Blood Plasma Non-esterified Fatty Acids Research Articles

Short communication: Predicting blood plasma non-esterified fatty acid and beta-hydroxybutyrate concentrations from cow milk-addressing systematic issues in modelling.

Negative energy status in early lactation is linked to a variety of metabolic disorders, reduced fertility, and decreased milk production. To improve the energy status of cows by breeding and management, the identification of negative energy status is crucial. While biomarkers such as non-esterified fatty acid (NEFA) concentration and beta-hydroxybutyrate (BHB) in blood plasma could be used to identify a negative energy state, measuring them directly from blood is both invasive and expensive. In this work, we developed prediction equations for blood plasma NEFA and BHB levels based on mid-IR spectral measurements of milk. The models were fitted using partial least squares regression and evaluated using both cross-validation and independent-herd validation. A total of 3183 spectral records from 606 lactations originating from three different herds were utilised. R2 values of 0.53 (RMSE=0.206mmol/l, RMSE of cross-validation (RMSECV) 0.217mmol/l) for NEFA and 0.63 (RMSE=0.326mmol/l, RMSECV=0.353mmol/l) for BHB were obtained. Furthermore, relatively similar prediction accuracies were found for BHB (RMSE of prediction (RMSEP) 0.411mmol/l and 0.422mmol/l) and NEFA (RMSEP=0.186mmol/l and 0.221mmol/l) when model training was done using two herds and validated on the third herd. The results from the model fits confirm that it is possible to build blood plasma BHB and NEFA models based on mid-IR spectra that are sufficiently accurate for practical use.

Effect of monensin on milk fatty acid profile in dairy cows and on the use of fatty acids for early diagnosis of elevated blood plasma concentrations of nonesterified fatty acids and hyperketonemia

This work examined the effects of precalving administration of continuous-release monensin capsule on postcalving milk fatty acid (FA) profile and on the accuracy of FA as a biomarker in the early identification of cows with elevated blood plasma nonesterified fatty acids (NEFA) and β-hydroxybutyrate (BHB) concentrations. Approximately 3 wk before expected calving, 203 multiparous Estonian Holstein cows were randomly divided into control (CO; n = 116) and experimental (MO; n = 87) groups, and a continuous-release capsule of monensin was administered to the MO cows. Blood samples were taken daily in the first 4 d postpartum, then on the sixth or seventh day in milk, twice in the second week, and thenceforth once per week until the end of the sixth week. Milk samples were taken once from 4 to 7 d in milk, twice in the second week, and thenceforth once per week. Blood samples were analyzed for NEFA and BHB, and milk was analyzed for FA concentrations. Cows with postpartum BHB concentrations ≥1.2 mmol/L at least once during the 6 wk were classified as hyperketonemic (HYK), and cows with NEFA concentrations ≥1.0 mmol/L as having elevated concentration of NEFA (NEFAH). The ability of FA to predict NEFAH and HYK cows was studied with logistic regression and receiver operating characteristic curve analysis and the identification accuracy was estimated by area under the receiver operating characteristic curve. For these analyses, we used FA measured on the ninth day after calving. Monensin administration affected FA mobilization and metabolism of the animals as blood NEFA were lower in the MO group on wk 1 and wk 3, and BHB values were considerably lower from wk 1 to wk 4 compared with the CO group. The FA dynamics were generally similar for MO and CO groups. Monensin administration resulted in higher concentrations of C15:0, C16:0, iso C17:0, anteiso C15:0, anteiso C17:0, total trans monounsaturated FA, and C18:2 cis-9,trans-11, and lower proportions of C18:0, C18:1 cis-9, and most of the iso FA. The identification accuracy of NEFAH and HYK cows was higher in the CO compared with the MO group and for the identification of HYK compared with NEFAH cows (0.75-0.77 vs. 0.78-0.80 in the CO group, and 0.61-0.66 vs. 0.68-0.75 in the MO group for NEFAH vs. HYK, respectively). For all FA, the threshold values to identify NEFAH and HYK cows were different in the CO and MO groups. Results suggest that specific threshold values for the identification of NEFAH and HYK cows could be applicable only within similar feeding conditions and rumen environment.

Mid-infrared spectroscopic analysis of raw milk to predict the blood nonesterified fatty acid concentrations in dairy cows

In high-yielding dairy cattle, severe postpartum negative energy balance is often associated with metabolic and infectious disorders that negatively affect production, fertility, and welfare. Mobilization of adipose tissue associated with negative energy balance is reflected through an increased level of nonesterified fatty acids (NEFA) in the blood plasma. Earlier, identification of negative energy balance through detection of increased blood plasma NEFA concentration required laborious and stressful blood sampling. More recently, attempts have been made to predict blood NEFA concentration from milk samples. In this study, we aimed to develop and validate a model to predict blood plasma NEFA concentration using the milk mid-infrared (MIR) spectra that are routinely measured in the context of milk recording. To this end, blood plasma and milk samples were collected in wk 2, 3, and 20 postpartum for 192 lactations in 3 herds. The blood plasma samples were taken in the morning, and representative milk samples were collected during the morning and evening milk sessions on the same day. To predict plasma NEFA concentration from the milk MIR spectra, partial least squares regression models were trained on part of the observations from the first herd. The models were then thoroughly validated on all other observations of the first herd and on the observations of the 2 independent herds to explore their robustness and wide applicability. The final model could accurately predict blood plasma NEFA concentrations <0.6 mmol/L with a root mean square error of prediction of <0.143 mmol/L. However, for blood plasma with >1.2 mmol/L NEFA, the model clearly underestimated the true level. Additionally, we found that morning blood plasma NEFA levels were predicted with significantly higher accuracy using MIR spectra of evening milk samples compared with MIR spectra of morning samples, with root mean square error of prediction values of, respectively, 0.182 and 0.197 mmol/L, and R2 values of 0.613 and 0.502. These results suggest a time delay between variations in blood plasma NEFA and related milk biomarkers. Based on the MIR spectra of evening milk samples, cows at risk for negative energy status, indicated by detrimental morning blood plasma NEFA levels (>0.6 mmol/L), could be identified with a sensitivity and specificity of, respectively, 0.831 and 0.800. As this model can be applied to millions of historical and future milk MIR spectra, it opens an opportunity for regular metabolic screening and improved resilience phenotyping.

Body and milk traits as indicators of dairy cow energy status in early lactation

The inclusion of feed intake and efficiency traits in dairy cow breeding goals can lead to increased risk of metabolic stress. An easy and inexpensive way to monitor postpartum energy status (ES) of cows is therefore needed. Cows' ES can be estimated by calculating the energy balance from energy intake and output and predicted by indicator traits such as change in body weight (ΔBW), change in body condition score (ΔBCS), milk fat:protein ratio (FPR), or milk fatty acid (FA) composition. In this study, we used blood plasma nonesterified fatty acids (NEFA) concentration as a biomarker for ES. We determined associations between NEFA concentration and ES indicators and evaluated the usefulness of body and milk traits alone, or together, in predicting ES of the cow. Data were collected from 2 research herds during 2013 to 2016 and included 137 Nordic Red dairy cows, all of which had a first lactation and 59 of which also had a second lactation. The data included daily body weight, milk yield, and feed intake and monthly BCS. Plasma samples for NEFA were collected twice in lactation wk 2 and 3 and once in wk 20. Milk samples for analysis of fat, protein, lactose, and FA concentrations were taken on the blood sampling days. Plasma NEFA concentration was higher in lactation wk 2 and 3 than in wk 20 (0.56 ± 0.30, 0.43 ± 0.22, and 0.13 ± 0.06 mmol/L, respectively; all means ± standard deviation). Among individual indicators, C18:1 cis-9 and the sum of C18:1 in milk had the highest correlations (r = 0.73) with NEFA. Seven multiple linear regression models for NEFA prediction were developed using stepwise selection. Of the models that included milk traits (other than milk FA) as well as body traits, the best fit was achieved by a model with milk yield, FPR, ΔBW, ΔBCS, FPR × ΔBW, and days in milk. The model resulted in a cross-validation coefficient of determination (R2cv) of 0.51 and a root mean squared error (RMSE) of 0.196 mmol/L. When only milk FA concentrations were considered in the model, NEFA prediction was more accurate using measurements from evening milk than from morning milk (R2cv = 0.61 vs. 0.53). The best model with milk traits contained FPR, C10:0, C14:0, C18:1 cis-9, C18:1 cis-9 × C14:0, and days in milk (R2cv = 0.62; RMSE = 0.177 mmol/L). The most advanced model using both milk and body traits gave a slightly better fit than the model with only milk traits (R2cv = 0.63; RMSE = 0.176 mmol/L). Our findings indicate that ES of cows in early lactation can be monitored with moderately high accuracy by routine milk measurements.

DYNAMIC OF CHANGES OF BLOOD PLASMA ENERGY METABOLISM PARAMETERS IN SUCKLING COWS DURING CALVING INTERVAL

In this study, effect of environmental condition changes during gazing period on energy metabolism parameters was investigated. Totally 40 Aberdeen Angus cows were selected for observation. Calving all of cows was situated into March. The feeding ration for the animals was comprised by pasture during the grazing period and corn silage, hay and granulated distiller’s grains during the winter period. At average age 9 days before calving, and subsequently 10, 81, 151, 189 and 273 days after calving, blood was sampled and analysed for glucose and NEFA (non-esterified fatty acid) concentrations on KONELAB T20xt automatic analyser (Thermo Fisher Scientific, Finland) and currently available commercial kits (Biovendor-Laboratorni medicina, Czech Republic). A rapid increase (p &lt; 0.05) of glucose concentration was detected in blood plasma of cows in period before calving to 81 days post partum. Average value of glucose concentration at 273 days postpartum was significant (p &lt; 0.05) lower comparing to day 189. The highest concentrations of NEFA in blood plasma of cows were found at 10 day postpartum. After that, during the persisted higher temperature period the NEFA concentration decreased significantly (p &lt; 0.01) till 189 days postpartum. At the end of monitored period concentration of NEFA in blood plasma significantly decreased (p &lt; 0.05). Changes of hot and cold season during the grazing period probably according to forage quality and had significant effects on blood plasma NEFA and glucose concentrations.

Milk fatty acids as possible biomarkers to early diagnose elevated concentrations of blood plasma nonesterified fatty acids in dairy cows

Most cows encounter a state of negative energy balance during the periparturient period, which may lead to metabolic disorders and impaired fertility. The aim of this study was to assess the potential of milk fatty acids as diagnostic tools of detrimental levels of blood plasma nonesterified fatty acids (NEFA), defined as NEFA concentrations beyond 0.6mmol/L, in a data set of 92 early lactating cows fed a glucogenic or lipogenic diet and subjected to 0-, 30-, or 60-d dry period before parturition. Milk was collected in wk 2, 3, 4, and 8 (n=368) and blood was sampled weekly from wk 2 to 8 after parturition. Milk was analyzed for milk fatty acids and blood plasma for NEFA. Data were classified as “at risk of detrimental blood plasma NEFA” (NEFA ≥0.6mmol/L) and “not at risk of detrimental blood plasma NEFA” (NEFA <0.6mmol/L). Concentrations of 45 milk fatty acids and milk fat C18:1 cis-9-to-C15:0 ratio were subjected to a discriminant analysis. Milk fat C18:1 cis-9 revealed the most discriminating variable to identify detrimental blood plasma NEFA. A false positive rate of 10% allowed us to diagnose 46% of the detrimental blood plasma NEFA cases based on a milk fat C18:1 cis-9 concentration of at least 230 g/kg of milk fatty acids. Additionally, it was assessed whether the milk fat C18:1 cis-9 concentrations of wk 2 could be used as an early warning for detrimental blood plasma NEFA risk during the first 8 wk in lactation. Cows with at least 240 g/kg of C18:1 cis-9 in milk fat had about 50% chance to encounter blood plasma NEFA values of 0.6mmol/L or more during the first 8 wk of lactation, with a false positive rate of 11.4%. Profit simulations were based on costs for cows suffering from detrimental blood plasma NEFA, and costs for preventive treatment based on daily dosing of propylene glycol for 3 wk. Given the relatively low incidence rate (8% of all observations), continuous monitoring of milk fatty acids during the first 8 wk of lactation to diagnose detrimental blood plasma NEFA does not seem cost effective. On the contrary, milk fat C18:1 cis-9 of the second lactation week could be an early warning of cows at risk of detrimental blood NEFA. In this case, selective treatment may be cost effective.

Plasma leptin, glucose and non-esterified fatty acid variations in dromedary camels exposed to prolonged periods of underfeeding or dehydration

The involvement of plasma leptin in the adaptation of dromedary camels to harsh conditions such as food or water shortages was studied through 2 experiments. In experiment 1, fourteen female camels were either fed at 68% of maintenance energy requirements (MER) during 112d (n=4) or overfed at 134% of MER during the first 56d and then underfed at 17% of MER the next 56d (OV-UN, n=5), or underfed and then overfed for the same durations and energy intake levels (UN-OV, n=5). Weekly plasma samples showed that leptin, glucose and non-esterified fatty acid (NEFA) concentrations were significantly modulated by energy intake level. NEFA increased sharply but transiently in underfed camels of the UN-OV or OV-UN groups, whereas glucose and leptin concentrations decreased with underfeeding and increased with overfeeding with more significant effects in camels that were previously overfed or underfed, respectively. In experiment 2 twelve female camels were either normally watered (n=6) or dehydrated (n=6) during 23d and then rehydrated during 4d. Dehydration specifically increased blood hematocrit, plasma NEFA and glucose whereas leptin decreased slightly. For both experiments, leptinemia was positively related to hump adipocyte volume. Taken together these results provide new data for a better understanding of lipid and energy metabolism in camels.

The Effect of B-Adrenergic Agonist Injection and Feeding Energy Level on Blood Plasma Non-Esterified Fatty Acids in The Lactating Dairy Ewe

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Sympathoadrenal activity during exercise in partial diabetic and diabetic rats.

Insulin-dependent diabetes mellitus is associated with altered fat and carbohydrate metabolism and disturbed sympathoadrenal functioning. The aim of this study was to investigate whether the short-term diabetic state alters the activity of the sympathoadrenal system and of the adrenal cortex during exercise. In addition, the possible reciprocal effects of a deviating sympathoadrenal functioning and an altered non-esterified fatty acid (NEFA) and glucose metabolism were investigated. Therefore, control rats, diabetic rats, and partial diabetic rats were submitted to swimming (15 minutes). Permanent heart catheters allowed frequent blood sampling without disturbing the animals. Blood glucose and plasma NEFA levels increased during exercise. partial diabetic animals showed similar effects as controls. In contrast, the glucose and NEFA increments were significantly higher in diabetic rats than in control and partial diabetic rats. During exercise, the very low insulin levels were unaltered in diabetic rats, whereas they decreased in the other groups. Exercise-induced elevations of plasma epinephrine and norepinephrine were similar in all groups, but were lower for corticosterone in the diabetic animals. Therefore, these results provide evidence that the exaggerated NEFA mobilization in diabetic rats is only the result of reduced inhibition of lipolysis by the relative lack of insulin. It is concluded that the short-term diabetic state does not alter the activity of the sympathetic nervous system during exercise, but lowers the activity of the adrenal cortex.

Technical note: an improved method to quantify nonesterified fatty acids in bovine plasma.

Nonesterified fatty acids (NEFA) are measured routinely in the study of nutrition and growth physiology. To improve the efficiency with which this variable is measured, a two-reaction, enzymatic-based assay was adapted and validated to quantify NEFA in bovine blood plasma using 96-well microtiter plates. The effects of incubation time, temperature, and of sample volume were examined in addition to possible interfering substances, recovery, reagent stability, and range of linearity. Incubation for 30 min at 21 degrees C for each of the two reactions resulted in maximal slope and r2 values (1.94 and .999, respectively). Percentage of recovery was 107% when using 5 microL and 100% when using 10 microL of heparinized bovine plasma in the assay. Uniform linear standard curves (r2 > .99) were obtained using reagents stored at 4 degrees C for 9 d. An equal volume of 1 mM acetate, propionate, valerate, and butyrate added to plasma did not affect results. The assay was linear from 125 to > 1,000 microEq/L. Intra- and interassay CV for the 5-microL sample volume were 3.6 and 3.7%, respectively. This modified assay provides results comparable to the standard assay yet reduces reagent and labor requirements and increases sample capacity.

The influence of digestible energy concentration of the diet on feed intake and rate of gain by beef steers

Eighty Hereford steers were fed diets with different levels of protected tallow. One-half of the steers received all-concentrate diets with 0–20% tallow, and the other half received 50% concentrate and 50% cubed alfalfa hay diets with 0–10% tallow. As the level of protected tallow in the diet increased, daily feed intake declined in both diets, but daily digestible energy (DE) intake declined then increased with the all-concentrate diets but increased then declined with the hay-concentrate diets. Digestible energy required per unit gain was not affected with the all-concentrate diets but increased then declined as the level of protected tallow was increased with the hay-concentrate diets. Carcass measurements were not influenced by the level of protected tallow in the all-concentrate diets. Carcass backfat declined with an increase in level of protected tallow in the hay-concentrate diets. Blood plasma nonesterified fatty acid levels increased as the level of protected tallow in the diet increased. Packed cell volume increased as protected tallow in the diet increased in the steers fed all-concentrate but not in those fed hay-concentrate. Blood glucose level was not affected by level of protected tallow in the diet. Key words: Steers, digestible energy concentration, protected tallow

Growth and carcass characteristics of lambs passively immunized with antibodies developed against ovine adipocyte plasma membranes.

Polyclonal antisera were collected from a mare immunized with ovine adipocyte plasma membranes. Ten crossbred wether lambs received three consecutive daily intraperitoneal injections of horse antisheep adipocyte plasma membrane immunoglobulin (ASIg) or nonimmune serum immunoglobulin (NSIg). Each injection delivered 1.5 ml serum Ig protein equivalent to either 53.4 mg ASIg or 51.1 mg NSIg per kilogram of live body weight. Lambs were housed in individual metabolic crates during the 28-d experiment and given ad libitum access to a pelleted, high-concentrate diet. Daily feed consumption was monitored individually over the experimental period and N retention was determined. Blood was collected on d 0, 4, 7, 14 and 28 for determination of nonesterified fatty acid (NEFA), triacylglycerides (TG) and hematocrit. At the conclusion of the experiment, lambs were slaughtered and carcasses were evaluated. Passive immunization against sheep adipocyte plasma membrane reduced (P less than .05) perirenal adipose tissue weight and decreased ether extract content of both subcutaneous and perirenal fat. Treatment tended to reduce average backfat thickness (24%) and estimated kidney pelvic fat (16%). Treatment with ASIg reduced (P less than .05) blood plasma NEFA but did not alter blood TG or hematocrit values. Average daily weight gain was lower (P less than .01) in the ASIg-treated group. However, the efficiency of carcass production, measured as carcass weight, was not affected by ASIg treatment.

Nutritional anestrus in beef cows: concentrations of glucose and nonesterified fatty acids in plasma and insulin in serum.

Twenty-two nonlactating Hereford cows exhibiting normal estrous cycles were fed either maintenance (M) or restricted (R) diets until most of the R cows became anestrus; R cows then were fed 160% of the M diet until estrous cycles resumed. Concentrations of progesterone, glucose, insulin and nonesterified fatty acids (NEFA) were determined in weekly blood samples. Blood also was collected frequently, before and after i.v. infusion of 300 ml of a 40% glucose solution, to evaluate responses in blood concentrations of glucose and insulin when cows were exhibiting normal estrous cycles, when R cows were initiating anestrus, during anestrus, and at the reinitiation of estrous cycles. Losses in BW and body condition score in R cows were associated with reduced (P less than .01) concentrations of glucose and insulin and greater (P less than .01) concentrations of NEFA in blood plasma compared with those of M cows. During normal estrous cycles, disappearance of infused glucose from plasma and concentrations of insulin in serum were similar for R and M cows. Glucose disappearance from plasma was retarded and serum concentrations of insulin remained increased for a longer time after glucose infusion in R at the start of anestrus compared with M cows (P less than .01). Similarly, during anestrus, the rate of glucose disappearance was slower for R cows (P less than .01). During refeeding of R cows, disappearance of infused glucose was similar for R and M cows. In conclusion, reduced concentrations of glucose and insulin and increased concentrations of NEFA in blood were associated with nutritional anestrus and the glucoregulatory effects of insulin were compromised during nutritional anestrus.

Diurnal metabolic profiles in hyperthyroidism.

Abstract. Hormone and metabolite profiles during a 12 h period of normal meals and activity were examined in nine hyperthyroid subjects with Graves' disease and sixteen matched controls. Six hyperthyroid subjects were restudied when euthyroid on carbimazole and thyroxine. Thyrotoxic patients had mild fasting hyperglycaemia (mean pL SEM blood glucose, 5·5 pL 0·2 v. 4·8 pL 0·1 mmol/l, P &lt; 0·01), elevated blood glycerol (0·15 pL 0·02 v. 0·08 pL 0·01 mmol/l, P &lt; 0·001) and elevated plasma non‐esterified fatty acid (NEFA) concentrations (0·91 pL 0·06 v. 0·58 pL 0·03 mmol/l, P &lt; 0·001) when compared to controls. Fasting blood concentrations of the gluconeogenic precursors lactate, pyruvate and alanine, blood ketone body concentrations and circulating insulin and growth hormone levels were similar in hyperthyroid and control subjects. Blood glucose responses to meals were exaggerated and the mean 12 h blood glucose was increased (6·1 pL 0·1 v. 5·5 pL 0·1 mmol/l, P &lt; 0·01) in hyperthyroidism. Similarly, hyperlactataemia and hyperpyruvicaemia were observed after meals. Blood ketone body, blood glycerol and plasma NEFA levels showed exaggerated pre‐prandial peaks and the mean 12 h values for blood glycerol (0·12 pL 0·01 v. 0·08 pL 0·01 mmol/l, P &lt; 0·01) and plasma NEFA (0·71 pL 0·03 v. 0·53 pL 0·04 mmol/l, P &lt; 0·01) were increased. Concentrations of insulin and growth hormone remained similar to control values throughout the study period. Blocking therapy with carbimazole and thyroid hormone replacement with thyroxine for 5–10 months suppressed blood glycerol, plasma NEFA and blood ketone body levels to normal or subnormal values but had no effect on the elevated blood glucose, blood lactate or blood pyruvate profiles.Graves' disease with hyperthyroidism is thus associated with abnormalities of carbohydrate metabolism which are not restored to normal by 5–10 months oral antithyroid therapy. The changes in lipid metabolism in hyperthyroidism are normalized by this treatment.

Stimulation of insulin secretion in man by medium chain triglycerides

The effects of an orally administered medium chain triglyceride (MCT) preparation on blood glucose, plasma non-esterified fatty acids (NEFA), and serum insulin levels have been determined in 10 normal volunteers and 9 patients with cirrhosis and evidence of portalsystemic communications. Blood glucose and plasma NEFA levels did not change appreciably in either group. Serum insulin levels increased significantly in the cirrhotic patients. It is suggested that MCT caused a modest stimulation of insulin secretion which was more easily detected in the cirrhotic patients because of (1) a reduced liver cell mass capable of binding insulin during its initial circulation through the liver, and (2) shunting around the liver through portalsystemic communications.

Observations on some extra-adrenal effects of corticotropin on carbohydrate and lipid metabolism in man

The possible significance of the reported extra-adrenal effects of corticotropin on lipid and carbohydrate metabolism has been studied in man. The fasting concentrations of blood sugar and pyruvate, and of plasma cholesterol and nonesterified fatty acids (NEFA) lay within normal limits in a group of patients with primary adrenal insufficiency, and the increase in non-esterified fatty acid levels during a 4 hour infusion of saline solution was significantly less than that observed in a group of control subjects. Since these patients might be expected to have elevated circulating levels of corticotropin it would appear that physiologic amounts of this hormone do not have significant extra-adrenal metabolic effects. In a group of patients with primary hypopituitarism and secondary adrenocortical insufficiency who might be presumed to have negligible levels of circulatory corticotropin, fasting concentrations of blood sugar, plasma NEFA and cholesterol were also normal. These values and the increase in NEFA levels during a 4 hour infusion of saline were similar to those found in the primary hypoadrenal group. In patients with primary adrenal insufficiency the intravenous infusion of pharmacologic amounts of corticotropin and of the synthetic polypeptide β 1–24 corticotropin were also without significant effects on these indices, as compared with the control infusion of normal saline solution.