Cannabinoid-1 receptor activation modulates lipid mobilization and adipogenesis in the adipose tissue of dairy cows.

Amplified adipose tissue (AT) lipolysis and suppressed lipogenesis characterize the periparturient period of dairy cows. The intensity of lipolysis recedes with the progression of lactation; however, when lipolysis is excessive and prolonged, disease risk is exacerbated and productivity compromised. Interventions that minimize lipolysis while maintaining adequate supply of energy and enhancing lipogenesis may improve periparturient cows' health and lactation performance. Cannabinoid-1 receptor (CB1R) activation in rodent AT enhances the lipogenic and adipogenic capacity of adipocytes, yet the effects in dairy cow AT remain unknown. Using a synthetic CB1R agonist and an antagonist, we determined the effects of CB1R stimulation on lipolysis, lipogenesis, and adipogenesis in the AT of dairy cows. Adipose tissue explants were collected from healthy, nonlactating and nongestating (NLNG; n = 6) or periparturient (n = 12) cows at 1 wk before parturition and at 2 and 3 wk postpartum (PP1 and PP2, respectively). Explants were treated with the ß-adrenergic agonist isoproterenol (1 µM) in the presence of the CB1R agonist arachidonyl-2'-chloroethylamide (ACEA) ± the CB1R antagonist rimonabant (RIM). Lipolysis was quantified based on glycerol release. We found that ACEA reduced lipolysis in NLNG cows; however, it did not exhibit a direct effect on AT lipolysis in periparturient cows. Inhibition of CB1R with RIM in postpartum cow AT did not alter lipolysis. To evaluate adipogenesis and lipogenesis, preadipocytes isolated from NLNG cows' AT were induced to differentiate in the presence or absence of ACEA ± RIM for 4 and 12 d. Live cell imaging, lipid accumulation, and expressions of key adipogenic and lipogenic markers were assessed. Preadipocytes treated with ACEA had higher adipogenesis, whereas ACEA+RIM reduced it. Adipocytes treated with ACEA and RIM for 12 d exhibited enhanced lipogenesis compared with untreated cells (control). Lipid content was reduced in ACEA+RIM but not with RIM alone. Collectively, our results support that lipolysis may be reduced by CB1R stimulation in NLNG cows but not in periparturient cows. In addition, our findings demonstrate that adipogenesis and lipogenesis are enhanced by activation of CB1R in the AT of NLNG dairy cows. In summary, we provide initial evidence which supports that the sensitivity of the AT endocannabinoid system to endocannabinoids, and its ability to modulate AT lipolysis, adipogenesis, and lipogenesis, vary based on dairy cows' lactation stage.

Symposium review: Mechanistic insights into adipose tissue inflammation and oxidative stress in periparturient dairy cows.

During the periparturient period, lipolysis in adipose tissue (AT) mobilizes fatty acid reserves to meet high energy needs of dairy cows. This physiological response is accompanied by the synthesis and secretion of a plethora of proteins (adipokines) and lipid products that modulate metabolic functions. In the AT, lipolysis generates free radicals (FR), including reactive oxygen species, and leads to a remodeling process characterized by an inflammatory response. In the AT of healthy cows with adequate lipolytic responses, antioxidant defenses neutralize FR, and the inflammation associated with remodeling is rapidly resolved. The control of these processes is orchestrated by numerous inflammatory and oxidative stress (OS)-related pathways identified by recent transcriptomic and proteomic analyses. For example, parturition and the onset of lactation enhance the transcription and translation of complement and acute-phase proteins and, at the same time, enrich antioxidant defenses that neutralize FR, including Nrf2. However, in cows with exacerbated and protracted lipolysis, the production of FR rapidly depletes antioxidant systems, and OS develops. The harmful effects of OS in AT include activating inflammatory responses and inhibiting insulin signaling within AT. By intensifying AT inflammation, OS impairs adipocyte response to insulin. This leads to a vicious circle where OS exacerbates AT lipolysis and inflammation, which further promotes OS. This review summarizes current knowledge on the mechanisms that modulate AT inflammatory responses and OS during the periparturient period of dairy cows.

Phosphoproteomic Analysis of Subcutaneous and Omental Adipose Tissue Reveals Increased Lipid Turnover in Dairy Cows Supplemented with Conjugated Linoleic Acid.

Phosphoproteomics is a cutting-edge technique that can be utilized to explore adipose tissue (AT) metabolism by quantifying the repertoire of phospho-peptides (PP) in AT. Dairy cows were supplemented with conjugated linoleic acid (CLA, n = 5) or a control diet (CON, n = 5) from 63 d prepartum to 63 d postpartum; cows were slaughtered at 63 d postpartum and AT was collected. We performed a quantitative phosphoproteomics analysis of subcutaneous (SC) and omental (OM) AT using nanoUPLC-MS/MS and examined the effects of CLA supplementation on the change in the phosphoproteome. A total of 5919 PP were detected in AT, and the abundance of 854 (14.4%) were differential between CON and CLA AT (p ≤ 0.05 and fold change ± 1.5). The abundance of 470 PP (7.9%) differed between OM and SC AT, and the interaction treatment vs. AT depot was significant for 205 PP (3.5% of total PP). The integrated phosphoproteome demonstrated the up- and downregulation of PP from proteins related to lipolysis and lipogenesis, and phosphorylation events in multiple pathways, including the regulation of lipolysis in adipocytes, mTOR signaling, insulin signaling, AMPK signaling, and glycolysis. The differential regulation of phosphosite on a serine residue (S777) of fatty acid synthase (FASN) in AT of CLA-supplemented cows was related to lipogenesis and with more phosphorylation sites compared to acetyl-coenzyme A synthetase (ACSS2). Increased protein phosphorylation was seen in acetyl-CoA carboxylase 1 (ACACA;8 PP), FASN (9 PP), hormone sensitive lipase (LIPE;6 PP), perilipin (PLIN;3 PP), and diacylglycerol lipase alpha (DAGLA;1 PP) in CLA vs. CON AT. The relative gene expression in the SC and OM AT revealed an increase in LIPE and FASN in CLA compared to CON AT. In addition, the expression of DAGLA, which is a lipid metabolism enzyme related to the endocannabinoid system, was 1.6-fold higher in CLA vs. CON AT, and the expression of the cannabinoid receptor CNR1 was reduced in CLA vs. CON AT. Immunoblots of SC and OM AT showed an increased abundance of FASN and a lower abundance of CB1 in CLA vs. CON. This study presents a complete map of the SC and the OM AT phosphoproteome in dairy cows following CLA supplementation and discloses many unknown phosphorylation sites, suggestive of increased lipid turnover in AT, for further functional investigation.

Biomarkers of fitness and welfare in dairy cattle: healthy productivity.

Milk production intensification has led to several unwanted aspects, such as sustainability issues and environmental pollution. Among these, increased milk outputs that have been achieved over the last 70 years have led to several health and pathophysiological conditions in high yielding dairy animals, including metabolic diseases that were uncommon in the past. Increased occurrence of diverse metabolic diseases in cattle and other domestic animals is a key feature of domestication that not only affects the animals' health and productivity, but also may have important and adverse health impacts on human consumers through the elevated use of drugs and antibiotics. These aspects will influence economical and ethical aspects in the near future. Therefore, finding and establishing proper biomarkers for early detection of metabolic diseases is of great interest. In the present review, recent work on the discovery of fitness, stress and welfare biomarkers in dairy cows is presented, focusing in particular on possible biomarkers of energy balance and oxidative stress in plasma and milk, and biomarkers of production-related diseases and decreased fertility.

Biomarkers of fitness and welfare in dairy animals: healthy living.

Increased animal productivity has reduced animal fitness, resulting in increased susceptibility to infectious and metabolic diseases, locomotion problems and subfertility. Future animal breeding strategies should focus on balancing high production levels with health status monitoring and improved welfare. Additionally, understanding how animals interact with their internal and external environment is essential for improving health, fitness, and welfare. In this context, the continuous validation of existing biomarkers and the discovery and field implementation of new biomarkers will enable us to understand the specific physiological process and regulatory mechanisms used by the organism to adapt to different environmental conditions. Thus, biomarkers may be used to monitor welfare and improve management and breeding strategies. In this article, we describe major achievements in the establishment of biomarkers in dairy cows and small ruminants. This review mainly focuses on the physiological biomarkers used to monitor animal responses to, and recovery from, environmental perturbations. We highlight future avenues for research in this field and present a timely positioning document to the scientific community.

Liver proteomic analysis of postpartum Holstein cows exposed to heat stress or cooling conditions during the dry period.

Heat stress negatively affects cow performance, compromises immune function, and increases susceptibility to metabolic disorders, particularly during the dry period and as cows transition from gestation to lactation. Metabolic adaptations of the liver are critical for successful transition, yet it is unclear how heat stress affects metabolic pathways within the liver at the proteomic level. The objective of this study was to investigate the liver proteome of postpartum cows that were cooled or heat stressed during the dry period to gain insight into how protein expression is altered by prior heat stress and may contribute to performance and disease outcomes. During the dry period, cows were either housed in shaded barns with fans and water soakers [cooled group (CL); n = 5] or in shaded barns lacking these cooling devices [heat-stressed group (HT); n = 5]. Liver biopsies were collected at 2 d postpartum, and protein content was analyzed by label-free quantitative shotgun proteomics (nanoscale liquid chromatography coupled to tandem mass spectrometry). In the most comprehensive bovine liver proteomics analysis completed to date, we identified 3,270 proteins, 75 of which were differentially expressed between HT and CL cows (fold change ±1.2). The top pathways differing between HT and CL cows were oxidative phosphorylation, mitochondrial dysfunction, farnesoid X receptor/retinoid X receptor (FXR/RXR) activation, and the methylmalonyl pathway. Cooling cows during the dry period likely improves ATP production, reduces oxidative stress, and prevents excessive accumulation of hepatic triglycerides and cholesterol, which may contribute to greater milk yield and lower susceptibility to transition-related diseases.

Defining the Adipose Tissue Proteome of Dairy Cows to Reveal Biomarkers Related to Peripartum Insulin Resistance and Metabolic Status.

Adipose tissue is a central regulator of metabolism in dairy cows; however, little is known about the association between various proteins in adipose tissue and the metabolic status of peripartum cows. Therefore, the objectives were to (1) examine total protein expression in adipose tissue of dairy cows and (2) identify biomarkers in adipose that are linked to insulin resistance and to cows' metabolic status. Adipose tissue biopsies were obtained from eight multiparous cows at -17 and +4 days relative to parturition. Proteins were analyzed by intensity-based, label-free, quantitative shotgun proteomics (nanoLC-MS/MS). Cows were divided into groups with insulin-resistant (IR) and insulin-sensitive (IS) adipose according to protein kinase B phosphorylation following insulin stimulation. Cows with IR adipose lost more body weight postpartum compared with IS cows. Differential expression of 143 out of 586 proteins was detected in prepartum versus postpartum adipose. Comparing IR to IS adipose revealed differential expression of 18.9% of the proteins; those related to lipolysis (hormone-sensitive lipase, perilipin, monoglycerol lipase) were increased in IR adipose. In conclusion, we found novel biomarkers related to IR in adipose and to metabolic status that could be used to characterize high-yielding dairy cows that are better adapted to peripartum metabolic stress.

Short communication: Concentrations of the mammalian lignan enterolactone in preovulatory follicles and the correlation with intrafollicular estradiol in dairy cows fed extruded flaxseed.

Flaxseed is a rich source of lignans that can be metabolized to the mammalian lignan enterolactone (EL), which may elicit weak estrogenic or antiestrogenic effects. The objectives of this study were to examine the effects of feeding an extruded flaxseed supplement to dairy cows on concentrations of EL in plasma and preovulatory follicles and the association with intrafollicular estradiol (E2). Twenty-four multiparous 256-d-pregnant Israeli Holstein cows were fed either a standard diet both pre- and postpartum (control; n=12) or provided with an extruded flaxseed supplement (n=12), at 7.9 and 9.2% of dry matter, pre- and postpartum, respectively. Follicular fluid (FF) aspirations were conducted at 84±16 d in lactation as follows: 7 to 8 d following behavioral estrus, cows were injected with prostaglandin F2α and 48h later follicles >7mm were aspirated. Follicles were regarded as preovulatory when the E2-to-progesterone ratio was >1. Plasma EL concentrations were not different between treatment groups; however, concentrations of EL in FF of preovulatory follicles were 1.7 times higher in extruded flaxseed-supplemented cows than in control. Across-treatment analysis revealed a positive correlation between concentrations of EL in plasma and in FF. In addition, intrafollicular EL concentrations were positively correlated with E2 concentrations (r=0.50), and with the intrafollicular E2-to-progesterone ratio. In conclusion, supplementing dairy cows with extruded flaxseed increased EL concentrations in preovulatory follicles. Intrafollicular EL was correlated with E2 concentrations; therefore, the possible effects of EL from flaxseed on follicular steroidogenesis should be considered.

Maternal supplementation with n-3 fatty acids affects placental lipid metabolism, inflammation, oxidative stress, the endocannabinoid system, and the neonate cytokine concentrations in dairy cows.

BACKGROUND: The placenta plays a crucial role in supporting and influencing fetal development. We compared the effects of prepartum supplementation with omega-3 (n-3) fatty acid (FA) sources, flaxseed oil (FLX) and fish oil (FO), on the expression of genes and proteins related to lipid metabolism, inflammation, oxidative stress, and the endocannabinoid system (ECS) in the expelled placenta, as well as on FA profile and inflammatory response of neonates. Late-pregnant Holstein dairy cows were supplemented with saturated fat (CTL), FLX, or FO. Placental cotyledons (n = 5) were collected immediately after expulsion, and extracted RNA and proteins were analyzed by RT-PCR and proteomic analysis. Neonatal blood was assessed for FA composition and concentrations of inflammatory markers. RESULTS: FO increased the gene expression of fatty acid binding protein 4 (FABP4), interleukin 10 (IL-10), catalase (CAT), cannabinoid receptor 1 (CNR1), and cannabinoid receptor 2 (CNR2) compared with CTL placenta. Gene expression of ECS-enzyme FA-amide hydrolase (FAAH) was lower in FLX and FO than in CTL. Proteomic analysis identified 3,974 proteins; of these, 51-59 were differentially abundant between treatments (P ≤ 0.05, |fold change| ≥ 1.5). Top canonical pathways enriched in FLX vs. CTL and in FO vs. CTL were triglyceride metabolism and inflammatory processes. Both n-3 FA increased the placental abundance of FA binding proteins (FABPs) 3 and 7. The abundance of CNR1 cannabinoid-receptor-interacting-protein-1 (CNRIP1) was reduced in FO vs. FLX. In silico modeling affirmed that bovine FABPs bind to endocannabinoids. The FLX increased the abundance of inflammatory CD44-antigen and secreted-phosphoprotein-1, whereas prostaglandin-endoperoxide synthase 2 was decreased in FO vs. CTL placenta. Maternal FO enriched neonatal plasma with n-3 FAs, and both FLX and FO reduced interleukin-6 concentrations compared with CTL. CONCLUSION: Maternal n-3 FA from FLX and FO differentially affected the bovine placenta; both enhanced lipid metabolism and modulated oxidative stress, however, FO increased some transcriptional ECS components, possibly related to the increased FABPs. Maternal FO induced a unique balance of pro- and anti-inflammatory components in the placenta. Taken together, different sources of n-3 FA during late pregnancy enhanced placental immune and metabolic processes, which may affect the neonatal immune system.

Glucose-6-Phosphate Dehydrogenase Activity in Milk May Serve as a Non-Invasive Metabolic Biomarker of Energy Balance in Postpartum Dairy Cows.

Negative energy balance (EB) postpartum is associated with adverse outcomes in dairy cows; therefore, non-invasive biomarkers to measure EB are of particular interest. We determined whether specific metabolites, oxidative stress indicators, enzyme activity, and fatty acid (FA) profiles in milk can serve as indicators of negative EB. Forty-two multiparous Holstein dairy cows were divided at calving into 2 groups: one was milked 3 times daily and the other, twice a day for the first 30 d in milk (DIM). Cows were classified retrospectively as being in either negative EB (NEB, n = 19; the mean EB during the first 21 DIM were less than the overall median of -2.8 Mcal/d), or in positive EB (PEB, n = 21; the mean EB was ≥-2.8 Mcal/d). The daily milk yield, feed intake, and body weight were recorded individually. Blood samples were analyzed for metabolites and stress biomarkers. Milk samples were taken twice weekly from 5 to 45 DIM to analyze the milk solids, the FA profile, glucose, glucose-6-P (G6P), G6P-dehydrogenase (G6PDH) activity, malic and lactic acids, malondialdehyde (MDA), and oxygen radical antioxidant capacity (ORAC). The NEB cows produced 10.5% more milk, and consumed 7.6% less dry matter than the PEB cows. The plasma glucose concentration was greater and ß-hydroxybutyrate was lower in the PEB vs. the NEB cows. The average concentrations of milk glucose, G6P, malic and lactic acids, and MDA did not differ between groups; however, the G6PDH activity was higher and ORAC tended to be higher in the milk of NEB vs. the PEB cows. The correlation between milk G6PDH activity and EB was significant (r = -0.39). The percentages of oleic acid and total unsaturated FA in milk were higher for the NEB vs. the PEB cows. These findings indicate that G6PDH activity in milk is associated with NEB and that it can serve as a non-invasive candidate biomarker of NEB in postpartum cows, that should be validated in future studies.

Alpha-linolenic acid modulates systemic and adipose tissue-specific insulin sensitivity, inflammation, and the endocannabinoid system in dairy cows.

Metabolic disorders are often linked to alterations in insulin signaling. Omega-3 (n-3) fatty acids modulate immunometabolic responses; thus, we examined the effects of peripartum n-3 on systemic and adipose tissue (AT)-specific insulin sensitivity, immune function, and the endocannabinoid system (ECS) in dairy cows. Cows were supplemented peripartum with saturated fat (CTL) or flaxseed supplement rich in alpha-linolenic acid (ALA). Blood immunometabolic biomarkers were examined, and at 5-8 d postpartum (PP), an intravenous glucose-tolerance-test (GTT) and AT biopsies were performed. Insulin sensitivity in AT was assessed by phosphoproteomics and proteomics. Peripartum n-3 reduced the plasma concentrations of Interleukin-6 (IL-6) and IL-17α, lowered the percentage of white blood cells PP, and reduced inflammatory proteins in AT. Systemic insulin sensitivity was higher in ALA than in CTL. In AT, the top canonical pathways, according to the differential phosphoproteome in ALA, were protein-kinase-A signaling and insulin-receptor signaling; network analysis and immunoblots validated the lower phosphorylation of protein kinase B (Akt), and lower abundance of insulin receptor, together suggesting reduced insulin sensitivity in ALA AT. The n-3 reduced the plasma concentrations of ECS-associated ligands, and lowered the abundances of cannabinoid-1-receptor and monoglycerol-lipase in peripheral blood mononuclear cells PP. Peripartum ALA supplementation in dairy cows improved systemic insulin sensitivity and immune function, reduced ECS components, and had tissue-specific effects on insulin-sensitivity in AT, possibly counter-balancing the systemic responses.

Proteomic analysis of adipose tissue revealing differentially abundant proteins in highly efficient mid-lactating dairy cows.

The improvement of nutrient utilization efficiency in dairy cows represents an important task in view of the current rising demand for animal products and sustainable resource usage. In this perspective, the identification of appropriate markers to identify the most efficient animals for dairy production becomes a crucial factor. Residual feed intake (RFI), which represents the difference between predicted and actual intake, is used to define the efficiency of cows. In this study, subcutaneous adipose tissue (AT) was collected from five high efficient (HEF) and five low efficient (LEF) mid-lactation Holstein dairy cows, that represented subgroups of the 20% lowest RFI values (HEF) and highest 20% RFI values (LEF), out of a cohort of 155 cows that were examined for feed efficiency at the individual dairy barn at Volcani Institute, Israel. Adipose samples were examined for proteomic analysis by nano-LC/MS-MS and gene expression by RT-PCR. A total of 101 differential proteins (P ≤ 0.05 and fold change ± 1.5) and two protein networks related to feed efficiency were found between HEF and LEF cows. Among the enriched top canonical pathways, FAT10 signaling, EIF2 signaling, Sirtuin signaling, Acute phase response signaling, Protein ubiquitination and mTOR signaling pathways were related to feed efficiency in AT. Furthermore, abundance of transferrin (TF; FC = 78.35, P = 0.02) enriched pathways, including mTOR signaling, LXR/RXR and FXR/RXR activation was found in AT of HEF cows. Relative mRNA expression of RBM39, which is involved in energy metabolism, was decreased in AT of HEF versus LEF. The relationship found between the AT proteins and/or metabolic pathways and the feed efficiency demonstrates that AT may reflect metabolic adaptations to high efficiency, and suggests that these proteins together with their metabolic mechanisms are suitable candidates as biomarkers to identify efficient cows for dairy production.

Effects of Environmental Heat Load on Endocannabinoid System Components in Adipose Tissue of High Yielding Dairy Cows.

Environmental heat load (HL) adversely affects the performance of dairy cows. The endocannabinoid system (ECS) regulates metabolism and the stress response, thus we hypothesized that HL may affect the ECS of dairy cows. Our objective was to determine the levels of endocannabinoids (eCBs) and gene and protein expressions of the ECS components in adipose tissue (AT) and plasma of early postpartum (PP) and late-lactation cows. In addition, we examined eCBs in milk, and studied the interaction of eCBs with bovine cannabinoids receptors CB1 and CB2. In the first experiment, plasma and AT were sampled from cows calving during summer (S, n = 9) or winter (W, n = 9). Dry matter intake (DMI) and energy balance (EB) were lower in S vs. W, and relative gene expressions of transient-receptor-potential-cation-channel-subfamily-V-member-1 (TRPV1), the cannabinoid receptors CNR1 (CB1) and CNR2 (CB2), and monoglyceride lipase (MGLL) were decreased in AT of S compared to W. Protein abundance of peroxisome proliferator-activated-receptor-alpha (PPAR-α) was decreased, while tumor-necrosis factor-α (TNF-α) was increased in AT of S vs. W. Other components of the ECS were not different between S and W calving cows. To study whether the degree of HL may affect the ECS, we performed a second experiment with 24 late-lactation cows that were either cooled (CL) or not cooled (heat-stressed; HS) during summer. DMI was lower in HS vs. CL, AT protein abundance of PPAR-α was lower, and TRPV1 tended to be lower in HS vs. CL, but other components of the ECS were not different between groups. Milk levels of 2-arachidonoylglycerol (2-AG) tended to increase in HS vs. CL. Additionally, modeling of the bovine cannabinoid receptors demonstrated their binding to anandamide and 2-AG. Environmental HL, possibly via lower intake, is associated with limited alterations in ECS components in AT of dairy cows.

Effects of omega-3 supplementation on components of the endocannabinoid system and metabolic and inflammatory responses in adipose and liver of peripartum dairy cows.

BACKGROUND: Dietary supplementation of omega-3 fatty acids can reduce the activation of the endocannabinoid system (ECS) by decreasing the availability of arachidonic acid, thus lowering endocannabinoids (eCBs) levels. The ECS is a modulator of energy metabolism, stress response and inflammation in mammals, yet there is little information on the roles of the ECS in transition dairy cows. During the periparturient period, the adipose tissue and liver are the main metabolic organs that participate in the adaptations of dairy cows to onset of lactation; however, exceeded adipose tissue lipolysis and accumulation of lipids in the liver have adverse effects on cows' physiology. Here we aimed to examine whether omega-3 supplementation during the transition period will modulate ECS activation and affect metabolic and inflammatory indices in postpartum dairy cows, by supplementing twenty-eight transition Holstein dairy cows with either saturated fat (CTL) or encapsulated flaxseed oil (FLX). Components of the ECS, metabolic and inflammatory markers were measured in blood, liver, and subcutaneous adipose tissue. RESULTS: FLX supplementation reduced feed intake by 8.1% (P < 0.01) and reduced plasma levels of arachidonic acid (by 44.2%; P = 0.02) and anandamide (by 49.7%; P = 0.03) postpartum compared to CTL. The mRNA transcription levels of the cannabinoid receptor 1 (CNR1/CB1) tended to be lower (2.5 folds) in white blood cells of FLX than in CTL (P = 0.10), and protein abundance of ECS enzyme monoacylglycerol lipase was higher in peripheral blood mononuclear cells of FLX than in CTL (P = 0.04). In adipose tissue, palmitoylethanolamide levels were lower in FLX than in CTL (by 61.5%; P = 0.02), relative mRNA transcription of lipogenic genes were higher, and the protein abundance of cannabinoid receptor 2 (P = 0.08) and monoacylglycerol lipase (P = 0.10) tended to be higher in FLX compared to CTL. Hepatic 2-arachidonoylglycerol tended to be higher (by 73.1%; P = 0.07), and interlukin-6 mRNA transcription level was 1.5 folds lower in liver of FLX than in CTL (P = 0.03). CONCLUSIONS: Nutritional supplementation of omega-3 fatty acids seems to partly modulate ECS activation, which could be related to lower feed intake. The altered ECS components in blood, adipose tissue and liver are associated with moderate modulations in lipid metabolism in the adipose and inflammation in liver of peripartum dairy cows.

Proteome dataset of peripheral blood mononuclear cells in postpartum dairy cows supplemented with different sources of omega-3 fatty acids.

This article contains raw and processed data related to research published by Kra et al. [1]. There is a scarce knowledge on the proteome of peripheral blood mononuclear cells (PBMC) during the transition period in dairy cows. In human research, proteomics PBMC is used in order to gain insight into inflammatory diseases and syndromes. Dietary fats, and specifically omega-3 (n-3) FA, can moderate the immune fluctuation caused by parturition through improvements of the immune function [2]. Therefore, this study aim was to characterize the changes that may occur in proteome of PBMC during transition, as influenced by different n-3 FA supplementation. Proteomics data of PBMC was obtained from postpartum dairy cows supplemented peripartum with either encapsulated saturated fat (CTL), encapsulated flaxseed oil that is enriched with ALA (α-linolenic acid; FLX) or encapsulated fish oil that is enriched with EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid; FO).The analysis was done by liquid chromatography-mass spectrometry from PBMCs protein extraction. The cells were collected from six cows per treatment during the 1st week postpartum. Quantification of differential abundance between groups was done using MS1 intensity based label-free. Label-free quantitative shotgun proteomics was used for characterization. This novel dataset of proteomics data from PBMC contains 3807 proteins; 44, 42 and 65 were differently abundant (P ≤ 0.05 and FC ± 1.5), in FLX vs. CTL, FO vs. CTL and FLX vs. FO, respectively; these findings are discussed in our recent research article (Kra et al., 2021). The present dataset of PBMC proteome adds new information regarding the effects of n-3 FA on the immune system, while providing reference for PBMC proteome in postpartum dairy cows.

Antioxidant Resveratrol Increases Lipolytic and Reduces Lipogenic Gene Expression under In Vitro Heat Stress Conditions in Dedifferentiated Adipocyte-Derived Progeny Cells from Dairy Cows.

Heat stress (HS) induces oxidative stress by increasing reactive oxygen species (ROS), and the polyphenol resveratrol (RSV) has been shown to have antioxidant properties by reducing ROS. Hence, we aimed to examine the effects of RSV, HS and their interaction on bovine adipocytes. We generated bovine dedifferentiated adipocyte-derived progeny (DFAT) cells from subcutaneous adipose tissue and examined the effects of RSV (100 µM), heat conditions: isothermal (ISO-37 °C), short heat (SH-41.2 °C for 1 h) and long HS (LH-41.2 °C for 16 h), and their interaction on gene expression in DFAT-cells. In medium of DFAT-cells treated with RSV, malondialdehyde levels were reduced and oxygen-radical absorbance-capacity levels were increased compared to control. Treating DFAT-cells with RSV increased the relative mRNA expression of stress-induced-phosphoprotein-1 (STIP1) and the expression of hormone-sensitive-lipase (LIPE) and perilipin-1 (PLIN1), whereas it reduced the expressions of fatty-acid-synthase (FASN) and of pro-inflammatory chemotactic-C-C-motif-chemokine-ligand-2 (CCL2) also under HS. Moreover, reduced protein abundance of FASN was found in RSV-treated DFAT-cells compared to controls. Molecular docking of RSV with FASN confirmed its possible binding to FASN active site. This work demonstrates that RSV has an antioxidant effect on bovine DFAT cells and may induce adipose lipolysis and reduce lipogenesis also under in vitro HS conditions.

A proposed modulatory role of the endocannabinoid system on adipose tissue metabolism and appetite in periparturient dairy cows.

To sustain the nutrient demands of rapid fetal growth, parturition, and milk synthesis, periparturient dairy cows mobilize adipose tissue fatty acid stores through lipolysis. This process induces an inflammatory response within AT that is resolved as lactation progresses; however, excessive and protracted lipolysis compounds the risk for metabolic and inflammatory diseases. The suppression of lipolytic action and inflammation, along with amplification of adipogenesis and lipogenesis, serve as prospective therapeutic targets for improving the health of periparturient dairy cows. Generally, the activation of cannabinoid receptors by endocannabinoids enhances adipogenesis and lipogenesis, suppresses lipolysis, and increases appetite in mammals. These biological effects of activating the endocannabinoid system open the possibility of harnessing the endocannabinoid system through nutritional intervention in dairy herds as a potential tool to improve dairy cows' health, although much is still to be revealed in this context. This review summarizes the current knowledge surrounding the components of the endocannabinoid system, elaborates on the metabolic effects of its activation, and explores the potential to modulate its activity in periparturient dairy cows.

Proteome dataset of liver from dairy cows experiencing negative or positive energy balance at early lactation.

This article contains raw and processed data related to research published by Swartz et al. [1]. We present proteomics data from liver of postpartum dairy cows that were obtained by liquid chromatography-mass spectrometry following protein extraction. Differential abundance between liver of cows experiencing either negative energy balance (NEB, n = 6) or positive energy balance (PEB, n = 4) at 17 ± 3 days in lactation was quantified using MS1 intensity based label-free. There is a paucity of studies examining the associations of NEB with the liver proteome in early lactation dairy cows. Therefore, our objective was to examine the differences in the liver proteome in periparturient dairy cows experiencing naturally occurring NEB compared to cows in PEB. In this study, multiparous Holstein dairy cows were milked either 2 or 3 times daily for the first 30 days in milk (DIM) to alter energy balance, and were classified retrospectively as NEB (n = 18) or PEB (n = 22). We collected liver biopsies from 10 cows (n = 5 from each milking frequency), that were retrospectively classified according to their energy balance (NEB, n = 6; PEB, n = 4). The liver proteome was characterized using label-free quantitative shotgun proteomics. This novel dataset contains 2,741 proteins were identified, and 68 of those were differentially abundant between NEB and PEB (P ≤ 0.05 and FC± 1.5); these findings are discussed in our recent research article [1]. The present dataset of liver proteome can be used as either biological markers for disease or therapeutic targets to improve metabolic adaptations to lactation in postpartum dairy cattle. Data are available via ProteomeXchange with identifier PXD028124.

Characterization of the liver proteome in dairy cows experiencing negative energy balance at early lactation.

Negative energy balance (NEB) is associated with metabolic disorders in early lactation dairy cows. Therefore, our objective was to characterize the liver proteome in cows experiencing either NEB or positive energy balance (PEB). Forty-two multiparous Holstein dairy cows were milked either 2 or 3 times daily for the first 30 days in milk (DIM) to alter EB, and were classified retrospectively as NEB (n = 18) or PEB (n = 22). Liver biopsies were collected from 10 cows (n = 5 from each milking frequency) at 17 ± 3 DIM (NEB, n = 6; PEB, n = 4). The liver proteome was characterized using label-free quantitative shotgun proteomics and Ingenuity Pathway Analysis used to identify key affected canonical pathways. Overall, 2741 proteins were identified, and 68 of those were differentially abundant (P ≤ 0.05 and FC ± 1.5). ENO3 (FC = 10.3, P < 0.01) and FABP5 (FC = -12.5, P = 0.045) were the most dramatically upregulated and downregulated proteins, respectively, in NEB cows. Numerous mitochondrial proteins (NDUFA5, NDUFS3, NDUFA6, COX7A2L, COX6C, and COA5) were differentially abundant. Canonical pathways associated with NEB were LPS/IL-1 mediated inhibition of RXR function, oxidative phosphorylation, and mitochondrial dysfunction. Additionally, cows experiencing NEB had less hepatic IL10 transcript abundance than PEB. Together, NEB was associated with altered hepatic inflammatory status, likely due to oxidative stress from mitochondrial dysfunction. SIGNIFICANCE: Our manuscript describes the associations of negative energy balance with the liver proteome in early lactation dairy cows, when metabolic stress and the incidence of diseases is increased. Specifically, we found associations of negative energy balance with shifts in hepatic protein abundance involved in fatty acid uptake, impaired anti-inflammatory responses, and mitochondrial dysfunction. Moving forward, differentially abundant proteins found in this study may be useful as either biological markers for disease or therapeutic targets to improve metabolic adaptations to lactation in postpartum dairy cattle.