Lipomobilization is essential for dairy cows to balance the energy requirement for milk production in early lactation. This study aimed to determine the role of hormone-sensitive lipase (HSL) and its activation by phosphorylation at Ser 660 (HSLp660) and 563 (HSLp563) in different adipose tissue depots as influenced by time and postpartum diet in dairy cows. Biopsy samples were obtained from s.c. (SCAT) and retroperitoneal (RPAT) adipose tissues of 20 Holstein cows 21 d prepartum, and 1 and 21 d postpartum. After d 1 postpartum, cows were randomly assigned to 2 groups (n=10). Groups received diets with either a concentrate-to-roughage ratio on a dry matter basis of 30:70% (low-concentrate, LC, group) or 60:40% (high-concentrate group), fed until the third biopsy sampling 21 d postpartum. Dry matter intake, milk yield, and milk composition were recorded. Blood samples were taken weekly, starting 21 d prepartum and analyzed for nonesterified fatty acids, β-hydroxybutyrate (BHBA), glucose, and insulin. Protein expression of HSL and its extent of phosphorylation in adipose tissue were measured by semiquantitative Western blotting. Total HSL expression was lower in both adipose tissues 1 d after calving compared with prepartum sampling (SCAT: 4.10±0.5 vs. 2.4±0.3; RPAT: 11.1±1.3 vs. 6.6±1.1). Phosphorylation at Ser 660 was higher 21 d postpartum compared with 21 d prepartum in RPAT (2.9±0.3 vs. 4.6±0.6). Phosphorylation at Ser 563 was higher 21 d postpartum than 21 d prepartum in SCAT (0.6±0.1 vs. 3.9±1.1), and in RPAT a difference was observed between 21 d prepartum and 1 d postpartum (1.0±0.1 vs. 3.3. ± 0.6). On d 21 postpartum, the LC group showed a lower extent of Ser 563 phosphorylation in RPAT (3.9±0.8 vs.10.0±1.9) and a higher concentration of serum BHBA (0.77±0.05 vs. 0.47±0.11) than did the high-concentrate group. An inhibitory influence of higher BHBA concentrations on HSL phosphorylation in the LC group could be a possible explanation. On comparing RPAT to SCAT, HSL expression and the extent of Ser 660 and 563 phosphorylation was higher in RPAT at 21 d prepartum (HSL: 4.1±0.5 vs. 11.1±1.2; HSLp660 1.3±0.2 vs. 2.9±0.3; HSLp563: 0.6±0.1 vs. 1.0±0.1). In conclusion, the postpartum feeding regimen influenced the phosphorylation pattern, especially in RPAT, implying a regulatory role for different phosphorylation sites in adaptive lipolysis of dairy cows. It is suggested that RPAT is more sensitive to periparturient challenges than is SCAT.

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.

Samples of peritoneal fluid and jugular venous blood were taken simultaneously from 95 clinically healthy Holstein-Friesian cows. The concentrations of total protein, albumin, glucose, cholesterol, fibrinogen, L-lactate and D-dimer, the activities of lactate dehydrogenase (LDH), alkaline phosphatase and creatine kinase, and the white blood cell count were determined in the samples. Light's criteria, the serum-ascites albumin gradient (SAAG) and the ratios of the concentration of each parameter in peritoneal fluid to its concentration in blood were calculated. The mean concentrations of total protein, albumin and D-dimer, the activity of LDH and the SAAG were different from the reference values for monogastric animals and human beings.

Background: Peritoneal fluid analysis in cattle traditionally includes the classic parameters despite the fact that they have only moderate diagnostic accuracy and often fail to identify the pathogenesis or etiological factors. Therefore additional parameters recently have been established to improve diagnostic precision. In a recent study, reference ranges for several of these parameters have been proposed in dairy cows.Hypothesis/Objectives: The aim of this observational study was to assess the diagnostic value of D-Dimer and other measurements of peritoneal fluid analysis in dairy cows with peritonitis.Animals: The study included 110 Holstein-Friesian cows grouped into cows with peritonitis (n 5 47) and cows without peritonitis (n 5 63).Methods: Peritoneal fluid was obtained by abdominocentesis. Total protein, albumin, glucose, cholesterol, fibrinogen, Llactate, D-Dimer, lactate dehydrogenase (LDH), alkaline phosphatase, creatine phosphokinase, white blood cell, and red blood cell were determined in peritoneal fluid and venous blood. Serum-ascites albumin gradient (SAAG) and ratios of peritoneal fluid-venous blood were calculated. Sensitivity (SN) and specificity (SP) were calculated and receiver operating characteristic curve analysis performed.Results: Peritoneal fluid D-Dimer was most accurate in diagnosing peritonitis in cows (SN and SP495.0%). Total protein concentration, LDH and LDH ratio, and SAAG had sensitivities between 49.0 and 67.1%, and specificities between 88.4 and 95.5%. A low-peritoneal fluid glucose concentration was found to be highly indicative of septic peritonitis.Conclusions and Clinical Importance: Measurement of the recently introduced parameters may increase the diagnostic value of peritoneal fluid analysis and provide additional specific information. Therefore these measurements should be included in the routine procedure.

A balanced lipolytic regulation in adipose tissues based on fine-tuning of prolipolytic and antilipolytic pathways is of vital importance to maintain the metabolic health in dairy cows. Antilipolytic pathways, such as the G protein-coupled receptor 109A (GPR109A)-mediated pathway and the insulin signaling pathway in bovine adipose tissues may be involved in prohibiting excessive lipomobilization by reducing triglycerol hydrolysis. This study aimed to evaluate the in vitro antilipolytic potential of the mentioned pathways in bovine adipose tissue explants. Therefore, subcutaneous and retroperitoneal adipose tissue samples (approximately 100mg) of German Holstein cows were treated for 90 min ex vivo with nicotinic acid (2, 8, or 32 μM), nicotinamide (2, 8, or 32 μM), β-hydroxybutyrate (0.2, 1, or 5mM), or insulin (12 mU/L), with a concurrent lipolytic challenge provoked with 1 μM isoproterenol. Lipolytic and antilipolytic responses of the adipose tissues were assessed by measuring free glycerol and nonesterified fatty acid release. To identify molecular components of the investigated antilipolytic pathways, protein abundance of GPR109A and the extent of hormone-sensitive lipase (HSL) phosphorylation at serine residue 563 were detected by Western blotting. Treatment with nicotinic acid or β-hydroxybutyrate decreased the lipolytic response in adipose tissue explants and concurrently reduced the extent of HSL phosphorylation, but treatment with nicotinamide or insulin did not. Subcutaneous adipose tissue constitutively expressed more GPR109A protein, but no other depot-specific differences were observed. This study provides evidence that the GPR109A-mediated pathway is functionally existent in bovine adipose tissues, and confirms that HSL phosphorylation at serine residue 563 is also important in antilipolytic regulation in vitro. This antilipolytic pathway may be involved in a balanced lipid mobilization in the dairy cow.

In response to negative energy balance, overconditioned cows mobilize more body fat than thin cows and subsequently are prone to develop metabolic disorders. Changes in adipose tissue (AT) metabolism are barely investigated in overconditioned cows. Therefore, the objective was to investigate the effect of increasing body condition on key regulator proteins of fat metabolism in subcutaneous AT and circulation of dairy cows. Nonlactating, nonpregnant dairy cows (n=8) investigated in the current study served as a model to elucidate the changes in the course of overcondition independent from physiological changes related to gestation, parturition, and lactation. Cows were fed diets with increasing portions of concentrate during the first 6wk of the experiment until 60% were reached, which was maintained for 9wk. Biopsy samples from AT of the subcutaneous tailhead region were collected every 8wk, whereas blood was sampled monthly. Within the experimental period cows had an average BW gain of 243±33.3 kg. Leptin and insulin concentrations were increased until wk 12. Based on serum concentrations of glucose, insulin, and nonesterified fatty acids, the surrogate indices for insulin sensitivity were calculated. High-concentrate feeding led to decreased quantitative insulin sensitivity check index and homeostasis model assessment due to high insulin and glucose concentrations indicating decreased insulin sensitivity. Adiponectin, an adipokine-promoting insulin sensitivity, decreased in subcutaneous AT, but remained unchanged in the circulation. The high-concentrate diet affected key enzymes reflecting AT metabolism such as AMP-activated protein kinase and hormone-sensitive lipase, both represented as the proportion of the phosphorylated protein to total protein, as well as fatty acid synthase. The extent of phosphorylation of AMP-activated protein kinase and the protein expression of fatty acid synthase were inversely regulated throughout the experimental period, whereas the extent of phosphorylation of hormone-sensitive lipase was consistently decreasing by the high-concentrate diet. Overcondition in nonpregnant, nonlactating dairy cows changed the expression of key regulator proteins of AT metabolism and circulation accompanied by impaired insulin sensitivity, which might increase the risk for metabolic disorders.

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The transition from pregnancy to lactation is characterized by major changes in glucose and adipose tissue metabolism. Anti- and prolipolytic pathways mediated via the hydroxycarboxylic acid receptors 1 (HCAR1) and 2 (HCAR2) and tumor necrosis factor-α receptor 1 (TNFR1), as well as the adipokines apelin and resistin, are likely involved in regulating these processes. This study aimed to determine the mRNA abundance of the aforementioned receptors in both subcutaneous and visceral adipose tissue, to characterize the adipokine concentrations in serum, and to test the effects of feeding diets with either high or low portions of concentrate and a concomitant niacin supplementation from late gestation to early lactation. Twenty pluriparous German Holstein cows were all kept on the same silage-based diet until d 42 antepartum, when they were allocated to 2 feeding groups: until d 1 antepartum, 10 animals each were assigned to either a high-concentrate (60:40 concentrate-to-roughage ratio) or a low-concentrate diet (30:70). Both groups were further subdivided into a control and a niacin group, the latter receiving 24 g/d of nicotinic acid from d -42 until 24. From d 1 to 24 postpartum, the concentrate portion was increased from 30 to 50% for all cows. Biopsies of subcutaneous (SCAT) and retroperitoneal adipose tissue (RPAT) were taken at d -42, 1, 21, and 100 relative to parturition. Blood samples were drawn along with the biopsies and on d -14, 3, 7, 14, and 42. The concentrations of the adipokines apelin and resistin in serum were measured via ELISA. The mRNA of the 3 receptors in AT was quantified as well as the protein abundance of HCAR2 by Western blot. The feeding regimen did not affect the variables examined. The concentrations of apelin remained fairly constant during the observation period, whereas the resistin concentrations increased toward parturition and decreased to precalving levels within 1 wk after calving. The mRNA abundance of HCAR1, HCAR2, and TNFR1 changed in SCAT and RPAT during the considered time period. For the HCAR2 protein, time-dependent changes were restricted to SCAT. The mRNA abundance of all receptors was greater in RPAT than in SCAT. The tissue-specific correlations observed between the receptors point to a link between these factors and may indicate different regulatory roles in the respective tissues. This study provides insight into the complex metabolic adaptations during the transition period and supports a differential regulation of lipolysis among SCAT and RPAT in dairy cows.