H. Sauerwein

Acute phase proteins in ruminants

The physiological response to infections and injuries involves local inflammation and the initiation of events leading to a systemic response, also called acute phase reaction (APR). This multiplicity of changes is distant from the site of injury, and includes fever, leukocytosis and quantitative and qualitative modification of a group of non-structurally related proteins present in blood and other biological fluids, collectively named Acute Phase Proteins (APP). Proteomic investigations of serum or plasma following natural or experimental infection frequently reveal substantial alterations in the APP, several of which are high abundance proteins in these fluids. The present review will focus on the results of recent research on ruminant APP. Highlight points will include: - The structure and the functions of the main APPs in ruminants, as well as the regulatory mechanisms that trigger their systemic and local expression in both physiological and pathological conditions.- The clinical aspects of APPs in ruminants, including the current and future application to veterinary diagnosis and animal production.- The APP in small and wildlife ruminants.- Alteration in APP detected by proteomic investigations.

Characterisation of the affinity of different anabolics and synthetic hormones to the human androgen receptor, human sex hormone binding globulin and to the bovine progestin receptor

For the steroidal growth promoters trenbolone acetate (TBA) and melengestrol acetate (MGA) neither the complete spectrum of biological activities nor the potential endocrine disrupting activity of their excreted metabolites in the environment is fully understood. The potency of these substances in [3H]‐dihydrotestosterone ([3H]‐DHT) displacement from the recombinant human androgen receptor (rhAR) and from human sex‐hormone binding globulin (hSHBG) was evaluated. In addition, the potency for [3H]‐ORG2058 displacement from the bovine uterine progestin receptor (bPR) was tested. For comparison, different anabolics and synthetic hormones were also tested for their binding affinities. For 17β‐trenbolone (17β‐TbOH), the active compound after TBA administration, an affinity the rhAR similar to dihydrotestosterone (DHT) and a slightly higher affinity to the bPR than progesterone were demonstrated. The affinity of the two major metabolites, 17α‐trenbolone and trendione, was reduced to less than 5% of the 17β‐TbOH‐value. The affinity of these three compounds and of MGA to the hSHBG was much lower compared with DHT. MGA showed a 5.3‐fold higher affinity than progesterone to the bPR but only a weak affinity to the rhAR. The major MGA metabolites have an affinity to the bPR between 85% and 28% of the affinity of progesterone. In consequence, MGA and TBA metabolites may be hor‐monally active substances, which will be present in edible tissues and in manure. We conclude that detailed investigations on biodegradation, distribution and bio‐efficacy of these substances are necessary.

Individual variability in physiological adaptation to metabolic stress during early lactation in dairy cows kept under equal conditions

This study was conducted to investigate individual metabolic and endocrine adaptation to lactation under conditions of identical housing and feeding conditions in high-yielding dairy cows. Forty-five cows were studied on a research farm under standardized but practical conditions. From wk 2 before calving until wk 14 postpartum, blood samples were collected at weekly intervals and assayed for blood chemistry and various metabolites and hormones. Body weight, BCS, and backfat thickness were also recorded weekly. Milk yield, milk composition, and feed intake and energy balance were accordingly measured during the postpartum phase. The animals were retrospectively classified according to their plasma concentration of beta-hydroxybutyrate (BHB): cows in which a BHB threshold of 1 mM was exceeded at least once during the experiment were classified as BHB positive (BHB+); cows with BHB values consistently below this threshold were classified as BHB negative (BHB -). Using this classification, differences for NEFA and glucose concentrations were observed, but the mean calculated energy balance did not differ between the groups during the experimental period (-22.2 MJ of NE(1)/d +/- 4.7 for BHB+ and -18.9 MJ of NE(1)/d +/- 4.9 for BHB-). In BHB+ cows, the peripartum decrease (P < 0.05) of BW, BCS, and backfat thickness was more pronounced than in BHB- cows. Mean milk yields did not differ between groups. However, BHB+ cows had greater milk fat and lesser milk protein contents (P < 0.05), resulting in a greater (P < 0.05) fat:protein ratio than in BHB- cows. Thus, to some extent, cows were able to compensate for the negative energy balance by adjustments in performance. Milk acetone concentrations followed BHB concentrations in blood. Insulin-like growth factor-I and leptin concentrations were greater (P < 0.05) in BHB- cows during the time of observation than in the BHB+ cows. Comparing the reproductive variables recorded (first increase of progesterone, first service conception rate, number of services per conception, interval from calving to first AI, interval from first AI to conception, and days open) between the 2 groups yielded no significant differences. Our findings imply that despite comparable energy balance, there is considerable individual variation of the adaptive ability of cows during early lactation based on a variety of metabolic and endocrine variables.

Metabolism and lactation performance in dairy cows fed a diet containing rumen-protected fat during the last twelve weeks of gestation.

Effects of dietary fat supplementation prepartum on liver lipids and metabolism in dairy cows are contradictory. Thus, we examined in 18 German Holstein cows (half-sib; first lactation 305-d milk yield >9,000 kg) whether dietary fat:carbohydrate ratio during the last trimester of gestation affects lipid metabolism and milk yield. The diets were formulated to be isoenergetic and isonitrogenous but differed in rumen-protected fat (FD; 28 and 46.5 g/kg of dry matter during far-off and close-up dry period; mainly C16:0 and C18:1) and starch concentration [carbohydrate diet (CD); 2.3 times as much starch as FD]. Diets were given ad libitum starting 12 wk before expected parturition. After parturition all cows were fed a single lactation diet ad libitum for 14 wk. With the FD treatment, dry matter intake was depressed prepartum, milk yield during first 4 wk of lactation was lower (36.9 vs. 41.0 kg/d), and postpartum energy balance during this period was more negative. During the first 4 wk, cows in the FD group had lower lactose percentage and yield but higher milk fat, whereas milk protein and fat yield as well as energy-corrected milk did not differ. Between wk 5 and 14, milk fat and milk protein percentage was lower in CD than in FD. Milk fat C14:0 was lower and C16:1 was higher in the FD group. For FD cows, plasma triacylglycerol, nonesterified fatty acids, and cholesterol concentrations were higher prepartum, whereas plasma beta-hydroxybutyrate and glucose concentrations were lower. During the first 10 d after parturition, plasma triacylglycerol concentration was higher in FD, and prepartum plasma glucose and cholesterol differences persisted during the first 14 wk of lactation. Irrespective of prepartum nutrient composition, concentrations of plasma leptin and subcutaneous fat leptin mRNA decreased between -10 d to +10 d relative to parturition, and liver lipids and glycogen reached maximum and minimal values, respectively, 10 d after parturition. Acetyl-coenzyme A carboxylase alpha mRNA abundance in subcutaneous fat decreased between -10 d to +1 d relative to parturition by 97%, whereas it was generally much lower in the liver and remained at a low level until wk 14 of lactation. In conclusion, feeding a diet containing rumen-protected fat during late lactation and dry period until calving negatively affected dry matter intake, energy balance, and milk yield during subsequent lactation, did not change acetyl-coenzyme A carboxylase alpha mRNA abundance in subcutaneous fat, and was not beneficial for liver lipid accumulation.

Transition period-related changes in the abundance of the mRNAs of adiponectin and its receptors, of visfatin, and of fatty acid binding receptors in adipose tissue of high-yielding dairy cows.

Adipose tissue expresses adipokines, which are involved in regulation of energy expenditure, lipid metabolism, and insulin sensitivity. To adapt for the transition from pregnancy to lactation, particularly in high-yielding dairy cows, adipokines, their receptors, and particular G-protein coupled receptors (GPRs) are of potential importance. Signaling by GPR 41 stimulates leptin release via activation by short-chain fatty acids; GPR 43/109A inhibits lipolysis, and GPR 109A thereby mediates the lipid-lowering effects of nicotinic acid and beta-hydroxybutyrate. The aim of this study was to compare the mRNA expression of adiponectin and visfatin, adiponectin receptors 1 and 2 (AdipoR1/2), leptin receptor (obRb), insulin receptor as of the aforementioned GPRs during the transition period in high-yielding dairy cows. Biopsies from subcutaneous fat and blood samples were obtained from 10 dairy cows 1 week before and 3 weeks after calving. For AdipoR1 and AdipoR2 mRNA abundance as well as for leptin concentrations in plasma, a reduction (P</=.05) was observed postpartum; for visfatin and putative GPR 109A mRNA abundance in adipose tissue, there was a trend (P<.1) for analogous changes. In contrast, the mRNA content of obRb and GPR 41 in adipose tissue was higher (P</=.05) in samples from early lactation than in those from late gestation. Our results indicate decreasing adiponectin sensitivity in adipose tissue after calving, which might be involved in the reduced insulin sensitivity of adipose tissue during early lactation. In addition, visfatin, GPR 41, and GPR 109A may further modulate insulin sensitivity.

Vitellogenin in carp (Cyprinus carpio) and perch (Perca fluviatilis): purification, characterization and development of an ELISA for the detection of estrogenic effects

Vitellogenin (VTG), a phospholipoglycoprotein precursor of egg yolk is synthesized and secreted in the liver in response to circulating estrogens in female fish. Thus, the presence of VTG in male fish is a useful biomarker to identify estrogenic activity of natural or anthropogenic substances in sewage effluents. We report the purification of carp (Cyprinus carpio) and perch (Perca fluviatilis) VTG with the subsequent development and characterization of a specific ELISA for VTG measurement. VTG was purified by combination of ion exchange chromatography and size exclusion chromatography. The purified proteins were used as antigen for antibody production, as standard and tracer in the assay. Carp VTG was stable and showed a characteristic double band after Western blotting of the purified protein and in serum samples, respectively. In perch samples, several bands with lower molecular weight were present and appeared to be degradation products of VTG. The development of the carp VTG ELISA led to a sensitive and valid test system with inter-assay coefficients of variation between 3.0 and 12.3%. In contrast to carp, the described ELISA for perch VTG showed a much higher inter-assay variation up to 24%, possibly attributable to fast protein degradation. In conclusion, the described two-step chromatography is a simple purification method for VTG. Immunological and electrophoretical test systems are valid methods to determine VTG in some species like carp, but for other species like perch in which VTG is not stable, these methods are not applicable.

Conjugated linoleic acids mediate insulin release through islet G protein coupled receptor FFA1/GPR40

Among dietary components, conjugated linoleic acids (CLAs) have attracted considerable attention as weight loss supplements in the Western world because they reduce fat stores and increase muscle mass. However, a number of adverse effects are also ascribed to the intake of CLAs such as aggravation of insulin resistance and the risk of developing diabetes. However, the mechanisms accounting for the effects of CLAs on glucose homeostasis are incompletely understood. Herein we provide evidence that CLAs specifically activate the cell surface receptor FFA1, an emerging therapeutic target to treat type 2 diabetes. Using different recombinant cellular systems engineered to stably express FFA1 and a set of diverse functional assays including the novel, label-free non-invasive dynamic mass redistribution technology (Corning® Epic® biosensor), both CLA isomers cis-9, trans-11-CLA and trans-10, cis-12-CLA were found to activate FFA1 in vitro at concentrations sufficient to also account for FFA1 activation in vivo. Each CLA isomer markedly increased glucose-stimulated insulin secretion in insulin-producing INS-1E cells that endogenously express FFA1 and in primary pancreatic β-cells of wild type but not FFA1−/− knock-out mice. Our findings establish a clear mechanistic link between CLAs and insulin production and identify the cell surface receptor FFA1 as a molecular target for CLAs, explaining their acute stimulatory effects on insulin secretion in vivo. CLAs are also revealed as insulinotropic components in widely used nutraceuticals, a finding with significant implication for development of FFA1 modulators to treat type 2 diabetes.

Technical note: Identification of reference genes for gene expression studies in different bovine tissues focusing on different fat depots

Selection of stable reference genes (REF) is important in real-time PCR data normalization. Bovine tissues such as the mammary gland, liver, muscle, and s.c. fat from the tail head have been thoroughly explored for stable REF, whereas fewer reports exist for other fat depots. Therefore, a suitable combination of REF was tested for different tissues of dairy cattle. Holstein dairy heifers (n = 25) were supplemented (100 g/d) with a control fat (n = 15) without conjugated linoleic acids or with rumen-protected conjugated linoleic acids (n = 10) from the day of calving until slaughter at 1, 42, or 105 d postpartum (n = 5, 10, and 10, respectively). Samples from 6 fat depots (omental, mesenterial, retroperitoneal, s.c. tail head, s.c. withers, and s.c. sternum), liver, semitendinosus muscle, and mammary gland were collected. The REF mRNA were quantified and their stability was analyzed using geNorm(plus). The 3 most stable REF in individual fat tissues and muscle were EMD (emerin), POLR2A (RNA polymerase II), and LRP10 (lipoprotein receptor-related protein 10); in mammary gland were MARVELD1 (marvel domain containing 1), EMD, and LRP10; and in liver were HPCAL1 (hippocalcin-like 1), LRP10, and EIF3K (Eukaryotic translation initiation factor 3). The 3 most stable REF in s.c. fat were EMD, LRP10, and EIF3K; in visceral fat were POLR2A, LRP10, and MARVELD1; and for all 6 adipose tissues were LRP10, EIF3K, and MARVELD1. When the mammary gland was added to the 6 adipose depots, at least 5 REF (LRP10, POLR2A, EIF3K, MARVELD1, and HPCAL1) were needed to reach the threshold of 0.15. Addition of liver to the above-mentioned tissues increased the V value. The data improve the comparison of gene expression between different fat depots. In each case, GAPDH had the lowest stability value.

Development of an enzyme immuno assay for the determination of porcine haptoglobin in various body fluids: testing the significance of meat juice measurements for quality monitoring programs.

Quantification of haptoglobin (Hp), an acute phase protein, in blood is presently discussed as being useful to monitor animal health. We developed an enzyme immuno assay (EIA) which is specific for porcine Hp, is not impaired by hemolytic samples and is sufficiently sensitive to be applied in meat juice. Hp was purified from porcine serum by affinity chromatography on hemoglobin Sepharose followed by gel filtration. A specific rabbit antiserum was obtained. In a competitive approach, biotinylated porcine Hp was used as tracer and incubated with Hp standard or sample in microtiter plates. The limit of detection was 0.02 mg/l, parallelism of sample dilutions was proven; recovery of Hp added to serum samples was 96.4 +/- 4.7%. The coefficients of intra and inter-assay variation were 3.3 (n=5) and 10.2% (n=16), respectively. Hp was reliably quantified in blood serum and plasma, whole blood, saliva and meat juice. For healthy pigs of different ages (4 weeks and 6 months), mean Hp concentrations of about 0.5-0.7 mg/ml were observed. To test the significance of Hp measurements in other matrices, samples were obtained from fattening pigs or from slaughter pigs. Blood serum or plasma was collected in parallel. In whole blood, Hp concentrations were about 40% lower than in plasma, but were closely related (n=24,r=0.85,P<0.001). Saliva Hp concentrations ranged between 0.3 and 3.0 microg/ml and were marginally related with blood plasma concentrations (n=93,r=0.35,P<0.001). From 106 hybrid slaughter pigs (100-110 kg) blood and muscle samples (diaphragmatic pillar, d.p.; m. brachiocephalicus, m.b.) were collected. Meat juice was obtained after freezing and thawing. Concentrations were 0.39+/-0.5 mg/ml in serum and 0.04+/-0.06 mg/ml in meat juice. Hp concentrations in blood were closely correlated with those in d.p. juice (P<0.001,r=0.750) and m.b. juice (P<0.001,r=0.776). In view of the many reports on Hp measurements being predictive for animal health even in the subclinical range, we conclude that Hp quantification in meat juice might be useful to assess meat quality at slaughter and further along the processing chain in terms of animal health.

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.