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In its climate protection law, Germany pursues the aim of achieving greenhouse gas neutrality by 2045. To approach this aim, the emissions from all sectors shall be reduced by 65 % by 2030 relative to 1990 and this includes mitigation of enteric methane (CH

Improvements in feed intake of dairy cows entering the early lactation period potentially decrease the risk of metabolic disorders, but before developing approaches targeting the intake level, mechanisms controlling and dysregulating energy balance and feed intake need to be understood. This review focuses on different inflammatory pathways interfering with the neuroendocrine system regulating feed intake of periparturient dairy cows. Subacute inflammation in various peripheral organs often occurs shortly before or after calving and is associated with increased pro-inflammatory cytokine levels. These cytokines are released into the circulation and sensed by neurons located in the hypothalamus, the key brain region regulating energy balance, to signal reduction in feed intake. Besides these peripheral humoral signals, glia cells in the brain may produce pro-inflammatory cytokines independent of peripheral inflammation. Preliminary results show intensive microglia activation in early lactation, suggesting their involvement in hypothalamic inflammation and the control of feed intake of dairy cows. On the other hand, pro-inflammatory cytokine-induced activation of the vagus nerve transmits signalling to the brain, but this pathway seems not exclusively necessary to signal feed intake reduction. Yet, less studied in dairy cows so far, the endocannabinoid system links inflammation and the hypothalamic control of feed intake. Distinct endocannabinoids exert anti-inflammatory action but also stimulate the posttranslational cleavage of neuronal proopiomelanocortin towards β-endorphin, an orexigen promoting feed intake. Plasma endocannabinoid concentrations and hypothalamic β-endorphin levels increase from late pregnancy to early lactation, but less is known about the regulation of the hypothalamic endocannabinoid system during the periparturient period of dairy cows. Dietary fatty acids may modulate the formation of endocannabinoids, which opens new avenues to improve metabolic health and immune status of dairy cows.

Dairy cows experience a negative energy balance due to increasing energy demands and insufficient voluntary feed intake in the transition from late pregnancy to early lactation. For supplying sufficient energy toward the conceptus and the mammary gland, insulin sensitivity in peripheral tissues is reduced leading to adipose tissue mobilization. Adiponectin, an insulin-sensitizing adipokine, is presumably related to energy metabolism and could play an important role in these metabolic adaptations. We hypothesize (1) that primiparous cows would differ from pluriparous cows in their circulating adiponectin concentrations during the transition from late pregnancy to early lactation and (2) that feeding different energy levels would affect the adiponectin concentrations during early lactation in dairy cows. For the first hypothesis, we examined 201 primiparous and 456 pluriparous Holstein dairy cows on three experimental farms. Ante partum, primiparous cows had lower adiponectin and greater NEFA concentrations than pluriparous cows, but vice versa post partum. Hence, adiponectin might be involved in the energy partitioning in primiparous cows (conceptus and lactation vs other still growing body tissues) with changing priorities from pregnancy to lactation. For the second hypothesis, 110 primiparous and 558 pluriparous Holstein and Simmental dairy cows in six experimental farms received either roughage with 6.1 or 6.5 MJ NEl/kg dry matter (adjusted with different amounts of wheat straw) ad libitum, combined with either 150 or 250 g concentrates/kg energy corrected milk. Greater amounts of concentrate lead to greater milk yield, but did not affect the blood variables. The higher energy level in the roughage led to greater glucose and IGF-1 but lower adiponectin in pluriparous cows. Further studies are needed to elucidate the mechanisms behind the roughage effect and its metabolic consequences.

Our aim was to compare the energy balance estimated (EBest) according to equations published by various energy feeding systems (German Society for Nutrition Physiology, French National Institute for Agricultural Research, and US National Research Council) and the EB calculated by use of calorimetrically measured heat production (EBhp) of 20 high-yielding (≥10,000 kg/305 d) German Holstein cows at -4 (pregnant, nonlactating) and 2 wk (early lactation) relative to parturition. In addition to heat production, feed and water intake, physical activity (including standing-lying behavior), body weight, body condition score, body temperature, plasma concentrations of fatty acids and β-hydroxybutyrate, milk yield, and milk composition were measured to characterize the metabolic status. The EBhp was balanced [2.74 ± 4.09 MJ of metabolizable energy (ME)/d; ±standard error] before calving, but strongly negative (-84.7 ± 7.48 MJ of ME/d) at wk 2 of lactation. At both time points, EBhp and EBest differed significantly. On average, the equations overestimated the antepartum EB by 33 MJ of ME/d and underestimated the postpartum negative EB by 67 MJ of ME/d, respectively. Because the same ME intake and energy-corrected milk values were used for calculation of EBest and EBhp in our study, we considered that the factors (0.488 to 0.534 MJ of ME/kg0.75) currently used to calculate the ME requirements for maintenance probably underestimate the needs of high-yielding dairy cows, particularly during early lactation. In accord, heat production values determined under standard conditions of thermoneutrality and locomotion restriction amounted to 0.76 ± 0.02 MJ of ME/kg0.75 (4 wk antepartum) and 1.02 ± 0.02 MJ of ME/kg0.75 (2 wk postpartum), respectively. The expected positive correlation between EBhp and DMI was observed in pregnant cows only; however, a bias of 26 MJ of ME/d between mean actual energy intake and ME intake predicted according to German Society for Nutrition Physiology was found in cows at wk 4 antepartum. At both investigated time points, mobilization of tissue energy reserves (reflected by plasma fatty acid concentration) was related to EBhp. In early lactating cows, metabolic body weight (kg0.75) and the percentage of milk fat showed the strongest correlation (correlation coefficient = -0.70 and -0.73) to EBhp. Our findings must be taken into account when experimental data are interpreted because the true energy status might be significantly overestimated when EBest is used.

Manure nitrogen (N) from cattle contributes to nitrous oxide and ammonia emissions and nitrate leaching. Measurement of manure N outputs on dairy farms is laborious, expensive, and impractical at large scales; therefore, models are needed to predict N excreted in urine and feces. Building robust prediction models requires extensive data from animals under different management systems worldwide. Thus, the study objectives were (1) to collate an international database of N excretion in feces and urine based on individual lactating dairy cow data from different continents; (2) to determine the suitability of key variables for predicting fecal, urinary, and total manure N excretion; and (3) to develop robust and reliable N excretion prediction models based on individual data from lactating dairy cows consuming various diets. A raw data set was created based on 5,483 individual cow observations, with 5,420 fecal N excretion and 3,621 urine N excretion measurements collected from 162 in vivo experiments conducted by 22 research institutes mostly located in Europe (n = 14) and North America (n = 5). A sequential approach was taken in developing models with increasing complexity by incrementally adding variables that had a significant individual effect on fecal, urinary, or total manure N excretion. Nitrogen excretion was predicted by fitting linear mixed models including experiment as a random effect. Simple models requiring dry matter intake (DMI) or N intake performed better for predicting fecal N excretion than simple models using diet nutrient composition or milk performance parameters. Simple models based on N intake performed better for urinary and total manure N excretion than those based on DMI, but simple models using milk urea N (MUN) and N intake performed even better for urinary N excretion. The full model predicting fecal N excretion had similar performance to simple models based on DMI but included several independent variables (DMI, diet crude protein content, diet neutral detergent fiber content, milk protein), depending on the location, and had root mean square prediction errors as a fraction of the observed mean values of 19.1% for intercontinental, 19.8% for European, and 17.7% for North American data sets. Complex total manure N excretion models based on N intake and MUN led to prediction errors of about 13.0% to 14.0%, which were comparable to models based on N intake alone. Intercepts and slopes of variables in optimal prediction equations developed on intercontinental, European, and North American bases differed from each other, and therefore region-specific models are preferred to predict N excretion. In conclusion, region-specific models that include information on DMI or N intake and MUN are required for good prediction of fecal, urinary, and total manure N excretion. In absence of intake data, region-specific complex equations using easily and routinely measured variables to predict fecal, urinary, or total manure N excretion may be used, but these equations have lower performance than equations based on intake., This study is part of the Global Network project, funded by the Joint Programming Initiative on Agriculture, Food Security and Climate Change (FACCE-JPI) and is an activity of the Feed and Nutrition Network, which is part of the Livestock Research Group of the Global Research Alliance for Agricultural Greenhouse Gases (https://globalresearchalliance.org; accessed May 10, 2021). Partial funding for the study was provided through USDA (Washington, DC) National Institute of Food and Agriculture (NIFA), awards 2014-67003-21979 and 2019-67019-29400, and Federal Appropriations under Project PEN 04539 and Accession Number 1000803 and FONDECYT/Regular Accession Number 1191476.

Improving feed utilization efficiency in dairy cattle could have positive economic and environmental effects that would support the sustainability of the dairy industry. Identifying key differences in metabolism between high and low feed-efficient animals is vital to enhancing feed conversion efficiency. Therefore, our objectives were (1) to determine whether cows grouped by either high or low feed efficiency have measurable differences in net fat and carbohydrate metabolism that account for differences in heat production (HP), and if so, whether these differences also exists under conditions of feed withdrawal when the effect of feeding on HP is minimized, and (2) to determine whether the abundance of mitochondria in the liver can be related to the high or low feed-efficient groups. Ten dairy cows from a herd of 15 (parity = 2) were retrospectively grouped into either a high (H) or a low (L) feed-efficient group (n = 5 per group) based on weekly energy-corrected milk (ECM) divided by dry mater intake (DMI) from wk 4 through 30 of lactation. Livers were biopsied at wk -4, 2, and 12, and blood was sampled weekly from wk -3 to 12 relative to parturition. Blood was subset to be analyzed for the transition period (wk -3 to 3) and from wk 4 to 12. In wk 5.70 ± 0.82 (mean ± SD) postpartum (PP), cows spent 2 d in respiration chambers (RC), in which CO2, O2, and CH4 gases were measured every 6 min for 24 h. Fatty acid oxidation (FOX), carbohydrate oxidation (COX), metabolic respiratory quotient (RQ), and HP were calculated from gas measurements for 23 h. Cows were fed ad libitum (AD-LIB) on d 1 and had feed withdrawn (RES, restricted diet) on d 2. Additional blood samples were taken at the end of the AD-LIB and RES feeding periods in the RC. During wk 4 to 30 PP, H had greater DMI/kg of metabolic body weight (BW0.75), ECM per kilogram of BW0.75 yield, and ECM/DMI ratio, compared with L, but a lower body condition score between wk 4 and 12 PP. In the RC period, we detected no differences in BW, DMI, or milk yield between groups. We also detected no significant group or group by feeding period interactions for plasma metabolites except for Revised Quantitative Insulin Sensitivity Check Index, which tended to have a group by feeding period interaction. The H group had lower HP and HP per kilogram of BW0.75 compared with L. Additionally, H had lower FOX and FOX per kilogram of BW0.75 compared with L during the AD-LIB period. Methane, CH4 per kilogram of BW0.75, and CH4 per kilogram of milk yield were lower in H compared with L, but, when adjusted for DMI, CH4/DMI did not differ between groups, nor did HP/DMI. Relative mitochondrial DNA copy numbers in the liver were lower in the L than in the H group. These results suggest that lower feed efficiency in dairy cows may result from fewer mitochondria per liver cell as well as a greater whole-body HP, which likely partially results from higher net fat oxidation.

High performing dairy cows experience distinct metabolic stress during periods of negative energy balance. Subclinical disorders of the cow’s energy metabolism facilitate failure of adaptational responses resulting in health problems and reduced performance. The autonomic nervous system (ANS) with its sympathetic and parasympathetic branches plays a predominant role in adaption to inadequate energy and/or fuel availability and mediation of the stress response. Therefore, we hypothesize that indices of heart rate variability (HRV) that reflect ANS activity and sympatho-vagal balance could be early markers of metabolic stress, and possibly useful to predict cows with compromised regulatory capacity. In this study we analysed the autonomic regulation and stress level of 10 pregnant dried-off German Holstein cows before, during and after a 10-h fasting period by using a wide range of HRV parameters. In addition heat production (HP), energy balance, feed intake, rumen fermentative activity, physical activity, non-esterified fatty acids, β-hydroxybutyric acid, cortisol and total ghrelin plasma concentrations, and body temperature (BT) were measured. In all cows fasting induced immediate regulatory adjustments including increased lipolysis (84%) and total ghrelin levels (179%), reduction of HP (−16%), standing time (−38%) and heart rate (−15%). However, by analysing frequency domain parameters of HRV (high-frequency (HF) and low-frequency (LF) components, ratio LF/HF) cows could be retrospectively assigned to groups reacting to food removal with increased or decreased activity of the parasympathetic branch of the ANS. Regression analysis reveals that under control conditions (feeding ad libitum) group differences were best predicted by the nonlinear domain HRV component Maxline (L MAX, R 2=0.76, threshold; TS=258). Compared with cows having L MAX values above TS (>L MAX: 348±17), those with L MAX values below TS (L MAX cows (18.5±0.4 and 47.3 kg/day). From the present study, it seems conceivable that L MAX can be used as a predictive marker to discover alterations in central autonomic regulation that might precede metabolic disturbances.

Free fatty acid receptors (FFAR) play significant roles in various physiological processes, including energy metabolism, through interaction with their ligands, fatty acids. To determine whether the receptors FFAR1 and FFAR2 are involved in the regulation of liver metabolism during the peripartal period, we selected 13 German Holstein multiparous dairy cows and grouped them as high β-hydroxybutyrate (H-BHB; n = 8) or low β-hydroxybutyrate (L-BHB; n = 5) according to their individual maximum plasma BHB concentration observed within wk 2 or 3 postpartum (H-BHB: >1 mmol/L and L-BHB

The evaluation of greenhouse gas mitigation strategies requires the quantitative assessment of individual methane production. Because methane measurement in respiration chambers is highly accurate, but also comprises various disadvantages such as limited capacity and high costs, the establishment of an indicator for estimating methane production of individual ruminants would provide an alternative to direct methane measurement. Methyl-coenzyme M reductase is involved in methanogenesis and the subunit α of methyl-coenzyme M reductase is encoded by the mcrA gene of rumen archaea. We therefore examined the relationship between methane emissions of Holstein dairy cows measured in respiration chambers with 2 different diets (high- and medium-concentrate diet) and the mcrA DNA and mcrA cDNA abundance determined from corresponding rumen fluid samples. Whole-body methane production per kilogram of dry matter intake and mcrA DNA normalized to the abundance of the rrs gene coding for 16S rRNA correlated significantly when using qmcrA primers. Use of qmcrA primers also revealed linear correlation between mcrA DNA copy number and methane yield. Regression analyses based on normalized mcrA cDNA abundances revealed no significant linear correlation with methane production per kilogram of dry matter intake. Furthermore, the correlations between normalized mcrA DNA abundance and the rumen fluid concentration of acetic and isobutyric acid were positive, whereas the correlations with propionic and lactic acid were negative. These data suggest that the mcrA DNA approach based on qmcrA primers could potentially be a molecular proxy for methane yield after further refinement.

Our objective was to compare the ranking of dairy cows according to their methane (CH) emissions as measured by a respiration chamber (RC) technique and the GreenFeed (GF) technique during 3 periods in second lactation. Two-day CH measurements in a RC performed in wk 3, 14, and 42 of lactation were flanked by GF measurements for 20 (period 1 [P1]), 35 (period 2 [P2]), and 35 (period 3 [P3]) days, respectively, before and after RC measurement. This gave the total duration of CH measurements using the GF system of 40, 70, and 70 d for P1, P2, and P3, respectively. Mean daily CH production (g/d) of the 8 dairy cows was 346, 439, and 430 using the RC technique and 338, 378, and 416 using the GF system during P1, P2, and P3, respectively. Average daily CH production determined by the GF technique was 2.4, 13.8, and 3.2% lower in P1, P2, and P3, respectively. Methane normalized to DMI continuously increased from P1 to P3 when measured in a RC, whereas it was lowest during P2 when measured by the GF method. Ranking of the cows according to CH production, CH/energy-corrected milk yield (ECM; CH/ECM), and CH/DMI differed between periods no matter which method was used. Cluster analysis including all 3 periods, however, identified the same cows with the highest and lowest CH production determined either by the RC technique or the GF system. In conclusion, multiple CH measurements at different stages of lactation are necessary for reliable discrimination of highest and lowest CH emitting cows and the GF system may be used to discriminate the extremes.

The liver plays a central role in adaptation for energy requirements around calving, and changes in the effects of insulin on hepatic energy metabolism contribute to metabolic adaptation in dairy cows. Hepatic insulin effects may depend on body fat mobilization. The objective of this study was to investigate the effects of insulin on the hepatic gene expression of enzymes involved in energy metabolism and factors related to nutrition partitioning in cows with high and low total liver fat concentration (LFC) after calving. Holstein cows were retrospectively grouped according to their LFC after calving as a proxy for body fat mobilization. Cows were classified as low (LLFC; LFC 24.4% fat/dry matter; n = 10) fat-mobilizing after calving. Euglycemic-hyperinsulinemic clamps [6 mU/(kg × min) of insulin for 6 h] were performed in wk 5 antepartum (ap) and wk 3 postpartum (pp). Before and at the end of the euglycemic-hyperinsulinemic clamps, liver biopsies were taken to measure the mRNA abundance of enzymes involved in carbohydrate and lipid metabolism, expression related to the somatotropic axis, and adrenergic and glucocorticoid receptors. The mRNA abundance of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase (PEPCK; PCK1), acyl-CoA-dehydrogenase very long chain (ACADVL), and hydroxyl-methyl-glutaryl-CoA-synthase 1 increased, but the mRNA abundance of solute carrier family 2 (SLC2A2 and SLC2A4), growth hormone receptor 1A (GHR1A), insulin-like growth factor 1 (IGF1), sterol regulatory element binding factor 1, adrenoceptor α 1A, and glucocorticoid receptor decreased from ap to pp. Insulin treatment was associated with decreased PCK1, mitochondrial PEPCK, glucose-6-phosphatase, propionyl-CoA-carboxylase α, carnitine-palmitoyl-transferase 1A, ACADVL, and insulin receptor mRNA, but increased IGF1 and SLC2A4 mRNA ap and pp and GHR1A mRNA pp. The mRNA abundance of SLC2A4 was greater, and the mRNA abundance of GHR1A and IGF1 tended to be lower in LLFC than in HLFC. Administration of insulin, albeit at a supraphysiological dose, was associated with inhibition of gene expression related to glucose production and β-oxidation, but we observed variable effects in the degree of insulin depression of individual genes. Insulin status is important for regulation of nutrient partitioning, but different LFC pp had very little influence on changes in hepatic gene expression following administration of insulin.

Hormones and metabolites act as satiety signals in the brain and play an important role in the control of feed intake (FI). These signals can reach the hypothalamus and brainstem, 2 major centers of FI regulation, via the blood stream or the cerebrospinal fluid (CSF). During the early lactation period of high-yielding dairy cows, the increase of FI is often insufficient. Recently, it has been demonstrated that insulin-like growth factors (IGF) may control FI. Thus, we asked in the present study if IGF-binding proteins (IGFBP) are regulated during the periparturient period and in response to feed restriction and therefore might affect FI as well. In addition, we specifically addressed conditional distribution of IGFBP in plasma and CSF. In one experiment, 10 multiparous German Holstein dairy cows were fed ad libitum and samples of CSF and plasma were obtained before morning feeding on d −20, −10, +1, +10, +20, and +40 relative to calving. In a second experiment, 7 cows in second mid-lactation were sampled for CSF and plasma after ad libitum feeding and again after feeding 50% of the previous ad libitum intake for 4 d. Intact IGFBP-2, IGFBP-3, and IGFBP-4 were detected in plasma by quantitative Western ligand blot analysis. In CSF, we were able to predominantly identify intact IGFBP-2 and a specific IGFBP-2 fragment containing detectable binding affinities for biotinylated IGF-II. Whereas plasma concentrations of IGFBP-2 and IGFBP-4 increased during the periparturient period, IGFBP-3 was unaffected over time. In CSF, concentrations of IGFBP-2, both intact and fragmented, were not affected during the periparturient period. Plasma IGF-I continuously decreased until calving but remained at a lower concentration in early lactation than in late pregnancy. Food restriction did not affect concentrations of IGF components present in plasma or CSF. We could show that the IGFBP profiles in plasma and CSF are clearly distinct and that changes in IGFBP in plasma do not simply correspond in the brain. We thus assume independent control of IGFBP distribution between plasma and CSF. Due to the known anorexic effect of IGF-I, elevated plasma concentrations of IGFBP-2 and IGFBP-4 during the postpartum period in conjunction with reduced plasma IGF-I concentrations may be interpreted as an endocrine response against negative energy balance in early lactation in dairy cows.

During early lactation, high-yielding dairy cows often show insufficient feed intake (FI) and, as a consequence, they enter into a negative energy balance associated with an altered pattern of plasma metabolites and hormones. These act as short- and long-term hunger or satiety signals in the brain and play an important role in the control of FI. Metabolites and hormones also occur in cerebrospinal fluid (CSF), which surrounds the hypothalamus and brainstem, 2 major centers of FI regulation. The CSF hormone and metabolite concentrations are mainly under control of the blood-brain barrier. Consequently, CSF hormone and metabolite concentrations differ from those in blood. However, the contribution of putative orexigenic and anorexigenic CSF signals possibly leading to insufficient FI of high-yielding dairy cows during early lactation has not been studied so far. Therefore, the aim of this study was to elucidate associations existing between both plasma and CSF hormones and metabolites during the periparturient period. Ten multiparous German Holstein dairy cows were fed ad libitum and samples of CSF from the spinal cord and blood from the jugular vein were withdrawn before morning feeding on d -20, -10, +1, +10, +20, and +40 relative to calving. Feed intake started to decrease from d 5 before calving and increased thereafter. Glucose, β-hydroxybutyrate (BHBA), cholesterol, nonesterified fatty acids, urea (all enzymatic), lactate (colorimetric), amino acids (HPLC), osmolality (osmometer), ghrelin (RIA), leptin (ELISA), and resistin (Western immunoblot) were measured in both CSF and plasma, whereas free fatty acids (gas chromatography-mass spectrometry) and volatile fatty acids (gas chromatography-flame-ionization detector) were determined in plasma only. Whereas leptin concentrations decreased after calving in both plasma and CSF, ghrelin concentrations were not altered, and abundances of total resistin and its hexamers decreased only in plasma. Although plasma concentrations of cholesterol and nonesterified fatty acids changed during the periparturient period, their concentrations were not affected in CSF. In contrast, CSF Gln concentration tended to increase until calving, whereas CSF concentrations of BHBA, α-aminobutyric acid, Cit, Gly, Ile, Val, and Leu were increased in early lactation compared with the preparturient period. Because Gln is known to serve as neuronal substrate generating ATP, Gln is suggested to act as a central anorexigenic signal shortly before parturition. Moreover, due to their known anorexic effect, BHBA and Leu may potentially act as central signals and thereby suppress a sufficient increase in FI during early lactation.

In addition to its role in energy storage, adipose tissue (AT) is an important endocrine organ and it secretes adipokines. The adipokine adiponectin improves insulin sensitivity by activation of its receptors AdipoR1 and AdipoR2. Lipolysis in AT is downregulated by the G-protein coupled receptor (GPR109A), which binds the endogenous ligand β-hydroxybutyrate (BHBA). Insulin sensitivity is reduced during the transition from late pregnancy to early lactation in dairy cattle and BHBA is increased postpartum, implying the involvement of the adiponectin system and GPR109A in this process. The aim of the current investigation was to study the effect of the genetic background of cows on the mRNA abundance of the adiponectin system, as well as GPR109A, in an F(2) population of 2 Charolais × German Holstein families. These families were deduced from full- and half-sibs sharing identical but reciprocal paternal and maternal Charolais grandfathers. The animals of the 2 families showed significant differences in fat accretion and milk secretion and were designated fat-type (high fat accretion but low milk production) and lean-type (low fat accretion but high milk production). The mRNA of the adiponectin system and GPR109A were quantified by real-time PCR in different fat depots (subcutaneous from back, mesenteric, kidney) and liver. The mRNA data were correlated with AT masses (intermuscular topside border fat, kidney, mesenteric, omental, total inner fat mass, total subcutaneous fat mass, and total fat mass) and blood parameters (glucose, nonesterified fatty acids, BHBA, urea, insulin, and glucagon). The abundance of adiponectin system mRNA was higher in discrete AT depots of fat-type cows [adiponectin mRNA in mesenteric fat (trend), AdipoR1 in kidney and mesenteric AT, and AdipoR2 in subcutaneous fat (trend)] than in lean-type cows. More GPR109A mRNA was found in kidney fat of the lean-type family than in that of the fat-type family. In liver, the abundance of AdipoR2 and GPR109A (trend) mRNA was higher in lean-type than in fat-type cows. Correlation analyses disclosed clear differences between the groups. In total, the results revealed obvious disparities for the mRNA targets between the 2 families with common but reciprocal paternal and maternal genetic backgrounds. Visceral AT mass of both families showed most correlations with the mRNA abundance of the target genes in different AT depots. The effect of adiponectin secretion, especially by visceral AT depots, on liver metabolism should be clarified in further studies.

A general phenomenon in peripartum mammals is the breakdown of (acquired) immunity. The incidence of parasite load, disease and inflammation often rise during the specific energetically demanding time of pregnancy and lactation. In this period, blood leukocytes display decreased DNA synthesis in response to mitogens in vitro. Leukocyte activation, the phase of the cell cycle preceding the DNA synthetic phase has hardly been investigated, but the few studies suggest that leukocyte activation may also be impaired by the limited energy/nutrient availability. Leukocyte activation is characterized by manifold processes, thus, we used the cellular oxygen consumption rate (OCR) as a measure of ATP turnover to support all these processes. We hypothesized that the activation of peripheral blood mononuclear cells (PBMC) - in terms of oxygen consumed over basal levels after in vitro stimulation - is altered by energy balance around parturition. We studied peripartum high-yielding dairy cows because they undergo substantial fluctuations in energy intake, energy output and body fat mass. We established a fluorescence-based test strategy allowing for long-term (≥24h) quantification of O(2)-consumption and studied the peripartum period from 5 weeks ante partum to 5 weeks postpartum. In addition, we determined cellular lactate production, DNA/RNA synthesis and cell size and zoo-technical parameters such as animal energy intake and milk yield were assessed, as well as selected plasma parameters, e.g. glucose concentration. The basal OCR of PBMC from pregnant, non-lactating cows (n=6, -5 weeks ante partum) was 1.19±0.15 nmol min(-1) (10(7)cells)(-1) and increased to maximum levels of 2.54±0.49 nmol min(-1) (10(7)cells)(-1) in phytohemagglutinin (PHA)-stimulated PBMC. The basal OCR did not change over the peripartum period. Whereas the activation indices, herein defined as the PHA-induced 24h-increase of OCR above baseline, amounted to 1.1±0.3, 4.2±0.3, 4.1±1.1, 2.1±0.3, and 2.7±0.5 at weeks -5, -1, +1, +2, and +5 relative to parturition, respectively. Because the activation index was positively correlated to plasma glucose levels and to energy balance during late pregnancy (week -5/week -1) and transition to lactation (week -1/week +2), we conclude that PBMC activation is modulated by energy/nutrient availability. In future studies, the activation index should aid the identification of causal mechanisms of disparity in PBMC activation, such as attenuated ion transport or macromolecule synthesis.

Archaeol (1,2-di- O -phytanyl- sn -glycerol) is a cell membrane lipid component of methanogens that has the potential to be used as a biomarker for methane production in ruminants. However, its analysis via gas chromatography–mass spectrometry (GC-MS) is challenging because of its molecular size and structure. In this study, 2 different sample preparation methods were tested, Soxhlet and sonication-aided extraction, and the methods were compared for extraction efficiency using the internal standard (IS; 1,2-di- o -hexadecyl- rac -glycerol). The extraction efficiency of the Soxhlet method for fecal archaeol was twice that of sonication. With the use of a high-temperature GC column, the retention times of IS and archaeol were 17.6 and 19.4 min, respectively, with a total run time of only 25 min. The molecule ions m / z 611.4 (IS) and m / z 725.8 (archaeol), or alternatively the fragment ion of the glycerol moiety m / z 130.0, were used for identification and quantification via GC-MS in positive chemical ionization mode. The intra-assay coefficients of variation for fecal archaeol measurements were 1.3% ( m / z 725.8) and 2.1% ( m / z 130.0) (n=3), respectively. Fecal archaeol quantifications did not differ between the use of the molecule or glycerol moiety ions (paired t -test, n=156). Archaeol concentrations tended to be 3.3% greater in samples stored at −20°C before drying compared with samples that were immediately dried after collection (paired t -test, n=5). The detection limit of archaeol was 0.5 µg/g of fecal dry matter (DM); no archaeol could be detected in feed samples. In different fractions of rumen fluid, archaeol levels ranged from 1.9 to 24.0 µg/g of DM. In 10 cows fed the same grass and corn silage/hay-based ration, diurnal variations of fecal archaeol levels (5 time points over 2 d) were cow dependent and ranged from 26.2 to 77.2 µg/g of DM (mean 48.4 µg/g of DM). Thus, within-animal variation in cows on the same diet was between 4 and 27%. We suggest that this finding is related to the amount and time of the latest feed intake event before the fecal sampling. Feeding pattern can determine the passage rate of digesta through the alimentary tract and thus the duration of contact time of archaea with their substrate.

Dairy cows undergo significant metabolic and endocrine changes during the transition from pregnancy to lactation, and impaired insulin action influences nutrient partitioning toward the fetus and the mammary gland. Because impaired insulin action during transition is thought to be related to elevated body condition and body fat mobilization, we hypothesized that over-conditioned cows with excessive body fat mobilization around calving may have impaired insulin metabolism compared with cows with low fat mobilization. Nineteen dairy cows were grouped according to their average concentration of total liver fat (LFC) after calving in low [LLFC; LFC 24.4% total fat/DM; n=10) fat-mobilizing cows. Blood samples were taken from wk 7 antepartum (ap) to wk 5 postpartum (pp) to determine plasma concentrations of glucose, insulin, glucagon, and adiponectin. We applied euglycemic-hyperinsulinemic (EGHIC) and hyperglycemic clamps (HGC) in wk 5 ap and wk 3 pp to measure insulin responsiveness in peripheral tissue and pancreatic insulin secretion during the transition period. Before and during the pp EGHIC, [(13)C6] glucose was infused to determine the rate of glucose appearance (GlucRa) and glucose oxidation (GOx). Body condition, back fat thickness, and energy-corrected milk were greater, but energy balance was lower in HLFC than in LLFC. Plasma concentrations of glucose, insulin, glucagon, and adiponectin decreased at calving, and this was followed by an immediate increase of glucagon and adiponectin after calving. Insulin concentrations ap were higher in HLFC than in LLFC cows, but the EGHIC indicated no differences in peripheral insulin responsiveness among cows ap and pp. However, GlucRa and GOx:GlucRa during the pp EGHIC were greater in HLFC than in LLFC cows. During HGC, pancreatic insulin secretion was lower, but the glucose infusion rate was higher pp than ap in both groups. Plasma concentrations of nonesterified fatty acids decreased during HGC and EGHIC, but in both clamps, pp nonesterified fatty acid concentrations did not reach the ap levels. The study demonstrated a minor influence of different degrees of body fat mobilization on insulin metabolism in cows during the transition period. The distinct decrease in the glucose-dependent release of insulin pp is the most striking finding that explains the impaired insulin action after calving, but does not explain differences in body fat mobilization between HLFC and LLFC cows.

We recently showed that the mRNA expression of genes encoding for specific nutrient sensing receptors, namely the free fatty acid receptors (FFAR) 1, 2, 3, and the hydroxycarboxylic acid receptor (HCAR) 2, undergo characteristic changes during the transition from late pregnancy to lactation in certain adipose tissues (AT) of dairy cows. We hypothesised that divergent energy intake achieved by feeding diets with either high or low portions of concentrate (60% v. 30% concentrate on a dry matter basis) will alter the mRNA expression of FFAR 1, 2, 3, as well as HCAR2 in subcutaneous (SCAT) and retroperitoneal AT (RPAT) of dairy cows in the first 3 weeks postpartum (p.p.). For this purpose, 20 multiparous German Holstein cows were allocated to either the high concentrate ration (HC, n=10) or the low concentrate ration (LC, n=10) from day 1 to 21 p.p. Serum samples and biopsies of SCAT (tail head) and RPAT (above the peritoneum) were obtained at day −21, 1 and 21 relative to parturition. The mRNA abundances were measured by quantitative PCR. The concentrations of short-chain fatty acid (SCFA) in serum were measured by gas chromatography-flame ionisation detector. The FFAR1 and FFAR2 mRNA abundance in RPAT was higher at day −21 compared to day 1. At day 21 p.p. the FFAR2 mRNA abundance was 2.5-fold higher in RPAT of the LC animals compared to the HC cows. The FFAR3 mRNA abundance tended to lower values in SCAT of the LC group at day 21. The HCAR2 mRNA abundance was neither affected by time nor by feeding in both AT. On day 21 p.p. the HC group had 1.7-fold greater serum concentrations of propionic acid and lower concentrations of acetic acid (trend: 1.2-fold lower) compared with the LC group. Positive correlations between the mRNA abundance of HCAR2 and peroxisome proliferator-activated receptor γ-2 (PPARG2) indicate a link between HCAR2 and PPARG2 in both AT. We observed an inverse regulation of FFAR2 and FFAR3 expression over time and both receptors also showed an inverse mRNA abundance as induced by different portions of concentrate. Thus, indicating divergent nutrient sensing of both receptors in AT during the transition period. We propose that the different manifestation of negative EB in both groups at day 21 after parturition affect at least FFAR2 expression in RPAT.

The high metabolic priority of the mammary gland for milk production, accompanied by limited feed intake around parturition results in a high propensity to mobilize body fat reserves. Under these conditions, fuel selection of many peripheral organs is switched, for example, from carbohydrate to fat utilization to spare glucose for milk production and to ensure partitioning of tissue- and dietary-derived nutrients toward the mammary gland. For example, muscle tissue uses nonesterified fatty acids (NEFA) but releases lactate and amino acids in a coordinated order, thereby providing precursors for milk synthesis or hepatic gluconeogenesis. Tissue metabolism and in concert, nutrient partitioning are controlled by the endocrine system involving a reduction in insulin secretion and systemic insulin sensitivity and orchestrated changes in plasma hormones such as insulin, adiponectin, insulin growth factor-I, growth hormone, glucagon, leptin, glucocorticoids, and catecholamines. However, the endocrine system is highly sensitive and responsive to an overload of fatty acids no matter if excessive NEFA supply originates from exogenous or endogenous sources. Feeding a diet containing rumen-protected fat from late lactation to calving and beyond exerts similar negative effects on energy intake, glucose and insulin concentrations as does a high extent of body fat mobilization around parturition in regard to the risk for ketosis and fatty liver development. High plasma NEFA concentrations are thought not to act directly at the brain level, but they increase the energy charge of the liver which is, signaled to the brain to diminish feed intake. Cows differing in fat mobilization during the transition phase differ in their hepatic energy charge, whole body fat oxidation, glucose metabolism, plasma ghrelin, and leptin concentrations and in feed intake several week before parturition. Hence, a high lipid load, no matter if stored, mobilized or fed, affects the endocrine system, metabolism, and feed intake, and increases the risk for metabolic disorders. Future research should focus on a timely parallel increase in feed intake and milk yield during early lactation to reduce the impact of body fat on feed intake, metabolic health, and negative energy balance.

Our objective was to investigate the quantitative and qualitative effects of propylene glycol (PG) allocation on postpartum adipose tissue mobilization in over-conditioned Holstein cows. Nine ruminally cannulated and arterially catheterized cows were, at parturition, randomly assigned to a ruminal pulse dose of either 500 g of tap water (n = 4) or 500 g of PG (n = 5) once a day. The PG was given with the morning feeding for 4 wk postpartum (treatment period), followed by a 4-wk follow-up period. All cows were fed the same prepartum and postpartum diets. At –16 (±3), 4 (±0), 15 (±1) and 29 (±2) days in milk (DIM), body composition was determined using the deuterium oxide dilution technique, liver and subcutaneous adipose tissue biopsies were collected, and mammary gland nutrient uptake was measured. Weekly blood samples were obtained during the experiment and daily blood samples were taken from –7 to 7 DIM. Postpartum feed intake and milk yield was not affected by PG allocation. The body content of lipid was not affected by treatment, but tended to decrease from 4 to 29 DIM with both treatments. Except for the first week postpartum, no difference in plasma nonesterified fatty acids concentration was noted between treatments in the treatment period. Yet, PG allocation resulted in decreased plasma concentrations of β-hydroxybutyrate (BHB) and increased plasma concentrations of glucose. In the follow-up period, plasma concentrations of nonesterified fatty acids, glucose, and BHB did not differ between treatments. Additionally, the change in abundance of proteins in adipose tissue biopsies from prepartum to 4 DIM was not affected by treatment. In conclusion, the different variables to assess body fat mobilization were concurrent and showed that a 4-wk postpartum PG allocation had limited effect on adipose tissue mobilization. The main effect was an enhanced glucogenic status with PG. No carry-over effect of PG allocation was recorded for production or plasma metabolites, and, hence, a new period of metabolic adaption to lactation seemed to occur with PG treatment after ceasing PG allocation. Thus, PG seemed to induce a 2-step adaption to lactation, reducing the immediate postpartum nadir and peak of plasma concentration of glucose and BHB, respectively; which is beneficial for postpartum cows at high risk of lipid-related metabolic diseases.

The beginning of lactation requires huge metabolic adaptations to meet increased energy demands for milk production of dairy cows. One of the adaptations is the mobilization of body reserves mainly from adipose tissue as reflected by increased plasma nonesterified fatty acid (NEFA) concentrations. The capacity of the liver for complete oxidation of NEFA is limited, leading to an increased formation of ketone bodies, reesterification, and accumulation of triglycerides in the liver. As the skeletal muscle also may oxidize fatty acids, it may help to decrease the fatty acid load on the liver. To test this hypothesis, 19 German Holstein cows were weekly blood sampled from 7 wk before until 5 wk after parturition to analyze plasma NEFA concentrations. Liver biopsies were obtained at d 3, 18, and 30 after parturition and, based on the mean liver fat content, cows were grouped to the 10 highest (HI) and 9 lowest (LO). In addition, muscle biopsies were obtained at d -17, 3, and 30 relative to parturition and used to quantify mRNA abundance of genes involved in fatty acid degradation. Plasma NEFA concentrations peaked after parturition and were 1.5-fold higher in HI than LO cows. Muscle carnitine palmitoyltransferase 1α and β mRNA was upregulated in early lactation. The mRNA abundance of muscle peroxisome proliferator-activated receptor γ (PPARG) increased in early lactation and was higher in HI than in LO cows, whereas the abundance of PPARA continuously decreased after parturition. The mRNA abundance of muscle PPARD, uncoupling protein 3, and the β-oxidative enzymes 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase, very long-chain acyl-CoA dehydrogenase, and 3-ketoacyl-CoA was greatest at d 3 after parturition, whereas the abundance of PPARγ coactivator 1α decreased after parturition. Our results indicate that around parturition, oxidation of fatty acids in skeletal muscle is highly activated, which may contribute to diminish the fatty acid load on the liver. The decline in muscle fatty acid oxidation within the first 4 wk of lactation accompanied with increased feed intake refer to greater supply of ruminally derived acetate, which as the preferred fuel of the muscle, saves long-chain fatty acids for milk fat production.

In addition to plasma metabolites and hormones participating as humoral signals in the control of feed intake, oxidative metabolic processes in peripheral organs also generate signals to terminate feeding. Although the degree of oxidation over longer periods is relatively constant, recent work suggests that the periprandial pattern of fuel oxidation is involved in regulating feeding behavior in the bovine. However, the association between periprandial oxidative metabolism and feed intake of dairy cows has not yet been studied. Therefore, the aim of this study was to elucidate possible associations existing between single feed intake events and whole-body net fat and net carbohydrate oxidation as well as their relation to plasma metabolite concentrations. To this end, 4 late-lactating cows equipped with jugular catheters were kept in respiratory chambers with continuous and simultaneous recording of gas exchange and feed intake. Animals were fed ad libitum (AL) for 24h and then feed restricted (RE) to 50% of the previous AL intake for a further 24h. Blood samples were collected hourly to analyze β-hydroxybutyrate (BHBA), glucose, nonesterified fatty acids (NEFA), insulin, and acylated ghrelin concentrations. Cross-correlation analysis revealed an offset ranging between 30 and 42 min between the maximum of a feed intake event and the lowest level of postprandial net fat oxidation (FOX(net)) and the maximum level of postprandial net carbohydrate oxidation (COX(net)), respectively. During the AL period, FOX(net) did not increase above -0.2g/min, whereas COX(net) did not decrease below 6g/min before the start of the next feed intake event. A strong inverse cross-correlation was obtained between COX(net) and plasma glucose concentration. Direct cross-correlations were observed between COXnet and insulin, between heat production and BHBA, between insulin and glucose, and between BHBA and ghrelin. We found no cross-correlation between FOX(net) and NEFA. During RE, FOX(net) increased with an exponential slope, exceeded the threshold of -0.2g/min as indicated by increasing plasma NEFA concentrations, and approached a maximum rate of 0.1g/min, whereas COX(net) decayed in an exponential manner, approaching a minimal COX(net) rate of about 2.5 g/min in all cows. Our novel findings suggest that, in late-lactating cows, postprandial increases in metabolic oxidative processes seem to signal suppression of feed intake, whereas preprandially an accelerated FOX(net) rate and a decelerated COX(net) rate initiate feed intake.

Modern animal breeding programs are largely based on biotechnological procedures, including AI and embryo transfer technology. Recent breakthroughs in reproductive technologies, such as somatic cell nuclear transfer and in vitro embryo production, and their combination with the emerging molecular genetic tools, will further advance progress and provide new opportunities for livestock breeding. This is urgently needed in light of the global challenges such as the ever-increasing human population, the limited resources of arable land, and the urgent environmental problems associated with farm animal production. Here, we focus on genomic breeding strategies and transgenic approaches for making farm animals more feed efficient. Based on studies in the mouse and rat model, we have identified a panel of genes that are critically involved in the regulation of feed uptake and that could contribute toward future breeding of farm animals with reduced environmental impact. We anticipate that genetically modified animals will play a significant role in shaping the future of feed-efficient and thus sustainable animal production, but will develop more slowly than the biomedical applications because of the complexity of the regulation of feed intake and metabolism.

A stereotaxic apparatus for access to the lateral brain ventricle of Holstein cows was constructed. This apparatus permits topographic orientation on the head of an anesthetized cow that has been placed in right lateral recumbency. After assessing the XY-plane in parallel to the frontal bone, correct coordinates in reference to the skullcap were adjusted. The lateral brain ventricles were accessed by drilling holes perpendicular to the XY-plane. The subsequent surgical implant of a tailor-made cannula guide enabled the repeatable placement of a catheter. This permanent catheter allowed the frequent withdrawal of cerebrospinal fluid from and injections into cerebral ventricles of nonanesthetized dairy cows. This method is critical for elucidating the role of central hormones, metabolites, and electrolytes in the regulation of diverse physiological processes and for the study of neuropathological disorders in cows.