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Title:Immunometabolism of methyl donors by dairy cattle during periods of negative nutrient balance and heat stress
Author(s):Coleman, Danielle N.
Director of Research:Loor, Juan J
Doctoral Committee Chair(s):Loor, Juan J
Doctoral Committee Member(s):Pan, Yuan-Xiang; Cardoso, Felipe; Drackley, James
Department / Program:Animal Sciences
Discipline:Animal Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Mammary Gland
Abstract:For dairy cows, the periparturient period and periods of heat stress (HS) are characterized by a negative nutrient balance (NNB), decreased milk production and altered immunometabolism. The first experiment examined the effects of post-ruminal choline supply during NNB on production and pathways of one-carbon metabolism. Ten primiparous rumen-cannulated cows (158 ± 24 DIM) were used in a replicated 5  5 Latin square design with 4 d treatment periods and 10 d of recovery. Treatments were unrestricted intake with abomasal infusion of water, restricted intake (R; 60% of net energy for lactation requirements) with abomasal infusion of water or R plus abomasal infusion of 6.25, 12.5, or 25 g/d choline ion. Liver tissue was collected at the end of each treatment period to measure the activity and mRNA abundance of enzymes and metabolite concentrations related to one-carbon metabolism. Abundance of proteins in mechanistic target of rapamycin (mTOR) signaling were also measured in liver tissue. Plasma was collected each d of the treatment period and used for plasma AA analysis. Concentrations of plasma biomarkers of inflammation, liver function and oxidative stress were measured on d 1, 3 and 5. Enhancing choline supply increased milk yields, but decreased liver triacylglycerol (TAG). The activity of betaine homocysteine methyltransferase increased with choline, while methionine synthase tended to increase, and cystathionine β-synthase was decreased. These changes were associated with increased liver and plasma Met. Changes in plasma biomarkers indicated an improvement in liver function, likely associated with improvements in hepatic TAG. Protein abundance of phosphorylated (p) EEF2 and EIF2A decreased with enhanced choline supply potentially helping to maintain or increase protein synthesis during NNB. Overall, the enhanced supply of choline during NNB increases flux through the Met cycle to regenerate Met and reduce liver TAG which is associated with an improvement in liver function. The second experiment examined the effects of rumen-protected Met (RPM) during HS on mTOR-related signaling proteins in the mammary gland. Thirty-two multiparous cows (184 ± 59 DIM) were assigned to an environmental treatment, and a dietary treatment [TMR with RPM (0.105% DM) or without (CON)] in a crossover design. There were 2 periods with 2 phases per period. In phase 1 (9 d), all cows were in thermoneutral conditions (TN) and fed ad libitum. During phase 2 (9 d), group 1 (n = 16) was exposed to HS using electric heat blankets while group 2 (n = 16) remained in TN but were pair-fed to HS counterparts. After a washout period (21 d), the study was repeated (period 2), with environmental treatments being inverted and dietary treatments remaining the same. Mammary tissue was collected at the end of phase 2 and protein was extracted for western blot analysis. An abundance of p-mTOR was greater with RPM and tended to be greater with HS. Control cows had a greater decrease in milk protein (%) during phase 2 (difference from phase 1) compared with RPM cows, suggesting that RPM supplementation during HS may support greater milk protein synthesis via mTOR activation. In the same experiment we investigated the effects of RPM during HS on the response of mammary gland explants to lipopolysaccharide (LPS). Twenty-five mg of tissue obtained from cows in the second experiment was incubated with 0 or 3 μg/mL of LPS for 2 h and then used to measure mRNA abundance of genes associated with immune and antioxidant responses and inflammation. Incubation with LPS increased the abundance of genes associated with inflammation, while HS decreased genes associated with antioxidant responses. Expression of NFKB1was greater in LPS-treated explants from non-HS compared with HS cows. These data indicate that HS reduced immune and antioxidant responses while RPM did not attenuate the inflammatory response induced by LPS in vitro. Lastly, as part of the HS experiment the effects of RPM supplementation during HS on mTOR and antioxidant signaling in liver explants was investigated. Fifty mg of liver tissue was obtained from cows at the end of each period 2 and incubated for 2 h at the lab and then protein was extracted for western blot analysis. Heat stress increased the abundance of cullin 3, an inhibitor of nuclear factor erythroid 2 like 2, was downregulated by HS, suggesting a decrease in antioxidant responses with HS. The ratio phosphorylated (p):total mTOR tended to be greater with RPM supplementation, suggesting an increase in mTOR activity. Heat stress also increased abundance of p-AKT and solute carrier family 2 member A4, suggesting an upregulation in insulin signaling, but this increase was not observed in explants from RPM supplemented cows. These data suggest that RPM supplementation may help promote mTOR signaling and maintain homeostasis in hepatic insulin signaling during an HS challenge. Overall, data indicated a beneficial effect of choline during NNB and Met during HS on immunometabolism in dairy cows.
Issue Date:2021-01-05
Rights Information:Copyright 2021 Danielle N. Coleman
Date Available in IDEALS:2021-09-17
Date Deposited:2021-05

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