Einfluss des Flavonols Quercetin auf Glucosestoffwechsel und Gesundheits- status beim neugeborenen Kalb

With birth, calves are confronted with a new, extrauterine environment and have to meet their energy demand autonomously. For this reason, it is essential that not only overall energy intake, but also endogenous glucose production work effectively, because glucose is one of the main energy sources during the first stage of life. With the intake of first colostrum calves ingest not only nutrients, but also a plethora of bioactive factors which support the maturation and development of metabolic processes. Colostrum is further essential to support the naïve immune system of newborn calves during the first weeks of life and to mediate passive immunity. Unfortunately, colostrum supply is often inadequate. To support the health status, flavonoids could play an important role in the upbringing of newborn calves. These are secondary plant metabolites with numerous attributed health-promoting properties. One of the most important flavonoids is quercetin, which is proven to possess antioxidative, anti-inflammatory and antimicrobial capacity and to modulate the intestinal microflora. However, quercetin also interacts with glucose metabolism by inhibiting intestinal carbohydrate absorption and reducing plasma glucose concentrations. For newborn calves, such an effect would be crucial. Therefore, it was the main objective of the present work to investigate the effects of an oral quercetin supplementation on the glucose metabolism of newborn calves, because this knowledge would be the prerequisite to consider the usage of quercetin as a health promoting feed additive in calf rearing. In the first part of the study, 28 newborn male Holstein Friesian calves were assigned to two dietary groups and fed colostrum or a milk-based formula with same macronutrient composition, but without bioactive factors, during the first two days of life. On d 2 of life, groups were subdivided into control and treatment groups, the latter receiving quercetin aglycone with meals during the first week of life. On d 3, intestinal xylose absorption was probed, on d 7, a tracer study was conducted to investigate the first pass uptake of glucose, and on d 8, a liver biopsy sample was taken and analyzed via PCR. The postabsorptive recovery rate of orally administered xylose and 13C6-labelled glucose in plasma was higher in calves that initially received colostrum, indicating a better intestinal absorption capacity and a lower splanchnic glucose extraction when compared to colostrum-deprived calves. Irrespective of quercetin supplementation, the mRNA abundance of hepatic mitochondrial phosphoenolpyruvate carboxykinase and several plasma metabolites were also reduced in formula-fed calves, pointing to a delayed maturation of metabolic pathways after colostrum deprivation. Although quercetin-fed calves initially showed higher peak concentrations of 13C6-glucose in plasma on d 7, this effect did not last for the following hours. Based on the finding that an oral quercetin supplementation does not seem to disadvantage the glucose metabolism in newborn calves, the health parameters recorded during the trial were evaluated in the second part of this work. Additionally, plasma samples of the calves were analyzed for concentrations of immunoglobulins, acute-phase proteins, as well as parameters of the antioxidative system. Furthermore, the expression of genes for some proinflammatory cytokines, antioxidative enzymes and acute-phase proteins was analyzed in cDNA samples generated from the liver biopsy tissue. Data indicate that an adequate initial colostrum supply supports neonatal health and prepares the calves very well to cope with the new environment. Colostrum-fed calves showed reduced signs of inflammation and were more vital than calves initially fed formula, further, the metabolic status was improved. In contrast to this, the quercetin supplementation did not induce any detectable health-promoting effects, a finding that underlines that results proven in vitro are not always transferable to the organism as a whole. In summary, it can be concluded from the present work that an oral quercetin supplemen-tation does not impair the glucose metabolism of newborn dairy calves. However, health-promoting properties cannot be reproduced in vivo, hence quercetin feeding cannot compensate for an inadequate initial colostrum supply.Mit der Geburt werden Kälber mit einer neuen, extrauterinen Umgebung konfrontiert und müssen ihren Energiebedarf selbstständig decken. Dafür ist es essentiell, dass nicht nur ausreichend Nährstoffe aufgenommen werden, sondern auch die endogene Glucosepro-duktion effizient funktioniert, da Glucose in der ersten Lebensphase als eine Hauptenergie-quelle dient. Mit der Aufnahme von Kolostrum werden neben Nährstoffen auch zahlreiche bioaktive Substanzen zugeführt, die die Reifung und Entwicklung von Stoffwechselprozessen fördern. Kolostrum ist außerdem essentiell, um das naive Immunsystem des Kalbes während der ersten Lebenswochen zu unterstützen und passive Immunität zu vermitteln. Leider ist die Kolostrumversorgung oft suboptimal. Um die Gesundheit zu fördern, könnte dem Einsatz von Flavonoiden hier eine wichtige Rolle zukommen. Es handelt sich dabei um sekundäre Pflanzenstoffe, denen zahlreiche gesundheitsfördernde Eigenschaften zugeschrieben werden. Einer der Hauptvertreter ist Quercetin, welches nachweislich antioxidativ, anti- inflammatorisch und antimikrobiell wirken und die Darmflora modulieren kann. Allerdings interagiert es auch mit dem Glucosestoffwechsel, wodurch die intestinale Aufnahme sowie der Blutglucosespiegel gesenkt werden. Für das Kalb wäre dies fatal. Somit war es das Hauptziel dieser Arbeit, die Wirkung einer oralen Quercetinsupplementierung auf den Glucosestoffwechsel neugeborener Kälber zu untersuchen, da die Kenntnis darüber die Voraussetzung ist, um den Einsatz von Quercetin als gesundheitsfördernden Futter-zusatzstoff in der Kälberaufzucht überhaupt in Betracht ziehen zu können. Zu diesem Zweck wurden 28 neugeborene männliche Kälber der Rasse Deutsche Holstein in zwei Fütterungsgruppen unterteilt und während der ersten beiden Lebenstage entweder mit Kolostrum oder einer Formula mit gleicher Makronährstoffzusammensetzung, jedoch ohne bioaktive Substanzen, gefüttert. Am 2. Lebenstag erfolgte eine Unterteilung in Kontroll- und Behandlungsgruppen, wobei letztere während der ersten Lebenswoche Quercetin mit der Fütterung erhielten. Am 3. Lebenstag wurde eine Xyloseabsorptionsstudie durchgeführt, am 7. Lebenstag erfolgte eine Tracerstudie zum first pass uptake von Glucose, und am 8. Tag wurde Lebergewebe mittels Biopsie entnommen und mit Hilfe von PCR analysiert. Die postabsorptive Wiederfindung von oral verabreichter Xylose und 13C6-markierter Glucose im Plasma war höher in initial Kolostrum-gefütterten Kälbern, was im Vergleich zu Formula-gefütterten Tieren auf eine bessere Absorptionskapazität sowie eine geringere Glucoseextraktion im Splanchnikusgewebe schließen lässt. Ferner waren die mRNA Abundanz der hepatischen mitochondrialen Phosphoenolpyruvatcarboxykinase sowie verschiedene Plasmametaboliten in den Formulagruppen unabhängig von der Quercetin-supplementierung reduziert, was auf eine verzögerte Reifung des Stoffwechsels nach Kolostrumentzug hindeutet. Obwohl die Quercetinsupplementierung am 7. Lebenstag den intestinalen Glucoseabsorptionspeak steigern konnte, verlor sich dieser Effekt im Verlauf der darauffolgenden Stunden. Basierend darauf, dass eine orale Quercetinsupplementierung den Glucosestoffwechsel bei neugeborenen Kälbern nicht nachteilig beeinflusst, wurden im zweiten Teil der Arbeit die während der Studie aufgenommenen Gesundheitsparameter genauer ausgewertet. Weiterhin wurden Plasmaproben der Tiere auf Konzentrationen von Immunglobulinen, Akute- Phase-Proteinen sowie auf Parameter des antioxidativen Systems untersucht und die Genexpression verschiedener proinflammatorischer Zytokine, antioxidativer Enzyme und akuter-Phase-Proteine in den zuvor angelegten cDNA Umschreibungen analysiert. Die Ergebnisse zeigten, dass die adäquate Versorgung mit Kolostrum während der ersten Lebenstage die Kälber gut für die erste Lebensphase ausstattet und die Kälbergesundheit fördert. Die Tiere zeigten verminderte Anzeichen von Inflammation und waren in den ersten Lebenstagen vitaler als Kälber der Formulagruppe, ferner war der metabolische Status verbessert. Die Quercetinsupplementierung hingegen brachte keine gesundheitsfördernden Effekte, was beweist, dass in vitro nachgewiesene Effekte nicht auf den Gesamtorganismus übertragbar sind. Zusammenfassend lässt sich aus dieser Arbeit ableiten, dass die Fütterung von Quercetin Aglykon beim neugeborenen Kalb zwar keine nachteiligen Effekte auf den Glucose-stoffwechsel hat. Dennoch sind die gesundheitsfördernden Eigenschaften nicht in vivo reproduzierbar, sodass der Einsatz von Quercetin als Futterzusatz keinesfalls eine unzureichende Kolostrumversorgung kompensieren kann

Quercetin Feeding in Newborn Dairy Calves Cannot Compensate Colostrum Deprivation: Study on Metabolic, Antioxidative and Inflammatory Traits

Immaturity of the neonatal immune system is causative for high morbidity in calves and colostrum intake is crucial for acquiring passive immunity. Pathogenesis is promoted by reactive oxygen species accumulating at birth if counter-regulation is inadequate. The flavonol quercetin exerts antioxidative and anti-inflammatory effects that may enhance neonatal health. The aim of this work was to study effects of quercetin feeding on metabolic, antioxidative and inflammatory parameters in neonatal calves to investigate whether quercetin could compensate for insufficient colostrum supply. Twenty-eight newborn calves were assigned to two dietary groups fed colostrum or milk-based formula on day 1 and 2 and milk replacer thereafter. From day 2 onwards, 7 calves per diet group were additionally fed quercetin aglycone (50 mg/(kg body weight x day)). Blood samples were taken repeatedly to measure plasma concentrations of flavonols, glucose, lactate, total protein, albumin, urea, non-esterified fatty acids, triglycerides, cholesterol, insulin, glucagon, cortisol, immunoglobulins, fibrinogen, haptoglobin and serum amyloid A. Trolox equivalent antioxidative capacity, ferric reducing ability of plasma, thiobarbituric acid reactive species and F2-isoprostanes were analyzed to evaluate plasma antioxidative status. Expression of tumor necrosis factor, interleukin-1a, interleukin-1ß, serum amyloid A, haptoglobin, fibrinogen, C-reactive protein, catalase, glutathione peroxidase and superoxide dismutase mRNA were measured in liver tissue on day 8. Plasma flavonol concentrations were detectable only after quercetin-feeding without differences between colostrum and formula feeding. Plasma glucose, lactate, total protein, immunoglobulins, triglycerides, cholesterol, trolox equivalent antioxidative capacity and thiobarbituric acid reactive species were higher after colostrum feeding. Body temperature, fecal fluidity and plasma concentrations of cortisol and haptoglobin were higher in formula- than in colostrum-fed groups. Hepatic mRNA expression of tumor necrosis factor was higher after quercetin feeding and expression of C-reactive protein was higher after formula feeding. Data confirm that colostrum improves neonatal health and indicate that quercetin feeding cannot compensate for insufficient colostrum supply

Effects of Feeding Milk Replacer Ad Libitum or in Restricted Amounts for the First Five Weeks of Life on the Growth, Metabolic Adaptation, and Immune Status of Newborn Calves

The pre-weaning period is critical for calf health and growth, and intensive milk feeding programs may assist postnatal development by improving body growth and organ maturation. The aim of the present work was to study the effects of ad libitum milk replacer (MR) feeding on the growth, metabolic adaptation, health, and immune status of newborn calves. Twenty-eight newborn Holstein and Holstein x Charolais crossbred calves were fed ad libitum (ADLIB) or in restricted amounts (6 liters per day; RES) during the first five weeks of life. The MR intake in the ADLIB treatment was gradually reduced at weeks 6 and 7, and all calves then received 6 liters of MR per day until day 60. Blood samples were collected to measure the plasma concentrations of metabolites, insulin, insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBP), immunoglobulins, and acute phase proteins. The expression of mRNA associated with both the somatotropic axis and gluconeogenic enzymes was measured in the liver on day 60. Intensive feeding improved MR intake and growth in ADLIB without influencing concentrate intake. Carcass weight, perirenal fat, and muscle mass were greater in ADLIB. Plasma concentrations of glucose, triglycerides, insulin, and IGF-I were greater, whereas plasma concentrations of β-hydroxybutyrate, total protein, albumin, urea, IGFBP-2 and -4, and fibrinogen were lower at distinct time points in ADLIB. The hepatic mRNA expression of cytosolic phosphoenolpyruvate carboxykinase was greater in ADLIB. Most metabolic and endocrine differences occurred during the MR feeding period, but a slightly greater concentrate intake was associated with increased plasma IGF-I and insulin at the end of the study. The immune and health status of the calves were not affected by MR feeding. However, increased plasma fibrinogen in the RES group suggested differences in the acute phase response

Flavonol concentrations in plasma.

<p>Stacked concentrations (<b>A</b>) and percentage composition (<b>B</b>) of flavonols in basal plasma samples of calves fed either colostrum (dark grey) or formula (light grey) on days 1 and 2 and supplemented with quercetin aglycone from day 2 to day 8 (50 mg/(kg body weight × day)). Flavonol metabolites: quercetin (without symbol), isorhamnetin (horizontal line), tamarixetin (crossline) and kaempferol (horizontal bold line). Data are presented as the least squares means. Different uppercase letters (A, B, C) symbolize differences in the total flavonol concentration within the same group on different days (<i>P</i> < 0.05). † tend to differ among groups on the same day (<i>P</i> < 0.10).</p

Antioxidative Status in Plasma.

<p>Plasma parameters for antioxidative capacity (<b>A</b>, <b>B</b>) and oxidative stress (<b>C</b>, <b>D</b>) in calves fed either colostrum (dark grey) or formula (light grey) on days 1 and 2 and supplemented with (crossline) or without (no symbol) quercetin on days 2–8 (50 mg/(kg body weight × day)). Data are presented as the least squares means ± standard errors. Bars with different lowercase letters (a, b) differ among groups within the same day (<i>P</i> < 0.05). TEAC, trolox equivalent antioxidative capacity (in trolox equivalents, TE); FRAP, ferric reducing ability of plasma (in ascorbic acid equivalents, ASCE); TBARS, thiobarbituric acid reactive species (in malondialdehyde equivalents, MDAE).</p

Feed Intake and Growth Performance.

<p>Colostrum and milk replacer (MR) (<b>A</b>) and concentrate intake (<b>B</b>), total metabolizable energy (ME) intake, body weight (<b>D</b>), and average daily gain (<b>E</b>) in calves fed MR either <i>ad libitum</i> (ADLIB, black square, straight line) or restrictively (RES, white circle, dashed line) for the first five weeks of life. MR intake in ADLIB was gradually reduced during weeks 6 and 7 to amounts fed in RES. The data are presented as the least squares means ± standard error. ^†Trend between groups <i>P</i> < 0.1; *different between groups <i>P</i> < 0.05; **different between groups <i>P</i> < 0.01; and ***different between groups <i>P</i> < 0.001.</p

Endocrine Status in Blood Plasma.

<p>The plasma concentrations of insulin (<b>A</b>), insulin-like growth factor 1 (IGF-I; <b>B</b>), and IGF-binding proteins (IGFBP)-2 (<b>C</b>), -3 (<b>D</b>), and -4 (<b>E</b>) in calves fed milk replacer (MR) either <i>ad libitum</i> (ADLIB, black square, straight line) or restrictively (RES, white circle, dashed line) for the first five weeks of life. MR intake in ADLIB was gradually reduced during weeks 6 and 7 to amounts fed in RES. The data are presented as the least squares means ± standard error. ^†Trend between between groups <i>P</i> < 0.1, *different between groups <i>P</i> < 0.05, **different between groups <i>P</i> < 0.01, and ***different between groups <i>P</i> < 0.001.</p

Carcass Composition.

<p>The weight of the hot carcass, liver, pancreas, fat depots, and distinct muscle cuts<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168974#t003fn002" target="_blank">¹</a> and the size of the musculus longissimus dorsi (MLD) in calves fed milk replacer (MR) either <i>ad libitum</i> (ADLIB) or restrictively (RES) for the first five weeks of life.</p

Metabolic Status in Blood Plasma.

<p>The plasma concentrations of glucose (<b>A</b>), lactate (<b>B</b>), beta-hydroxybutyrate (BHB; <b>C</b>), non-esterified fatty acids (NEFA; <b>D</b>), triglycerides (<b>E</b>), and cholesterol (<b>F</b>) in calves fed milk replacer (MR) either <i>ad libitum</i> (ADLIB, black square, straight line) or restrictively (RES, white circle, dashed line) for the first five weeks of life. MR intake in ADLIB was gradually reduced during weeks 6 and 7 to amounts fed in RES. The data are presented as the least squares means ± standard error. ^†Trend between groups <i>P</i> < 0.1, *different between groups <i>P</i> < 0.05, **different between groups <i>P</i> < 0.01, and ***different between groups <i>P</i> < 0.001.</p