P = 0.087, respectively). CONCLUSIONS: AP has antimicrobial effects in vitro and stimulated the expression of proinflammatory cytokines in Caco-2 cells. The additive had no effect on growth in healthy piglets but increased the immune response in LPS-treated animals. In addition, AP appeared to have antioxidative effects in vivo. Therefore, AP merits testing as a future alternative to antibiotics in animal husbandry.

The phytochemical investigation of Agrimonia eupatoria L. and Agrimonia procera Wallr. as valid sources of Agrimoniae herba--The pharmacopoeial plant material.

The agrimony herb is a traditional plant drug, which is commonly used as a mildly astringent agent. According to European Pharmacopoeia, the only source of this plant drug is Agrimonia eupatoria. By contrast the German Commission E pharmacopoeial monograph used to allow Agrimonia procera to be used as a second valid source of Agrimoniae herba. Several studies have been conducted on the phytochemical composition of common agrimony. The data on the phytochemistry of A. procera are scarce. The aim of the present study was an in-depth phytochemical comparison of A. eupatoria and A. procera in the context of the pharmacopoeial monograph of A. herba. The comparison of two agrimony species showed that there are no significant qualitative differences. The quantitative HPLC analysis revealed that fragrant agrimony is a much better source of agrimoniin than common agrimony. This difference could not be detected using the pharmacopoeial method of quantification for the total tannin content. The present study has shown for the first time the possible use of apigenin-C-glycosides (vitexin and isovitexin) as chemotaxonomic markers for distinguishing both agrimony species. The potential chemical markers such as apigenin-7-O-glucoside and high agrimoniin content were also suggested for fragrant agrimony. Based on the data obtained, A. procera should be considered as a valid source of pharmacopoeial plant material.

UVB exposure of farm animals: study on a food-based strategy to bridge the gap between current vitamin D intakes and dietary targets.

Vitamin D deficiency is a global health problem. This study aimed to investigate the efficacy of ultraviolet (UV) B radiation for improving vitamin D3 content of eggs and meat. In a two-factorial design hens that received diets with 0 (-D3) or 3,000 IU (+D3) vitamin D3/kg were non-exposed (-UVB) or exposed to UVB radiation (+UVB) for 3 h daily over 4 weeks. Data show that UVB radiation was very effective in raising the vitamin D3 content of egg yolk and meat. Egg yolk from +UVB/-D3 hens had a higher vitamin D3 content (17.5±7.2 µg/100 g dry matter (DM)) than those from the -UVB/+D3 group (5.2±2.4 µg/100 g DM, p<0.01). Vitamin D3 content in egg yolk of vitamin D3-supplemented hens could be further increased by UVB radiation (32.4±10.9 µg/100 g DM). The content of 25-hydroxyvitamin D3 (25(OH)D3) in the egg yolk also increased in response to UVB, although less pronounced than vitamin D3. Meat revealed about 4-fold higher vitamin D3 contents in response to UVB than to dietary vitamin D3 (p<0.001). In conclusion, exposure of hens to UVB is an efficient approach to provide consumers with vitamin D3-enriched foods from animal sources.

Effects of dietary benzoic acid and sodium-benzoate on performance, nitrogen and mineral balance and hippuric acid excretion of piglets.

The objective of this study was to compare the effects of sodium-benzoate (NaB) with those of benzoic acid (BAc) on growth performance of piglets as well as nutrient digestibility, nitrogen and mineral balance, urinary pH, and the urinary excretion of BAc and hippuric acid (HAc). The study was conducted with 120 weaning piglets (6.5 kg body weight), divided in four groups (15 replicates of two piglets each), which received (1) a basal diet (Control), or the basal diet supplemented with (2) 4 g NaB per kg (Group 4NaB), (3) 3.5 g BAc per kg (Group 3.5BAc) or (4) 5 g BAc per kg (Group 5BAc). Performance data were monitored over a 42-day period. Urine and faeces were collected from day 28-33 in metabolic cages with five piglets per treatment. Piglets of Groups 3.5BAc and 5BAc had similarly a considerably improved average daily gain and feed intake (p < 0.05). Performance of Group 4NaB was not significantly different from the other groups. Compared to the Control, the nitrogen retention was only improved in Group 5BAc (p < 0.05); the other groups showed intermediate values. In the supplemented groups, most of the BAc was excreted as HAc in urine, but only Groups 3.5BAc and 5BAc had reduced urinary pH (p < 0.05). Daily intake and faecal and urinary excretion of P and Ca were not affected by the treatment. The molar excess of Na in Group 4NaB was reflected by higher renal excretion of Na compared to the other groups (p < 0.05).

Influence of broccoli extract and various essential oils on performance and expression of xenobiotic- and antioxidant enzymes in broiler chickens.

The aim of our present study was to examine the regulation of xenobiotic- and antioxidant enzymes by phytogenic feed additives in the intestine and the liver of broilers. A total of 240 male Ross-308 broiler chickens (1 d old) were fed a commercial starter diet for 2 weeks. On day 15, the birds were assigned to six treatment groups of forty birds each. The control (Con) group was fed a diet without any additive for 3 weeks. The diet of group sulforaphane (SFN) contained broccoli extract providing 0.075 g/kg SFN, whereas the diets of the other four groups contained 0.15 g/kg essential oils from turmeric (Cuo), oregano (Oo), thyme and rosemary (Ro). Weight gain and feed conversion were slightly impaired by Cuo and Oo. In the jejunum SFN, Cuo and Ro increased the expression of xenobiotic enzymes (epoxide hydrolases 1 and 2 and aflatoxin B1 aldehyde reductase) and of the antioxidant enzyme haeme oxygenase regulated by an 'antioxidant response element' (ARE) compared to group Con. In contrast to our expectations in the liver, the expression of these enzymes was decreased by all the additives. Nevertheless, all the additives increased the Trolox equivalent antioxidant capacity of the jejunum and the liver and reduced Fe-induced lipid peroxidation in the liver. We conclude that the up-regulation of ARE genes in the small intestine reduces oxidative stress in the organism and represents a novel mechanism by which phytogenic feed additives improve the health of farm animals.

Effect of L-carnitine on the hepatic transcript profile in piglets as animal model.

BACKGROUND: Carnitine has attracted scientific interest due to several health-related effects, like protection against neurodegeneration, mitochondrial decay, and oxidative stress as well as improvement of glucose tolerance and insulin sensitivity. The mechanisms underlying most of the health-related effects of carnitine are largely unknown. METHODS: To gain insight into mechanisms through which carnitine exerts its beneficial metabolic effects, we fed piglets either a control or a carnitine supplemented diet, and analysed the transcriptome in the liver. RESULTS: Transcript profiling revealed 563 genes to be differentially expressed in liver by carnitine supplementation. Clustering analysis of the identified genes revealed that most of the top-ranked annotation term clusters were dealing with metabolic processes. Representative genes of these clusters which were significantly up-regulated by carnitine were involved in cellular fatty acid uptake, fatty acid activation, fatty acid ß-oxidation, glucose uptake, and glycolysis. In contrast, genes involved in gluconeogenesis were down-regulated by carnitine. Moreover, clustering analysis identified genes involved in the insulin signaling cascade to be significantly associated with carnitine supplementation. Furthermore, clustering analysis revealed that biological processes dealing with posttranscriptional RNA processing were significantly associated with carnitine supplementation. CONCLUSION: The data suggest that carnitine supplementation has beneficial effects on lipid and glucose homeostasis by inducing genes involved in fatty acid catabolism and glycolysis and repressing genes involved in gluconeogenesis.

Dietary L-carnitine alters gene expression in skeletal muscle of piglets.

SCOPE: Carnitine improves protein accretion, muscle mass, and protein:fat accretion in piglets. The underlying mechanisms, however, are largely unknown. METHODS AND RESULTS: To gain insight into mechanisms through which carnitine exerts these effects, we fed piglets either a control or a carnitine-supplemented diet, and analyzed the transcriptome in skeletal muscle. Carnitine concentrations in plasma and muscle were about four-fold higher in the carnitine group when compared to the control group. Transcript profiling revealed 211 genes to be differentially expressed in muscle by carnitine supplementation. The identified genes were mainly involved in molecular processes such as cytoskeletal protein binding, insulin-like growth factor (IGF) binding, transcription factor activity, and insulin receptor binding. Identified genes with the molecular function transcription factor activity encoded primarily transcription factors, most of which were down-regulated by carnitine, including pro-apoptotic transcription factors such as proto-oncogene c-fos, proto-oncogene c-jun and activating transcription factor 3. Furthermore, atrophy-related genes such as atrogin-1, MuRF1, and DRE1 were significantly down-regulated by carnitine. IGF signalling and insulin signalling were identified as significantly up-regulated regulatory pathways in the carnitine group. CONCLUSION: Carnitine may have beneficial effects on skeletal muscle mass through stimulating the anabolic IGF-1 pathway and suppressing pro-apoptotic and atrophy-related genes, which are involved in apoptosis of muscle fibers and proteolysis of muscle proteins, respectively.

Supplementation of L-carnitine in pigs: absorption of carnitine and effect on plasma and tissue carnitine concentrations.

This study was performed to investigate the bioavailability of carnitine supplements and their effects on the carnitine status of pigs. Seven groups of young pigs with an average body weight of 10 kg were fed a basal diet or the same diets supplemented with 25, 50, 100, 200, 500 or 1000 mg of L-carnitine per kg for 20 days. Absorption rate of the supplemented carnitine in the small intestine, assessed by the use of titanium dioxide as an indigestible indicator, was greater than 95% for the lower doses (25, 50, 100 mg/kg) and greater than 90% for the higher doses (200, 500, 1000 mg/kg). Supplementation of carnitine caused a dose-dependent increase of free carnitine, acetyl and total carnitine concentrations in plasma, liver, kidney, heart and skeletal muscle. At the highest dose of 1000 mg/kg, plasma and tissue total carnitine concentrations were 3- to 6-fold higher than in the unsupplemented control group. In conclusion, the present study shows that young pigs have a high capacity to absorb carnitine from the diet. It is also shown that plasma and tissue carnitine concentrations in young pigs can be markedly increased by supplementation of carnitine.

Effect of xylanase on apparent ileal and total tract digestibility of nutrients and energy of rye in young pigs.

A digestibility experiment was carried out on weanling piglets to study the effect of an enzyme complex with predominant xylanase activity on apparent ileal (AID) and apparent total tract digestibility of nutrients and energy. The enzyme was supplemented at four levels (0, 50, 100 and 200 mg/kg) to a diet containing 96% rye. There were significant effects of the added enzyme on AID of dry matter, organic matter and crude fibre, and on apparent total tract digestibility of dry matter, organic matter and energy. However, the improvements in the digestibility were rather small. Except for galactose, there was a significant response in AID of all non-starch polysaccharide constituents to enzyme supplementation, the greatest effect being found at 100 mg/kg. The improvement in AID of arabinose + xylose (685%) was much higher than that of the remaining sugars (110%). AID of galactose was negative in all dietary treatments, presumably due to its high concentration in endogenous secretions. There was a significant response in AID of the sum of essential and total amino acids to the increased level of the enzyme. It is concluded that the enzyme complex is efficient in degrading dietary fibre components, thus improving the digestibility of organic matter, amino acids and energy.

Clofibrate treatment up-regulates novel organic cation transporter (OCTN)-2 in tissues of pigs as a model of non-proliferating species.

Recent studies have shown that treatment of rodents with agonists of peroxisome proliferator-activated receptor (PPAR)-alpha causes an up-regulation of novel organic cation transporter (OCTN)-2, a carnitine transporter, and increases carnitine concentration in the liver. This study was performed to investigate whether such effects occur also in pigs which like humans have a lower expression of PPAR alpha and are less responsive to treatment with PPAR alpha agonists than rodents. An experiment with 18 pigs was performed which were fed a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had higher relative mRNA concentrations of OCTN2 in liver (3.1-fold), skeletal muscle (1.5-fold) and epithelial cells from small intestine (1.8-fold) than control pigs (P<0.05). Pigs treated with clofibrate had also higher concentrations of free and total carnitine in the liver and a higher concentration of free carnitine in skeletal muscle than control pigs (P<0.05). Concentrations of gamma-butyrobetaine, the precursor of endogenous formation of carnitine, in liver, muscle and plasma did not differ between both groups; the activity of gamma-butyrobetaine dioxygenase, the rate limiting enzyme of carnitine synthesis, in the liver was lower in pigs treated with clofibrate than in control pigs (P<0.05). This study shows for the first time that treatment with a PPAR alpha agonist causes an up-regulation of OCTN2 in liver, muscle and enterocytes from small intestine of pigs. This in turn increases carnitine concentrations in liver and muscle probably by enhancing carnitine uptake into cells.

Clofibrate causes an upregulation of PPAR-{alpha} target genes but does not alter expression of SREBP target genes in liver and adipose tissue of pigs.

This study investigated the effect of clofibrate treatment on expression of target genes of peroxisome proliferator-activated receptor (PPAR)-alpha and various genes of the lipid metabolism in liver and adipose tissue of pigs. An experiment with 18 pigs was performed in which pigs were fed either a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had heavier livers, moderately increased mRNA concentrations of various PPAR-alpha target genes in liver and adipose tissue, a higher concentration of 3-hydroxybutyrate, and markedly lower concentrations of triglycerides and cholesterol in plasma and lipoproteins than control pigs (P < 0.05). mRNA concentrations of sterol regulatory element-binding proteins (SREBP)-1 and -2, insulin-induced genes (Insig)-1 and Insig-2, and the SREBP target genes acetyl-CoA carboxylase, 3-methyl-3-hydroxyglutaryl-CoA reductase, and low-density lipoprotein receptor in liver and adipose tissue and mRNA concentrations of apolipoproteins A-I, A-II, and C-III in the liver were not different between both groups of pigs. In conclusion, this study shows that clofibrate treatment activates PPAR-alpha in liver and adipose tissue and has a strong hypotriglyceridemic and hypocholesterolemic effect in pigs. The finding that mRNA concentrations of some proteins responsible for the hypolipidemic action of fibrates in humans were not altered suggests that there were certain differences in the mode of action compared with humans. It is also shown that PPAR-alpha activation by clofibrate does not affect hepatic expression of SREBP target genes involved in synthesis of triglycerides and cholesterol homeostasis in liver and adipose tissue of pigs.

Clofibrate increases hepatic triiodothyronine (T3)- and thyroxine (T4)-glucuronosyltransferase activities and lowers plasma T3 and T4 concentrations in pigs.

In rats, clofibrate acts as a microsomal enzyme inducer and disrupts the metabolism of thyroid hormones by increasing hepatic glucuronidation of thyroxine. Whether similar effects occur in the pig has not yet been investigated. This study was performed to investigate the effect of clofibrate treatment on metabolism of thyroid hormones in pigs. To this end, an experiment with 18 pigs, which were assigned to two groups, was performed. One group received a control diet, and the other group was fed the same diet supplemented with 5 g of clofibrate/kg for 28 days. Pigs treated with clofibrate had higher hepatic activities of T(3)- and T(4)-UDP glucuronosyltransferases (UGT) and lower concentrations of total and free T(4) and total T(3) in plasma than control pigs (P < 0.05). Weights and histology of the thyroid gland (epithelial height, follicle lumen diameter) did not differ between the two groups, but pigs treated with clofibrate had higher mRNA concentrations of various genes in the thyroid responsive to thyroid-stimulating hormone (TSH) such as TSH receptor, sodium iodine symporter, thyroid peroxidase, and cathepsin B than control pigs (P < 0.05). Pigs treated with clofibrate also had lower hepatic mRNA concentrations of proteins involved in plasma thyroid hormone transport [thyroxine-binding globulin (P < 0.10), transthyretin (P < 0.05), and albumin (P < 0.05)] and thyroid hormone receptor alpha(1) (P < 0.05) than control pigs. In conclusion, this study shows that clofibrate treatment induces a strong activation of T(3)- and T(4)-UGT in pigs, leading to increased glucuronidation and markedly reduced plasma concentrations of these hormones, accompanied by a moderate stimulation of thyroid function.

Nutrient composition and concentrations of immunoglobulins in milk of sows supplemented with L-carnitine.

Recent studies have shown that L-carnitine supplementation of sows increases growth of their piglets during the suckling period. In this study, the composition of the milk of sows supplemented with L-carnitine was determined to find out whether an altered milk composition could account for the increased growth rates of the piglets. Milk of 13 control sows and 14 sows supplemented with L-carnitine (125 mg/d during pregnancy, 250 mg/d during lactation) was collected 5-8 h after birth (colostrum) and on days 10 and 20 of lactation. Concentrations of fat and lactose and the energy content in milk at day 10 and 20 did not differ between both groups of sows. Sows supplemented with L-carnitine had a higher concentration of protein in colostrum (p < 0.05) while concentrations of fat, lactose, immunoglobulins G, M and A as well as the energy content in colostrum did not differ between both groups of sows. These findings show that milk composition does not play a major role for the increased postnatal growth of piglets from sows supplemented with L-carnitine observed in recent studies.

L-carnitine supplementation of sows during pregnancy improves the suckling behaviour of their offspring.

It has been shown that L-carnitine supplementation of sows increases their milk production and the postnatal growth of the suckling piglets. To test the hypothesis that this effect is due to an improved suckling behaviour of the piglets, two experiments with sows were performed. Two groups of thirteen or ten sows each (in experiments 1 and 2, respectively) were fed diets with or without supplemental L-carnitine during pregnancy (125 mg/d) and lactation (250 mg/d). After birth, the litters of all sows were standardised to equal sizes of eleven and nine piglets per litter in experiments 1 and 2, respectively. In experiment 1, the piglets of L-carnitine-supplemented sows had a higher total suckling time per day on days 3, 6 and 9, and greater weight gains during the suckling period, than the piglets of control sows (P<0.05). In experiment 2, all litters were taken away from their mothers and switched to other sows. Half of the control sows and half of the L-carnitine-supplemented sows were given litters born to control sows, the other half of each group being given litters born to L-carnitine-supplemented sows. Piglets born to L-carnitine-supplemented sows had a higher total suckling time per day on day 3 and greater body weight gains during the first 14 d compared with piglets born to control sows (P<0.05). This study shows that piglets born to sows supplemented with L-carnitine are able to suckle for longer, which enables them to obtain more milk and grow faster than piglets born to control sows.

Body composition, muscle fibre characteristics and postnatal growth capacity of pigs born from sows supplemented with L-carnitine.

Recently, it has been shown that supplementation of sows with L-carnitine increases their plasma concentrations of insulin-like growth factor (IGF)-I, and it has been hypothesized that this may stimulate fetal myogenesis. This study was performed to investigate whether piglets of sows supplemented with L-carnitine differ in muscle fibre characteristics, chemical body composition and postnatal growth capability from pigs of control sows. Muscle fibre characteristics and chemical body composition were determined at weaning in 21 piglets of control sows and 21 piglets of sows treated with L-carnitine with similar body weights; postnatal growth capability was determined from weaning until slaughter at a body weight of 118 kg in 80 pigs of control sows and 80 pigs of sows treated with L-camitine which had also similar body weights at weaning. Piglets of sows supplemented with L-carnitine did not differ in number, area, diameter and type (percentages of slow twitch oxidative + fast twitch oxidative fibres, and fast twitch glycolytic fibres) of muscle fibres in m. longissimus dorsi and m. semitendinosus and in chermical body composition (concentrations of dry matter, crude protein, crude fat) from piglets of control sows. Postnatal growth capability (body weight gains, feed conversion ratio) from weaning to slaughter as well as carcass composition (carcass yield, meat thickness, fat thickness) was also not different between pigs of sows treated with L-carnitine and pigs of control sows. In conclusion, data of this study do not support the hypothesis that L-carnitine supplementation of sows during pregnancy enhances fetal muscle fibre development and increases postnatal growth capability of the offspring.

Concentrations of cholesterol oxidation products in raw, heat-processed and frozen-stored meat of broiler chickens fed diets differing in the type of fat and vitamin E concentrations.

The present study was performed to investigate the effect of dietary fat and vitamin E on concentrations of cholesterol oxidation products (COP) in broiler muscle. A total of 144 1-d-old broiler chicks were fed diets with either palm oil, soyabean oil or linseed oil and vitamin E concentrations of 20, 40 or 200 mg/kg for 35 d. COP concentrations were analysed in raw, heat-processed (180 degrees C, 20 min) and frozen-stored (-20 degrees C, 6 months) breast and thigh muscles. COP concentrations were influenced by dietary vitamin E concentration, dietary fat, treatment and type of muscle (P<0.001). Increasing the dietary vitamin E concentration generally reduced the concentration of COP. This effect was strongest in broilers fed linseed oil and weakest in broilers fed palm oil; the effect of vitamin E was also stronger in heated muscles than in raw or frozen-stored muscles. Moreover, the concentration of COP in thigh muscle was more strongly influenced by dietary vitamin E than that in breast muscle. COP concentrations in muscles were on average highest in broilers fed linseed oil and lowest in broilers fed palm oil, but the effect of the dietary fat also depended on the vitamin E concentration, the treatment and the type of muscle. In conclusion, our study shows that dietary fat and vitamin E influence the concentrations of total COP in broiler muscle. However, the effects of these factors were not only influenced by interactions between each other, but also depended on the treatment of the muscle and the type of muscle.

Supplementation of sows with L-carnitine during pregnancy and lactation improves growth of the piglets during the suckling period through increased milk production.

Recent studies showed that piglets of sows fed diets supplemented with L-carnitine grow faster during the suckling period than piglets of control sows fed diets without L-carnitine. This study was undertaken to investigate the effect of L-carnitine supplementation in sows on milk production and milk constituents. An experiment was performed in which two groups of 20 gilts each were fed diets with or without supplemental L-carnitine during pregnancy (0 vs. 125 mg L-carnitine daily/sow) and lactation (0 vs. 250 mg L-carnitine daily/sow). The experiment was continued over two reproductive cycles. L-carnitine-treated sows had larger litters (P<0.01) and higher litter weights (P<0.05) than control sows. Piglets of L-carnitine-treated sows had lower birth weights (P<0.05) but grew faster during the suckling period (P<0.01) and were heavier (P<0.05) at weaning than piglets of control sows. L-carnitine-treated sows had higher milk yields on d 11 and 18 of lactation than control sows (P<0.05). Milk of L-carnitine-treated sows had higher concentrations of total and free carnitine than milk of control sows (P<0.001); concentrations of fat, protein and lactose and the amounts of gross energy in the milk did not differ between the two groups of sows. The amounts of protein (P<0.05) and lactose (P<0.05) were higher in L-carnitine-treated sows than in control sows; the amount of energy secreted with the milk tended to be higher in carnitine-treated sows than in control sows (P<0.10). The study suggests that piglets of carnitine-treated sows grow faster during the suckling period than those of control sows because they ingest more nutrients and energy with the milk.