Dietary L-Carnitine Affects Leukocyte Count and Function in Dairy Cows Around Parturition.

In early lactation, an energy deficit leading to a negative energy balance (NEB) is associated with increased susceptibility to disease and has been shown to be an important factor during transition in dairy cows. L-carnitine as a key factor in the mitochondrial transport of fatty acids and subsequently for ß-oxidation and energy release is known to modulate mitochondrial biogenesis and thus influence metabolism and immune system. In the current study, we characterized hematological changes around parturition and investigated the potential effects of dietary L-carnitine supplementation on immune cell functions. For this approach, dairy cows were assigned either to a control (CON, n = 30) or an L-carnitine group [CAR, n = 29, 25 g rumen-protected L-carnitine per cow and day (d)]. Blood samples were taken from d 42 ante partum (ap) until d 110 post-partum (pp), with special focus and frequent sampling from 0.5 to72 h post-calving to clarify the impact of L-carnitine supplementation on leukocyte count, formation of reactive oxygen species (ROS) in polymorphonuclear cells (PMN) and peripheral mononuclear cells (PBMC) and their phagocytosis activity. Blood cortisol concentration and the capacity of PBMC proliferation was also investigated. All populations of leukocytes were changed during the peripartal period, especially granulocytes showed a characteristic increase up to 4 h pp. L-carnitine supplementation resulted in increased levels of eosinophils which was particularly pronounced one day before to 4 h pp, indicating a possible enhanced support for tissue repair and recovery. Non-supplemented cows showed a higher phagocytic activity in PBMC as well as a higher phagocytic capacity of PMN during the most demanding period around parturition, which may relate to a decrease in plasma levels of non-esterified fatty acids reported previously. L-carnitine, on the other hand, led to an increased efficiency to form ROS in stimulated PMN. Finally, a short period around calving proved to be a sensitive period in which L-carnitine administration was effective.

Dietary l-carnitine Supplementation Modifies the Lipopolysaccharide-Induced Acute Phase Reaction in Dairy Cows.

l-carnitine plays an important role in energy metabolism through supporting the transport of activated fatty acids to the subcellular site of ß-oxidation. An acute phase reaction (APR) is known as an energy consuming process. Lipopolysaccharides (LPS) are often used in animal models to study intervention measures during innate immune responses such as APR. Thus, the aim of the study was to investigate the effects of dietary l-carnitine supplementation during an LPS-induced APR in mid-lactating German Holstein cows. Animals were assigned to a control (CON, n = 26) or l-carnitine group (CAR, n = 27, 25 g rumen-protected l-carnitine/cow/d) and received an intravenous injection of LPS (0.5 µg/kg body weight) at day 111 post-partum. Blood samples were collected from day 1 pre-injection until day 14 post-injection (pi). From 0.5 h pi until 72 h pi blood samplings and clinical examinations were performed in short intervals. Clinical signs of the APR were not altered in group CAR except rumen motility which increased at a lower level compared to the CON group after a period of atonia. Group CAR maintained a higher insulin level compared to group CON even up to 72 h pi which might support glucose utilization following an APR.

Effects of a Dietary L-Carnitine Supplementation on Performance, Energy Metabolism and Recovery from Calving in Dairy Cows.

Dairy cows are metabolically challenged during the transition period. Furthermore, the process of parturition represents an energy-consuming process. The degree of negative energy balance and recovery from calving also depends on the efficiency of mitochondrial energy generation. At this point, L-carnitine plays an important role for the transfer of fatty acids to the site of their mitochondrial utilisation. A control (n = 30) and an L-carnitine group (n = 29, 25 g rumen-protected L-carnitine per cow and day) were created and blood samples were taken from day 42 ante partum (ap) until day 110 post-partum (pp) to clarify the impact of L-carnitine supplementation on dairy cows, especially during the transition period and early puerperium. Blood and clinical parameters were recorded in high resolution from 0.5 h to 72 h pp. L-carnitine-supplemented cows had higher amounts of milk fat in early lactation and higher triacylglyceride concentrations in plasma ap, indicating increased efficiency of fat oxidation. However, neither recovery from calving nor energy balance and lipomobilisation were influenced by L-carnitine.

Gain and loss of subcutaneous and abdominal fat depot mass from late pregnancy to 100 days in milk in German Holsteins.

This research paper addresses the hypothesis that in times of negative energy balance around parturition in dairy cattle, lipids stored in adipocytes are mobilised in a more intensive manner out of the abdominal depots than out of the subcutaneous adipose tissues. Furthermore, the impact of niacin supplementation and energy density of the ration on adipose tissue mass gain and loss was assessed. Absolute masses of subcutaneous (SCAT), retroperitoneal (RPAT), omental (OMAT), mesenterial (MAT) and abdominal adipose tissue as a whole (AAT) were estimated by ultrasonography at -42, 3, 21 and 100 DIM. Absolute and relative daily gain during dry period (-42 to 3 DIM) and loss in fresh cow period (3 to 21 DIM) and early lactation period (22 to 100 DIM) were calculated. Feeding regime neither by niacin nor by energy density exerted any effect on adipose tissue masses. The AAT was always bigger than SCAT, but RPAT, OMAT and MAT did not differ amongst each other. All depot masses showed similar patterns with an increase during dry period and a decrease after calving. In fresh cow period AAT absolutely and relatively lost more mass than SCAT. This confirms that AAT is more intensively mobilised than SCAT during that time span. Further absolute daily gain during dry period was strongly negatively correlated with absolute daily loss during fresh cow period. This underlines the impact of individual body condition on adipose mobilisation in periparturient dairy cows. According to these results, it has to be taken into account that the largest amount of fat mobilised in the fresh cow period origins from AAT. This might impact the pattern of adipose derived metabolites and metabolic effectors interacting in physiological and deregulated adaptation to negative energy balance.

Insulin Signaling in Liver and Adipose Tissues in Periparturient Dairy Cows Supplemented with Dietary Nicotinic Acid.

The glucose homeostasis in dairy cattle is very well controlled, in line with the metabolic adaptation during the periparturient period. Former studies showed that nicotinic acid (NA) lowered plasma non-esterified fatty acids (NEFA) concentrations and increased insulin sensitivity in dairy cows. Thus, the purpose of this study was to investigate whether the expression of proteins involved in hepatic and adipose insulin signaling and protein expression of hepatic glucose transporter 2 (GLUT2) were affected by dietary NA and dietary concentrate intake in periparturient dairy cows. Twenty pluriparous German Holstein cows were fed with the same diet from about 21 days before the expected calving date (d-21) to calving. After calving, cows were randomly assigned in 4 groups and fed with diets different in concentrate proportion ("HC" with 60:40% or "LC" with 30:70% concentrate-to-roughage ratio) and supplemented with NA (24 g/day) (NA) or without (CON) until d21. Biopsy samples were taken from the liver, subcutaneous (SCAT) and retroperitoneal (RPAT) adipose tissues at d-21 and d21. Protein expression of insulin signaling molecules (insulin receptor (INSR), phosphatidylinositol-3-kinase (PI3K), protein kinase Cζ (PKCζ)) and hepatic GLUT2 was measured by Western Blotting. The ratio of protein expression at d21/at d-21 was calculated and statistically evaluated for the effects of time and diet. Cows in HC had significantly higher dietary energy intake than cows in LC. In RPAT a decrease in PI3K and PKCζ expression was found in all groups, irrespectively of diet. In the liver, the GLUT2 expression was significantly lower in cows in NA compared with cows in CON. In conclusion, insulin signaling might be decreased in RPAT over time without any effect of diet. NA was able to modulate hepatic GLUT2 expression, but its physiological role is unclear.

Associations between Forkhead Box O1 (FoxO1) Expression and Indicators of Hepatic Glucose Production in Transition Dairy Cows Supplemented with Dietary Nicotinic Acid.

Forkhead box protein O1 (FoxO1) is a transcription factor which promotes hepatic glucose production (HGP) by up-regulating the transcription of gluconeogenic enzymes in monogastric species. The activity of FoxO1 is inhibited by insulin-induced phosphorylation. The aims of the present study were to find associations between FoxO1 expression and variables associated with HGP as affected by feeding regimen in dairy cows during the transition period. Twenty one healthy German Holstein cows were allocated to four groups (LC-CON, HC-CON, LC-NA with 5 cows/group and HC-NA with 6 cows/group, respectively). Cows received 0 (LC-CON and HC-CON) or 24 (LC-NA and HC-NA) g/d nicotinic acid with high (HC) or low (LC) concentrate proportion from -42 days (-41.8 + 4.8; mean + standard deviation) relative to expected calving date (d-42) to d24. Liver biopsy was taken at d-42, 1, 21, and 100. The total protein expression of FoxO1 (tFoxO1) and the extent of phosphorylation of FoxO1 at serine 256 (pFoxO1) were analysed semiquantitatively by Western Blotting. The expression of hepatic mRNA of FoxO1 and seven genes associated with HGP was measured by real-time RT-PCR. Mixed model and Pearson's correlation were used for statistical evaluation with the level of significance at P<0.05. No dietary effect was observed either on feed intake, energy balance, or on the concentration of blood metabolites. Neither time nor diet affected the expression of FoxO1 total protein and mRNA. A NA × concentrate interaction was found in pFoxO1. However, no corresponding dietary effect was found in the mRNA expression of investigated genes. Different patterns of correlations between FoxO1-related variables and investigated indicators for HGP were found at d21 and 100. The results indicated that the regulation of HGP did not take place on the levels of mRNA and protein expression and the phosphorylation of FoxO1 in dairy cows in early lactation.

Effects of Prepartum Dietary Energy Level and Nicotinic Acid Supplementation on Immunological, Hematological and Biochemical Parameters of Periparturient Dairy Cows Differing in Parity.

The periparturient period is critical according to health, productivity and profitability. As this period is fundamental for the success of the lactation period, the interest in improving periparturient health by dietary supplements increased in recent years. The present study investigated the effects of feeding nicotinic acid (NA) combined with varying dietary energy densities on immunological, hematological and biochemical parameters of periparturient cows differing in parity. Thirty-six multiparous and 20 primiparous dairy cows were enrolled in the study 42 days before expected parturition date until 100 days postpartum with the half of the cows being supplemented with 24 g of NA/d. After parturition a diet with 30% concentrate was fed to all cows which was followed by different concentrate escalation strategies. Dietary NA supplementation was ceased on day 24 postpartum. Dietary NA increased (P = 0.010) serum nicotinamide concentrations (mean of 3.35 ± 1.65 µg/mL), whereas NA could not be detected. Present data emphasize that periparturient cows are faced with major physiological challenges and that both parity-groups have different prerequisites to adapt to those changes irrespective of NA supplementation. The overfeeding of energy to cows which were similar in body condition score had only minor effects on periparturient immune system function and the metabolism of those cows.

Effects of an energy-dense diet and nicotinic acid supplementation on production and metabolic variables of primiparous or multiparous cows in periparturient period.

It is well observed that feeding energy-dense diets in dairy cows during the dry period can cause metabolic imbalances after parturition. Especially dairy cows with high body condition score (BCS) and fed an energy-dense diet were prone to develop production diseases due to metabolic disturbances postpartum. An experiment was conducted to determine the effects of an energy-dense diet and nicotinic acid (NA) on production and metabolic variables of primiparous and multiparous cows in late pregnancy and early lactation which were not pre-selected for high BCS. Thirty-six multiparous and 20 primiparous German Holstein cows with equal body conditions were fed with energy-dense (60% concentrate/40% roughage mixture; HC group) or adequate (30% concentrate/70% roughage mixture; LC group) diets prepartum. After parturition, concentrate proportion was dropped to 30% for all HC and LC groups and was increased to 50% within 16 days for LC and within 24 days for HC cows. In addition, half of the cows per group received 24 g NA supplement per day and cow aimed to attenuate the lipid mobilisation postpartum. Feeding energy-dense diets to late-pregnant dairy cows elevated the dry matter (p < 0.001) and energy intake (p < 0.001) as well as the energy balance (p < 0.001) without affecting the BCS (p = 0.265) during this period. However, this did not result in any metabolic deviation postpartum as the effects of prepartum concentrate feeding were not carried over into postpartum period. Multiparous cows responded more profoundly to energy-dense feeding prepartum compared with primiparous cows, and parity-related differences in the transition from late pregnancy to lactation were obvious pre- and postpartum. The supplementation with 24 g NA did not reveal any effect on energy metabolism. This study clearly showed that energy-dense feeding prepartum did not result in metabolic imbalances postpartum in multiparous and primiparous cows not selected for high BCS. A genetic predisposition for an anabolic metabolic status as indicated by high BCS may be crucial for developing production diseases at the onset of lactation.

Supplementation with conjugated linoleic acids extends the adiponectin deficit during early lactation in dairy cows.

Decreasing insulin sensitivity (IS) in peripheral tissues allows for partitioning nutrients towards the mammary gland. In dairy cows, extensive lipid mobilization and continued insulin resistance (IR) are typical for early lactation. Adiponectin, an adipokine, promotes IS. Supplementation with conjugated linoleic acids (CLA) in rodents and humans reduces fat mass whereby IR and hyperinsulinemia may occur. In dairy cows, CLA reduce milk fat, whereas body fat, serum free fatty acids and leptin are not affected. We aimed to investigate the effects of CLA supplementation on serum and adipose tissue (AT) adiponectin concentrations in dairy cows during the lactation driven and parity modulated changes of metabolism. High yielding cows (n=33) were allocated on day 1 post partum to either 100 g/day of a CLA mixture or a control fat supplement (CON) until day 182 post partum. Blood and subcutaneous (sc) AT (AT) biopsy samples were collected until day 252 post partum to measure adiponectin. Serum adiponectin decreased from day 21 pre partum reaching a nadir at calving and thereafter increased gradually. The distribution of adiponectin molecular weight forms was neither affected by time, parity nor treatment. Cows receiving CLA had decreased serum adiponectin concentrations whereby primiparous cows responded about 4 weeks earlier than multiparous cows. The time course of adiponectin concentrations in sc AT (corrected for residual blood) was similar to serum concentrations, without differences between CLA and CON. CLA supplementation attenuated the post partum increase of circulating adiponectin thus acting towards prolongation of peripartal IR and drain of nutrients towards the mammary gland.

Longitudinal profiling of the tissue-specific expression of genes related with insulin sensitivity in dairy cows during lactation focusing on different fat depots.

In dairy cows the milk associated energy output in early lactation exceeds the input via voluntary feed intake. To spare glucose for mammary lactose synthesis, peripheral insulin sensitivity (IS) is reduced and fat mobilization is stimulated. For these processes a link between IS and the endocrine functions of adipose tissue (AT) is likely; we thus aimed to characterise the mRNA expression from bovine AT derived proteins and receptors that are related to IS according to the literature in metabolically active tissues plus systemic IS throughout lactation. Conjugated linoleic acids (CLA) reduce milk fat thus decreasing the milk drain of energy and potentially dampening lipolysis, but may also affect IS. Subcutaneous (s.c.) AT and liver from pluriparous cows receiving either control fat or CLA supplement (100 g/day from 1 to 182 days in milk each) were biopsied covering week -3 to 36 relative to parturition. In an additional trial with primiparous cows treated analogously and slaughtered on days in milk 1, 42 or 105, samples from liver, udder, skeletal muscle and 3 visceral and 3 s.c. AT were obtained and assayed for mRNA abundance of adiponectin, its receptors, leptin, leptin receptor, PPARγ, PPARγ2, IL-6, and TNF-α. In pluriparous animals, the mRNA abundance of most of the target genes decreased after parturition in s.c. AT but increased in liver. In primiparous cows, AT depot specific differences were mostly related to retroperitoneal AT; adiponectin receptor 1 and TNF-α were affected predominantly. CLA effects in primiparous cows were largely limited to decreased PPARγ2 mRNA abundance in udder tissue. In pluriparous cows, insulin secretion was increased by CLA resulting in decreased systemic IS but without consistent changes in tissue target mRNA abundance. The temporal gene expression profiles from the adipokines and related receptors support their coactive function in adapting to the needs of lactation.

Effects of niacin supplementation and dietary concentrate proportion on body temperature, ruminal pH and milk performance of primiparous dairy cows.

The objective of this study was to investigate the effects of niacin and dietary concentrate proportion on body temperature, ruminal pH and milk production of dairy cows. In a 2 × 2 factorial design, 20 primiparous Holstein cows (179 ± 12 days in milk) were assigned to four dietary treatments aimed to receive either 0 or 24 g niacin and 30% (low) or 60% (high) concentrate with the rest being a partial mixed ration (PMR) composed of 60% corn and 40% grass silage (on dry matter basis). Ambient temperature and relative humidity were determined and combined by the calculation of temperature humidity index. Respiration rates, rectal, skin and subcutaneous temperatures were measured. Milk production and composition were determined. Ruminal pH and temperature were recorded at a frequency of 5 min using wireless devices for continuous intra-ruminal measurement (boluses). pH values were corrected for pH sensor drift. The climatic conditions varied considerably but temporarily indicated mild heat stress. Niacin did not affect skin, rectal and subcutaneous temperatures but tended to increase respiration rates. High concentrate reduced skin temperatures at rump, thigh and neck by 0.1-0.3°C. Due to the technical disturbances, not all bolus data could be subjected to statistical evaluation. However, both niacin and high concentrate influenced mean ruminal pH. High concentrate increased the time spent with a pH below 5.6 and ruminal temperatures (0.2-0.3°C). Niacin and high concentrate enhanced milk, protein and lactose yield but reduced milk fat and protein content. Milk fat yield was slightly reduced by high concentrate but increased due to niacin supplementation. In conclusion, niacin did not affect body temperature but stimulated milk performance. High concentrate partially influenced body temperatures and had beneficial effects on milk production.

Effects of long-term supplementation of dairy cow diets with rumen-protected conjugated linoleic acids (CLA) on performance, metabolic parameters and fatty acid profile in milk fat.

The supplementation of conjugated linoleic acids (CLA) to the rations of dairy cows represents an opportunity to reduce the content of milk fat. Therefore, CLA have the potential beneficial effect of reducing energy requirements of the early lactating cow. The present study aimed at the examination of long-term and posttreatment effects of dietary CLA intake on performance, variables of energy metabolism-like plasma levels of non esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB), and fatty acid profile in milk fat. Forty-six pregnant German Holstein cows were assigned to one of three dietary treatments: (1) 100 g/ d of control fat supplement (CON), (2) 50 g/d of control fat supplement and 50 g/ d of CLA supplement (CLA-1) and (3) 100 g/d of CLA supplement (CLA-2). The lipid-encapsulated CLA supplement consisted of approximately 10% of trans-10, cis-12 CLA and cis-9, trans-11 CLA each. The experiment started 1 d after calving and continued for about 38 weeks, divided into a supplementation (26 weeks) and a depletion period (12 weeks). Over the first 7 weeks of treatment, 11 and 16% reductions in dry matter intake compared to control were observed for the cows fed CLA-1 and CLA-2 supplements respectively. Consequently, the calculated energy balance for these two CLA groups was lower compared to the control. Plasma levels of NEFA and BHB remained unaffected. Later in lactation the highest CLA supplementation resulted in a reduction of milk fat content of 0.7%. However, no reduction in milk fat yield, and accordingly no milk fat depression (MFD), could be shown. The trans-10, cis-12 CLA in milk fat increased with increasing dietary CLA supplementation in a dose-dependent manner. The proportion of C16 in milk fat was decreased by the highest CLA supplementation. With the exception of an increase in plasma glucose level in the CLA-2 group, no post-treatment effects were observed. Overall, under the conditions of the present study no improvement in the calculated energy balance by CLA supplementation could be shown for the entire evaluation period.

Assessment of reference values for copper and zinc in blood serum of first and second lactating dairy cows.

The influence of different copper and zinc contents in rations on blood serum concentrations in dependence on feeding (Groups A and B) and lactation (Lactation 1 and 2) was tested in a feeding trial with 60 German Holstein cows. All animals received a diet based on maize and grass silage ad libitum. 30 cows received a concentrate supplemented with copper and zinc as recommended (Group A), whereas the other 30 animals were offered a concentrate with roughly double the amount of copper and zinc (Group B). Blood samples were taken several times during the lactation to analyse serum concentrations of copper and zinc. Copper serum concentration was influenced neither by the different feeding (11.7 mumol/L in Group A and 12.3 mumol/L in Group B) nor by the lactation (12.0 mumol/L in Lactation 1 and 12.1 mumol/L in Lactation 2). Zinc serum concentration was significantly influenced as well as by feeding (14.1 mumol/L in Group B and 12.5 mumol/L in Group A) and lactation (14.2 mumol/L in the second lactation and 12.8 mumol/L for first lactating cows). For an exact diagnosis of trace element supply, blood serum is a not qualified indicator; other sources (feedstuffs, liver, hair) must also be investigated.

[Influence of different copper and zinc supply on their concentrations in blood serum, liver and hair of dairy cows]. / Einfluss unterschiedlicher Kupfer- und Zinkversorgung auf deren Konzentrationen in Blutserum, Leber und Deckhaar bei Milchkühen.

In a feeding trial at the Institute of Animal Nutrition of the Federal Research Institute of Animal Health (FLI) over 12 weeks with 20 cows of the German Holstein Breed the influence of different copper and zinc contents in the ration on their concentration in blood serum, liver and hair was tested. All animals received a diet based on maize- and grass silage ad libitum. The animals were divided in two groups with 10 cows each; group A received a concentrate according to their milk yield with a copper and zinc content as recommended (GfE 2001), whereas group B was offered a concentrate with roughly the double amount of copper and zinc. At the beginning and at the end of the trial a sample of blood, pigmented hair and a liver bioptate was taken from all animals to evaluate the incorporation of copper and zinc in these tissues. In serum and pigmented hair the copper concentrations did not differ between the two groups [13.4 for Group A and 12.5 micromol/L for Group B in serum respectively 6.8 (Group A) and 7.4 mg/kg DM (Group B) in pigmented hair]. Only the copper concentration in the liver was influenced by the different feeding. The higher copper content for group B resulted in a significantly higher copper concentration in the liver (506 mg/kg DM compared to 383 mg/kg DM). The liver is the best indicator organ for a sufficient copper supply. An increase in the zinc content in the ration resulted neither in higher zinc concentrations in serum (15.1 in Group B in comparison to 13.4 micromol/L for Group A) nor in higher zinc concentrations in liver (140 for Group B and 112 mg/kg DM for Group A) and pigmented hair (130 in Group A and 123 mg/kg DM in Group B). There is a significant correlation between copper intake and copper concentration in the liver (r = 0.46), whereas the correlation between zinc intake and zinc concentration in the liver is only tendencially (r = 0.23). The three tested samples serum, liver and cow hair are not qualified to reflect exactly a sufficient zinc supply.