Predicting CO2 production of lactating dairy cows from animal, dietary, and production traits using an international dataset.

Automated measurements of the ratio of concentrations of methane and carbon dioxide, [CH4]:[CO2], in breath from individual animals (the so-called "sniffer technique") and estimated CO2 production can be used to estimate CH4 production, provided that CO2 production can be reliably calculated. This would allow CH4 production from individual cows to be estimated in large cohorts of cows, whereby ranking of cows according to their CH4 production might become possible and their values could be used for breeding of low CH4-emitting animals. Estimates of CO2 production are typically based on predictions of heat production, which can be calculated from body weight (BW), energy-corrected milk yield, and days of pregnancy. The objectives of the present study were to develop predictions of CO2 production directly from milk production, dietary, and animal variables, and furthermore to develop different models to be used for different scenarios, depending on available data. An international dataset with 2,244 records from individual lactating cows including CO2 production and associated traits, as dry matter intake (DMI), diet composition, BW, milk production and composition, days in milk, and days pregnant, was compiled to constitute the training dataset. Research location and experiment nested within research location were included as random intercepts. The method of CO2 production measurement (respiration chamber [RC] or GreenFeed [GF]) was confounded with research location, and therefore excluded from the model. In total, 3 models were developed based on the current training dataset: model 1 ("best model"), where all significant traits were included; model 2 ("on-farm model"), where DMI was excluded; and model 3 ("reduced on-farm model"), where both DMI and BW were excluded. Evaluation on test dat sets with either RC data (n = 103), GF data without additives (n = 478), or GF data only including observations where nitrate, 3-nitrooxypropanol (3-NOP), or a combination of nitrate and 3-NOP were fed to the cows (GF+: n = 295), showed good precision of the 3 models, illustrated by low slope bias both in absolute values (-0.22 to 0.097) and in percentage (0.049 to 4.89) of mean square error (MSE). However, the mean bias (MB) indicated systematic overprediction and underprediction of CO2 production when the models were evaluated on the GF and the RC test datasets, respectively. To address this bias, the 3 models were evaluated on a modified test dataset, where the CO2 production (g/d) was adjusted by subtracting (where measurements were obtained by RC) or adding absolute MB (where measurements were obtained by GF) from evaluation of the specific model on RC, GF, and GF+ test datasets. With this modification, the absolute values of MB and MB as percentage of MSE became negligible. In conclusion, the 3 models were precise in predicting CO2 production from lactating dairy cows.

Influence of lactation stage on heat production and macronutrient oxidation in dairy cows during a 24-hour fasting period.

Understanding nutrient utilization and partitioning is essential for advancing the efficiency of dairy cattle. Our objective was to determine if dairy cows exposed to a 24-h fasting period differ in heat production (HP) and macronutrient oxidation at different stages of lactation. Twelve primiparous, lactating German Holstein dairy cows were used in a longitudinal study design spanning from 2013 to 2014. Dairy cows were housed in respiration chambers during 3 stages of the lactation cycle: early (mean ± SD; 28.8 ± 6.42 d), mid- (89.4 ± 4.52 d), and late (293 ± 7.76 d) lactation. Individual CO2, O2, and CH4 gas exchanges were measured every 6 min for two 24-h periods, an ad libitum period and fasting period (RES). Blood was sampled at the start and end of the RES period. Gas measurements were used to calculate HP, net carbohydrate oxidation (COX), and net fat oxidation (FOX). Measurements were corrected with metabolic BW (kg of BW0.75; cBW). The RES period for each stage of lactation was further subdivided into the start (RESstart) and end (RESend) by averaging the first and last 2 h of the RES period. The net change was calculated as RESend - RESstart. All energy variables differed among lactation stage within the RES period except for HP/cBW. As expected, COX, COX/cBW, COX/HP, HP, and HP/cBW, were greater at the RESstart compared with RESend, whereas FOX, FOX/cBW, and FOX/HP were greater at the RESend except for FOX and FOX/cBW during mid lactation, which was only a tendency for a difference. The net change for COX, COX/cBW, HP, HP/cBW, and FOX/cBW did not differ among stages of lactation. Despite detecting a tendency for a difference among stage of lactation for FOX, pairwise analysis revealed no differences. Plasma triglyceride, urea, and nonesterified fatty acid concentrations were greater at RESend than RESstart. The net change for plasma glucose, urea, ß-hydroxybutyrate, and nonesterified fatty acid concentrations were greater in early than late lactation. Our results demonstrate that despite differences in absolute measurements of energy variables and plasma metabolites, the change in whole-body macronutrient oxidation and HP as cows' transition from a fed-like state to a starvation-like state during a 24-h fasting period is consistent throughout lactation.

Influence of dietary fiber content and horn status on thermoregulatory responses of Brown Swiss dairy cows under thermoneutral and short-term heat stress conditions.

In the present experiment, 10 horned and 10 disbudded mid-lactating Brown Swiss cows were included in a crossover feeding trial with a hay or hay and concentrate diet. The effects of dietary neutral detergent fiber (NDF) content and horn status on thermoregulatory responses under thermoneutral and short-term heat stress conditions were studied, as both are considered to ease the cow's thermoregulation under an environmental heat load. Cows received either ad libitum hay and alfalfa pellets (85:15, C-, NDF content: 41.0%) or restricted amounts of hay and concentrate (70:30, C+, NDF content: 34.5%). The level of restriction applied with the C+ diet was determined from pre-experimental ad libitum intakes, ensuring that both diets provided the same intake of net energy for lactation (NEL). For data collection, cows were housed in respiration chambers for 5 d. The climatic conditions were 10°C and 60% relative humidity (RH), considered thermoneutral (TN) conditions (temperature-humidity index (THI): 52) for d 1 and 2, and 25°C and 70% RH, considered heat stress (HS) conditions (THI: 74), for d 4 and 5. On d 3, the temperature and RH were increased gradually. Compared with TN, HS conditions increased the water intake, skin temperature, respiration and heart rates, and endogenous heat production. They did not affect body temperature, feed intake, or milk production. Lowering dietary fiber content via concentrate supplementation lowered methane and increased carbon dioxide production. It did not mitigate physiological responses to HS. Although the responses of horned and disbudded cows were generally similar, the slower respiration rates of horned cows under HS conditions indicate a possible, albeit minor, role of the horn in thermoregulation. In conclusion, future investigations on nutritional strategies must be conducted to mitigate mild heat stress.

Prediction of nitrogen excretion from data on dairy cows fed a wide range of diets compiled in an intercontinental database: A meta-analysis.

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.

Differences in net fat oxidation, heat production, and liver mitochondrial DNA copy numbers between high and low feed-efficient dairy cows.

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.

Comparative analyses of estimated and calorimetrically determined energy balance in high-yielding dairy cows.

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 ß-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.

Short communication: Development of an equation for estimating methane emissions of dairy cows from milk Fourier transform mid-infrared spectra by using reference data obtained exclusively from respiration chambers.

Evaluation and mitigation of enteric methane (CH4) emissions from ruminant livestock, in particular from dairy cows, have acquired global importance for sustainable, climate-smart cattle production. Based on CH4 reference measurements obtained with the SF6 tracer technique to determine ruminal CH4 production, a current equation permits evaluation of individual daily CH4 emissions of dairy cows based on milk Fourier transform mid-infrared (FT-MIR) spectra. However, the respiration chamber (RC) technique is considered to be more accurate than SF6 to measure CH4 production from cattle. This study aimed to develop an equation that allows estimating CH4 emissions of lactating cows recorded in an RC from corresponding milk FT-MIR spectra and to challenge its robustness and relevance through validation processes and its application on a milk spectral database. This would permit confirming the conclusions drawn with the existing equation based on SF6 reference measurements regarding the potential to estimate daily CH4 emissions of dairy cows from milk FT-MIR spectra. A total of 584 RC reference CH4 measurements (mean ± standard deviation of 400 ± 72 g of CH4/d) and corresponding standardized milk mid-infrared spectra were obtained from 148 individual lactating cows between 7 and 321 d in milk in 5 European countries (Germany, Switzerland, Denmark, France, and Northern Ireland). The developed equation based on RC measurements showed calibration and cross-validation coefficients of determination of 0.65 and 0.57, respectively, which is lower than those obtained earlier by the equation based on 532 SF6 measurements (0.74 and 0.70, respectively). This means that the RC-based model is unable to explain the variability observed in the corresponding reference data as well as the SF6-based model. The standard errors of calibration and cross-validation were lower for the RC model (43 and 47 g/d vs. 66 and 70 g/d for the SF6 version, respectively), indicating that the model based on RC data was closer to actual values. The root mean squared error (RMSE) of calibration of 42 g/d represents only 10% of the overall daily CH4 production, which is 23 g/d lower than the RMSE for the SF6-based equation. During the external validation step an RMSE of 62 g/d was observed. When the RC equation was applied to a standardized spectral database of milk recordings collected in the Walloon region of Belgium between January 2012 and December 2017 (1,515,137 spectra from 132,658 lactating cows in 1,176 different herds), an average ± standard deviation of 446 ± 51 g of CH4/d was estimated, which is consistent with the range of the values measured using both RC and SF6 techniques. This study confirmed that milk FT-MIR spectra could be used as a potential proxy to estimate daily CH4 emissions from dairy cows provided that the variability to predict is covered by the model.

Symposium review: Uncertainties in enteric methane inventories, measurement techniques, and prediction models.

Ruminant production systems are important contributors to anthropogenic methane (CH4) emissions, but there are large uncertainties in national and global livestock CH4 inventories. Sources of uncertainty in enteric CH4 emissions include animal inventories, feed dry matter intake (DMI), ingredient and chemical composition of the diets, and CH4 emission factors. There is also significant uncertainty associated with enteric CH4 measurements. The most widely used techniques are respiration chambers, the sulfur hexafluoride (SF6) tracer technique, and the automated head-chamber system (GreenFeed; C-Lock Inc., Rapid City, SD). All 3 methods have been successfully used in a large number of experiments with dairy or beef cattle in various environmental conditions, although studies that compare techniques have reported inconsistent results. Although different types of models have been developed to predict enteric CH4 emissions, relatively simple empirical (statistical) models have been commonly used for inventory purposes because of their broad applicability and ease of use compared with more detailed empirical and process-based mechanistic models. However, extant empirical models used to predict enteric CH4 emissions suffer from narrow spatial focus, limited observations, and limitations of the statistical technique used. Therefore, prediction models must be developed from robust data sets that can only be generated through collaboration of scientists across the world. To achieve high prediction accuracy, these data sets should encompass a wide range of diets and production systems within regions and globally. Overall, enteric CH4 prediction models are based on various animal or feed characteristic inputs but are dominated by DMI in one form or another. As a result, accurate prediction of DMI is essential for accurate prediction of livestock CH4 emissions. Analysis of a large data set of individual dairy cattle data showed that simplified enteric CH4 prediction models based on DMI alone or DMI and limited feed- or animal-related inputs can predict average CH4 emission with a similar accuracy to more complex empirical models. These simplified models can be reliably used for emission inventory purposes.

Short communication: Free fatty acid receptors FFAR1 and FFAR2 during the peripartal period in liver of dairy cows grouped by their postpartum plasma ß-hydroxybutyrate concentrations.

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 ß-hydroxybutyrate (H-BHB; n = 8) or low ß-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: <0.77 mmol/L). The selected cows had a milk yield of more than 10,000 kg/305 d during a previous lactation. The cows were fed a total mixed ration according to their requirements during the far-off dry period [5.9 MJ of net energy for lactation (NEL)/kg of dry matter (DM), crude protein (CP) 126 g/kg of DM], close-up dry period (6.5 MJ of NEL/kg of DM, CP 137 g/kg of DM), and lactation (7 MJ of NEL/kg of DM, CP 163 g/kg of DM). Blood samples were taken weekly, from d -34 to d 40 relative to parturition. Liver biopsies were taken on d -34, -17, 3, 18, and 30 relative to parturition and at slaughter (d 40). The protein abundance of FFAR1 was lower during the whole peripartal period in the H-BHB group. The abundance of FFAR2 increased over time and tended to be higher in H-BHB cows. The abundance of FFAR1 might be associated with imbalances of liver metabolism in peripartal dairy cows.

Variable liver fat concentration as a proxy for body fat mobilization postpartum has minor effects on insulin-induced changes in hepatic gene expression related to energy metabolism in dairy cows.

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% fat/dry matter; n = 9) and high (HLFC; 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 ß-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.

Technical note: Analytical refinements of the methane indicator archaeol in bovine feces, rumen fluid, and feedstuffs.

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.

Insulin-dependent glucose metabolism in dairy cows with variable fat mobilization around calving.

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% total fat/dry matter (DM); n=9] and high (HLFC; 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.

Effect of propylene glycol on adipose tissue mobilization in postpartum over-conditioned Holstein cows.

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 500g of tap water (n=4) or 500g 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 ß-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.

Methyl-coenzyme M reductase A as an indicator to estimate methane production from dairy cows.

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.

Effects of parturition and feed restriction on concentrations and distribution of the insulin-like growth factor-binding proteins in plasma and cerebrospinal fluid of dairy cows.

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.

Fuel feeds function: Energy balance and bovine peripheral blood mononuclear cell activation.

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.

Abundance of adiponectin system and G-protein coupled receptor GPR109A mRNA in adipose tissue and liver of F2 offspring cows of Charolais × German Holstein crosses that differ in body fat accumulation.

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 ß-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.

Concentrations of hormones and metabolites in cerebrospinal fluid and plasma of dairy cows during the periparturient period.

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, ß-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.

Increased muscle fatty acid oxidation in dairy cows with intensive body fat mobilization during early lactation.

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 ß 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 ß-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.

Short-term feed intake is regulated by macronutrient oxidation in lactating Holstein cows.

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 ß-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.