Methyl donor supply to heat stress-challenged polymorphonuclear leukocytes from lactating Holstein cows enhances 1-carbon metabolism, immune response, and cytoprotective gene network abundance

[EN] Mechanisms controlling immune function of dairy cows are dysregulated during heat stress (HS). Methyl donor supply-methionine (Met) and choline (Chop-positively modulates innate immune function, particularly antioxidant systems of polymorphonuclear leukocytes (PMN). The objective of this study was to investigate the effect of Met and Chol supply in vitro on mRNA abundance of genes related to 1-carbon metabolism, inflammation, and immune function in short-term cultures of PMN isolated from mid-lactating Holstein cows in response to heat challenge. Blood PMN were isolated from 5 Holstein cows (153 +/- 5 d postpartum, 34.63 +/- 2.73 kg/d of milk production; mean +/- SD). The PMN were incubated for 2 h at thermal-neutral (37 degrees C; TN) or heat stress (42 degrees C; HS) temperatures with 3 levels of Chol (0, 400, or 800 mu g/mL) or 3 ratios of Lys:Met (Met; 3.6:1, 2.9:1, or 2.4:1). Supernatant concentrations of IL-1 beta, IL-6, and tumor necrosis factor-alpha were measured via bovine-specific ELISA. Fold-changes in mRNA abundance were calculated separately for Chol and Met treatments to obtain the fold-change response at 42 degrees C (HS) relative to 37 degrees C (TN). Data were subjected to ANOVA using PROC MIXED in SAS (SAS Institute Inc., Cary, NC). Orthogonal contrasts were used to determine the linear or quadratic effect of Met and Chol for mRNA fold-change and supernatant cytokine concentrations. Compared with PMN receiving 0 mu g of Chol/mL, heat-stressed PMN supplemented with Chol at 400 or 800 mu g/mL had greater fold-change in abundance of CBS, CSAD, GSS, GSR, and GPX1. Among genes associated with inflammation and immune function, fold-change in abundance of TLR2, TLR4, IRAK1, IL1B, and IL10 increased with 400 and 800 mu g of Chol/mL compared with PMN receiving 0 mu g of Chol/mL. Fold-change in abundance of SAHH decreased linearly at increasing levels of Met supply. A linear effect was detected for MPO, NFKB1, and SOD1 due to greater fold-change in abundance when Met was increased to reach Lys: Met ratios of 2.9:1 and 2.4:1. Although increasing Chol supply upregulated BAX, BCL2, and HSP70, increased Met supply only upregulated BAX. Under HS conditions, enhancing PMN supply of Chol to 400 mu g/mL effectively increased fold-change in abundance of genes involved in antioxidant production (conferring cellular processes protection from free radicals and reactive oxygen species), inflammatory signaling, and innate immunity. Although similar outcomes were obtained with Met supply at Lys:Met ratios of 2.9:1 and 2.4:1, the response was less pronounced. Both Chol and Met supply enhanced the cytoprotective characteristics of PMN through upregulation of heat shock proteins. Overall, the modulatory effects detected in the present experiment highlight an opportunity to use Met and particularly Chol supplementation during thermal stress.M. Vailati-Riboni was supported in part by Hatch funds under project ILLU-538-914, National Institute of Food and Agriculture (Washington, DC). The authors declare no conflict of interest.Lopreiato, V.; Vailati-Riboni, M.; Parys, C.; Fernández Martínez, CJ.; Minuti, A.; Loor, J. (2020). Methyl donor supply to heat stress-challenged polymorphonuclear leukocytes from lactating Holstein cows enhances 1-carbon metabolism, immune response, and cytoprotective gene network abundance. Journal of Dairy Science. 103(11):10477-10493. https://doi.org/10.3168/jds.2020-18638S104771049310311Abdelmegeid, M. K., Vailati-Riboni, M., Alharthi, A., Batistel, F., & Loor, J. J. (2017). Supplemental methionine, choline, or taurine alter in vitro gene network expression of polymorphonuclear leukocytes from neonatal Holstein calves. 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Can lipid supplementation modulate inflammatory state and immune response in periparturient goats? A case study on hepatic and adipose miRNA expression

As a new perspective in controlling cellular pathways, the aim of the trial was to investigate the expression of miRNA implicated in adipogenesis and metabolic and endocrine functions in periparturient dairy goat fed saturated or unsaturated fatty acids. Hepatic and adipose tissue samples were obtained from twenty-three second-parity twins-diagnosed alpine dairy goats were fed either calcium stearate (ST, n:7), fish oil (FO, n:8) or a control diet without any fat supplement (C, n:8). Dietary treatments lasted from one week before (30g/head/d of fatty acids) to three weeks after kidding (50g/head/d of fatty acids). ST provided 26% C16:0 and 69.4% C18:0 while FO provided 10.4% EPA and 7.8% DHA. The expression of miR-26b and 155 (adipose infiltration of immune cells), miR-99a, 145 and 221 (inflammation and lipolysis) and miR-143 and 378 (pro-adipogenic function) was performed by RT-PCR on hepatic and adipose biopsies collected on day -7 and 7 and 21 from kidding. Data were statistically analyzed by MIXED and GLM procedures of SAS. No diet effect was found for all the miRNA considered, but a significant effect of time for miR-155 (P= 0.028) and a tendency for miR-221 (P=0.083) were found with increased values from -7 to +21d. In particular, significant higher miR-155 expression (P<0.01) from -7 to 7d (-0.17 and 0.27, respectively) proved an inflammatory status in the first week after kidding in all the groups. Obtained results for both miRNA-155 and miRNA-221 over the time copes with our previous findings on metabolic, productive parameters and mRNA expression of genes related to lipid metabolism and inflammatory response (Farina et al., 2016). These outcomes consolidate the hypothesis that lipogenesis takes place in dairy goat in the two weeks around calving, but it is reduced when compared to dairy cow (Khan et al., 2013)

Molecular networks of insulin signaling and amino acid metabolism in subcutaneous adipose tissue are altered by body condition in periparturient Holstein cows

[EN] Peripartal cows mobilize not only body fat but also body protein to satisfy their energy requirements. The objective of this study was to determine the effect of prepartum BCS on blood biomarkers related to energy and nitrogen metabolism, and mRNA and protein abundance associated with AA metabolism and insulin signaling in subcutaneous adipose tissue (SAT) in peripartal cows. Twenty-two multiparous Holstein cows were retrospectively classified into a high BCS (HBCS; n = 11, BCS >= 3.5) or normal BCS (NBCS; n = 11, BCS <= 3.17) group at d 28 before expected parturition. Cows were fed the same diet as a total mixed ration before parturition and were fed the same lactation diet postpartum. Blood samples collected at -10, 7, 15, and 30 d relative to parturition were used for analyses of biomarkers associated with energy and nitrogen metabolism. Biopsies of SAT harvested at -15, 7, and 30 d relative to parturition were used for mRNA (real timePCR) and protein abundance (Western blotting) assays. Data were subjected to ANOVA using the MIXED procedure of SAS (v. 9.4; SAS institute Inc., Cary, NC), with P <= 0.05 being the threshold for significance. Cows in HBCS had greater overall plasma nonesterified fatty acid concentrations, due to marked increases at 7 and 15 d postpartum. This response was similar (BCS x Day effect) to protein abundance of phosphorylated (p) protein kinase B (p-AKT), the insulin-induced glucose transporter (SLC2A4), and the sodium-coupled neutral AA transporter (SLC38A1). Abundance of these proteins was lower at -15 d compared with NBCS cows, and either increased (SLC2A4, SLC38A1) or did not change (p-AKT) at 7 d postpartum in IIBCS. Unlike protein abundance, however, overall mRNA abundances of the high-affinity cationic (SLC7A1), proton-coupled (SLC96A1), and sodium-coupled amino acid transporters (SLC,98,42) were greater in IIBCS than NBCS cows, due to upregulation in the postpartum phase. Those responses were similar to protein abundance of p-mTOR, which increased (BCS x Day effect) at 7 d in HBCS compared with NBCS cows. mRNA abundance of argininosuccinate lyase (ASL) and arginase 1 (ARG1) also was greater overall in HBCS cows. Together, these responses suggested impaired insulin signaling, coupled with greater postpartum AA transport rate and urea cycle activity in SAT of HBCS cows. An in vitro study using adipocyte and macrophage cocultures stimulated with various concentrations of fatty acids could provide some insights into the role of immune cells in modulating adipose tissue immunometabolic status, including insulin resistance and AA metabolism.Y. Liang is a recipient of a doctoral fellowship from the China Scholarship Council (CSC, Beijing, China) to perform his PhD studies at the University of Illinois (Urbana). A. S. Alharthi received a fellowship from King Saud University (Riyadh, Saudi Arabia) to perform his PhD studies at the University of Illinois (Urbana). A. A. Elolimy was recipient of a fellowship from the Higher Education Ministry (Cairo, Egypt) to perform his PhD studies at the University of Illinois (Urbana). We thank Perdue AgriBusiness (Salisbury, MD) for the donation of ProvAAL2 AADvantage during the course of the experiment. The authors declare no conflicts of interest.Liang, Y.; Alharthi, A.; Elolimy, A.; Bucktrout, R.; Lopreiato, V.; Cortes, I.; Xu, C.... (2020). Molecular networks of insulin signaling and amino acid metabolism in subcutaneous adipose tissue are altered by body condition in periparturient Holstein cows. 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Transcriptional regulation of lipid metabolism and inflammation in transition dairy goats by fish oil and stearate

To better understand the interaction between saturated or unsaturated fatty acids and its effect on expression of genes involved in subcutaneous adipose tissue metabolism, 23 second parity alpine dairy goats were enrolled in the experiment and fed either a non fat-supplemented basal diet (C; n=8), the basal diet supplemented with stearic acid (ST; n=7) or the basal diet supplemented with fish oil (FO; n=8). 30g/head/d supplemental fatty acids during the dry period and 50g/head/d during lactation were delivered starting one week before parturition up to 21 days in milk. Subcutaneous adipose tissue samples were harvested at day -7, 7 and 21 relative to kidding and mRNA levels of genes involved in inflammation were measured via qPCR. Data were analyzed using the MIXED procedure of SAS. No significant effects for treatment were observed, however eight genes were significant for time. HP and SAA3 expression peaked at day 7 postpartum, to then return at prepartum level around 21 d relative to kidding, while IL8, IL10, and IL18 expression constantly increased along the transition period. Vice versa, expression of IL1\u3b2, IL6R, and RXRA decreased in response to kidding, with a subsequent increase at day 21. The obtained results led us to hypothesize that goats face a postponed lipomobilization after kidding, probably related to their reduced production. The next step will involve the analysis of miRNA related to immune cell infiltration, adipocyte inflammation and lipolysis and positive regulation of adipogenesis to better understand the complex network of lipid metabolism in periparturient goats

Body condition alters glutathione and nuclear factor erythroid 2-like 2 (NFE2L2)-related antioxidant network abundance in subcutaneous adipose tissue of periparturient Holstein cows

[EN] Dairy cows with high body condition score (BCS) in late prepartum are more susceptible to oxidative stress (OS). Nuclear factor erythroid 2-like 2 (NFE2L2) is a major antioxidant transcription factor. We investigated the effect of precalving BCS on blood biomarkers associated with OS, inflammation, and liver function, along with mRNA and protein abundance of targets related to NFE2L2 and glutathione (GSH) metabolism in s.c. adipose tissue (SAT) of periparturient dairy cows. Twenty-two multiparous Holstein cows were retrospectively classified into a high BCS (HBCS; n = 11, BCS ¿3.5) or normal BCS (NBCS; n = 11, BCS ¿3.17) on d 28 before parturition. Cows were fed a corn silage- and wheat straw-based total mixed ration during late prepartum, and a corn silage- and alfalfa hay-based total mixed ration postpartum. Blood samples obtained at ¿10, 7, 15, and 30 d relative to parturition were used for analyses of biomarkers associated with inflammation, including albumin, ceruloplasmin, haptoglobin, and myeloperoxidase, as well as OS, including ferric reducing ability of plasma (FRAP), reactive oxygen species (ROS), and ß-carotene. Adipose biopsies harvested at ¿15, 7, and 30 d relative to parturition were analyzed for mRNA (real-time quantitative PCR) and protein abundance (Western blotting) of targets associated with the antioxidant transcription regulator nuclear factor, NFE2L2, and GSH metabolism pathway. In addition, concentrations of GSH, ROS and malondialdehyde were measured. High BCS cows had lower prepartum dry matter intake expressed as a percentage of body weight along with greater BCS loss between ¿4 and 4 wk relative to parturition. Plasma concentrations of ROS and FRAP increased after parturition regardless of treatment. Compared with NBCS, HBCS cows had greater concentrations of FRAP at d 7 postpartum, which coincided with peak values in those cows. In addition, NBCS cows experienced a marked decrease in plasma ROS after d 7 postpartum, while HBCS cows maintained a constant concentration by d 30 postpartum. Overall, ROS concentrations in SAT were greater in HBCS cows. However, overall mRNA abundance of NFE2L2 was lower and cullin 3 (CUL3), a negative regulator of NFE2L2, was greater in HBCS cows. Although HBCS cows had greater overall total protein abundance of NFE2L2 in SAT, ratio of phosphorylated NFE2L2 to total NFE2L2 was lower, suggesting a decrease in the activity of this antioxidant system. Overall, mRNA abundance of the GSH metabolism-related genes glutathione reductase (GSR), glutathione peroxidase 1 (GPX1), and transaldolase 1 (TALDO1), along with protein abundance of glutathione S-transferase mu 1 (GSTM1), were greater in HBCS cows. Data suggest that HBCS cows might experience greater systemic OS after parturition, while increased abundance of mRNA and protein components of the GSH metabolism pathway in SAT might help alleviate tissue oxidant status. Data underscored the importance of antioxidant mechanisms at the tissue level. Thus, targeting these pathways in SAT during the periparturient period via nutrition might help control tissue remodeling while allowing optimal performance.Y. Liang is a recipient of a doctoral fellowship from China Scholarship Council (CSC, Beijing, China). A. S. Alharthi received a fellowship from King Saud University to perform his PhD studies at the University of Illinois (Urbana). A. A. Elolimy was recipient of a fellowship from Higher Education Ministry, Egypt to perform his Ph.D. studies at the University of Illinois (Urbana). We thank Perdue AgriBusiness (Salisbury, MD) for the donation of ProvAAL2 AADvantage during the course of the experiment. The authors have not stated any conflicts of interest.Liang, Y.; Alharthi, A.; Bucktrout, R.; Elolimy, A.; Lopreiato, V.; Martinez-Cortes, I.; Xu, C.... (2020). Body condition alters glutathione and nuclear factor erythroid 2-like 2 (NFE2L2)-related antioxidant network abundance in subcutaneous adipose tissue of periparturient Holstein cows. Journal of Dairy Science. 103(7):6439-6453. https://doi.org/10.3168/jds.2019-17813S643964531037Alharthi, A., Zhou, Z., Lopreiato, V., Trevisi, E., & Loor, J. J. (2018). Body condition score prior to parturition is associated with plasma and adipose tissue biomarkers of lipid metabolism and inflammation in Holstein cows. Journal of Animal Science and Biotechnology, 9(1). doi:10.1186/s40104-017-0221-1Aquilano, K., Baldelli, S., & Ciriolo, M. R. (2014). Glutathione: new roles in redox signaling for an old antioxidant. 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Hepatic and subcutaneous adipose lipid metabolism genes modulation by dietary fish oil and stearate in transition goats

The objective of the experiment was to understand the interaction between saturated or unsaturated fatty acids and genes involved in lipid metabolism in liver and subcutaneous adipose tissue. With this purpose, further gene expression assays were performed on obtained adipose and liver samples from a previous in vivo study where expression levels of ADIPOQ, LPIN1, LPL, PPARG, SREBF1 and THRSP were already determined. The study consisted on the administration of either a no fat-supplemented, or a stearic acid or fish oil supplemented diets to dairy goats from the last week of gestation until 21 days after kidding. Fat-supplied goats received 30g/head/d extra fatty acids during the dry period and 50g/head/d during lactation. Liver and subcutaneous adipose tissue samples were harvested at day 7, 7 and 21 relative to kidding and immediately snap frozen in liquid nitrogen. At the present moment, quantitative real-time RT-PCR of ACAT1, MSMO1, CPT1, IL6 on liver and ACACA, LEP, LPL, FASN, IL6 and PLIN2 on adipose tissue are running. Data obtained will be analysed using the MIXED procedure of SAS and results may increase the knowledge on the mechanism of action of saturated or unsaturated dietary fatty acid sources in the fatty acid metabolism changes during transition in dairy goats

Development of a dynamic energy-partitioning model for enteric methane emissions and milk production in goats using energy balance data from indirect calorimetry studies

[EN] The main objective of this study was to develop a dynamic energy balance model for dairy goats to describe and quantify energy partitioning between energy used for work (milk) and that lost to the environment. Increasing worldwide concerns regarding livestock contribution to global warming underscore the importance of improving energy efficiency utilization in dairy goats by reducing energy losses in feces, urine and methane (CH4). A dynamic model of CH(4)emissions from experimental energy balance data in goats is proposed and parameterized (n= 48 individual animal observations). The model includes DM intake, NDF and lipid content of the diet as explanatory variables for CH(4)emissions. An additional data set (n= 122 individual animals) from eight energy balance experiments was used to evaluate the model. The model adequately (root MS prediction error,RMSPE) represented energy in milk (E-milk;RMSPE = 5.6%), heat production (HP;RMSPE = 4.3%) and CH(4)emissions (E-CH4; RMSPE = 11.9%). Residual analysis indicated that most of the prediction errors were due to unexplained variations with small mean and slope bias. Some mean bias was detected for HP (1.12%) and E-CH4(1.27%) but was around zero for E-milk (0.14%). The slope bias was zero for HP (0.01%) and close to zero for E-milk (0.10%) and E-CH4(0.22%). Random bias was >98% for E-CH4, HP and E-milk, indicating non-systematic errors and that mechanisms in the model are properly represented. As predicted energy increased, the model tended to underpredict E-CH(4)and E-milk. The model is a first step toward a mechanistic description of nutrient use by goats and is useful as a research tool for investigating energy partitioning during lactation. 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