Basal Non-esterified Fatty Acid Research Articles

Dietary Betaine Impacts Metabolic Responses to Moderate Heat Exposure in Sheep.

Dietary betaine supplementation can ameliorate physiological responses to heat exposure (HE) in sheep. This experiment measured metabolic responses to glucose (intravenous glucose tolerance, IVGTT), insulin (insulin tolerance test, ITT), and adrenocorticotropic hormone (ACTH) challenges in Merino ewes (n = 36, 39.7 kg) maintained at thermoneutral (TN, 21 °C) or HE (18-43 °C) and supplemented with either 0, 2, or 4 g/day dietary betaine (n = 6 per group). Sheep had ad libitum access to water and were pair-fed such that the intake of the TN sheep mimicked that of the HE sheep. After 21 days of treatment, sheep were fitted with jugular catheters and subjected to consecutive daily challenges (IVGTT, ITT, and ACTH, d 21-23, respectively), followed by skeletal muscle and subcutaneous adipose tissue biopsy collections for gene expression analysis (d 24). The HE-treated sheep had a greater insulin:glucose ratio (p = 0.033), a greater estimated homeostatic model assessment of insulin resistance (HOMAIR; p = 0.029), and a reduced revised quantitative insulin sensitivity check index (RQUICKI; p = 0.015). Sheep fed betaine (2 + 4 g/day) had a greater basal plasma insulin (p = 0.017) and a reduced basal non-esterified fatty acid (NEFA; p = 0.036) concentration, while the RQUICKI was reduced (p = 0.001) in sheep fed betaine. The results suggested that betaine supplementation alters lipid metabolism by potentially improving insulin signaling, although these responses differ between TN and HE conditions. There was no other impact of temperature or dietary treatments on the tissue gene expressions measured. Our results support the notion that betaine, in part, acts to modify lipid metabolism.

Pregnancy but Not Moderate Undernutrition Attenuates Insulin Suppression of Fat Mobilization in Sheep , ,

Nonpregnant and late-pregnant ditocous ewes were fed either to maintain zero energy balance in maternal tissues (fed) or at 50% of this level (underfed) for several weeks. Plasma concentrations of nonesterified fatty acids (NEFA) and glycerol were measured under basal conditions and during infusion of various doses of insulin while maintaining euglycemia (hyperinsulinemic, euglycemic clamp technique). Pregnancy and undernutrition separately increased basal plasma NEFA concentration in an additive manner; plasma glycerol was increased by pregnancy but unaffected by undernutrition. The molar ratio of NEFA to glycerol was significantly greater in underfed ewes. Analysis of dose-response relations between plasma insulin and metabolites during insulin infusions showed that maximally insulin-suppressed concentrations of NEFA and glycerol were significantly greater in pregnant than in nonpregnant ewes but were unaffected by undernutrition. Neither pregnancy nor undernutrition affected the maximally insulin-suppressed NEFA to glycerol ratio, or the plasma insulin concentration for 50% maximal responses to insulin of plasma NEFA, plasma glycerol, or the plasma NEFA to glycerol ratio. Thus, even in ewes at or close to zero energy balance, pregnancy seems to reduce adipose responsiveness but not sensitivity to the antilipolytic effect of insulin. This is another manifestation of the normal development of insulin resistance in maternal tissues during late pregnancy.

Modelling beef cows’ individual response to short nutrient restriction in different lactation stages

Short-term nutrient restrictions can occur naturally in extensive beef cattle production systems due to low feed quality or availability. The aims of the study were to (1) model the curves of milk yield, plasma non-esterified fatty acids (NEFAs) and β-hydroxybutyrate (BHB) contents of beef cows in response to short nutritional challenges throughout lactation; (2) identify clusters of cows with different response profiles; (3) quantify differences in cows’ response between the clusters and lactation stages. Data of BW, body condition score (BCS), milk yield, NEFA, and BHB plasma concentration from 31 adult beef cows (626 ± 48 kg at calving) were used to study the effect of 4-day feed restriction repeated over months 2, 3 and 4 of lactation. On each month, all cows received a single diet calculated to meet the requirements of the average cow: 100 % requirements for 4 days (d-4 to d-1, basal period), 55 % requirements on the next 4 days (d0 to d3, restriction period) and 100 % requirements for 4 days (d4 to d7, refeeding period). Natural cubic splines were used to model the response of milk yield, NEFA and BHB to restriction and refeeding in the 3 months. The new response variables [baseline value, peak value, days to peak and to regain baseline, and areas under the curve (AUC) during restriction and refeeding] were used to cluster cows according to their metabolic response (MR) into two groups: Low MR and High MR. The month of lactation affected all the traits, and basal values decreased as lactation advanced. Cows from both clusters had similar BW and BCS values, but those in the High MR cluster had higher basal milk yield, NEFA and BHB contents, and responded more intensely to restriction, with more marked peaks and AUCs. Reaction times were similar, and baseline values recovered during refeeding in both clusters. Our results suggest that the response was driven by cows’ milk potential rather than size or body reserves, and despite high-responding cattle’s higher milk yield, they were able to activate metabolic pathways to respond to and recover from the challenge.

Rescue Potential of Supportive Embryo Culture Conditions on Bovine Embryos Derived from Metabolically Compromised Oocytes.

Elevated non-esterified fatty acid (NEFA), predominantly palmitic acid (PA), concentrations in blood and follicular fluid are a common feature in maternal metabolic disorders such as obesity. This has a direct negative impact on oocyte developmental competence and the resulting blastocyst quality. We use NEFA-exposure during bovine oocyte in vitro maturation (IVM) as a model to mimic oocyte maturation under maternal metabolic stress conditions. However, the impact of supportive embryo culture conditions on these metabolically compromised zygotes are not known yet. We investigated if the addition of anti-apoptotic, antioxidative and mitogenic factors (namely, Insulin-Transferrin-Selenium (ITS) or serum) to embryo culture media would rescue development and important embryo quality parameters (cell proliferation, apoptosis, cellular metabolism and gene expression patterns) of bovine embryos derived from high PA- or high NEFA-exposed oocytes when compared to controls (exposed to basal NEFA concentrations). ITS supplementation during in vitro culture of PA-exposed oocytes supported the development of lower quality embryos during earlier development. However, surviving blastocysts were of inferior quality. In contrast, addition of serum to the culture medium did not improve developmental competence of PA-exposed oocytes. Furthermore, surviving embryos displayed higher apoptotic cell indices and an aberrant cellular metabolism. We conclude that some supportive embryo culture supplements like ITS and serum may increase IVF success rates of metabolically compromised oocytes but this may increase the risk of reduced embryo quality and may thus have other long-term consequences.

Oocyte maturation under lipotoxic conditions induces carryover transcriptomic and functional alterations during post-hatching development of good-quality blastocysts: novel insights from a bovine embryo-transfer model.

Does oocyte maturation under lipolytic conditions have detrimental carry-over effects on post-hatching embryo development of good-quality blastocysts after transfer? Surviving, morphologically normal blastocysts derived from bovine oocytes that matured under lipotoxic conditions exhibit long-lasting cellular dysfunction at the transcriptomic and metabolic levels, which coincides with retarded post-hatching embryo development. There is increasing evidence showing that following maturation in pathophysiologically relevant lipotoxic conditions (as in obesity or metabolic syndrome), surviving blastocysts of good (transferable) morphological quality have persistent transcriptomic and epigenetic alteration even when in vitro embryo culture takes place under standard conditions. However, very little is known about subsequent development in the uterus after transfer. Bovine oocytes were matured in vitro in the presence of pathophysiologically relevant, high non-esterified fatty acid (NEFA) concentrations (HIGH PA), or in basal NEFA concentrations (BASAL) as a physiological control. Eight healthy multiparous non-lactating Holstein cows were used for embryo transfers. Good-quality blastocysts (pools of eight) were transferred per cow, and cows were crossed over for treatments in the next replicate. Embryos were recovered 7days later and assessed for post-hatching development, phenotypic features and gene expression profile. Blastocysts from solvent-free and NEFA-free maturation (CONTROL) were also tested for comparison. Recovered Day 14 embryos were morphologically assessed and dissected into embryonic disk (ED) and extraembryonic tissue (EXT). Samples of EXT were cultured for 24h to assess cellular metabolic activity (glucose and pyruvate consumption and lactate production) and embryos' ability to signal for maternal recognition of pregnancy (interferon-τ secretion; IFN-τ). ED and EXT samples were subjected to RNA sequencing to evaluate the genome-wide transcriptome patterns. The embryo recovery rate at Day 14 p.i. was not significantly different among treatment groups (P> 0.1). However, higher proportions of HIGH PA embryos were retarded in growth (in spherical stage) compared to the more elongated tubular stage embryos in the BASAL group (P< 0.05). Focusing on the normally developed tubular embryos in both groups, HIGH PA exposure resulted in altered cellular metabolism and altered transcriptome profile particularly in pathways related to redox-regulating mechanisms, apoptosis, cellular growth, interaction and differentiation, energy metabolism and epigenetic mechanisms, compared to BASAL embryos. Maturation under BASAL conditions did not have any significant effects on post-hatching development and cellular functions compared to CONTROL. The datasets of RNA sequencing analysis are available in the NCBI's Gene Expression Omnibus (GEO) repository, series accession number GSE127889 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127889). Datasets of differentially expressed genes and their gene ontology functions are available in the Mendeley datasets at http://dx.doi.org/10.17632/my2z7dvk9j.2. The bovine model was used here to allow non-invasive embryo transfer and post-hatching recovery on Day 14. There are physiological differences in some characteristics of post-hatching embryo development between human and cows, such as embryo elongation and trophoblastic invasion. However, the main carry-over effects of oocyte maturation under lipolytic conditions described here are evident at the cellular level and therefore may also occur during post-hatching development in other species including humans. In addition, post-hatching development was studied here under a healthy uterine environment to focus on carry-over effects originating from the oocyte, whereas additional detrimental effects may be induced by maternal metabolic disorders due to adverse changes in the uterine microenvironment. RNA sequencing results were not verified by qPCR, and no solvent control was included. Our observations may increase the awareness of the importance of maternal metabolic stress at the level of the preovulatory oocyte in relation to carry-over effects that may persist in the transferrable embryos. It should further stimulate new research about preventive and protective strategies to optimize maternal metabolic health around conception to maximize embryo viability and thus fertility outcome. This study was supported by the Flemish Research Fund (FWO grant 11L8716N and FWO project 42/FAO10300/6541). The authors declare there are no conflicts of interest.

Correlated response in body condition and energy mobilisation in rabbits selected for litter size variability

A divergent selection experiment on litter size variability (high and low lines) was performed in rabbits over seven generations. The aim of this study was to evaluate the correlated responses to selection in body condition and fat reserves mobilisation. Litter size variability was estimated as phenotypic variance of litter size within female after correcting for the year-season and the parity-lactation status effects. A total of 226 females were used in this study, of which 158 females were used to measure body condition and energy mobilisation. Body condition was measured as BW and perirenal fat thickness. Females were stimulated with the adrenergic isoproterenol. Mobilisation capacity of fat reserves was measured by the lipolytic potential, defined as the increment in non-esterified fatty acids (NEFA) levels from basal concentration until adrenergic stimulation at mating, delivery and 10 days after delivery of the second reproductive cycle. Females were classified as survivor or non-survivor when they were culled for sanitary reasons or died before the third kindling. Data were analysed using Bayesian methodology. Survivor females presented higher BW than the non-survivor females at delivery (238 g, P=1.00) and 10 days after delivery (276 g, P=1.00). They also showed higher perirenal fat thickness at 10 days after delivery (0.62 mm, P=1.00). At delivery, basal NEFA levels was lower in survivor than non-survivor females (−0.18 mmol/l, P=1.00), but their lipolytic potential (∆NEFA) was higher (0.08 mmol/l, P=0.94). Body weight was similar between lines in survivor females. Perirenal fat thickness was lower in the high line than in the low line at delivery (−0.23 mm, P=0.90) and 10 days after delivery (−0.28 mm, P=0.92). The high line exhibited higher NEFA (0.10 mmol/l, P=0.93) and lower ∆NEFA (−0.08 mmol/l, P=0.92) than the low line at delivery. The low line showed a favourable correlated response to selection on body condition and fat reserves mobilisation. In conclusion, the low line selected for litter size variability seems to adapt better to adverse conditions, as it has a greater capacity to mobilise energy reserves at delivery than the high line. Females that adequately manage their body reserves and perform energy mobilisation correctly have a lower risk of dying or being culled.

Alterations in plasma non-esterified fatty acid (NEFA) kinetics and relationship with insulin resistance in polycystic ovary syndrome.

Are non-esterified fatty acid (NEFA) kinetics altered in women with polycystic ovary syndrome (PCOS)? Women with PCOS, particularly obese subjects, have dysregulated plasma NEFA kinetics in response to changes in plasma insulin and glucose levels, which are associated with insulin resistance (IR) independently of the fasting plasma NEFA levels. Elevated plasma NEFA levels are associated with IR in many disorders, although the homeostasis of NEFA kinetics and its relationship to IR in women with PCOS is unknown. We prospectively compared insulin sensitivity and NEFA kinetics in 29 PCOS and 29 healthy controls women matched for BMI. This study was conducted in a tertiary institution. Plasma NEFA, glucose and insulin levels were assessed during a modified frequently sampled intravenous glucose tolerance test (mFSIVGTT). Minimal models were used to assess insulin sensitivity (Si) and NEFA kinetics (i.e. model-derived initial plasma NEFA level [NEFA0], phi constant [Φ], reflecting glucose-mediated inhibition of lipolysis and measures of maximum rate of lipolysis [SFFA] and NEFA uptake from plasma [KFFA]). The study provides new evidence that women with PCOS have defective NEFA kinetics characterized by: (i) lower basal plasma NEFA levels, measured directly and modeled (NEFA0), and (ii) a greater glucose-mediated inhibition of lipolysis in the remote or interstitial space (reflected by a lower affinity constant [Φ]). There were no differences, however, in the maximal rates of adipose tissue lipolysis (SFFA) and the rate at which NEFA leaves the plasma pool (KFFA). The differences observed in NEFA kinetics were exacerbated, and almost exclusively observed, in the obese PCOS subjects. Our study did not study NEFA subtypes. It was also cross-sectional and based on women affected by PCOS as defined by the 1990 National Institutes of Health (NIH) criteria (i.e. Phenotypes A and B) and identified in the clinical setting. Consequently, extrapolation of the present data to other phenotypes of PCOS should be made with caution. Furthermore, our data is exploratory and therefore requires validation with a larger sample size. Dysfunction in NEFA kinetics may be a marker of metabolic dysfunction in nondiabetic obese women with PCOS and may be more important than simply assessing circulating NEFA levels at a single point in time for understanding the mechanism(s) underlying the IR of PCOS. This work was supported by NIH grants R01-DK073632 and R01-HD29364 to R.A.; a Career Development Award from MD Medical Group, Moscow, RF, to D.L. and Augusta University funds to Y.-H.C. RA serves as consultant to Ansh Labs, Medtronics, Spruce Biosciences and Latitude Capital. U.E., Z.A., D.L., R.M., Y.-H.C., R.C.B. and Y.D.I.C. have no competing interests to declare. Not applicable.

Effects of dietary energy allowance and decline in dry matter intake during the dry period on responses to glucose and insulin in transition dairy cows

We assessed whether high energy intake during the early dry period [144% of metabolizable energy (ME) requirements/d] followed by a gradual restriction of energy intake in the close-up dry period (119% of ME/d; HEI) impaired whole-body insulin sensitivity compared with a controlled energy intake (100% of ME/d; CEI) throughout the 6-wk dry period. Multiparous Ayrshire dairy cows (n = 16) were blocked by body weight, body condition score, and expected date of parturition and were used in a randomized complete block design until 10 d after parturition. Cows were fed either HEI or CEI diets based on grass silage during the first 3 wk of the dry period and grass silage supplemented with a commercial concentrate (30% of ME intake) during the final 3 wk of gestation. After calving, all cows were fed grass silage ad libitum and an increasing amount of commercial concentrate (maximum 9 kg at d 10 postpartum). Intravenous glucose tolerance tests (IVGTT) and intravenous insulin challenges were performed -10 ± 5 d (n = 15) and +10 ± 1 d (n = 14) relative to parturition. Following glucose injection, we did not find any treatment effects on glucose and insulin responses. The prepartal nonesterified fatty acid (NEFA) response of the HEI group was blunted, basal NEFA and the decrement of NEFA were smaller, and the area under the response curve (AUC) of NEFA was less negative in HEI cows than in CEI cows. The NEFA response reversed after parturition; the NEFA AUC of the HEI group was more negative than that of the CEI group. We did not find similar responses after insulin injection. Across the treatments, NEFA AUC correlated strongly with the basal NEFA concentration during the IVGTT pre- and postpartum. Calculated and model-based indices characterizing the overall glucose tolerance and β-cell function and the insulin sensitivity were higher after parturition than during the dry period. Consistent with the lower basal insulin, the acute insulin release after the glucose infusion was smaller in postpartal IVGTT than in prepartal IVGTT. The results suggest that whole-body insulin sensitivity of the cows increased after parturition. However, the role of peripheral insulin sensitivity in the regulation of glucose partitioning seems to be minor relative to the major change in insulin secretion and clearance during the periparturient period.

Relationship between body condition and energy mobilization in rabbit does

&lt;p&gt;The present work was performed to examine the relationships between measures of body condition and energy mobilization in rabbit does. The variables studied were body weight (BW), perirenal fat thickness (PFT), basal non-esterified fatty acid concentration (NEFA&lt;sub&gt;b&lt;/sub&gt;) and non-esterified fatty acids after lipolysis stimulation by isoproterenol (NEFA&lt;sub&gt;r&lt;/sub&gt;). The effect of time of measuring (at mating, delivery and 10 d after delivery) was estimated in 157 primiparous does. Correlations between body condition components were estimated and a principal component analysis performed. The does decreased BW (6%) and PFT (3%), and increased NEFA&lt;sub&gt;b&lt;/sub&gt; (25%) and NEFA&lt;sub&gt;r&lt;/sub&gt; (16%) from mating to delivery. Later, NEFA&lt;sub&gt;b&lt;/sub&gt; and NEFA&lt;sub&gt;r&lt;/sub&gt; decreased around 20% from delivery to 10 d after delivery without changing perirenal fat thickness. All BW and PFT lay in the first principal component, and all NEFA traits lay in the second component, showing low correlations with body condition measurements. Both NEFA traits showed high positive correlations when measured at the same time (0.65, 0.72 and 0.69), but low correlations when measured at different times (0.09, to 0.20). We conclude that although body weight and perirenal fat thickness are good predictors of body condition, NEFA should be used when an accurate measurement of energetic mobilization is needed, due to their low correlation.&lt;/p&gt;

Maternal metabolic stress may affect oviduct gatekeeper function.

We hypothesized that elevated non-esterified fatty acids (NEFA) modify in vitro bovine oviduct epithelial cell (BOEC) metabolism and barrier function. Hereto, BOECs were studied in a polarized system with 24-h treatments at Day 9: (1) control (0 µM NEFA + 0% EtOH), (2) solvent control (0 µM NEFA + 0.45% EtOH), (3) basal NEFA (720 µM NEFA + 0.45% EtOH in the basal compartment) and (4) apical NEFA (720 µM NEFA + 0.45% EtOH in the apical compartment). FITC-albumin was used for monolayer permeability assessment and related to transepithelial electric resistance (TER). Fatty acid (FA), glucose, lactate and pyruvate concentrations were measured in spent medium. Intracellular lipid droplets (LD) and FA uptake were studied using Bodipy 493/503 and immunolabelling of FA transporters (FAT/CD36, FABP3 and CAV1). BOEC-mRNA was retrieved for qRT-PCR. Results revealed that apical NEFA reduced relative TER increase (46.85%) during treatment and increased FITC-albumin flux (27.59%) compared to other treatments. In basal NEFA, FAs were transferred to the apical compartment as free FAs: mostly palmitic and oleic acid increased respectively 56.0 and 33.5% of initial FA concentrations. Apical NEFA allowed no FA transfer, but induced LD accumulation and upregulated FA transporter expression (↑CD36, ↑FABP3 and ↑CAV1). Gene expression in apical NEFA indicated increased anti-apoptotic (↑BCL2) and anti-oxidative (↑SOD1) capacity, upregulated lipid metabolism (↑CPT1, ↑ACSL1 and ↓ACACA) and FA uptake (↑CAV1). All treatments had similar carbohydrate metabolism and oviduct function-specific gene expression (OVGP1, ESR1 and FOXJ1). Overall, elevated NEFAs affected BOEC metabolism and barrier function differently depending on NEFA exposure side. Data substantiate the concept of the oviduct as a gatekeeper that may actively alter early embryonic developmental conditions.

Acute Hypercortisolemia Exerts Depot-Specific Effects on Abdominal and Femoral Adipose Tissue Function.

Context:Glucocorticoids have pleiotropic metabolic functions, and acute glucocorticoid excess affects fatty acid metabolism, increasing systemic lipolysis. Whether glucocorticoids exert adipose tissue depot-specific effects remains unclear.Objective:To provide an in vivo assessment of femoral and abdominal adipose tissue responses to acute glucocorticoid administration.Design and Outcome Measures:Nine healthy male volunteers were studied on two occasions, after a hydrocortisone infusion (0.2 mg/kg/min for 14 hours) and a saline infusion, respectively, given in randomized double-blind order. The subjects were studied in the fasting state and after a 75-g glucose drink with an in vivo assessment of femoral adipose tissue blood flow (ATBF) using radioactive xenon washout and of lipolysis and glucose uptake using the arteriovenous difference technique. In a separate study (same infusion design), eight additional healthy male subjects underwent assessment of fasting abdominal ATBF and lipolysis only. Lipolysis was assessed as the net release of nonesterified fatty acids (NEFAs) from femoral and abdominal subcutaneous adipose tissue.Results:Acute hypercortisolemia significantly increased basal and postprandial ATBF in femoral adipose tissue, but the femoral net NEFA release did not change. In abdominal adipose tissue, hypercortisolemia induced substantial increases in basal ATBF and NEFA release.Conclusions:Acute hypercortisolemia induces differential lipolysis and ATBF responses in abdominal and femoral adipose tissue, suggesting depot-specific glucocorticoid effects. Abdominal, but not femoral, adipose tissue contributes to the hypercortisolemia-induced systemic NEFA increase, with likely contributions from other adipose tissue sources and intravascular triglyceride hydrolysis.

186 THE EFFECT OF SHORT VERSUS LONG TERM ELEVATED NONESTERIFIED FATTY ACID CONCENTRATIONS DURING MURINE IN VITRO FOLLICLE GROWTH ON OOCYTE DEVELOPMENTAL COMPETENCE

Metabolic disorders, like obesity and type 2 diabetes are characterised by lipolysis-linked elevated nonesterified fatty acid (NEFA) concentrations. Exposure to high NEFA concentrations during the final phase of bovine in vitro oocyte maturation (24 h) impairs oocyte developmental competence and subsequent embryo quality. However, because elevated NEFA concentrations in vivo are often present for a longer period of time, our recent research focused on a more in vivo-like long-term NEFA exposure (12 d) of whole murine follicles in vitro. The model covers follicular growth from the early secondary to the antral stage in vitro, including final oocyte maturation after an ovulatory stimulus (OS). Results showed an altered follicular growth and physiology (steroid synthesis, gene expression) and a subsequent reduced oocyte developmental competence. However, it remains unclear what specific time frame in follicular development is the most sensitive to such metabolic insult. Therefore, we hypothesised that chronic elevated NEFA concentrations throughout follicle growth affect oocyte developmental competence more severely than short-term NEFA exposure limited to the final phase of oocyte maturation. The aim was to study the effect of elevated NEFA concentrations 1) during the final phase of follicular oocyte maturation (after the OS) and 2) during the whole period of in vitro murine follicle growth until the antral stage, including final oocyte maturation. Early secondary follicles, isolated from the ovaries of 13-day-old B6CBAF1 mice, were cultured in vitro until the antral stage (3 replicates). Follicles were exposed to: BASAL NEFA mix for 12 days (BASAL-BASAL, control); HIGH stearic acid (SA) for 12 days (SA-SA); HIGH NEFA mix for 12 days (NEFA-NEFA); HIGH SA after the OS (BASAL-SA); and HIGH NEFA mix after the OS (BASAL-NEFA). Oocytes were isolated out of antral follicles 20 hours after the OS, routinely fertilized and presumptive zygotes cultured. Cleavage (n cleaved zygotes/n oocytes) and blastocyst (n blastocysts/n oocytes) rates were documented and analysed by means of binary logistic regression. Cleavage rate was reduced for BASAL-BASAL (37%) compared to BASAL-NEFA embryos (52%; P = 0.045). The BASAL-NEFA treatment (43%) presented with a higher blastocyst percentage than BASAL-BASAL (23%; P = 0.004), NEFA-NEFA (26%; P = 0.037) and SA-SA (15%; P = 0.001) treatments. The BASAL-SA (30%) treatment performed better than the SA-SA treatment (P = 0.049). Even though BASAL-BASAL (control) embryo development was surprisingly low, the results indicate that long-term NEFA exposure during follicle growth in vitro affects oocyte developmental competence more severely than an exposure limited to the final phase of maturation after the OS. The results thus emphasise that the maternal micro-environment throughout follicular growth and not only during final oocyte maturation is essential for optimal oocyte quality.

Evaluation of the nutritional status, insulin resistance and cardiovascular risk in a population of adolescents in the cities of Granada and Almeria (Spain)

The first objective of this study was to evaluate the nutritional status and insulin resistance index in a population of adolescents as calculated by Homeostatic Model Assessment (HOMA). The second objective was to establish correlations between the nutritional status of the subjects, the possible existence of insulin resistance, and the risk of high blood pressure. POPULATION SAMPLE AND METHODOLOGY: The sample was composed of 1001 adolescents, 9-17 years of age, from 18 schools in the provinces of Granada and Almeria. Their nutritional status was determined by means of anthropometric evaluation. For the metabolic study, a blood sample was collected from each subject by venipuncture. An analysis was performed of the basal glucose and insulin levels as well as the Homeostatic Model Assessment- Insulin Resistance (HOMAIR) index. Also evaluated were the levels of glycosylated haemoglobin (HbA1c), serum lipoprotein (a), and non-esterified fatty acids (NEFAs). Insulin resistance was calculated with the formula, proposed by Matthews et al. (1985) : HOMA-IR = (insulin[mmol/L] x glucose[mU/L])/22.5. The evaluation of the nutritional status of the subjects reflected a progressive increase in the values of anthropometric variables as the nutritional status of the subjects worsened. The results of this study showed, regardless of age and gender, 85.01% of the subjects were of normal weight, whereas 9.99% were overweight, and 4.99% were obese. The metabolic study reflected that in comparison to normal-weight and overweight students, obese students had significantly higher serum levels (p < 0,0001) of HbA1c, basal insulin, basal glycemia, basal NEFA, lipoprotein (a), and HOMA-IR. Obesity was found to be a serious health problem in the population of adolescents studied, especially given the high cardiovascular risk that is characteristic of this condition. As reflected in the results of this study, obesity led to the premature development of metabolic disorders, which generally do not appear until adulthood.

Effects of heat stress on the response to metabolic challenges in growing pigs

Heat stress (HS) decreases feed intake but unexplainably increases plasma insulin parameters in a variety of animal models. Whether HS alters post‐absorptive carbohydrate and lipid metabolism in growing pigs is unknown. Seven crossbred gilts (53±6 kg BW) were subjected to 2 periods: thermoneutral (TN; 20°C, 36% RH) for 4d, and HS (32°C, 23% RH) for 8d. All pigs received an intravenous glucose tolerance test (GTT) on d2 of TN and d1 and 6 of HS; and an epinephrine challenge (EC) on d3 of TN and d2 and 7 of HS. Heat stress increased rectal temperatures (P&lt;0.01; 39.4 vs 40.4°C) and respiratory rates (P&lt;0.01; 30 vs 94 bpm), and reduced feed intake by 37% (P&lt;0.01). Heat stress decreased (P&lt;0.05) basal glucose and insulin concentrations by 14% and 50%, respectively. Basal nonesterified fatty acids (NEFA) were initially elevated during HS (92%), but progressively declined to TN levels by d3 (P&lt;0.01). On d1 HS, glucose disposal and insulin response to a GTT decreased (49% and 24%, respectively) compared to TN. By d6 HS, the insulin response remained unchanged, but glucose disposal increased compared to d1 (P&lt;0.01). On d2, NEFA response to an EC did not differ from TN, but was reduced (53%; P&lt;0.01) by d7. Glucose response to the EC did not change over time (P&gt;;0.05). These data suggest that, despite a low plane of nutrition, decreased adipose tissue utilization and increased glucose oxidation might be key acclimation mechanisms to heat stress.

Effects of abomasal infusion of tallow or camelina oil on responses to glucose and insulin in dairy cows during late pregnancy

Late pregnancy is associated with moderate insulin resistance in ruminants. Reduced suppression of lipolysis by insulin facilitates mobilization of nonesterified fatty acids (NEFA) from adipose tissue, resulting in elevated plasma NEFA concentrations. Decrease in dry matter intake (DMI) before parturition leads to accelerated lipomobilization and increases plasma NEFA, which may further impair insulin sensitivity. The aim of the study was to evaluate the effects of elevation of plasma NEFA concentration by abomasal infusions tallow (TAL) or camelina oil (CAM) on whole-body responses to exogenous glucose and insulin. We further assessed whether CAM, rich in C18:3n-3, enhances whole-body insulin sensitivity compared with TAL. Six late-pregnant, second-parity, rumen-cannulated dry Ayrshire dairy cows fed grass silage to meet 95% of metabolizable energy requirements were used in a replicated 3×3 Latin square with 5-d periods and 5 recovery days between each period. Treatments consisted of abomasal infusion of 500mL/d (430g of lipids/d) of water (control), TAL, or CAM administered in 10 equal doses daily. Intravenous glucose tolerance test (IVGTT) and i.v. insulin challenge (IC) were performed on d 5 after 98 and 108h of treatment infusions, respectively. Infusion of lipids increased basal plasma NEFA concentrations on d 5 (CAM: 0.25; TAL: 0.28; control: 0.17mmol/L). Following glucose injection, the rate of glucose clearance (CR) was lower in lipid-treated cows (CAM: 1.34; TAL: 1.48; control: 1.74%/min) and time to reach half-maximal glucose concentration (T1/2) was longer (CAM: 54; TAL: 47; control: 42min). Similar responses were observed after insulin injection. Increased plasma NEFA concentration tended to decrease insulin secretion in IVGTT. Infusion of CAM increased plasma C18:3n-3 content (CAM: 26.4; TAL: 16.1; control: 20.9 g/100g of fatty acids). Data suggest that CAM had an insulin-sensitizing effect, because the disposition index and insulin sensitivity index, derived from minimal model analysis, were higher in CAM than in TAL during IVGTT, and lower insulin concentrations during IC led to similar glucose clearance in CAM as in TAL. These results indicate that elevated plasma NEFA concentration per se induces whole-body insulin resistance in late-pregnant dry cows.

Effect of total sleep deprivation on postprandial metabolic and insulin responses in shift workers and non-shift workers

Epidemiological studies have shown that shift workers are at a greater risk of developing cardiovascular disease which may, in part, be related to metabolic and hormonal changes. Partial sleep deprivation, a common consequence of rotating shift work, has been shown to affect glucose tolerance and insulin sensitivity. The current study investigated the effects of one night of total sleep deprivation, as a proxy for the first night shift, on postprandial glucose, insulin and lipid (triacylglycerols (TAGs) and non-esterified fatty acids (NEFAs)) responses under controlled laboratory conditions in shift workers and non-shift workers. Eleven experienced shift workers (35.7+/-7.2 years, mean+/-s.d.) who had worked in shifts for 8.7+/-5.25 years were matched with 13 non-shift workers who had worked for 32.8+/-6.4 years. After an adaptation night and a baseline sleep night, volunteers were kept awake for 30.5 h, followed by a nap (4 h) and recovery sleep. Blood samples were taken prior to and after a standard breakfast following baseline sleep, total sleep deprivation and recovery sleep. Basal TAG levels prior to the standard breakfast were significantly lower after sleep deprivation, indicating higher energy expenditure. Basal NEFA levels were significantly lower after recovery sleep. Postprandial insulin and TAG responses were significantly increased, and the NEFA response was decreased after recovery sleep, suggestive of insulin insensitivity. Although there were no overall significant differences between non-shift workers and shift workers, non-shift workers showed significantly higher basal insulin levels, lower basal NEFA levels, and an increased postprandial insulin and a decreased NEFA response after recovery sleep. In future, the reasons for these inter-group differences are to be investigated.

358 THE CONSEQUENCE OF HIGH NON-ESTERIFIED FATTY ACID CONCENTRATIONS DURING OOCYTE IN VITRO MATURATION ON BOVINE EMBRYO QUALITY

High non-esterified fatty acid (NEFA) concentrations in the blood, associated with negative energy balance (NEB), obesity or diabetes Type II are known to alter the follicular micro-environment. These environmental changes have been associated with disappointing fertility outcome through disabled ovarian cell function and oocyte’s developmental competence. Our hypothesis was that elevated NEFA concentrations during final oocyte maturation might hamper the quality of the pre-implantation embryo as well. To assess embryo quality, the present study focused on total cell number and apoptotic index in 7-day-old embryos. Applied NEFA concentrations in the maturation medium were based on analyses in the follicular fluid of high yielding dairy cows early post partum during NEB. Bovine cumulus-oocyte complexes were exposed to 1) physiological NEFA concentrations, i.e. a combination of basal palmitic (25 μM), stearic (50 μM) and oleic acid (75 μM) concentrations (CONTROL), 2) elevated NEFA concentrations, i.e. a combination of high palmitic (75 μM), stearic (150 μM) and oleic acid (200 μM) concentrations (HIGH COMBI) and 3) elevated palmitic acid (75 μM) concentrations (HIGH PA). Palmitic acid has been recognized as a major saturated fatty acid in terms of cellular toxicity. After serum-free in vitro maturation (24 h) and fertilization (22 h), zygotes were cultured in SOF medium with 5% serum for 7 days. Blastocysts were evaluated for developmental competence, total cell number (by Propidium Iodide staining) and apoptotic index (by TUNEL detection kit). In total, 684 oocytes were cultured in 3 replicates. Data were analyzed with binary logistic regression and a mixed model ANOVA. Preliminary research showed that maturation in a combination of basal NEFA concentrations has no effect on oocyte’s developmental competence compared to the standard serum free maturation system. In the present study, maturation in HIGH COMBI resulted in significantly lower blastocyst rates (21.4%) compared to CONTROL (30.1%) (P = 0.03). No significant effect of HIGH PA maturation on blastocyst rate (24.1%) could be found. Also total cell number tended to be lower in the HIGH COMBI (104.7 ± 26.1) compared to CONTROL (125.8 ± 29.4) (P = 0.08). The apoptotic index was significantly increased in the HIGH PA group (0.22 ± 0.12) compared to the CONTROL group (0.11 ± 0.07) (P = 0.02) and tended to be higher than the HIGH COMBI group (0.14 ± 0.12) (P = 0.06). Maternal metabolic conditions, leading to increased lipolysis and high NEFA concentrations, can hamper fertility through a reduction of the oocyte’s developmental competence. The data of the present study furthermore suggest that elevated NEFAs might induce a negative carry over effect from the oocyte during its maturation to the embryo quality. This may ultimately lead to embryonic mortality and thus to a disappointing fertility outcome. Veerle Van Hoeck is supported by the Special Research Fund, University of Antwerp (Grant 22590).

Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatotropin

Heat stress is detrimental to dairy production and affects numerous variables including feed intake and milk production. It is unclear, however, whether decreased milk yield is primarily due to the associated reduction in feed intake or the cumulative effects of heat stress on feed intake, metabolism, and physiology of dairy cattle. To distinguish between direct (not mediated by feed intake) and indirect (mediated by feed intake) effects of heat stress on physiological and metabolic indices, Holstein cows (n=6) housed in thermal neutral conditions were pair-fed (PF) to match the nutrient intake of heat-stressed cows (HS; n=6). All cows were subjected to 2 experimental periods: 1) thermal neutral and ad libitum intake for 9 d (P1) and 2) HS or PF for 9 d (P2). Heat-stress conditions were cyclical with daily temperatures ranging from 29.7 to 39.2°C. During P1 and P2 all cows received i.v. challenges of epinephrine (d 6 of each period), and growth hormone releasing factor (GRF; d 7 of each period), and had circulating somatotropin (ST) profiles characterized (every 15min for 6h on d 8 of each period). During P2, HS cows were hyperthermic for the entire day and peak differences in rectal temperatures and respiration rates occurred in the afternoon (38.7 to 40.2°C and 46 to 82 breaths/min, respectively). Heat stress decreased dry matter intake by greater than 35% and, by design, PF cows had similar reduced intakes. Heat stress and PF decreased milk yield, although the pattern and magnitude (40 and 21%, respectively) differed between treatments. The reduction in dry matter intake caused by HS accounted for only approximately 35% of the decrease in milk production. Both HS and PF cows entered into negative energy balance, but only PF cows had increased (approximately 120%) basal nonesterified fatty acid (NEFA) concentrations. Both PF and HS cows had decreased (7%) plasma glucose levels. The NEFA response to epinephrine did not differ between treatments but was increased (greater than 50%) in all cows during P2. During P2, HS (but not PF) cows had a modest reduction (16%) in plasma insulin-like growth factor-I. Neither treatment nor period had an effect on the ST response to GRF and there was little or no treatment effect on mean ST levels or pulsatility characteristics, but both HS and PF cows had reduced mean ST concentrations during P2. In summary, reduced nutrient intake accounted for just 35% of the HS-induced decrease in milk yield, and modest changes in the somatotropic axis may have contributed to a portion of the remainder. Differences in basal NEFA between PF and HS cows suggest a shift in postabsorptive metabolism and nutrient partitioning that may explain the additional reduction in milk yield in cows experiencing a thermal load.

Effects of a supplemental yeast culture on heat-stressed lactating Holstein cows

Multiparous, lactating Holstein cows (n=23; 120±30 d in milk, 690±67kg of body weight) housed in climatic chambers were randomly assigned to 1 of 2 dietary treatments: a diet containing a novel yeast culture formulation (YC) for heat stress (n=12, 10 g/d) or a control diet (n=11). The trial length was 28 d and consisted of a 7-d thermal neutral period (TN; 18°C, 20% humidity) followed by 21 d of heat stress (HS; cyclical daily temperatures ranging from 29.4 to 37.8°C and 20% humidity). Cows were individually fed a total mixed ration consisting primarily of alfalfa hay and steam-flaked corn. During TN, the YC feeding had no effect on production variables or most body temperature indices. During HS, all body temperature indices increased and YC had no effect on rump surface temperature, respiration rate, or sweating rates. Cows fed YC had lower rectal temperatures at 1200 and 1800h (40.29 vs. 40.02°C and 40.35 vs. 40.12±0.07°C, respectively) compared with control-fed cows. Cows fed both diets lost body weight (42kg) during HS, but there were no differences between diets. Control-fed cows had increased dry matter intake (DMI) and milk yield (19.1 vs. 17.9±0.5kg/d and 32.15 vs. 29.15±0.02kg/d, respectively) compared with YC-fed cows, but intake and milk production were similar between diets when evaluated on a body weight basis. Heat stress progressively decreased DMI (29%) and milk yield, with milk production reaching a nadir (33%) in the third week. Heat stress decreased milk protein (7%) and lactose (5%) levels, but did not alter milk fat content. Heat-stressed cows were in calculated negative energy balance (−1.91±0.70 Mcal/d) and this was unaffected by diet. Independent of diet, HS decreased plasma glucose (11%), but neither diet nor HS altered basal nonesterified fatty acid levels. Heat stress increased plasma urea N concentrations (11.5 vs. 14.8±0.4mg/dL). Despite YC-fed cows having slightly reduced body temperatures indices, feeding YC did not prevent the negative effects of HS.

Long-term pioglitazone treatment enhances lipolysis in rat adipose tissue

The insulin-sensitizing effects of thiazolidinediones are believed to depend at least in part on reductions in circulating levels of nonesterified fatty acids (NEFA). The mechanisms that mediate the reductions in NEFA are not fully understood and could involve reductions in adipose tissue lipolysis, increases in glyceroneogenesis and NEFA reesterification in triglycerides in adipose tissue and increases in NEFA metabolism by oxidative tissues. In a congenic strain of spontaneously hypertensive rats that fed a high-sucrose diet to promote features of the metabolic syndrome, we studied the effects of chronic pioglitazone treatment over 4 months on adipose tissue lipolysis and NEFA metabolism. We observed significant increases in basal and adrenaline-stimulated NEFA and glycerol release, and near-total suppression of NEFA reesterification in epididymal adipose tissue isolated from rats chronically treated with pioglitazone. However, pioglitazone-treated rats also exhibited significant increases in mitochondrial DNA levels in adipose tissue (3.2-fold increase, P=0.001) and potentially greater sensitivity to the antilipolytic effects of insulin than untreated controls. In addition, chronic pioglitazone treatment was associated with increased palmitate oxidation in soleus muscle, reduced fasting levels of serum NEFA and triglycerides, as well as reduced serum levels of insulin and increased serum levels of adiponectin. Despite suppressing NEFA reesterification and increasing basal and adrenaline-stimulated lipolysis, chronic pioglitazone treatment may decrease circulating NEFA levels in part by increasing adipose tissue sensitivity to the antilipolytic effects of insulin and by enhancing NEFA oxidation in skeletal muscle.