Higher Non-esterified Fatty Acids Research Articles

L-Histidine attenuates NEFA-induced inflammatory responses by suppressing Gab2 expression

Non-esterified fatty acids (NEFAs), key to energy metabolism, may become pathogenic at elevated levels, potentially eliciting immune reactions. Our laboratory's findings of reduced L-histidine in ketotic states, induced by heightened NEFA concentrations, suggest an interrelation with NEFA metabolism. This observation necessitates further investigation into the mitigating role of L-histidine on the deleterious effects of NEFAs. Our study unveiled that elevated NEFA concentrations hinder the proliferation of Bovine Mammary Epithelial Cells (BMECs) and provoke inflammation in a dose-responsive manner. Delving into L-histidine's influence on BMECs, RNA sequencing revealed 2124 genes differentially expressed between control and L-histidine-treated cells, with notable enrichment in pathways linked to proliferation and immunity, such as cell cycle and TNF signaling pathways. Further analysis showed that L-histidine treatment positively correlated with an increase in EdU-555-positive cell rate and significantly suppressed IL-6 and IL-8 levels (p < 0.05) compared to controls. Crucially, concurrent treatment with high NEFA and L-histidine normalized the number of EdU-555-positive cells and cytokine expression to control levels. Investigating the underlying mechanisms, Gab2 (Grb2-associated binder 2) emerged as a central player; L-histidine notably reduced Gab2 expression, while NEFA had the opposite effect (p < 0.05). Gab2 overexpression escalated nitric oxide (NO) production and IL6 and IL8 expression. However, L-histidine addition to Gab2-overexpressing cells resulted in NO concentrations indistinguishable from controls. Our findings collectively indicate that L-histidine can counteract NEFA-induced inflammation in BMECs by inhibiting Gab2 expression, highlighting its therapeutic potential against NEFA-related metabolic disturbances.

Prepartum behaviors as early indicators for postpartum energy associated biomarkers status in Holstein dairy cows.

Our main aim was to investigate the predictive value of prepartum behaviors such as total daily rumination (TDR), total daily activity (TDA) and dry matter intake (DMI) as early indicators to detect cows at risk for hyperketonemia (HYK), hypoglycemia (HYG) or high non-esterified fatty acid (NEFA) status in the first (wk1) and second week (wk2) postpartum. In a case control study, 64 Holstein cows were enrolled 3 weeks before the expected time of calving and monitored until 15 days in milk (DIM). Postpartum blood samples were taken at D3 and D6 for wk1 and at D12 and D15 for wk2 to measure beta-hydroxybutyrate, NEFA and glucose concentration. Ear-mounted accelerometers were used to measure TDR and TDA. DMI and milk yield were obtained from farm records. Relationships between the average daily rate of change in prepartum TDR (ΔTDR), TDA (ΔTDA), and DMI (ΔDMI) with postpartum HYK, HYG and NEFA status in wk1 and wk2 post-partum were evaluated using linear regression models. Models were adjusted for potential confounding variables, and covariates retained in the final models were determined by backward selection. No evidence was found to support the premise that prepartum ΔTDR, ΔTDA or ΔDMI predicted postpartum HYK, HYG or NEFA status in wk1 or in wk2. Overall, prepartum ΔTDR, ΔTDA and ΔDMI were not effective predictors of HYK, HYG or NEFA status in the first 2 weeks postpartum.

Effect of high NEFA concentration on lipid metabolism disorders in hepatocytes based on lipidomics.

Introduction: High concentrations of nonesterified fatty acids (NEFA) is the key of characteristic of fatty liver in dairy cows. Therefore, the aim of this study was to investigate the effect of high concentration of NEFA on lipid metabolism in hepatocytes through the lipidomic approach and molecular biology techniques. Methods: Stimulate AML-12 cells with different concentrations of NEFA, observe the cellular lipid accumulation, and select 0.6mM NEFA stimulation concentration for subsequent experiments. Collect cells for lipidomics analysis. Results: High concentration of NEFA (0.6-2.4mM) significantly reduced the cell viability in a concentration-dependent manner, indicating that high concentrations of NEFA have lipotoxicity on hepatocytes. In addition, NEFA promoted triglycerides (TAG) accumulation, increased the mRNA expression of the lipogenic molecules SREBP1c and FASN, and decreased the mRNA expression of lipolytic molecules CPT1A and HSL in hepatocytes. Mechanistically, high concentration of NEFA induced lipid metabolism disorders in hepatocytes by regulating metabolic pathways such as glycerol phospholipid metabolism, glycosyl phosphatidylinositol anchored biosynthesis, triglyceride metabolism, sphingolipid metabolism, and inositol phosphate metabolism. Discussion: High concentration of NEFA is lipotoxic to cells, promoting lipid accumulation. LPE (18:2), LPE (18:3), LPE (18:1) via glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, glycerolipid metabolism, sphingolipid metabolism, and inositol phosphate metabolism, indicating their potential regulation role in the pathogenesis of fatty liver.

Effects of dry-off or continuous lactation in Alpine and Saanen dairy goats carrying single or double kids on peripartum metabolic profile, performances, and milk composition

Dairy goat’s lactation persistence forces farmers at limiting nutrient supply to reduce yield at dry-off. Omitting dry period could be a solution, but metabolic effects of this practice have never been tested. Eight Alpine (AL) and 12 Saanen (SA) goats approaching their second kidding blocked by breed and number of kids carried (single - SIN - or double - DOU) were allocated to one out of two groups (4 AL and 6 SA; 5 SIN and 5 DOU in each group). At −42 ± 7 days from kidding (DFK), they were either dried off (DR) or milked continuously until kidding (CL). Body condition score (BCS) was assessed, and blood samples were collected at −10, −3, 5, 12, and 29 DFK to determine metabolic profile. Milk yield and composition were assessed at −56, 7, 31, 62, and 97 DFK. Compared with DR, CL had higher plasma reactive oxygen metabolites and liver enzymes. Compared with DR counterparts, AL-CL had higher nonesterified fatty acids (NEFA) at −10, whereas SA-CL had lower NEFA at −3 DFK. CL goats had lower BCS, higher plasma beta-hydroxybutyrate (BHB) and urea before kidding, but higher glucose at −3 and 5, lower NEFA at 5 and 12, and higher BCS at 29 DFK. CL goats had lower haptoglobin and myeloperoxidase at −3 and 5 DFK, paired with higher albumin, cholesterol, and paraoxonase at 12 DFK. Omitting dry period mitigated the inflammatory condition around kidding in dairy goats, possibly accounting for an improved energy balance in early lactation, despite body reserve mobilisation prepartum was greater under continuous lactation.

Immunological Profile and Markers of Endothelial Dysfunction in Elderly Patients with Cognitive Impairments.

The process of aging is accompanied by a dynamic restructuring of the immune response, a phenomenon known as immunosenescence. Further, damage to the endothelium can be both a cause and a consequence of many diseases, especially in elderly people. The purpose of this study was to carry out immunological and biochemical profiling of elderly people with acute ischemic stroke (AIS), chronic cerebral circulation insufficiency (CCCI), prediabetes or newly diagnosed type II diabetes mellitus (DM), and subcortical ischemic vascular dementia (SIVD). Socio-demographic, lifestyle, and cognitive data were obtained. Biochemical, hematological, and immunological analyses were carried out, and extracellular vesicles (EVs) with endothelial CD markers were assessed. The greatest number of significant deviations from conditionally healthy donors (HDs) of the same age were registered in the SIVD group, a total of 20, of which 12 were specific and six were non-specific but with maximal differences (as compared to the other three groups) from the HDs group. The non-specific deviations were for the MOCA (Montreal Cognitive Impairment Scale), the MMSE (Mini Mental State Examination) and life satisfaction self-assessment scores, a decrease of albumin levels, and ADAMTS13 (a Disintegrin and Metalloproteinase with a Thrombospondin Type 1 motif, member 13) activity, and an increase of the VWF (von Willebrand factor) level. Considering the significant changes in immunological parameters (mostly Th17-like cells) and endothelial CD markers (CD144 and CD34), vascular repair was impaired to the greatest extent in the DM group. The AIS patients showed 12 significant deviations from the HD controls, including three specific to this group. These were high NEFAs (non-esterified fatty acids) and CD31 and CD147 markers of EVs. The lowest number of deviations were registered in the CCCI group, nine in total. There were significant changes from the HD controls with no specifics to this group, and just one non-specific with a maximal difference from the control parameters, which was α1-AGP (alpha 1 acid glycoprotein, orosomucoid). Besides the DM patients, impairments of vascular repair were also registered in the CCCI and AIS patients, with a complete absence of such in patients with dementia (SIVD group). On the other hand, microvascular damage seemed to be maximal in the latter group, considering the biochemical indicators VWF and ADAMTS13. In the DM patients, a maximum immune response was registered, mainly with Th17-like cells. In the CCCI group, the reaction was not as pronounced compared to other groups of patients, which may indicate the initial stages and/or compensatory nature of organic changes (remodeling). At the same time, immunological and biochemical deviations in SIVD patients indicated a persistent remodeling in microvessels, chronic inflammation, and a significant decrease in the anabolic function of the liver and other tissues. The data obtained support two interrelated assumptions. Taking into account the primary biochemical factors that trigger the pathological processes associated with vascular pathology and related diseases, the first assumption is that purine degradation in skeletal muscle may be a major factor in the production of uric acid, followed by its production by non-muscle cells, the main of which are endothelial cells. Another assumption is that therapeutic factors that increase the levels of endothelial progenitor cells may have a therapeutic effect in reducing the risk of cerebrovascular disease and related neurodegenerative diseases.

Lipopolysaccharide-binding protein in follicular fluid is associated with the follicular inflammatory status and granulosa cell steroidogenesis in dairy cows.

Metabolic stress and subsequent hepatic dysfunction in high-producing dairy cows are associated with inflammatory diseases and declining fertility. Lipopolysaccharide (LPS)-binding protein (LBP) is produced by hepatocytes and controls the immune response, suggesting that it is involved in the pathophysiology of inflammation-related attenuation of reproductive functions during metabolic stress. This study investigated the effect of LBP on the inflammatory status, oocyte quality, and steroidogenesis in the follicular microenvironment of dairy cows. Using bovine ovaries obtained from a slaughterhouse, follicular fluid and granulosa cells were collected from large follicles to evaluate the follicular status of metabolism, inflammation, and steroidogenesis. Cumulus-oocyte complexes were aspirated from small follicles and subjected to in vitro embryo production. The results showed that follicular fluid LBP concentrations were significantly higher in cows with fatty livers and hepatitis than in those with healthy livers. Follicular fluid LBP and LPS concentrations were negatively correlated, whereas LPS concentration showed a positive correlation with the concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyric acid in follicular fluid. The blastulation rate of oocytes after in vitro fertilization was impaired in cows in which coexisting large follicles had high NEFA levels. Follicular fluid NEFA concentration was negatively correlated with granulosa cell expression of the estradiol (E2) synthesis-related gene (CYP19A1). Follicular fluid LBP concentration was positively correlated with follicular fluid E2 concentration and granulosa cell CYP19A1 expression. In conclusion, follicular fluid LBP may be associated with favorable conditions in the follicular microenvironment, including low LPS levels and high E2 production by granulosa cells.

Comparison of Uterine Involution and the Resumption of Ovarian Cyclicity between Lame and Sound Holstein Cows

The aim of the study was to examine the effect of lameness and energy status on the involution of the uterus and the resumption of ovarian cyclicity in dairy cows. Lame (lameness score of four and the presence of hoof lesions, n = 22) and sound (normal gait and the absence of hoof lesions, n = 25) multiparous cows with healthy puerperium were enrolled simultaneously in the study and were monitored from day 10 antepartum (ap) to day 50 post-partum (pp). Ultrasonography of the cervix, the formerly gravid uterine horn and the ovarian structures was performed on d 8, 11, 14, 23, 30, and 42 pp. Blood sampling for progesterone, β-hydroxybutyrate (BHBA), and non-esterified fatty acids (NEFAs) was used to assess cyclicity and energy status. Lame compared to sound cows had higher NEFA concentrations on day 14 pp (0.54 ± 0.05 vs. 0.37 ± 0.05, respectively, p = 0.005), delayed involution of the cervix and the formerly pregnant uterine horn (p = 0.0003 and p = 0.02, respectively), lower ovulation rates within the experimental period (63.6% vs. 88%, respectively, p = 0.05), and higher rates of atresia or cyst formation on day 50 pp (36.4% vs. 12%, respectively, p = 0.05). Independently of lameness status, cows with high NEFA concentrations had lower ovulation rates within the experimental period (65.5% vs. 94.4%, p = 0.02), lower normal ovarian activity on day 50 pp (58.6% vs. 88.9%, p = 0.03), and higher rates of atresia or cyst formation on day 50 pp (34.5% vs. 5.6%, p = 0.02) compared to cows with optimal NEFA concentrations. Furthermore, an interaction between lameness and increased NEFA concentrations was observed regarding the ovulation rate within the experimental period and the percentage of atresia or cyst formation on day 50 pp. Sound cows with low NEFA levels had the lowest mean cervical diameter compared to cows with lameness (both with elevated and optimal NEFA concentrations, p = 0.009 and p = 0.002, respectively). Conclusively, lameness during puerperium negatively affected ovarian function and uterine involution. These effects were exacerbated (through interaction or cumulation) in relation to elevated NEFA concentrations.

Effects of blood sample collection and preparation methods on concentrations of glucose and nonesterified fatty acids in dairy cattle

Measurement of concentrations of glucose and nonesterified fatty acids (NEFA) in blood is common in nutrition and physiology studies. Proper collection and preparation conditions of the blood have been less well studied in dairy cattle. The objective of this experiment was to determine concentrations of glucose and NEFA in blood prepared with different anticoagulants (heparin vs. EDTA), use of fluoride as a glycolysis inhibitor, time until centrifugation (<30 min to 2.5 h), and plasma versus serum. Blood samples were obtained from 30 lactating cows and 15 milk-fed calves into 5 evacuated test tubes. Three of the tubes contained K3 EDTA and 1 tube contained heparin as anticoagulants to prepare plasma, and the fifth tube was a serum separator tube. One of the EDTA tubes was inverted and divided into 2 tubes containing NaF. One of the tubes with NaF and 1 EDTA tube were centrifuged within 30 min of collection and the others were held on ice for another 2 h before centrifugation. The heparin tube and the serum separator tube were centrifuged within 30 min. Glucose and NEFA were measured in the samples using enzymatic kits. Data were divided into 2 data sets representing normal dairy cow values or elevated values for glucose and NEFA. For glucose concentrations, results indicated that fluoride decreased concentrations, that a 2-h holding time before centrifugation did not affect results, and that serum and EDTA plasma resulted in lower glucose than heparin plasma. For low NEFA concentrations, addition of fluoride to the EDTA tubes resulted in a significant decrease of NEFA concentration. The effect of time sitting before centrifugation was significant for low NEFA samples, but contrary to our expectations, the effect was to decrease NEFA rather than increase it. Heparin as an anticoagulant did not affect NEFA concentrations in the low NEFA samples relative to EDTA. Heparin resulted in lower NEFA than serum in low NEFA samples. Serum and EDTA plasma resulted in similar NEFA concentrations. In the high NEFA samples, experimental power was limited due to the small sample size, but fluoride and time did not affect NEFA concentrations. Heparin tended to result in greater NEFA relative to EDTA. Serum produced greater NEFA values compared with EDTA but did not differ from heparin plasma. While differences among preparation methods were small and of limited biological significance, these results provide guidance for collection and processing of blood samples intended for glucose and NEFA assay.

L-carnitine enhances developmental potential of bovine oocytes matured under high lipid concentrations in vitro

Maternal obesity elevates non-esterified fatty acids (NEFA) follicular concentrations. Bovine cumulus-oocyte complexes (COCs) matured in vitro under high NEFA have altered metabolism and reduced quality. Systemically, obesity promotes altered mitochondrial metabolism linked to L-carnitine insufficiency. We hypothesized that L-carnitine supplementation during IVM of bovine COCs in the presence of high NEFA would lessen the negative effects of exposure to excessive lipids on embryonic development and oxidative stress. COCs were collected from abattoir ovaries and matured in four groups: CON (control), LC (3 mM L-carnitine), HN (high NEFA: 200uM oleic, 150uM palmitic and 75uM stearic acid), and HNLC (HN and LC). Mature oocytes were assayed for aerobic and anaerobic metabolism utilizing oxygen and pH microsensors or fertilized in vitro (D0). Cleavage (D3) and blastocyst (D7, D8) rates were assessed. D3 embryos with ≥ 4 cells were stained for cytosolic and mitochondrial ROS. D8 blastocysts were assayed for gene transcript abundance of metabolic enzymes. Oocyte metabolism was not affected by IVM treatment. D3 formation of embryos with ≥ 4 cells were lower in LC or HN than CON or HNLC; blastocyst rates were greater for CON and lower for HN than LC and HNLC. D3 embryo mitochondrial and cytosolic ROS were reduced in HNLC when compared to other groups. IVM in HN altered blastocyst gene transcript abundance when compared to CON, but not LC or HNLC. In conclusion, supplementation with L-carnitine protects oocytes exposed to high NEFA during IVM and improves their developmental competence, suggesting that high lipid exposure may lead to L-carnitine insufficiency in bovine oocytes.

Albumin and other metabolic parameters as potential indicators of purulent vaginal discharge in dairy cows during the transition period.

The aims of this study were to evaluate metabolic profiles obtained at -14, 14, and 28 days in milk (DIM), and to identify potential predictive biomarkers of Holstein dairy cows with purulent vaginal discharge (PVD) at 28 DIM. The body condition score (BCS) and hematocrit (Hct) were evaluated, and a metabolic profile test (MPT) was performed at -14, 14, and 28 DIM using serum samples. Cows at 28 DIM were classified using a vaginoscopy and divided into groups of healthy cows (n=89) and cows with PVD (n=31). Albumin (Alb), total cholesterol (TCho), calcium (Ca) and, magnesium (Mg) levels were lower in cows with PVD than in healthy cows at 14 DIM. At 28 DIM, levels of Alb, TCho, Ca, blood urea nitrogen (BUN), Mg, and Hct were lower in cows with PVD. A multivariate stepwise logistic regression analysis showed that higher non-esterified fatty acids (NEFA; odds ratios; OR=4.47; P<0.01), lower Alb (OR=0.07; P<0.01) and lower TCho (OR=0.99; P=0.08) at 14 DIM, and lower Hct (OR=0.83; P=0.05), lower Alb (OR=0.12; P<0.01), and lower BUN (OR=0.74; P=0.02) at 28 DIM were significantly associated with PVD. In conclusion, serum Alb levels was a potential indicator associated with PVD, reflecting dietary protein deficiency preceding disease. Our findings suggest that MPT should be considered to monitor health status during the postpartum period for early diagnosis of PVD.

Effect of body condition score at calving on transition success in Nili Ravi buffaloes

Body condition score (BCS) at calving is a vital indicator of the effectiveness of the beginning of lactation in dairy animals. The purpose of this study was to examine the effect of BCS at calving on milk production and transition success in dairy buffaloes. Thirty-six (36) Nili Ravi buffaloes were enrolled at 40 days of expected calving and followed through 90 days of lactation. The buffaloes were categorized into three groups according to their BCS (on a scale of 1-5 with 0.25 increments) as follows: 1) low, buffaloes with BCS ≤ 3.0; 2) medium, buffaloes with BCS 3.25-3.5; and 3) high, buffaloes with BCS ≥ 3.75. All buffaloes were fed a similar diet ad libitum. The lactation diet had increased concentrate allowance according to milk yield. The results revealed that the BCS at calving did not affect milk yield; however, fat percentage (fat%) was lower in the low-BCS group. Dry matter intake (DMI) was similar among the treatment groups, although post-calving BCS loss was greater in the high-BCS group compared to the medium- and the low-BCS groups. Similarly, the buffaloes in the high-BCS group had higher non-esterified fatty acids (NEFA) concentration compared to the low- and medium-BCS groups. No cases of metabolic disorders were observed during the study. The present results suggest that the buffaloes in the medium-BCS group appeared to perform better compared to the low- and the high-BCS groups with respect to milk fat% and blood NEFA concentration.

Body Condition Scoring and Serum Metabolic Profiles vis-a-vis Parity in Postpartum Surti Buffaloes

Background: Lactating animals tend to lose body tissue reserves in support of lactation and move into a negative energy balance that leads to a higher incidence of metabolic disorders and compromised productive performance. Despite several efforts to assess the influence of parity on BCS and metabolic profiles in postpartum buffaloes, systematic information is less. The current study was conducted to evaluate the effect of parity on metabolic profile and changes in BCS in postpartum Surti buffaloes. Methods: In the present study conducted from September-2017 to May-2018, fourteen postpartum Surti buffaloes were selected and grouped based on parity as primiparous and multiparous (2nd to 5th lactation). The blood sample was collected from all the selected animals on the day of calving, 15th, 30th and 60th day post calving. Serum biochemical parameters viz., glucose, total protein, blood urea nitrogen (BUN), cholesterol and Non-esterified fatty acids (NEFA) were analysed. The body condition score (BCS) was assessed from the day of calving up to 60th day of the study at 15-day interval. Result: The study revealed that the BCS was significantly higher in multiparous buffaloes as compared to primiparous group. Among biochemical parameters, glucose was significantly higher in primiparous group as compared to their multiparous counterparts. Parity did not have a significant influence on serum total protein, BUN, cholesterol and NEFA. However, NEFA was slightly higher in multiparous group of Surti buffaloes. High NEFA and low serum glucose indicated a greater degree of negative energy balance in multiparous buffaloes due to higher milk output.

Metabolic and antioxidant status during transition is associated with changes in the granulosa cell transcriptome in the preovulatory follicle in high-producing dairy cows at the time of breeding

In this study, we hypothesized that early postpartum (pp) metabolic and oxidative stress conditions in dairy cows (particularly those with severe negative energy balance, NEB) are associated with long-term changes in granulosa cell (GC) functions in the preovulatory follicle at the time of breeding. Blood samples were collected at wk 2 and wk 8 pp from 47 healthy multiparous cows. Follicular fluid (FF) and GC were collected from the preovulatory follicle after estrous synchronization at wk 8. Several metabolic and antioxidant parameters were measured in blood and FF, and their correlations were studied. Subsequently, 27 representative GC samples were selected for RNA sequencing analysis. The GC gene expression data of LH-responsive genes and the estradiol:progesterone ratio in FF were used to identify pre- and post-LH surge cohorts. We compared the transcriptomic profile of subgroups of cows within the highest and lowest quartiles (Q4 vs. Q1) of each parameter, focusing on the pre-LH surge cohort (n = 16, at least 3 in each subgroup). Differentially expressed genes (DEG: adjusted P-value < 0.05, 5% false discovery rate) were determined using DESeq2 analysis and were functionally annotated. Blood and FF β-carotene and vitamin E concentrations at wk 2, but not at wk 8, were associated with the most pronounced transcriptomic differences in the GC, with up to 341 DEG indicative for lower catabolism, increased oxidoreductase activity and signaling cascades that are known to enhance oocyte developmental competence, increased responsiveness to LH, and a higher steroidogenic activity. In contrast, elevated blood NEFA concentrations at wk 2 (and not at wk 8) were associated with a long-term carryover effect detectable in the GC transcriptome at wk 8 (64 DEG). These genes are related to response to lipids and ketones, oxidative stress, and immune responses, which suggests persistent cellular stress and oxidative damage. This effect was more pronounced in cows with antioxidant deficiencies at wk 8 (up to 148 DEG), with more genes involved in oxidative stress-dependent responses, apoptosis, autophagy and catabolic processes, and mitochondrial damage. Interestingly, within the severe NEB cows (high blood NEFA at wk 2), blood antioxidant concentrations (high vs. low) at wk 8 were associated with up to 194 DEG involved in activation of meiosis and other signaling pathways, indicating a better oocyte supportive capacity. This suggests that the cow antioxidant profile at the time of breeding might alleviate, at least in part, the effect of NEB on GC functions. In conclusion, these results provide further evidence that the metabolic and oxidative stress in dairy cows early postpartum can have long-term effects on GC functions in preovulatory follicles at the time of breeding. The interplay between the effects of antioxidants and NEFA illustrated here might be useful to develop intervention strategies to minimize the effect of severe NEB on fertility.

Effects of captopril on glucose metabolism and autophagy in liver and muscle from mice with type 1 diabetes and diet-induced obesity

Impaired metabolic functions underlie the pathophysiology of diabetes and obesity. The renin-angiotensin system (RAS) is one pathway related to the pathophysiology of both diseases. RAS activation in metabolically active tissues exerts pro-inflammatory effects via angiotensin II (Ang II), linked to dysfunction in cellular processes such as autophagy, which is associated with obesity and diabetes. Here, we determined whether RAS is involved in metabolic dysregulations in a Type 1 Diabetes (T1D) mouse model, treated with captopril, and in an obesity mouse model (Agt-Tg) that overexpresses angiotensinogen (Agt) in adipose tissue. T1D mice had lower plasma leptin, resistin and higher non-esterified fatty acids (NEFA) compared to wild type (Wt) mice, even under captopril treatment. Further, mRNA levels for Agt, At1, Insr, and Beclin1 were upregulated in muscle and liver of T1D mice with captopril compared to Wt. Moreover, autophagy markers LC3 and p62 proteins were decreased, regardless of captopril treatment in the liver from T1D mice. In obese Wt mice, captopril increased muscle Irs1 gene levels. Further, captopril reduced mRNA levels of At1, Insr, Ampk, Beclin1, Atg12, and Lc3 in the liver from both Wt and Agt-Tg mice, while Agt, At1, Insr, and Atg12 expression was reduced in Agt-Tg mice without captopril treatment. Irs1 expression was decreased in the liver from obese Wt mice treated with captopril. Our results suggest that captopril treatment upregulates components of RAS, insulin signaling, and autophagy in both muscle and liver, indicating potential utility of captopril in targeting both insulin sensitivity and autophagy in diabetes and obesity.

P-035 Exposure to high concentrations of non-esterified fatty acids impairs bovine sperm quality

Abstract Study question Do high concentrations of non-esterified fatty acids (NEFA) affect sperm quality? Summary answer In vitro exposure to high NEFA concentrations induced detrimental effects on acrosome integrity, mitochondrial membrane potential, plasma membrane integrity, and DNA damage in bovine spermatozoa. What is known already High NEFA levels present in obese individuals and in cows experiencing negative energy balance has been linked with impaired reproductive success. In vitro bovine models have showed that a high NEFA microenvironment (i.e. steric acid [SA], palmitic acid [PA], oleic acid [OA]) during oocyte maturation (Reproduction 2013, 145:33-44), fertilisation (ESHRE meeting 2021, P-176), and embryo culture (BMC Genomics 2016, 17:1004) is detrimental for blastocyst formation. However, the effect of high NEFA levels on sperm quality has been less explored. Study design, size, duration Bovine sperm from two bulls was prepared to a maximum concentration of 20x106/mL then incubated in in vitro fertilisation (IVF) medium for 4 h under different NEFA levels representing physiological (Control-1[C1], 28 μM SA, 23 μM PA, 21 μM OA) and pathophysiological (High-NEFA, 280μM SA, 230μM PA, 210μM OA), serum levels found in cows experiencing negative energy balance) concentrations. A second control (C2) group contained solvent. Participants/materials, setting, methods Fluorescence assays (Toxicity 2018, 393:52-50) were used to evaluate simultaneously acrosome integrity (FITC-PSA), mitochondrial membrane potential (JC-1) and plasma membrane integrity (DRAQ7, Hoechst 33342). Spermatozoa (n = 800 per group, four replicates) were assessed with epifluorescence microscopy and Ziess ZEN software. Sperm DNA damage was also assessed with the Halosperm G2® kit (C1; n = 812, C2; n = 879, High-NEFA; n = 930, six replicates). Data were arcsine transformed before analysis (ANOVA, followed by LSD post hoc test). Main results and the role of chance There was no significant difference between the control groups or between bulls in any of the variables analysed. The percentage (mean ± SEM) of spermatozoa with intact acrosome was higher in the control groups compared with high-NEFA (C1=64.50 ± 6.34%, C2=69.13 ± 4.08%, High-NEFA=49.75 ± 6.14%), but it reached statistical significance only when compared to C2 (C2 vs High-NEFA P = 0.037, C1 vs High-NEFA P = 0.096). Similarly, the percentage of spermatozoa with low mitochondrial membrane potential was increased following exposure to high concentrations of NEFA (C1=50.88 ± 5.69%, C2=45.00 ± 4.03%, High-NEFA=61.62 ± 3.38%), but a significant difference was only observed when compared to C2 (C2 vs High-NEFA P = 0.027, C1 vs High-NEFA P = 0.124). The high-NEFA group showed a higher plasma membrane damage (59.50 ± 1.06%) compared to controls (C1=47.50 ± 4.07% P = 0.01, C2=46.75 ± 1.79% P = 0.008). DNA damage was also increased in the High-NEFA group (20.00 ± 0.64%) compared to controls (C1=8.90 ± 0.71% P &amp;lt; 0.001, C2=6.82 ± 0.78% P &amp;lt; 0.001). Limitations, reasons for caution In vitro animal models do not reflect accurately in vivo conditions and cannot be directly extrapolated to humans, but they are useful for the development of conceptual models that could eventually be tested in the target species (e.g. obese individuals). Wider implications of the findings Our results indicate that high NEFA concentrations can impair sperm quality, and given that sperm with DNA damage can achieve fertilization (Human Reproduction 2010, 25:1594-1608), our data partially explain the decreased blastocyst formation previously observed in this model of high NEFA exposure (ESHRE meeting 2021, P-176). Trial registration number 000

Exposure to non-esterified fatty acids in vitro results in changes in the ovarian and follicular environment in cattle

Negative energy balance (NEB) in the postpartum period of dairy cows is associated with reduced fertility to insemination later in lactation. We hypothesized that elevated non-esterified fatty acids (NEFA) levels that occur during NEB result in accumulation of fatty acids within the ovarian tissue and preantral follicles, causing changes in ovarian gene expression that would indicate a response to injury. We performed ovarian cortex culture and oocyte maturation in medium containing a combination of palmitic, oleic and stearic acid (NEFA). Ovarian cortex was subjected to RNA sequencing and lipid content analysis via Nile Red staining and gas chromatography; oocytes were analyzed for maturation rate and mitochondrial mass and localization following in vitro maturation (IVM). Accumulation of lipids associated with the plasma membrane was increased in granulosa cells of preantral follicles exposed to NEFA in vitro; RNA sequencing revealed changes in biological functions associated with metabolic disease, stimulation of an inflammatory response, and reduction in glucose uptake. Oocyte maturation under high NEFA compromised nuclear, but not cytoplasmic maturation. These data demonstrate that exposure to NEFA in vitro affects the ovary, preantral follicles and cumulus-oocyte complexes, and provides further insight into the potential links between metabolic imbalance and infertility.

Major Nutritional Metabolic Alterations Influencing the Reproductive System of Postpartum Dairy Cows.

Early successful conception of postpartum dairy cows is crucial in determining the optimum reproductive efficiency and profitability in modern dairy farming. Due to the inherent high production potential of modern dairy cows, the extra stress burden of peri-parturient events, and associated endocrine and metabolic changes causes negative energy balance (NEBAL) in postpartum cows. The occurrence of NEBAL is associated with excessive fat mobilization in the form of non-esterified fatty acids (NEFAs). The phenomenon of NEFA mobilization furthers with occurrence of ketosis and fatty liver in postpartum dairy cows. High NEFAs and ketones are negatively associated with health and reproductive processes. An additional burden of hypocalcemia, ruminal acidosis, and high protein metabolism in postpartum cows presents further consequences for health and reproductive performance of postpartum dairy cows. This review intends to comprehend these major nutritional metabolic alterations, their mechanisms of influence on the reproduction process, and relevant mitigation strategies.

Leptin Concentration, Obesity, and Plasma Non-esterified Fatty Acid Levels in Children.

The association between obesity and higher non-esterified fatty acid (NEFA) levels has been established in adults. In contrast, lower NEFA levels have been described in children with obesity although the reason behind this association remains unclear. Leptin, which regulates body weight and plays a role in lipolysis, could be involved in this relationship. We evaluated the influence of leptin in the association between obesity and NEFA concentrations in children, analyzing two cohorts including 684 6- to 8-year-olds and 836 12- to 16-year-old children, respectively. After adjusting by leptin, insulin levels remained significantly higher in adolescents with obesity as compared with levels in those without obesity. However, insulin levels showed no differences between prepubertal children with and without obesity. The significantly lower NEFA concentrations observed in 6- to 8-year-old girls with obesity disappeared when comparing NEFA levels between girls with and without obesity after adjusting by leptin. We report an influence of leptin levels on the association between obesity and insulin and NEFA in young children that is not observed in adolescents. Our findings add information about factors that may contribute to explain the lower NEFA levels described in prepubertal children with obesity.

Sirtuin 3 inhibits nuclear factor-κB signaling activated by a fatty acid challenge in bovine mammary epithelial cells

Susceptibility to mastitis is highest during the peripartal (transition) period and is often concomitant with other comorbidities such as ketosis. Although infection with pathogenic microorganisms and immune-dysfunction around calving clearly play key roles in mastitis development, other metabolic factors also contribute. Sirtuin 3 (SIRT3), a mitochondrial deacetylase regulating energy and redox homeostasis, antagonizes the lipotoxic effects of nonesterified fatty acids (NEFA). Thus, we hypothesized that increases in circulating NEFA concentrations, as observed in the transition period, provokes inflammatory responses that can be reversed via activation of SIRT3. Here we aimed to study (1) proinflammatory NF-κB signaling and SIRT3 abundance in mammary tissue of ketotic cows and healthy controls, and (2) the effect of SIRT3 on NF-κB activation in bovine mammary epithelial cells (BMEC) treated with high levels of NEFA. The mammary gland biopsy samples were from a previous study, which included 15 healthy cows and 15 ketotic cows. Primary BMEC were isolated from 3 healthy Holstein cows with collagenase III digestion. Purified BMEC were incubated with or without SIRT3 overexpression adenovirus for 48 h, then treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 24 h. Mammary tissue of ketotic cows was associated with lower protein abundance of SIRT3 along with greater NF-κB P65 phosphorylation levels (p-NF-κB P65), p-NF-κB P65:NF-κB P65 ratio, and mRNA abundance of IL1B and IL6. In BMEC, exogenous NEFA dose-dependently reduced protein abundance of SIRT3, but increased p-NF-κB P65, p-NF-κB P65:NF-κB P65 ratio, and mRNA abundance of IL1B and IL6. Compared with green fluorescent protein adenovirus vector + NEFA, overexpression of SIRT3 in NEFA-treated BMEC downregulated p-NF-κB P65 and mRNA abundance of IL1B and IL6. Immunofluorescence indicated that overexpression of SIRT3 inhibited nuclear translocation of NF-κB P65. Overall, our data demonstrated that ketosis is associated with a reduction in SIRT3 abundance and activation of NF-κB signaling in the mammary gland. In vitro data provided evidence that high NEFA concentrations inhibit SIRT3, which contributes to enhanced NF-κB signaling including nuclear translocation and a pro-inflammatory response. The data suggest a promising role of SIRT3 as a target for helping alleviate localized inflammation of the mammary gland resulting from exposure to high concentrations of NEFA.

P–176 Effects of high non-esterified fatty acids exclusively during bovine in vitro fertilization on cell lineage allocation of blastocysts

Abstract Study question Are there effects of high non-esterified fatty acids exclusively during bovine in vitro fertilization on cell lineage allocation of blastocysts? Summary answer Under the conditions of the present study, high exposure to NEFA during bovine IVF significantly decreases embryo production and alters cell allocation of resultant blastocysts. What is known already Cattle models have shown that a high exposure to non-esterified fatty acids (NEFA) such as steric acid (SA), palmitic acid (PA]) and oleic acid (OA) during in vitro oocyte maturation and embryo development can disrupt both embryo formation and quality. However, the fertilization process per se have been less studied, which is needed to identify developmental stages were potential therapies could be develop to ameliorate NEFA toxicity during the periconceptional period Study design, size, duration Day–8 blastocysts were immunostained for CDX2, a transcription factor involved in trophectoderm differentiation, to examine cell allocation of blastocysts derived from oocytes fertilised under high NEFA levels. VF (19 h) was carried with different NEFA levels(4 replicates) representing physiological (Control–1[C1], 28μM SA, 23μM PA, 21μM OA) and pathophysiological (NEFA, 280μM SA, 230μM PA, 210μM OA) relevant concentrations. A second control (C2) group contained solvent. Blastocysts (C1; n = 14, C2; n = 12, NEFA; n = 8) Participants/materials, setting, methods All blastocysts were examined by confocal microscopy and cell counting was done with the Imaris software. Data were analysed by ANOVA (mean±SEM) with percentage data arcsine transformed before analysis. Main results and the role of chance Blastocyst formation was decreased by high NEFA levels (C1=25.6±2.7%, C2=26.0±2.3%, NEFA=9.4±0.4%, P &amp;lt; 0.001) which was associated with a decreased cleavage rate (C1=70.1±6.5%, C2=71.5±3.1%, NEFA=42.5±4.1%, P = 0.006) and an increase in embryo degeneration (C1=47.6±3.5%, C2=47.7±5.8%, NEFA=63.0±4.9%, P = 0.05). A lower total cell (TC) the number was observed in high NEFA-derived blastocyst (C1=125.2±6.6, C2=132.3±8.4, NEFA=67.3±5.6, P &amp;lt; 0.001) associated with a low cell number in both the trophectoderm (CDX2 positive cells, C1=90.2±5.9, C2=96.7±6.4, NEFA=41.3±4.1, P &amp;lt; 0.001) and the inner cell mass (ICM, C1=35.0±2.4, C2=35.7±3.5, NEFA=26.0±2.2, P &amp;lt; 0.001). Furthermore, high NEFA-derived blastocyst showed an increased allocation of cells towards the ICM (ICM/TC proportion, C1=28.2±1.7%, C2=26.9±1.8%, NEFA=39.1±2.2%, P &amp;lt; 0.001) Limitations, reasons for caution It will be better if the number of blastocysts reached increases. Wider implications of the findings: such research can be widely applied to the human model due to the similarities between both specie. Trial registration number Royal Embassy of Saudi Arabia Cultural Bureau