Source: UNIVERSITY OF ILLINOIS submitted to NRP
METABOLIC RELATIONSHIPS IN SUPPLY OF NUTRIENTS FOR LACTATING COWS (NC-1009)
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1002047
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
NC-_old2040
Project Start Date
Nov 21, 2013
Project End Date
Sep 30, 2018
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
Animal Sciences
Non Technical Summary
Research objectives in our lab include understanding the biochemical and physiological adaptations of metabolism in major tissues such as liver, adipose, and mammary during the dry period (nonlactating period of late pregnancy; generally 6 to 8 weeks before expected calving) through the first 3 weeks after calving (i.e. the 'transition period'). Nutritional management during the dry period can alter the amount of fat deposited in the animal, which upon calving can lead to excessive mobilization of fatty acids and contribute to deposition of fat in liver. This deposition of fat compromises liver function and contributes to metabolic disorders (such as ketosis) and infectious diseases that cows experience during the transition period. During the transition period typically 40 to 50% of all cows in commercial dairies suffer from one or more health disorders. These are highly interrelated and a major contributing factor is likely some compromise of liver function due to fat infiltration, and also a weakened immune system. Management practices such as dry period nutrition and pharmaceutical approaches that would minimize fat accumulation in the liver should improve animal well-being and profitability of dairy enterprises. Another important goal in our research program is to identify mechanisms regulating milk fat and protein synthesis in the mammary gland, and particularly the role of long-chain fatty acids and essential amino acids. As an additional goal, we will evaluate the transcriptome and microbiome associated with healthy and clinically mastitic mammary gland tissue in lactating goats. The use of cutting-edge technologies that allow an examination of large numbers of genes, proteins, and metabolites simultaneously offer a tremendous advantage in terms of generating new knowledge in a more holistic fashion, i.e. physiological functions in the animal are not regulated by single genes or proteins but by the coordinated interaction among many genes, proteins, and metabolites. The projects outlined will generate an integrated view of the multiple adaptations of the cow to nutritional management and other environmental factors. Such knowledge would be helpful in identifying targets that can be manipulated via nutrition or other means to decrease the risk of disease during the transition period. That will be beneficial in terms of enhancing the efficiency of milk production in farms.
Animal Health Component
(N/A)
Research Effort Categories
Basic
75%
Applied
(N/A)
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3023410101025%
3043410104075%
Knowledge Area
302 - Nutrient Utilization in Animals; 304 - Animal Genome;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1040 - Molecular biology; 1010 - Nutrition and metabolism;
Goals / Objectives
To quantify supply, availability, and interaction of nutrients and bioactive compounds utilized for efficient milk production while reducing environmental impact. To identify and quantify molecular, cellular, and organismal signals that regulate partitioning and efficient conversion of nutrients to milk.
Project Methods
In the first project designed to study molecular regulation of insulin sensitivity, dry non-pregnant Holstein cows were randomly assigned to treatments in a randomized block design based on body weight. Cows were assigned to treatments for a period of 64 days. During the first 21 days all cows received the same control diet. Between day 22 to 60 half of the cows were assigned to another diet and half of the cows continued on the control. Between day 36 and 49 all cows received a daily injection of the insulin-sensitizing drug Thiazolidinedione (TZD). Thus, they received the drug treatment for up to 14 days. The last 14 days (50 to 64 days) served as the 'washout' period. Treatments were as follows: 1) A ration containing low energy and high fiber (high-straw) fed ad libitum for 64 days; 2) Treatment 1 fed for 21 days followed by a moderate-energy ration fed ad libitum from day 22 to 64; 3) Same as treatment 1 but injected with 4 mg/kg BW TZD for days 36 to 49; and 4) Same as treatment 2 but injected with 4 mg/kg BW TZD for days 36 to 49. Cow dry matter intake was determined daily throughout the entire study. Body weights were obtained twice weekly, and body condition scores assigned throughout the study. Blood (20 to 50 mL) was sampled by puncture of a tail vein twice weekly during days 0 to 21, thrice weekly during days 22 to 35, four times per week during days 36 to 49, and thrice weekly from days 50 to 64. Samples will be used for determination of non-esterified fatty acids (NEFA), hydroxybutyrate (BHBA), glucose, insulin, urea, creatinine, aspartate aminotransferase (GGT; index of liver damage), gamma-glutamyl transferase (GOT; index of liver damage), haptoglobin, ceruloplasmin, albumin, total cholesterol, total bilirubin, serum Amyloid A (SAA), paraoxonase, metabolites of nitric oxide (nitrates and nitrites), vitamin A (i.e., retinol binding protein as index of negative acute phase proteins), reactive oxygen metabolites, vitamin E, beta-carotene, myeloperoxydases, and total antioxidants. Liver, adipose, and hind-leg muscle tissue from each cow was biopsied at d 35 (before TZD administration), 42, 49, and 56 +/-2 days. Adipose biopsies will be used to study transcriptome adaptations to treatments and protein expression of PPARgamma. Liver and muscle biopsies will be used for composition (lipid) and transcriptome analyses. Gene expression data will be analyzed and interpreted through bioinformatics analysis. Blood and dry matter intake data will be evaluated in the context of the molecular data. A second objective of this project involves the evaluation of long-term effects of feeding to meet or exceed dietary energy requirements on tissue mass at slaughter. As part of that project, we will determine gene and protein expression in mesenteric and subcutaneous fat as well as hypothalamus. A third objective of this project involves the in vitro challenge of subcutaneous and mesenteric adipose tissue with cytokines or insulin to determine the responsiveness of the tissues to the challenges. Data generated will include mRNA expression and protein expression of target molecules. Additional studies with focus on liver will involve transcriptome, metabolome, and bioinformatics analyses in response to various environmental challenges including heat stress and postpartal ketosis. We will perform in vitro studies with MacT cells or primary goat mammary cells to evaluate molecular pathways regulating milk fat and milk protein synthesis in response to long-chain fatty acids and essential amino acids. We will rely on gene expression analysis and target protein expression analysis. Lastly, we will use high-througput sequencing technology to evaluate the microbiome and transcriptome in mammary gland of goats with and without clinical mastitis. These data will be analyzed using our bioinformatics approaches to uncover unique patterns of microbes associated with healthy and disease tissue as well as the underlying molecular pathways.

Progress 11/21/13 to 09/30/18

Outputs
Target Audience:Fellow investigators and nutrition and veterinary professionals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students and other scholars had the opportunity to train on cutting-edge techniques. How have the results been disseminated to communities of interest?Through the publication of peer-reviewed manuscripts. What do you plan to do during the next reporting period to accomplish the goals?Continue pursuing additional avenues of inquiry for enhancing our understanding based on the results obtained.

Impacts
What was accomplished under these goals? Enhancing the post-ruminal supply of Methionine to obtain a Lys-to-Met ratio of 2.8:1 in the metabolizable protein during the periparturient period and early lactation is an effective approach to help mitigate oxidative stress and inflammation as well as enhance liver and neutrophil function in dairy cows. Data indicated that cows with body condition score (BCS) of 3.25 or lower before calving experienced greater alterations in systemic inflammation and basal lipolysis without excessive increases in free fatty acid (NEFA) plasma concentrations. These physiologic alterations could be related to incidence of disorders after parturition. Shifts in ruminal bacteria poulations and digestive enzyme activities during the peripartal period could, at least in part, be part of the mechanism associated with better feed efficiency in dairy cows. In vitro data indicate that an increase in Valine supply at an ideal ratio of Lys:Met could further enhance milk protein synthesis by altering intracellular concentrations of essential AA and metabolites that could play a regulatory role, increasing phosphorylation status of mTORC1 and key signaling proteins, and upregulation of AA transporters. Phosphorylation of the oxidative stress transcription regulator NFE2L2 and downstream signaling leading to postpartal upregulation of genes associated with oxidative stress and inflammation in the mammary gland seem to be key components of normal cellular function to maintain proper redox homeostasis. However, if the longitudinal increases in mRNA and protein abundance of these antioxidant mechanisms are a reflection of cellular oxidative stress, then the likelihood of protein and DNA damage would be greater and might be one factor compromising cell viability and potentially lactation persistency. In vivo data revealed the potential for maternal supply of Met during late pregnancy through either greater dry matter intake or Methionine itself to elicit some changes in blood neutrophil function during early postnatal life, partly through changes in mRNA expression encompassing cell adhesion and chemotaxis, oxidative stress, Toll-like receptor signaling, and Met metabolism.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Dong, X., Zhou, Z., Saremi, B., Helmbrecht, A., Wang, Z. and Loor, J.J. 2018. Varying the ratio of Lys:Met while maintaining the ratios of Thr:Phe, Lys:Thr, Lys:His, and Lys:Val alters mammary cellular metabolites, mammalian target of rapamycin signaling, and gene transcription. J. Dairy Sci. 2018 Feb;101(2):1708-1718. doi: 10.3168/jds.2017-13351. Epub 2017 Dec 14.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Abdelmegeid, M.K., Elolimy, A.A., Zhou, Z., Lopreiato, V., McCann, J.C. and Loor, J.J. 2018. Rumen-protected methionine during the peripartal period in dairy cows and its effects on abundance of major species of ruminal bacteria. J. Animal Sciences and Biotechnology. Feb 7;9:17. doi: 10.1186/s40104-018-0230-8. eCollection 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Dong, X., Zhou, Z., Wang, L., Saremi, B., Helmbrecht, A., Wang, Z., Loor, J.J. 2018. Increasing the availability of threonine, isoleucine, valine, and leucine relative to lysine while maintaining an ideal ratio of lysine:methionine alters mammary cellular metabolites, mammalian target of rapamycin signaling, and gene transcription. J. Dairy Sci. 2018 Jun;101(6):5502-5514. doi: 10.3168/jds.2017-13707. Epub 2018 Mar 15.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Batistel, F., Arroyo, J.M., Garces, C.I.M., Trevisi, E., Parys, C., Ballou, M.A., Cardoso, F.C. and Loor, J.J. 2018. Ethyl-cellulose rumen-protected methionine alleviates inflammation and oxidative stress and improves neutrophil function during the periparturient period and early lactation in Holstein dairy cows. J. Dairy Sci. 2018 Jan;101(1):480-490. doi: 10.3168/jds.2017-13185.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Alharthi, A., Zhou, Z., Lopreiato, V., Trevisi, E. and 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. J. Anim Sci Biotechnol. 2018 Jan 15;9:12. doi: 10.1186/s40104-017-0221-1. eCollection 2018.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Ma, Y.F., Wu, Z.H., Gao, M. and Loor, J.J. 2018. Nuclear factor erythroid 2-related factor 2 antioxidant response element pathways protect bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro. J. Dairy Sci. 2018 Jun;101(6):5329-5344. doi: 10.3168/jds.2017-14128. Epub 2018 Mar 21.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Ma, Y.F., Wu, Z.H., Gao, M. and Loor, J.J. 2018. Nuclear factor erythroid 2-related factor 2-antioxidant activation through the action of ataxia telangiectasia-mutated serine/threonine kinase is essential to counteract oxidative stress in bovine mammary epithelial cells. J. Dairy Sci. 2018 Jun;101(6):5317-5328. doi: 10.3168/jds.2017-13954. Epub 2018 Mar 28.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Han, L.Q., Zhou, Z., Ma, Y., Batistel, F., Osorio, J.S. and Loor, J.J. 2018. Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks. J. Dairy Sci. 2018 Jul;101(7):6511-6522. doi: 10.3168/jds.2017-14257. Epub 2018 Apr 19.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Han, L., Batistel, F., Ma, Y., Alharthi, A.S.M., Parys, C. and Loor, J.J. 2018. Methionine supply alters mammary gland antioxidant gene networks via phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) protein in dairy cows during the periparturient period. J. Dairy Science. Sep;101(9):8505-8512. doi: 10.3168/jds.2017-14206. Epub 2018 Jun 13.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Xu, T., Alharthi, A.S.M., Batistel, F., Helmbrecht, A., Parys, C., Trevisi, E., Shen, X. and Loor, J.J. 2018. Hepatic phosphorylation status of serine/threonine kinase 1, mammalian target of rapamycin signaling proteins, and growth rate in Holstein heifer calves in response to maternal supply of methionine. J. Dairy Sci. 2018 Sep;101(9):8476-8491. doi: 10.3168/jds.2018-14378. Epub 2018 Jun 13.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Jacometo, C.B., Alharthi, A.S., Zhou, Z., Luchini, D. and Loor, J.J. 2018. Maternal supply of methionine during late pregnancy is associated with changes in immune function and abundance of microRNA and mRNA in Holstein calf polymorphonuclear leukocytes. J. Dairy Sci. 2018 Sep;101(9):8146-8158. doi: 10.3168/jds.2018-14428. Epub 2018 Jun 13.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Members of the target audience included fellow investigators and nutrition and veterinary professionals. Changes/Problems:We are in the process of analyzing neonatal calf liver RNA sequencing data from animals born to cows receiving control or enhanced methionine supply during the last thirty days of pregnancy. We are also in the process of running RNA sequencing of ovine hypothalamus tissue from animals fed increasing levels of calcium propionate. Those data will be used to evaluate the control of feed intake. What opportunities for training and professional development has the project provided?The PI spent approximately 10% of his time working on the data interpretation and drafting the manuscripts in conjunctionwith three PhD students. The students were trained in several molecular techniques that allowed them to generate data.These graduate students had the opportunity to learn all aspects of scientific writing. How have the results been disseminated to communities of interest?Peer-reviewed manuscripts were published. What do you plan to do during the next reporting period to accomplish the goals?We will continue working with in vivo and in vitro systems to better understand the signalling pathways thatcoordinate adaptations to lactation, help regulate milk fat and protein synthesis, and also neutrophil function. Additional emphasis will be placed on the relationship between the dairy calf gut microbiome and efficiency of nutrient utilization as defined by residual feed intake (RFI).

Impacts
What was accomplished under these goals? Under Goal Two: During the peripartal period, proper acclimatization of rumen microorganisms to variations in nutritional management can facilitate the transition into lactation.Using samples collected from Holstein cows on days -14, -7, 10, 20, and 28 relative to parturitionwe observed strikingly lower inter-animal variations in the composition of the ruminal microbiota. GeneraRuminococcusandButyrivibrio, which are considered major fibrolytic rumen dwellers, were overrepresented in the prepartal rumen ecosystem. In contrast, increased postpartal voluntary DMI was associated with enrichment of bacterial genera mainly consisting of proteolytic, amylolytic, and lactate-producer species (includingPrevotella, Streptococcus, andLactobacillus). These, together with the postpartal enrichment of energy metabolism pathways, suggested a degree of acclimatization of the ruminal microbiota to harvest energy from the carbohydrate-dense lactation diet. In addition, correlations between ruminal microbiota and parameters such as milk yield and milk composition underscored the metabolic contribution of this microbial community to the cow's performance and production. Five neonatal Holstein calves (threeweeksold) were used for PMNL isolation and in vitro culture. The selected genes were related to the 1-carbon and Met cycles, cell signaling and cytokine mediators, inflammation, antimicrobial and killing mechanism associated genes, immune mediators, adhesion, and pathogen recognition. The results indicated that supplementation of Met, choline, and taurine increased homocysteine synthesis through upregulation of SAHH. Furthermore, the lower expression of CXCR1, IL10, IL6, IRAK1, NFKB1, NR3C1, SELL, TLR4, and TNFA indicated that all treatments mitigated the inflammatory activation of blood PMNL. As indicated by the modulation of GCLC and GPX1, choline and taurine supplementation also affected the antioxidant system. However, data indicate that oversupplementation could alter the inflammatory and oxidative status, suggesting the existence of cytotoxicity thresholds. Overall, multiple biological processes in calf PMNL related to inflammatory response and cytoprotection against oxidative stress were affected by Met, choline, and taurine supplementation. These data underscore an important role of these compounds in pre-weaning calf nutritional management. Long-term feeding of high-grain diets to dairy cows often results in systemic inflammation characterized by alterations in acute-phase proteins and other biomarkers, both in plasma and immune-responsive tissues like the liver. The molecular and systemic changes that characterize an acute grain feeding challenge remain unclear. The current study involved sixHolstein and sixJersey cows in a replicated two× twoLatin square. Periods (10 days) were divided into fourstages (S): S1, days 1 to 3, served as baseline with total mixed ration (TMR) ad libitum; S2, day 4, served as restricted feeding, with cows offered 50% of the average daily intake observed in S1; S3, day 5, a grain challenge was performed, in which cows were fed a TMR ad libitum without (CON) or with an additional pellet wheat-barley (1:1; HIG) at 20% of dry matter intake top-dressed onto the TMR; S4, days 6 to 10served as recovery during which cows were allowed ad libitum access to the TMR. Among the 28 biomarkers analyzed in blood 12 hours after grain challenge on day 5, the concentrations of fatty acids and bilirubin increased in HIG Holstein but not Jersey cows. In Holsteins, feeding HIG also increased total protein and albumin while decreasing ceruloplasmin, myeloperoxidase, and alkaline phosphatase concentrations. At the molecular level, hepatic genes associated with inflammation (IL1B, IL6, TNF, TLR4, MYD88, and NFKB1) were upregulated in Holstein cows fed HIG versus CON. Despite such response, expression of the acute-phase proteins SAA and HP in Holsteins fed HIG compared with CON was markedly downregulated. In Holsteins fed HIG versus CON, the marked downregulation of SCD, ELOVL6, and MTTP along with upregulated CPT1A, ACOX1, and APOA5 indicated alterations in fatty acid and lipoprotein metabolism during grain challenge. Genes related to ketogenesis (HMGCS2 and ACAT1) were upregulated in Jerseys, and gluconeogenic genes (PDK4 and PCK1) were upregulated in Holstein cows fed HIG, suggesting alterations in ketone body and glucose production. Expression of phosphorylated p70S6K1, RPS6, and 4EBP1 proteins, as well as total mechanistic target of rapamycin (mTOR) protein, decreased in Holsteins fed HIG, whereas phosphorylated mTOR and 4EBP1 proteins increased in Jerseys fed HIG. From a metabolic and inflammatory biomarker standpoint, data indicate that Jersey cows better tolerated the acute grain challenge. Alterations in mTOR signaling proteins in both Jerseys and Holsteins fed HIG suggest a potential role for exogenous AA in the hepatic adaptations to grain challenge. It remains to be determined if these acute responses to a grain challenge can elicit long-term liver dysfunction, which could negatively affect welfare of the cow. Feeding a higher-energy diet by increasing cereal grains at the expense of forage during the last 3 to 4 weeksprepartum is a traditional approach to help the rumen "adapt" to the traditional diets fed at the onset of lactation. Increasing grain/concentrate in the diet changes ruminal fermentation and in sheep and goats elicits marked changes in mRNA expression of immune-related genes in ruminal epithelium. Whether such changes at the epithelial and systemic levels occur in dairy cows when the dietary energy content increases at a fixed level of concentrate is unknown. Fourteen nonpregnant, nonlactating Holstein cows were fed a control lower-energy (CON, 1.30 Mcal/kg of dry matter) diet to meet 100% of estimated nutrient requirements for three weeks, after which half of the cows were assigned to a higher-energy diet (OVE, 1.60 Mcal/kg of dry matter) and half of the cows continued on CON for 6 weeks. Levels of forage and concentrate for CON and OVE were 80 and 79% and 20 and 21%, respectively. Plasma samples were collected one daybefore slaughter to examine biomarkers of metabolism, liver function, inflammation, and oxidative stress. The reticulo-rumen mass was recorded at slaughter, and samples of epithelium were harvested from all cows. The expression of 29 genes associated with tight junctions, immune function, and nutrient transport (volatile fatty acids, urea, and trace minerals) was examined. Overfeeding energy led to consistently greater dry matter intake over time, and lowered plasma concentrations of haptoglobin, paraoxonase, bilirubin, fatty acids, and myeloperoxidase (secreted by neutrophils). In contrast, OVE resulted in greater hydroxybutyrate and cholesterol concentrations. A greater reticulo-rumen mass in cows fed OVE did not alter genes associated with tight junctions (CDLN1, CDNL4, OCLN, TJP1), immune function (IL1B, IL10, NFKB1, TLR2, TLR4, TNF), oxidative stress (SOD1, SOD2), or most nutrient transporters. However, feeding OVE upregulated the acute-phase protein SAA3 by 3.5-fold and downregulated a volatile fatty acid transporter (SLC16A1) and a Fe and Cu transporter (SLC11A2). The lack of effect on mRNA expression along with lower plasma concentrations of inflammation biomarkers indicates that long-term intake of a higher-energy diet ad libitum was not detrimental to ruminal epithelium integrity. In that context, a protective function of SAA3 could be envisioned with a role in opsonizing gram-negative bacteria that produce endotoxins. The long-term control of volatile fatty acid absorption and trace minerals from the rumen in cows overfed energy does not seem to be controlled at the gene transcription level. The relevance of these findings to the nutritional management of pregnant dry cows merits further research.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Derakhshani, H., Tun, H.M., Cardoso, F.C., Plaizier, J.C., Khafipour, E. and Loor, J.J. 2017. Linking peripartal dynamics of ruminal microbiota to dietary changes and production parameters. Front Microbiol. 2017 Jan 12;7:2143.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Abdelmegeid, M.K., Vailati-Riboni, M., Alharthi, A., Batistel, F. and Loor, J.J. 2017. Supplemental methionine, choline, or taurine alter in vitro gene network expression of polymorphonuclear leukocytes from neonatal Holstein calves. J. Dairy Sci. 2017 Apr;100(4):3155-3165.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Jacometo, C.B., Zhou, Z., Luchini, D., Corr�a, M.N. and Loor, J.J. 2017. Maternal supplementation with rumen-protected methionine increases prepartal plasma methionine concentration and alters hepatic mRNA abundance of 1-carbon, methionine, and transsulfuration pathways in neonatal Holstein calves. J. Dairy Sci. 2017 Apr;100(4):3209-3219.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Bulgari, O., Dong, X., Roca, A.L., Caroli, A.M. and Loor, J.J. 2017. Innate immune responses induced by lipopolysaccharide and lipoteichoic acid in primary goat mammary epithelial cells. J. Anim. Sci. Biotechnol. 2017 Apr 1;8:29.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Batistel, F., Osorio, J.S., Tariq, M.R., Li, C., Caputo, J., Socha, M.T. and Loor, J.J. 2017. Peripheral leukocyte and endometrium molecular biomarkers of inflammation and oxidative stress are altered in peripartal dairy cows supplemented with Zn, Mn, and Cu from amino acid complexes and Co from Co glucoheptonate. J. Anim. Sci. Biotechnol. 2017 May 1;8:33.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Agrawal, A., Khan, M.J., Graugnard, D.E., Vailati-Riboni, M., Rodriguez-Zas, S.L., Osorio, J.S. and Loor, J.J. 2017. Prepartal energy intake alters blood polymorphonuclear leukocyte transcriptome during the peripartal period in Holstein cows. Bioinform. Biol. Insights. 2017 Apr 28;11:1177932217704667.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Hosseini, A., Salman, M., Zhou, Z., Drackley, J.K., Trevisi, E. and Loor, J.J. 2017. Level of dietary energy and 2,4-thiazolidinedione alter molecular and systemic biomarkers of inflammation and liver function in Holstein cows. J. Anim. Sci. Biotechnol. 2017 Aug 1;8:64.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Xu, T., Cardoso, F.C., Pineda, A., Trevisi, E., Shen, X., Rosa, F., Osorio, J.S. and Loor, J.J. 2017. Grain challenge affects systemic and hepatic molecular biomarkers of inflammation, stress, and metabolic responses to a greater extent in Holstein than Jersey cows. J. Dairy Sci. 2017 Nov;100(11):9153-9162.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Arroyo, J.M., Hosseini, A., Zhou, Z., Alharthi, A., Trevisi, E., Osorio, J.S. and Loor, J.J. 2017. Reticulo-rumen mass, epithelium gene expression, and systemic biomarkers of metabolism and inflammation in Holstein dairy cows fed a high-energy diet. J. Dairy Sci. 2017 Nov;100(11):9352-9360.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience: Nothing Reported Changes/Problems:We are in the process of evaluating changes in the hypothalamic transcriptome in sheep fed incremental levels of propionic acid. We also are planning to perform high-throughput gene transcription analysis in liver tissue of neonatal calves from cows fed a control diet or a rumen-protected methionine diet. What opportunities for training and professional development has the project provided?The PI spent approximately 10% of his time working on the data interpretation and drafting the manuscripts in conjunction with three PhD students and several visiting scholars. The students and visiting scholars were trained in conducting gene transcription analysis that allowed them to generate data. These scholars had the opportunity to learn all aspects of scientific writing. How have the results been disseminated to communities of interest?Peer-reviewed manuscripts were published. What do you plan to do during the next reporting period to accomplish the goals?We will continue working with in vivo systems utilizing tissue samples to better understand the signalling and metabolic pathways that coordinate physiological responses in cows and neonatal calves.

Impacts
What was accomplished under these goals? Under Goal #2: To gather preliminary data on the potential importance of FGF1, co-regulated genes, and upstream metabolic genes, we examined the hepatic mRNA expression in response to nutrition and inflammation in dairy cows. In experiment 1, induction of ketosis through feed restriction on day 5 postpartum upregulated FGF21, its co-receptor KLB, and PPARA but only elicited a numerical increase in serum FGF21 concentration. In experiment 2, cows in control (CON) or receiving 50 g/d of L-carnitine (C50) from -14 through 21 days had increased FGF21, PPARA, and NFIL3 on day 10 compared with day 2 postpartum. In contrast, compared with CON and C50, 100 g/d L-carnitine (C100) resulted in lower FGF21, KLB, ANGPTL4, and ARNTL expression on day 10. In experiment 3, cows were fed during the dry period either a higher-energy (OVE; 1.62 Mcal/kg DM) or lower-energy (CON; 1.34 Mcal/kg DM) diet and received 0 (OVE:N, CON:N) or 200 μg of LPS (OVE:Y, CON:Y) into the mammary gland at day 7 postpartum. For FGF21 mRNA expression in CON, the LPS challenge (CON:Y) prevented a decrease in expression between days 7 and 14 postpartum such that cows in CON:N had a 4-fold lower expression on day 14 compared with day 7. The inflammatory stimulus induced by LPS in CON:Y resulted in upregulation of PPARA on day 14 to a similar level as cows in OVE:N. In OVE:Y, expression of PPARA was lower than CON:N on day 7 and remained unchanged on day 14. On day 7, LPS led to a 4-fold greater serum FGF21 only in OVE but not in CON cows. In fact, OVE:Y reached the same serum FGF21 concentration as CON:N, suggesting a carryover effect of dietary energy level on signaling mechanisms within liver. Seven multiparous Holstein cows with a ruminal fistula were used to investigate the changes in rumen microbiota, gene expression of the ruminal epithelium, and blood biomarkers of metabolism and inflammation during the transition period. Samples of ruminal digesta, biopsies of ruminal epithelium, and blood were obtained during -14 through 28 days in milk (DIM). A total of 35 genes associated with metabolism, transport, inflammation, and signaling were evaluated by quantitative reverse transcription-PCR. Among metabolic-related genes, expression of HMGCS2 increased gradually from -14 to a peak at 28 DIM, underscoring its central role in epithelial ketogenesis. The decrease of glucose and the increase of nonesterified fatty acids and β-hydroxybutyrate in the blood after calving confirmed the state of negative energy balance. Similarly, increases in bilirubin and decreases in albumin concentrations after calving were indicative of alterations in liver function and inflammation. Despite those systemic signs, lower postpartal expression of TLR2, TLR4, CD45, and NFKB1 indicated the absence of inflammation within the epithelium. Alternatively, these could reflect an adaptation to react against inducers of the immune system arising in the rumen (e.g., bacterial endotoxins). The downregulation of RXRA, INSR, and RPS6KB1 between -14 and 10 DIM indicated a possible increase in insulin resistance. However, the upregulation of IRS1 during the same time frame could serve to restore sensitivity to insulin of the epithelium as a way to preserve its proliferative capacity. The upregulation of TGFB1 from -14 and 10 DIM coupled with upregulation of both EGFR and EREG from 10 to 28 DIM indicated the existence of 2 waves of epithelial proliferation. However, the downregulation of TGFBR1 from -14 through 28 DIM indicated some degree of cell proliferation arrest. The downregulation of OCLN and TJP1 from -14 to 10 DIM indicated a loss of tight-junction integrity. The gradual upregulation of membrane transporters MCT1 and UTB to peak levels at 28 DIM reflected the higher intake and fermentability of the lactation diet. In addition, those changes in the diet after calving resulted in an increase of butyrate and a decrease of ruminal pH and acetate, which partly explain the increase of Anaerovibrio lipolytica, Prevotella bryantii, and Megasphaera elsdenii and the decrease of fibrolytic bacteria (Fibrobacter succinogenes, Butyrivibrio proteoclasticus). The availability of Met in metabolizable protein (MP) of a wide range of diets for dairy cows is low. During late pregnancy and early lactation, in particular, suboptimal Met in MP limits its use for mammary and liver metabolism and also for the synthesis of S-adenosylmethionine, which is essential for many biological processes, including DNA methylation. The latter is an epigenetic modification involved in the regulation of gene expression, hence, tissue function. Thirty-nine Holstein cows were fed throughout the peripartal period (-21 days to 30 days in milk) a basal control (CON) diet (n=14) with no Met supplementation, CON plus MetaSmart (MS; Adisseo NA, Alpharetta, GA; n=12), or CON plus Smartamine M (SM; Adisseo NA; n=13). The total mixed ration dry matter for the close-up and lactation diets was measured weekly, then the Met supplements were adjusted daily and top-dressed over the total mixed ration at a rate of 0.19 (MS) or 0.07% (SM) on a dry matter basis. Liver tissue was collected on -10, 7, and 21 days for global DNA and peroxisome proliferator-activated receptor alpha (PPARα) promoter region-specific methylation. Several PPARα target and putative target genes associated with carnitine synthesis and uptake, fatty acid metabolism, hepatokines, and carbohydrate metabolism were also studied. Data were analyzed using PROC MIXED of SASwith the preplanned contrast CON versus SM + MS. Global hepatic DNA methylation on day 21 postpartum was lower in Met-supplemented cows than CON. However, of 2 primers used encompassing 4 to 12 CpG sites in the promoter region of bovine PPARA, greater methylation occurred in the region encompassing -1,538 to -1,418 from the transcription start site in cows supplemented with Met. Overall expression of PPARA was greater in Met-supplemented cows than CON. Concomitantly, PPARA-target genes, such as ANGPTL4, FGF21, and PCK1, were also upregulated overall by Met supplementation. The upregulation of PPARα target genes indicates that supplemental Met, likely through the synthesis of S-adenosylmethionine, activated PPARA-regulated signaling pathways. The hoof digital cushion is a complex structure composed of adipose tissue beneath the distal phalanx, i.e. axial, middle, and abaxial fat pad. The major role of these fat depots is dampening compression of the corium underneath the cushion. The study aimed to determine expression of target genes and fatty acid profiles in the hoof of non-pregnant dry Holstein cows fed low (CON) or high-energy (OVE) diets. The middle fat pad of the hoof digital cushion was collected soon after slaughter. Despite the lack of effect on expression of the transcription regulators SREBF1 and PPARG, the expression of the lipogenic enzymes ACACA, FASN, SCD, and DGAT2 was upregulated with OVE. Along with the upregulation of G6PD and IDH1, important for NADPH synthesis during lipogenesis, and the basal glucose transporter SLC2A1, these data indicated a pro-lipogenic response in the digital cushion with OVE. The expression of the lipid droplet-associated protein PLIN2 was upregulated while expression of lipolytic enzymes (ATGL, ABDH5, and LIPE) only tended to be upregulated with OVE. Therefore, OVE induced lipogenesis, lipid droplet formation, and lipolysis, albeit to different extents. Although concentration of monounsaturated fatty acids (MUFA) did not differ, among the polyunsaturated fatty acids (PUFA), the concentration of 20:5n3 was lower with OVE. Among the saturated fatty acids, 20:0 concentration was greater with OVE. Although data indicated that the hoof digital cushion metabolic transcriptome is responsive to higher-energy diets, this did not translate into marked differences in the fatty acid composition.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Akbar, H., Batistel, F., Drackley, J.K. and Loor, J.J. 2015. Alterations in hepatic FGF21, co-regulated genes, and upstream metabolic genes in response to nutrition, ketosis and inflammation in peripartal holstein cows. PLoS One. 2015 Oct 9;10(10):e0139963. doi: 10.1371/journal.pone.0139963.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Jacometo, C.B., Osorio, J.S., Socha, M., Corr�a, M.N., Piccioli-Cappelli, F., Trevisi, E. and Loor, J.J. 2015. Maternal consumption of organic trace minerals alters calf systemic and neutrophil mRNA and microRNA indicators of inflammation and oxidative stress. J. Dairy Sci. 2015 Nov;98(11):7717-29. doi: 10.3168/jds.2015-9359.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Hosseini, A., Tariq, M.R., Trindade da Rosa, F., Kesser, J., Iqbal, Z., Mora, O., Sauerwein, H., Drackley, J.K., Trevisi, E. and Loor, J.J. 2015. Insulin sensitivity in adipose and skeletal muscle tissue of dairy cows in response to dietary energy level and 2,4-thiazolidinedione (TZD). PLoS One. 2015 Nov 16;10(11):e0142633. doi: 10.1371/journal.pone.0142633.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Minuti, A., Palladino, A., Khan, M.J., Alqarni, S., Agrawal, A., Piccioli-Capelli, F., Hidalgo, F., Cardoso, F.C., Trevisi, E. and Loor, J.J. 2015. Abundance of ruminal bacteria, epithelial gene expression, and systemic biomarkers of metabolism and inflammation are altered during the peripartal period in dairy cows. J. Dairy Sci. 2015 Dec;98(12):8940-51. doi: 10.3168/jds.2015-9722.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Mois�, S.J., Shike, D.W., Shoup, L. and Loor, J.J. 2016. Maternal plane of nutrition during late-gestation and weaning age alter steer calf longissimus muscle adipogenic microRNA and target gene expression. Lipids. 2016 Jan;51(1):123-38. doi: 10.1007/s11745-015-4092-y.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Osorio, J.S., Jacometo, C.B., Zhou, Z., Luchini, D., Cardoso, F.C. and Loor, J.J. 2016. Hepatic global DNA and peroxisome proliferator-activated receptor alpha promoter methylation are altered in peripartal dairy cows fed rumen-protected methionine. J. Dairy Sci. 2016 Jan;99(1):234-44. doi: 10.3168/jds.2015-10157.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Li, C., Batistel, F., Osorio, J.S., Drackley, J.K., Luchini, D. and Loor, J.J. 2016. Peripartal rumen-protected methionine supplementation to higher energy diets elicits positive effects on blood neutrophil gene networks, performance and liver lipid content in dairy cows. J. Anim. Sci. Biotechnol. 2016 Mar 9;7:18. doi: 10.1186/s40104-016-0077-9.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Iqbal, Z.M., Akbar, H., Hosseini, A., Bichi Ruspoli Forteguerri, E., Osorio, J.S. and Loor, J.J. 2016. Digital cushion fatty acid composition and lipid metabolism gene network expression in Holstein dairy cows fed a high-energy diet. PLoS One. 2016 Jul 21;11(7):e0159536. doi: 10.1371/journal.pone.0159536.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Jacometo, C.B., Zhou, Z., Luchini, D., Trevisi, E., Corr�a, M.N., Loor, J.J. 2016. Maternal rumen-protected methionine supplementation and its effect on blood and liver biomarkers of energy metabolism, inflammation, and oxidative stress in neonatal Holstein calves. J. Dairy Sci. 2016 Aug;99(8):6753-63. doi: 10.3168/jds.2016-11018.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Li, S., Hosseini, A., Danes, M., Jacometo, C., Liu, J. and Loor, J.J. 2016. Essential amino acid ratios and mTOR affect lipogenic gene networks and miRNA expression in bovine mammary epithelial cells. J. Anim. Sci. Biotechnol. 2016 Aug 3;7:44. doi: 10.1186/s40104-016-0104-x.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Fellow investigators and nutrition and veterinary professionals. Changes/Problems:We are in the process of analyzing themicrobiome associated with healthy and clinically-mastitic mammary teats in lactating goats at different stages of the lactation cycle. We are also planning to perfrom RNA sequencing on RNA samples from milk cells from healthy goats or goats with clinical mastitis. What opportunities for training and professional development has the project provided?The PI spent approximately 10% of his time working on the data interpretation and drafting the manuscriptsin conjunction with threePhD students. The studentswere trained in several molecular techniques that allowed them to generatedata. These graduate students had the opportunity to learn all aspects ofscientific writing. How have the results been disseminated to communities of interest?Peer-reviewed manuscripts were published. What do you plan to do during the next reporting period to accomplish the goals?We will continue working with in vitro systems utilizing bovine cells to better understand the signallingpathways that coordinate milk fat and protein synthesis regulation, and also neutrophil function.

Impacts
What was accomplished under these goals? Under Goal #2: The prepartal dietary energy level is tightly correlated with the degree of tissue mobilization that the animal experiences around parturition (giving birth). To better understand the link between the dry period dietary energy management and the inflammatory status around parturition, 12 multiparous Holstein cows were fed for the entire dry period either a high-wheat straw/lower-energy diet to supply at least 100% of the calculated net energy for lactation (NEL) (control, CON) or a higher-energy diet to supply >140% of NEL (overfed, OVE). The blood was sampled throughout the transition period for biomarker analyses. Liver tissue samples were taken on days -14, 7, 14, and 30 relative to parturition for triacylglycerol (TAG) composition and gene expression analysis. Fifty genes involved in inflammation, endoplasmic reticulum (ER),oxidative stress, and cell cycle and growth were evaluated. Although blood biomarkers did not reveal signs of a greater inflammatory status compared with OVE, CON cows had a greater activation of the intrahepatic unfolded protein response prepartum. However, postpartum mRNA profiling indicated that the OVE group experienced a mild but sustained level of ER stress, with higher oxidative stress and impairment of antioxidant mechanisms. After parturition, inflammation-related genes were upregulated in OVE cows compared with CON. However, CON cows experienced a gradual increase in expression of key inflammatory transcription regulators up to 30 days postpartum which agreed with the lower plasma albumin and cholesterol, suggesting an inflammatory state. Data underscored that ER stress is not necessarily linked with inflammation during the peripartal period. Gene expression data also suggest that prepartum overnutrition could have negative effects on normal cell cycle activity. Overall, allowing cows to overconsume energy prepartum increased the hepatic pro-inflammatory response prepartum and up to the point of parturition. Subsequently, cows fed the lower-energy diet experienced a gradual increase in the inflammatory response. The lack of differences between groups in voluntary feed intake and lactation capacity suggests that nutritional management prepartum triggers different mechanisms that affect ER and oxidative stress along with inflammation. Although no clinical disorders were detected, these alterations expose animals to the development of immuno-metabolic disorders. The mechanisms of insulin signalling in adipose tissue and skeletal muscle of dairy cows as it relates to plane of dietary energy are still not well understood.The effects of dietary energy level and 2,4-thiazolidinedione (TZD) injection on feed intake, body fatness, blood biomarkers and TZD concentrations, genes related to insulin sensitivity in adipose tissue (AT) and skeletal muscle, and peroxisome proliferator-activated receptor gamma (PPARG) protein in subcutaneous AT (SAT) were evaluated in Holstein cows. Fourteen nonpregnant nonlactating cows were fed a control low-energy (CON, 1.30 Mcal/kg) diet to meet 100% of estimated nutrient requirements for 3 weeks, after which half of the cows were assigned to a higher-energy diet (OVE, 1.60 Mcal/kg) and half of the cows continued on CON for 6 weeks. All cows received an intravenous injection of TZD starting 2 weeks after initiation of dietary treatments and for an additional 2 weeks, which served as the washout period. Cows fed OVE had greater energy intake and body mass than CON, and TZD had no effect during the administration period. The OVE cows had greater TZD clearance rate than CON cows. The lower concentration of nonesterified fatty acids (NEFA) and greater concentration of insulin in blood of OVE cows before TZD injection indicated positive energy balance and higher insulin sensitivity. Administration of TZD increased blood concentrations of glucose, insulin, and beta-hydroxybutyrate (BHBA) at 2 to 4 weeks after diet initiation, while the concentration of NEFA and adiponectin (ADIPOQ) remained unchanged during TZD. The TZD upregulated the mRNA expression of PPARG and its targets FASN and SREBF1 in SAT, but also SUMO1 and UBC9 which encode sumoylation proteins known to down-regulate PPARG expression and curtail adipogenesis. Therefore, a post-translational response to control PPARG gene expression in SAT could be a counteregulatory mechanism to restrain adipogenesis. The OVE cows had greater expression of the insulin sensitivity-related genes IRS1, SLC2A4, INSR, SCD, INSIG1, DGAT2, and ADIPOQ in SAT. In skeletal muscle, where PPARA and its targets orchestrate carbohydrate metabolism and fatty acid oxidation, the OVE cows had greater glyceroneogenesis (higher mRNA expression of PC and PCK1), whereas CON cows had greater glucose transport (SLC2A4). Administration of TZD increased triacylglycerol concentration and altered expression of carbohydrate- and fatty acid oxidation-related genes in skeletal muscle. Results indicate that overfeeding did not affect insulin sensitivity in nonpregnant, nonlactating dairy cows. The bovine PPARG receptor appears TZD-responsive, with its activation potentially leading to greater adipogenesis and lipogenesis in SAT, while differentially regulating glucose homeostasis and fatty acid oxidation in skeletal muscle. Targeting PPARG via dietary nutraceuticals while avoiding excessive fat deposition might improve insulin sensitivity in dairy cows during times such as the peripartal period when the onset of lactation naturally decreases systemic insulin release and sensitivity in tissues such as AT. During the dry period, cows can easily overconsume higher-grain diets, a scenario that could impair immune function during the peripartal period. Objectives were to investigate the effects of energy overfeeding on expression profile of genes associated with inflammation, lipid metabolism, and neutrophil function, in 12 multiparous Holstein cows (n=6/dietary group) fed control [CON, 1.34 Mcal/kg of dry matter (DM)] or higher-energy (HE, 1.62 Mcal/kg of DM) diets during the last 45 d of pregnancy. Blood was collected to evaluate 43 genes in polymorphonuclear neutrophil leukocytes (PMNL) isolated at -14, 7, and 14 d relative to parturition. We detected greater expression of inflammatory-related cytokines (IL1B, STAT3, NFKB1) and eicosanoid synthesis (ALOX5AP and PLA2G4A) in HE cows than in CON cows. Around parturition, all cows had a close balance in mRNA expression of the pro-inflammatory IL1B and the anti-inflammatory IL10, with greater expression of both in cows fed HE than CON. The expression of CCL2, LEPR, TLR4, IL6, and LTC4S was undetectable. Cows in the HE group had greater expression of genes involved in PMNL adhesion, motility, migration, and phagocytosis, which was similar to expression of genes related to the pro-inflammatory cytokine. This response suggests that HE cows experienced a chronic state of inflammation. The greater expression of G6PD in HE cows could have been associated with the greater plasma insulin, which would have diverted glucose to other tissues. Cows fed the HE diet also had greater expression of transcription factors involved in metabolism of long-chain fatty acids (PPARD, RXRA), suggesting that immune cells might be predisposed to use endogenous ligands such as nonesterified fatty acids available in the circulation when glucose is in high demand for milk synthesis. The lower overall expression of SLC2A1 postpartum than prepartum supports this suggestion. Targeting interleukin-1β signaling might be of value in terms of controlling the inflammatory response around calving. The present study revealed that overfeeding cows during late pregnancy results in activation, ahead of parturition, of PMNL responses associated with stress and inflammation. These adaptations observed in PMNL did not seem to be detrimental for production.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Akbar, H., Batistel, F., Drackley, J.K. and Loor, J.J. 2015. Alterations in hepatic FGF21, co-regulated genes, and upstream metabolic genes in response to nutrition, ketosis and inflammation in peripartal holstein cows. PLoS One. 10:e0139963.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Hosseini, A., Tariq, M.R., Trindade da Rosa, F., Kesser, J., Iqbal, Z., Mora, O., Sauerwein, H., Drackley, J.K., Trevisi, E. and Loor, J.J. 2015. Insulin sensitivity in adipose and skeletal muscle tissue of dairy cows in response to dietary energy level and 2,4-thiazolidinedione (TZD). PLoS One. 10:e0142633.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Akbar, H., Grala, T.M., Vailati Riboni, M., Cardoso, F.C., Verkerk, G., McGowan, J., Macdonald, K., Webster, J., Schutz, K., Meier, S., Matthews, L., Roche, J.R. and Loor, J.J. 2015. Body condition score at calving affects systemic and hepatic transcriptome indicators of inflammation and nutrient metabolism in grazing dairy cows. J Dairy Sci. 98:1019-32.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Minuti, A., Zhou, Z., Graugnard, D.E., Rodriguez-Zas, S.L., Palladino, A.R., Cardoso, F.C., Trevisi, E. and Loor, J.J. 2015. Acute mammary and liver transcriptome responses after an intramammary Escherichia coli lipopolysaccharide challenge in postpartal dairy cows. Physiol Rep. 3. pii: e12388.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Wang, M., Zhou, Z., Khan, M.J., Gao, J. and Loor, J.J. 2015. Clock circadian regulator (CLOCK) gene network expression patterns in bovine adipose, liver, and mammary gland at 3 time points during the transition from pregnancy into lactation. J Dairy Sci. 98:4601-12.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Zhou, Z. Bu, D.P., Vailati Riboni, M., Khan, M.J., Graugnard, D.E., Luo, J., Cardoso, F.C. and Loor, J.J. 2015. Prepartal dietary energy level affects peripartal bovine blood neutrophil metabolic, antioxidant, and inflammatory gene expression. J Dairy Sci. 98:5492-505.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Shahzad, K., Akbar, H., Vailati-Riboni, M., Basiric�, L., Morera, P., Rodriguez-Zas, S.L., Nardone, A., Bernabucci, U. and Loor, J.J. 2015. The effect of calving in the summer on the hepatic transcriptome of Holstein cows during the peripartal period. J. Dairy Sci. 98:5401-13.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Khan, M.J., Jacometo, C.B., Riboni, M.V., Trevisi, E., Graugnard, D.E., Corr�a, M.N. and Loor, J.J. 2015. Stress and inflammatory gene networks in bovine liver are altered by plane of dietary energy during late pregnancy. Funct. Integr. Genomics. 15:563-76.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Jacometo, C.B., Osorio, J.S., Socha, M., Corr�a, M.N., Piccioli-Cappelli, F., Trevisi, E. and Loor, J.J. 2015. Maternal consumption of organic trace minerals alters calf systemic and neutrophil mRNA and microRNA indicators of inflammation and oxidative stress. J. Dairy Sci. 98:7717-29.
  • Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Minuti, A., Palladino, A., Khan, M.J., Alqarni, S., Agrawal, A., Piccioli-Capelli, F., Hidalgo, F., Cardoso, F.C., Trevisi, E. and Loor, J.J. 2015. Abundance of ruminal bacteria, epithelial gene expression, and systemic biomarkers of metabolism and inflammation are altered during the peripartal period in dairy cows. J. Dairy Sci. 98:8940-51.


Progress 11/21/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: As an additional goal, we will evaluate the microbiome associated with healthy and clinically mastitic mammary teats in lactating goats at different stages of the lactation cycle. We will also perform in vitro studies with goat mammary cells to study the response to infection. The microbiome will be studied using sequencing technology, and the in vitro transcriptome via RNAseq or microarrays. Functional bioinformatics analyses will be perfromed on the resulting data. What opportunities for training and professional development has the project provided? The PI spent approximately 5% of his time developing the experimental design in conjunction with two PhD students. They conducted the experiments and in the process were trained in several molceular techniques that allowed them to generate data. The graduate students involved had the opportunity to write an abstract, build poster presentations, and present their findings at the annual JAM meeting in Kansas City in July of 2014. How have the results been disseminated to communities of interest? Peer-reviewed manuscripts were published. What do you plan to do during the next reporting period to accomplish the goals? We will continue working with in vitro systems utilizing bovine and goat mammary cells to better understand the signalling pathways that coordinate milk fat and protein synthesis regulation.

Impacts
What was accomplished under these goals? Under Goal #2: Hepatic metabolic gene networks were studied in dairy cattle fed control (CON, 1.34 Mcal/kg) or higher energy (overfed (OVE), 1.62 Mcal/kg) diets during the last 45 days of pregnancy. A total of 57 target genes encompassing PPARα-targets/co-regulators, hepatokines, growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis, lipogenesis, and lipoprotein metabolism were evaluated on -14, 7, 14, and 30 days around parturition. OVE versus CON cows were in more negative energy balance (NEB) postpartum and had greater serum non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), and liver triacylglycerol (TAG) concentrations. Milk synthesis rate did not differ. Liver from OVE cows responded to postpartal NEB by up-regulating expression of PPARα-targets in the fatty acid oxidation and ketogenesis pathways, along with gluconeogenic genes. Hepatokines (fibroblast growth factor 21 (FGF21), angiopoietin-like 4 (ANGPTL4)) and apolipoprotein A-V (APOA5) were up-regulated postpartum to a greater extent in OVE than CON. OVE led to greater blood insulin prepartum, lower NEFA:insulin, and greater lipogenic gene expression suggesting insulin sensitivity was not impaired. A lack of change in APOB, MTTP, and PNPLA3 coupled with upregulation of PLIN2 postpartum in cows fed OVE contributed to TAG accumulation. Postpartal responses in NEFA and FGF21 with OVE support a role of this hepatokine in diminishing adipose insulin sensitivity. Our objective was to determine the effects of overfeeding energy on gene expression in mesenteric (MAT), omental (OAT), and subcutaneous (SAT) adipose tissue (AT) from nonpregnant and nonlactating Holstein cows. Eighteen cows were randomly assigned to either a low energy [LE, net energy for lactation (NE(L)) = 1.35 Mcal/kg of dry matter (DM)] or high energy (HE, NE(L) = 1.62 Mcal/kg of DM) diets for 8 wk. Cows were then euthanized and subsamples of MAT, OAT, and SAT were harvested for transcript profiling via quantitative PCR of 34 genes involved in lipogenesis, triacylglycerol (TAG) synthesis, lipolysis, lactate signaling, transcription regulation, and inflammation. The interaction of dietary energy and AT depot was only significant for LPL, which indicated a consistent response among the 3 sites. The expression of key genes related to de novo fatty acid synthesis (FASN) and desaturation (SCD) was upregulated by HE compared with LE. Other genes associated with those processes, such as ACLY, ACACA, ELOVL6, FABP4, GPAM, and LPIN1, were numerically upregulated by HE. The expression of lipolytic (PNPLA2 and ABHD5) genes was upregulated and the antilypolytic lactate receptor HCAR1 was downregulated with HE compared with LE. The putative transcription regulator THRSP was upregulated and the transcription regulator PPARG tended to be upregulated by HE, whereas SREBF1 was downregulated. Among adipocytokines, HE tended to upregulate the expression of CCL2, whereas IL6R was downregulated. Overall, results indicated that overfeeding energy may increase AT mass at least in part by stimulating transcription of the network encompassing key genes associated with de novo synthesis. In response to energy overfeeding, the expression of PPARG rather than SREBF1 was closely associated with most adipogenic or lipogenic genes. However, the transcriptional activity of these regulators needs to be verified to confirm their role in the regulation of adipogenesis or lipogenesis in bovine AT. Overfeeding energy also may predispose cows to greater lipolytic potential by stimulating expression of TAG hydrolysis genes while inhibiting signaling via hydroxycarboxylic acid receptor (HCAR1), which is a novel antilipolytic regulator. Our results do not support an overt inflammatory response in adipose tissues in response to an 8-wk energy overfeeding. As part fo the study above we also determined the effects of energy overfeeding on gene expression in mesenteric (MAT), omental (OAT), and subcutaneous (SAT) adipose tissue (AT) from nonpregnant and nonlactating Holstein cows. Eighteen cows were randomly assigned to either a controlled energy [LE, net energy for lactation (NE(L)) = 1.35 Mcal/kg of dry matter (DM)] or moderate energy-overfed group (HE, NE(L) = 1.62 Mcal/kg of DM) for 8 wk. Cows were then euthanized and subsamples of MAT, OAT, and SAT were harvested for transcript profiling via quantitative PCR of 34 genes involved in lipogenesis, triacylglycerol (TAG) synthesis, lactate signaling, hepatokine signaling, lipolysis, transcription regulation, and inflammation. The interaction of dietary energy and adipose depot was not significant for any gene analyzed except LPL, which indicated a consistent response to diet. Expression of ACACA and FASN was greater in SAT than MAT, whereas expression of SCD and ADFP were greatest in SAT, intermediate in OAT, and lowest in MAT. However, the 2 visceral depots had greater expression of THRSP, ACLY, LPL, FABP4, GPAM, and LPIN1 compared with SAT. The transcription factor SREBF1 was more highly expressed in MAT and SAT than in OAT. The expression of PNPLA2 was greater in visceral AT sites than in SAT, but other lipolysis-related genes were not differentially expressed among AT depots. Visceral AT depots had greater expression of LEP, ADIPOQ, and SAA3 compared with SAT. Moreover, MAT had greater expression than SAT of proinflammatory cytokines (IL1B and IL6), IL6 receptor (IL6R), and chemokines (CCL2 and CCL5). However, TNF expression was greatest in SAT, lowest in OAT, and intermediate in MAT. Overall, results indicated that visceral AT might be more active in uptake of preformed long-chain fatty acids than SAT, whereas de novo fatty acid synthesis could make a greater contribution to the intracellular pool of fatty acids in SAT than in visceral AT. The visceral AT compared with SAT seem to have a greater capacity for expression (and potentially for secretion) of proinflammatory cytokines; thus, excessive accumulation of visceral lipid due to a long-term overfeeding energy may be detrimental to liver function and overall health of dairy cows, particularly during the transition period.

Publications

  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Khan, M.J., Jacometo, C.B., Graugnard, D.E., Corr�a, M.N., Schmitt, E., Cardoso, F. and Loor, J.J. Overfeeding dairy cattle during late-pregnancy alters hepatic PPAR alpha-regulated pathways including hepatokines: Impact on metabolism and peripheral insulin sensitivity. Gene Regul Syst Bio. 3;8:97-111.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Ji, P., Drackley, J.K., Khan, M.J. and Loor, J.J. Overfeeding energy upregulates peroxisome proliferator-activated receptor (PPAR) alpha-controlled adipogenic and lipolytic gene networks but does not affect proinflammatory markers in visceral and subcutaneous adipose depots of Holstein cows. J. Dairy Sci. 97(6):3431-40.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Ji, P., Drackley, J.K., Khan, M.J. and Loor, J.J. Inflammation and lipid metabolism-related gene network expression in visceral and subcutaneous adipose depots of Holstein cows. J. Dairy Sci. 97(6):3441-8.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Moyes, K.M., Graugnard, D.E., Khan, M.J., Mukesh, M. and Loor, J.J. Postpartal immunometabolic gene network expression and function in blood neutrophils are altered in response to prepartal energy intake and postpartal intramammary inflammatory challenge. J. Dairy Sci. 97(4):2165-77.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Seo, J., Osorio, J.S., Schmitt, E., Corr�a, M.N., Bertoni, G., Trevisi, E. and Loor, J.J. Hepatic purinergic signaling gene network expression and its relationship with inflammation and oxidative stress biomarkers in blood from peripartal dairy cattle. J. Dairy Sci. 97(2):861-73.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Wang, P., Drackley, J.K., Stamey-Lanier, J.A., Keisler, D. and Loor, J.J. Effects of level of nutrient intake and age on mammalian target of rapamycin, insulin, and insulin-like growth factor-1 gene network expression in skeletal muscle of young Holstein calves. J. Dairy Sci. 97(1):383-91.