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Major Advances in Fundamental Dairy Cattle Nutrition

Article  in  Journal of Dairy Science · May 2006

DOI: 10.3168/jds.S0022-0302(06)72200-7 · Source: PubMed

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Major Advances in Fundamental Dairy Cattle Nutrition

J. K. Drackley,*1 S. S. Donkin,† and C. K. Reynolds‡*Department of Animal Sciences, University of Illinois, Urbana 61801†Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-2054‡Department of Animal Sciences, The Ohio State University, OARDC, Wooster, 44696-4076

ABSTRACT

Fundamental nutrition seeks to describe the complexbiochemical reactions involved in assimilation and pro-cessing of nutrients by various tissues and organs, andto quantify nutrient movement (flux) through those pro-cesses. Over the last 25 yr, considerable progress hasbeen made in increasing our understanding of metabo-lism in dairy cattle. Major advances have been madeat all levels of biological organization, including thewhole animal, organ systems, tissues, cells, and mole-cules. At the whole-animal level, progress has beenmade in delineating metabolism during late pregnancyand the transition to lactation, as well as in whole-bodyuse of energy-yielding substrates and amino acids forgrowth in young calves. An explosion of research usingmulticatheterization techniques has led to better quan-titative descriptions of nutrient use by tissues of theportal-drained viscera (digestive tract, pancreas, andassociated adipose tissues) and liver. Isolated tissuepreparations have provided important information onthe interrelationships among glucose, fatty acid, andamino acid metabolism in liver, adipose tissue, andmammary gland, as well as the regulation of these path-ways during different physiological states. Finally, thelast 25 yr has witnessed the birth of “molecular biology”approaches to understanding fundamental nutrition.Although measurements of mRNA abundance for pro-teins of interest already have provided new insightsinto regulation of metabolism, the next 25 yr will likelysee remarkable advances as these techniques continueto be applied to problems of dairy cattle biology. Integra-tion of the “omics” technologies (functional genomics,proteomics, and metabolomics) with measurements oftissue metabolism obtained by other methods is a par-ticularly exciting prospect for the future. The resultshould be improved animal health and well being, moreefficient dairy production, and better models to predictnutritional requirements and provide rations to meetthose requirements.

Received June 8, 2005.Accepted June 10, 2005.1Corresponding author: drackley@uiuc.edu

Key words: metabolism, nutrition, molecular biology,splanchnic tissue

INTRODUCTION

Dairy cattle nutrition can be defined broadly as theuse of the components of feeds for the processes of main-tenance, growth, reproduction, lactation, and health.Applied nutrition is the selection and proportioning offeedstuffs and ingredients to supply the correctamounts and balance of nutrients required for optimalproductive and reproductive performance. Fundamen-tal nutrition is the series of biochemical reactions usedin the body during the assimilation and processing ofnutrients to meet the physiological needs of the animal.Fundamental and applied nutrition are equally im-portant in determining optimal feeding and manage-ment strategies for dairy cattle for health and produc-tion. This review will mainly highlight aspects of basicor fundamental nutrition that are referred to as metab-olism, which encompasses many disciplines includingbiochemistry, physiology, and molecular biology.

Twenty-five years ago, the nutrients required bydairy cattle were known and most of their biochemicalfunctions had been established. Nutrient requirementand diet formulation systems, however, were stilllargely based on “black box” concepts such as net energyand crude protein. In the 75th anniversary issue of theJournal of Dairy Science, Paul Moe from the USDABeltsville research station reviewed progress in energymetabolism research in dairy cattle over the preceding25 yr. In summarizing, Moe suggested areas of dairynutrition research for the next 25 yr that he believedwould enable the formulation of diets for modern dairycows that allowed them to achieve their maximal poten-tial for milk production with minimal stresses. To for-mulate diets that promoted maximal intake and effec-tive absorption and metabolism of nutrients, he empha-sized the need for greater understanding of thequantitative relationships among diet, the end productsof digestion, and animal performance, and specificallythe effects of individual nutrients on milk componentproduction and nutrient partitioning.

The ensuing 25-yr period has seen noteworthy prog-ress in opening the black boxes of net energy and crude

protein. One indication that our understanding of fun-damental nutrition has advanced is the progression ofconcepts and models in the two editions of NutrientRequirements of Dairy Cattle published by the NationalResearch Council (NRC) Subcommittee on Dairy CattleNutrition during this period. The 1989 edition movedtoward a more mechanistic approach with the estab-lishment of undegraded intake protein and degradedintake protein, as well as providing equations and sim-ple software. The 2001 version is a significant step for-ward, and has begun to move the field toward moremechanistic definition of nutrient requirements. In thisedition, a dynamic model of nutrient requirements wasdeveloped that is based largely on fundamental knowl-edge of metabolism and physiology. Along with othermodels of dairy cattle nutrition (e.g., Cornell Net Carbo-hydrate and Protein System) and metabolism (e.g., theUC-Davis “Molly”), the NRC publication demonstratesthat our quantitative knowledge of fundamental nutri-tion, and how to use that knowledge to better feed dairycattle, has improved markedly during the last 25 yr.

In this article, our aim is to highlight areas wherewe believe major advances in understanding have beenmade toward the challenge put forward by Moe 25 yrago, and that have led the field to the current state ofknowledge. Many of these accomplishments have beenmade using traditional and classic nutritional or bio-chemical procedures; on the other hand, major advanceshave also been made possible by techniques that didnot exist or were in their infancy 25 yr ago.

PROGRESS IN UNDERSTANDINGWHOLE-ANIMAL METABOLISM

Over the last 25 yr, considerable progress has beenmade in aggregating knowledge from various biologicallevels (whole animal, organ system, tissues, cells, mole-cules) into a more complete picture of metabolism inthe whole animal, as affected by its genetic background,physiological state, environment, and diet. The dairyproduction enterprise requires accurate models of nu-trient metabolism for cattle at all stages of the life cycle,from birth to maturity. Although our knowledge is farfrom complete, we now have more useful mechanisticmodels to predict and evaluate performance of bothlactating and growing cattle that incorporate an im-proved fundamental understanding of metabolism. Inparticular, the last 25 yr has seen significant progressin describing metabolism during several key life-cyclestages that were inadequately understood previously.A few key examples follow.

A number of research groups have tackled the diffi-cult challenge of defining fundamental requirementsfor nutrients in cows during late pregnancy and into

early lactation. Previously, requirements for energyand protein for pregnancy had been established largelybased on differences in calorimetric measurements andbalance studies between pregnant and nonpregnantcows, with a few small and incomplete studies of concep-tus growth. Bell and colleagues at Cornell Universityundertook a comprehensive slaughter study to quantifythe accumulation of nutrients in the developing fetusand maternal reproductive tissues of Holstein cows atincreasing days of pregnancy. Their data set includedmeasurements of energy, protein, and minerals andprovided quantitative estimates of the rates of accretionwith advancing pregnancy. Through their studies, thenutrient requirements of pregnancy have been muchmore clearly defined, and that knowledge has been in-corporated into newer nutritional models includingNRC (2001).

A major area of emphasis and progress during thelast quarter-century has been to understand the rapidlychanging nutritional requirements and nutrient metab-olism during the “transition period” from late gestationto early lactation. Before the 1980s, the transition pe-riod was largely ignored, and even avoided, by research-ers because of the extreme variability in animal re-sponses and the frequent incidence of health problems.Several research groups had been working on variousaspects of dry period nutrition and metabolism beforethe 1980s. However, groundbreaking studies from thelaboratory of Ric Grummer at the University of Wiscon-sin were instrumental in focusing research and fieldattention to the need to consider these cows as distinctphysiological and metabolic entities. The Wisconsinstudies established that declining DM intake (DMI) inthe last few days before parturition was a major factorin setting into motion a series of events that includedincreased body fat mobilization, increased uptake ofnonesterified fatty acids (NEFA) by liver, and accumu-lation of triacylglycerol (TG) in liver. Along with keystudies dealing with nutritional maintenance of bloodcalcium by Elliott Block, Jesse Goff, Ron Horst, andDave Beede, among others, these studies createdawareness of the importance of the transition periodboth to dairy profitability and to animal well-being.Investigations into the biology of transition or peripart-urient cows has flourished across North America inseveral research groups (University of Alberta, CornellUniversity, University of Guelph, University of Illinois,Michigan State University, National Animal DiseaseCenter, The Ohio State University, Purdue University,and the University of Wisconsin, among others) as wellas other groups worldwide. Research over the last de-cade has begun to dissect the metabolic basis for adap-tations in liver, adipose tissue, skeletal muscle, bone,and mammary gland during the transition period. Prog-

ress in understanding the unique physiology of the tran-sition cow has led to improved understanding of nutri-ent requirements, although optimal nutrition and feed-ing practices are far from resolved.

Another life-cycle stage where significant progresshas been made is in better defining nutrient metabolismand requirements in the preruminant calf. Over thelast few years, researchers at Cornell University andthe University of Illinois undertook a reevaluation ofnutrients required for growth, for several reasons.First, the 1989 NRC publication did not adequatelydescribe requirements for calves, and the specificationstherein did not accurately predict growth in the field.Second, both the genetic makeup of modern calves andthe ingredient makeup of their diets had undergonemarked changes, as genetics led to leaner cattle andwhey proteins replaced dried skim milk in milk re-placers. Finally, the accelerating evolution of the dairyindustry to larger herd size and the advent of customcalf-rearing enterprises contributed to increase atten-tion on the calf and its fundamental biology.

Van Amburgh and colleagues at Cornell Universityaimed to extend their models for heifer growth (devel-oped earlier) to the young preruminant calf. Illinoisresearchers sought to better define the relationshipsbetween supplies of digestible protein and energy. Bothgroups used a combination of balance trials and thecomparative slaughter approach to determine changesin composition of visceral tissues and carcass duringearly growth. Measurements of the composition of bodytissue at different rates of growth and as affected bydietary composition have confirmed many of the princi-ples established by the 2001 NRC committee, and haveprovided a framework for subsequent improvements inrequirement systems for energy and nitrogen in calvesfed milk replacer. Moreover, this research demon-strated that body composition can be markedly affectedby the ratio of protein to energy, and served as a re-minder that conventional calf-rearing systems havelimited nutrient intake and lean growth far below theinherent capacity of the animal. The improved descrip-tion of the most fundamental concepts of growth—thatis, the metabolic use of dietary protein and energy-yielding nutrients for deposition of body tissue—hasalready led to several practical systems to capitalize onthe remarkable growth potential of the very young calf.

In addition to these advances in life-cycle metabo-lism, advances have been made in several other areasof fundamental nutrition. The most important variablein nutrient requirement and rationing models is DMI.Progress in understanding the regulation of DMI hasoccurred, both through whole-animal modeling ap-proaches as well as more mechanistic studies of neural,endocrine, and metabolic influences. Several groups

have made fundamental advances in understanding ru-men function and its role in provision of nutrients. Ad-vances include a more complete understanding of themicrobial population responsible for digestion of fibrousand nonfibrous carbohydrates and the synthesis of mi-crobial protein, enhanced descriptive chemistry of feedcomponents, and more robust models of how the rumenmicrobial population digests those components.

The preceding 25 yr has seen an explosion of knowl-edge in whole-animal metabolism of fatty acids. In thelate 1970s, research on fat nutrition and use by rumi-nants surged, so that research from the 1980s to thepresent has resulted in a fairly complete picture of howdairy cattle digest, absorb, and metabolize fatty acidsof dietary and endogenous origin. In the last 15 yr,research and understanding in this area has beengreatly stimulated by the discovery by M. W. Pariza atthe University of Wisconsin of the potent anticarcino-genic effects of conjugated linoleic acid (CLA), whichis a group of isomers of linoleic acid produced in therumen and in animal tissues. The cis-9, trans-11 isomerof CLA is responsible for the anticancer effects. Becausethe rumen microbial population is the source of CLAor its precursor, trans monounsaturated fatty acids,research has been refocused intensively on lipid nutri-tion and metabolism. Several research groups led byDale Bauman at Cornell University, Rich Erdman andBev Teter at the University of Maryland, and Joe Herb-ein at Virginia Tech University discovered the powerfulsuppressive effects of various microbially derived fattyacids having trans-10 double bonds on fat synthesis inmammary gland and adipose tissue. These findings,which were a by-product of the search for ways to en-hance the content of the anticarcinogenic CLA isomersin milk and beef, confirmed the hypothesis of Carl Davisand Dick Brown in the late 1960s that trans fatty acidsproduced in the rumen of cows fed high-grain dietsmight be responsible for milk fat depression.

Advances have been made in whole-animal aspectsof amino acid metabolism, ranging from prediction ofintestinal supply to tissue metabolism to detailed mech-anistic models published by large modeling teams.Work by several groups to unravel the tissue require-ments for amino acids has continued and progressed,so that better estimates of amino acid requirements formilk production and other functions can now be made.This area likely will receive renewed research focusover the next 25 yr as demands for more efficient useof dietary nitrogen and less nitrogen excretion into theenvironment continue to intensify.

Finally, advancements have been made in fundamen-tal nutrition of minerals and vitamins. A major achieve-ment has been in understanding factors that controlcalcium mobilization from bone during times of in-

creased tissue calcium demand and dietary calcium in-sufficiency. In particular, the role of dietary cation-anion balance and vitamin D in regulating this processhas led to fundamental changes in how cows are man-aged during the transition from dry period to lactation.A reexamination of phosphorus requirements and me-tabolism is moving the field toward lower dietary supplyof P, thereby improving environmental outcomes. Sev-eral groups have revisited the metabolic role of certainB vitamins, such as biotin and folic acid, during thetransition period and have provided evidence that thelong-held view that ruminants need no supplementalB vitamins may be incorrect for certain key life-cyclestages. Significant progress in understanding the meta-bolic roles of trace minerals and vitamins, especiallyvitamin E and selenium, in immune processes are dis-cussed elsewhere in this issue.

Much of the research described in this section hasused traditional nutritional and biochemical tools, suchas dose-response feeding trials, balance studies, intesti-nally cannulated animals, radioisotope tracer studies,and concentrations of metabolites and hormones inblood. Although these techniques have provided invalu-able information, techniques that were not available ornot perfected 25 yr ago have pushed back the bound-aries of knowledge on organ systems and cellular regu-lation of metabolism.

PROGRESS IN UNDERSTANDING NUTRIENTABSORPTION AND METABOLISM

BY SPLANCHNIC TISSUES

Paul Moe contended 25 yr ago that a greater under-standing of the quantitative relationships among diet,the end products of digestion, and animal performance,along with mathematical models describing the effectsof diet on nutrient absorption and metabolism, wereneeded to improve feeding systems and to better predictresponses to diet composition. This vision, shared bymany ruminant nutritionists at the time, spurred asubstantial body of research that quantified nutrientfluxes across the splanchnic tissues of ruminants andalso fostered development of mathematical models ofnutrient flow and metabolism in the dairy cow, suchas the UC-Davis model “Molly” and other mechanisticmodels of nutrient metabolism. The emphasis on effectsof nutrient supply on productive response reflected thebelief shared by many dairy nutrition researchers thatnutrient supply is a direct determinant of productiveresponse. In effect, this defines the “push” side of thelong-running “push vs. pull” debate, which is derivedin part from the desire to formulate diets for early lacta-tion cows that minimize the extent to which milk nutri-ent output is derived from body tissue. Here, we will

consider progress over the last 25 yr in developing tech-niques and acquiring measurements of nutrient absorp-tion and delivery to peripheral tissues in dairy cowsand other ruminants, the development of mathematicalmodels of nutrient use in dairy cows, and our under-standing of the environmental by genetic interactions(push vs. pull) that determine production and nutrientpartitioning in modern dairy cows.

In his review 25 yr ago, Moe highlighted the consider-able progress made in using calorimetry to develop adatabase of energy metabolism measurements thatwere used for the subsequent refinement of existingfeeding standards for dairy cows. However, his view atthe time was that measurements of the absorption ofspecific products of digestion were crucial for furtherimprovements to be made in those feeding standards.In a sense, many felt that the existing “black-box” sys-tems of rationing energy and protein needed to be re-placed with nutrient-based systems before the “HolyGrail” of predicting performance and milk compositioncould be reached. At the time, use of duodenal cannula-tion techniques to measure nutrient flow from the ru-men was yielding vitally important data describing siteof digestion in cattle. In the subsequent 25 yr, the data-base of measurements of ruminal and postruminal di-gestion of protein, starch, and fiber has grown tremen-dously, although descriptions of nutrients actually dis-appearing from the small intestine of lactating dairycows were, and still are, lacking. This in part reflectsthe difficulty of surgically establishing and successfullymaintaining the ileal cannulas required to quantify dis-appearance in the small intestine.

As an alternative to measuring nutrient disappear-ance from the lumen of the gut, the apparent absorptionof nutrients into the portal vein can be quantified usingmulticatheterization techniques pioneered in sheep byE. N. Bergman from Cornell University in the 1960sand 1970s, and advanced in cattle in the 1970s and1980s by D. B. Baird and colleagues at Compton inEngland and G. B. Huntington and colleagues at USDAin Beltsville, MD. These techniques enable the mea-surement of venous-arterial concentration differencesand blood flow across the tissues of the portal-drainedviscera (PDV; including the gastrointestinal tract, pan-creas, spleen and associated adipose) or sections of thePDV, such as the section of the intestines and adiposedrained by the anterior mesenteric vein (the mesen-teric-drained viscera). These measurements equate tothe net amount of specific nutrients, or their metabo-lites, absorbed into the portal vein and available toother body tissues after metabolism by the tissues in-cluded in the venous drainage (Figure 1).

All portal vein blood must traverse the liver beforereaching the heart and then the rest of the body; conse-

Figure 1. Stylized view of the splanchnic vasculature; arrows show direction of blood flow.

quently, liver metabolism has an immediate impact onthe availability of many nutrients absorbed into theportal vein. By placing a catheter into a hepatic vein,measurements of net liver flux can also be obtained.Together, the PDV and liver are often referred to asthe “total splanchnic” tissues. In quantitative terms,the net release of an absorbed nutrient across thesetissues represents the net availability of that nutrientfor other body functions, such as milk synthesis. Onthe other hand, net removal of a nutrient across thetotal splanchnic bed represents the net use of nutrientsderived from other body tissues, as can occur for lactatein early lactation, because it is used for glucose synthe-sis in the liver. In this case, a negative lactate fluxindicates that liver uptake is greater than the net ap-pearance across the PDV.

To properly interpret and integrate measurementsof net nutrient flux across the splanchnic tissues withnutrient metabolism by other tissues, it is critical to becognizant at all times that these are net measurements.For example, net flux of glucose across the PDV of dairycows is often zero or slightly negative, but this doesnot mean there is no glucose absorption, as one mightinitially conclude. Indeed, the low recovery of starchprovided postruminally to dairy cows as increased netabsorption of glucose across the PDV led many to thelogical conclusion that the amount of starch absorbedfrom the small intestine as glucose was low, or thatthere was substantial loss of glucose during absorptiondue to use by small intestinal enterocytes. However, anet flux of zero also may mean that the amount ofglucose removed from arterial blood by the PDV is equalto the amount of glucose absorbed by the small intestineand released into the portal vein. Indeed, recent studiesby D. L. Harmon and colleagues at the University of

Kentucky have found that increases in glucose absorp-tion into the portal vein are balanced in part by in-creases in the use of glucose from arterial blood. Theuse of arterial glucose by the PDV accounts for as muchas 25% of whole body turnover, in part because of thesubstantial adipose tissue included in the PDV.

To obtain measurements of true rates of nutrientabsorption and release into venous blood, and removalfrom arterial blood, researchers can combine measure-ments of blood flow and venous-arterial difference withnutrient-labeling methodologies. The combination ofmulticatheterization and labeling techniques providesa powerful tool for measuring the complexities of metab-olism of a nutrient in vivo under varying dietary andphysiological conditions. Depending on the nutrient la-beled, measurements of whole body and tissue fluxescan be obtained, as well as amounts of the nutrientoxidized and the interconversions with specific metabo-lites on a whole-body and specific-tissue basis. Histori-cally, radioactive isotopes were used to label specificnutrients and the products of their metabolism anddetermine rates of unidirectional or gross metabolismacross specific tissues. More recently, the developmentof mass spectrometry for measurements of stable iso-tope enrichment and the commercial availability of avariety of metabolites labeled with stable isotopes hasprovided methods for isotopic labeling that do not posea health and safety risk. These events have led to thevirtual replacement of radioisotopes with stable iso-topes in studies of nutrient turnover and flux acrosssplanchnic tissues.

The last 25 yr has seen remarkable progress bothin the improvement and refinement of techniques formeasuring nutrient absorption and metabolism by thesplanchnic tissues, and in the use of the technique to

quantify the impact of dietary composition and physio-logical state on nutrient absorption and metabolism. Ingeneral, the technique has progressed from the stagewhere very short-lived catheter patency meant thatmany studies were conducted in a limited number ofanimals within days after surgery, with obvious impli-cations for the robustness and applicability of the dataobtained. With improvements in techniques, technolog-ies, and experience of the researchers, the approach hasprogressed such that multicatheterization preparationsin dairy cattle enable viable measurements of the ef-fects of physiological state, dietary composition, or stra-tegic nutrient supply to the lumen of the gut via infu-sions over periods of months, and in many cases formultiple lactations. Although some innovations in bloodflow measurement have occurred, the improved successand viability of the approach for measurements ofsplanchnic metabolism most likely are not a conse-quence of any specific new technologies or catheter ma-terials, but to a large extent reflect the experience oflaboratories that have made a sustained commitmentto application of the techniques.

The NRC system for rationing energy to dairy cowsusing net energy of lactation was based on a statisticalintegration of roughly 500 measurements of energy bal-ance obtained using respiration calorimetry. During thelast 25 yr, many more measurements of net nutrientabsorption and metabolism by the splanchnic tissuesof cattle and sheep have been obtained, although farfewer measurements are available for lactating dairycows. In contrast to the Beltsville data set describingenergy metabolism, which was obtained using similarexperimental techniques at one location, measure-ments of splanchnic metabolism from individual trialsand laboratories are subject to considerable experimen-tal variance that must be accounted for in any mathe-matical integration of the data. Sources of variationinclude factors such as catheter sampling tip place-ment, sampling frequency, animal management, andanalytical techniques. However, one overriding conclu-sion from the available data is the extremely intensemetabolic activity of the splanchnic tissues. In studiesin which both splanchnic and whole body oxygen con-sumption have been measured simultaneously, thePDV and liver account for 40 to 55% of body oxygenconsumption, with the 2 tissue beds accounting forroughly equal portions. In addition, oxidative metabo-lism of splanchnic tissues accounts for a large portionof heat increment, as well as differences in the efficiencyof energy use between forages and concentrates. Theintense oxidative metabolism of these tissues is high-lighted by the fact that the PDV and liver account foronly 10 and 3%, respectively, of body mass. This highrate of metabolism reflects the numerous service func-

tions performed, including digestion and absorption,synthesis of large amounts of protein, and, in liver,glucose and urea synthesis. High rates of oxygen con-sumption and carbon dioxide production require highrates of blood flow; blood flow through the liver of adairy cow in early lactation can be nearly 3,000 L/h.

The intense metabolic activity of these tissues, andtheir anatomical location between sites of digestion andentry of nutrients into the arterial blood pool, has ledmany to assume that there must be considerable metab-olism of absorbed nutrients, which therefore limits theiravailability to other body tissues such as mammarygland. This assumption is supported by measurementsof net nutrient absorption across the PDV, which typi-cally demonstrate smaller quantities of individual nu-trients (VFA, glucose, amino acids) absorbed into theportal vein than have disappeared from the lumen ofthe gut. However, it is important to remember that thePDV represents a heterogeneous collection of tissues,the majority of which do not have access to nutrientsduring their absorption, but must rely on arterial bloodto provide the nutrients they require. The high rate ofblood flow across splanchnic tissues means that thesetissues receive 40% or more of cardiac output, and thushave access to the same proportion of the nutrient poolin arterial blood. When the extraction of nutrients fromarterial blood is accounted for, rates of absorption ofVFA, glucose, and essential amino acids from the lumenaccount for a much greater portion of net absorption,which indicates considerably less metabolism duringnutrient absorption than previously assumed. Al-though a substantial use of some nonessential aminoacids and VFA occurs during their absorption, theirnitrogen and carbon are in part repackaged as alanineand ketone bodies, which are made available to othertissues. Although in liver there may be a substantialextraction of individual amino acids from blood, thislargely represents use of amino acids derived from arte-rial blood, rather than metabolism of amino acids dur-ing their first pass through the liver after absorptioninto the portal vein. Absorbed propionate and n-buty-rate are extensively removed by the liver, but arelargely repackaged as glucose and β-hydroxybutyrate,respectively, which are released to the periphery.

The observation that splanchnic tissues derive themajority of their nutrient requirements from the arte-rial pool has important implications for mathematicalmodels of nutrient metabolism. Rather than restrictingentry of nutrients, it now appears that the splanchnictissues largely compete with other body tissues for nu-trients from the same arterial blood pool. Consequently,metabolism of nutrients in PDV and liver will be subjectto the same regulatory controls as for other tissues, andis in part determined by the supply of nutrients from the

diet relative to demand. Therefore, for future models ofnutrient use to predict the use of absorbed nutrients,the propensity of the mammary gland and other tissuesfor productive nutrient use, or the “pull” effect, mustbe represented mathematically. Numerous studies ofthe use of amino acids supplied via postruminal or in-travenous infusion have found that unless amino acidsare limiting production, the provision of supplementalessential amino acids has a greater effect on liver ureaproduction and urinary nitrogen excretion than on milkprotein output. This should not be surprising to anyonewith a rudimentary knowledge of amino acid nutritionin nonruminants. Similarly, increased supply of glucoseand other energy substrates does not necessarily “push”more milk component yield, but may increase body en-ergy deposition instead. The response will very muchdepend on the productive and physiological state of thecow, as demonstrated, for example, by the effects ofexogenous bovine somatotropin administration.

Over the last 25 yr, measurements of splanchnic me-tabolism have illuminated the “black box” of nutrientabsorption and metabolism within the cow, and an in-creasingly robust database of nutrient flux across thePDV and liver with which to parameterize mechanisticmodels of nutrient use has been generated. These mea-surements are crucial to our understanding of the ef-fects of diet composition on nutrient absorption andliver metabolism. For example, patterns of VFA absorp-tion into blood can only be assessed using multicathe-terization techniques, as the liver removes most of theVFA absorbed during first-pass metabolism. Similarly,patterns of gut and pancreatic hormone release intothe portal vein are not necessarily reflected by changesin their peripheral concentrations due to extensive liverremoval from portal vein blood. When mathematicalmodels fail to predict patterns of nutrient metabolismobserved in vivo, e.g., net acetate release by the liver,then reasons for that failure can be addressed by includ-ing additional levels of control at appropriate points inenzymatic pathways.

Multicatheterization techniques have also been usedto address hypotheses relevant to specific aspects ofdigestion and metabolism in ruminants. A nonexhaus-tive list of examples include the considerable body ofwork addressing the capacity for postruminal starchdigestion and glucose absorption, the extent of VFAmetabolism by ruminal tissues, the effect of increasedammonia absorption on liver metabolism, rates of nitro-gen cycling between the PDV and liver, and studies offactors influencing amino acid absorption and metabo-lism by the PDV and liver of lactating dairy cows. Al-though many important research hypotheses have beenaddressed by these studies, in many cases their greatestcontribution is the enhanced understanding they pro-

vide of basic metabolic processes, which may have morelong-term benefit than the answers they provided tothe questions that originally justified the research.

PROGRESS IN UNDERSTANDINGTISSUE METABOLISM

In parallel with the major advancements provided bymulticatheterization studies, the past quarter-centuryhas seen progress in characterizing and quantifyingnutrient metabolism by the tissues of key organs, in-cluding the mammary gland, adipose tissue, and liver.Two major outcomes of this research have been realizedthat complement nutrient flux data. One is the quanti-tative integration of endogenous nutrients provided bymobilization of lipids from adipose tissue and proteinfrom skeletal muscle and reproductive tract with di-etary nutrients provided by the digestive tract. Thesecond is the quantitative modeling of responses of themammary gland and other tissues to differences in nu-trient supply brought about by metabolic events in sup-port tissues.

In vitro tissue approaches can provide useful esti-mates of in vivo enzymatic activities and responses tocontrol mechanisms. For example, R. L. Baldwin andcolleagues at the University of California at Davis usedin vitro techniques to better establish fundamental nu-trient use by the mammary gland. As one example, theyused slices of mammary tissue incubated in vitro withvarious concentrations and combinations of key sub-strate molecules such as glucose, acetate, and lactate.By using a range of concentrations of substrates thatwere labeled with radioactive tracers in different molec-ular positions, they were able to provide quantitativeestimates of kinetic constants for metabolism of sub-strates through various important pathways such asthe citric acid cycle, the pentose phosphate pathway,and de novo lipogenesis. These kinetic data were thenused to expand their mathematical models of mammarymetabolism. This group also used arteriovenous concen-tration differences in blood across the mammary glandto provide estimates of uptake and metabolism of aminoacids and energy-yielding substrates by the mam-mary gland.

Metabolic activities of tissues that support milk pro-duction can also be determined using in vitro tech-niques. During the 1980s, John McNamara and col-leagues at Washington State University established anextensive quantitative description of adipose tissue me-tabolism over the entire lactation cycle, and how func-tions of lipid synthesis and mobilization are affected bystage of lactation, parity, and diet composition. Theirapproach was to measure responses of subcutaneousadipose tissue obtained by repeated biopsy across de-

fined timepoints in lactation from cows of different ge-netic backgrounds and fed at different energetic intensi-ties. Use of tissue slices incubated with radiolabeledsubstrates showed, for example, that fatty acid synthe-sis from acetate at 60 d of lactation was lower for cowsof high genetic merit for milk production than for thoseof lower genetic merit. Lipolytic responses generallywere higher throughout lactation than during the dryperiod and were enhanced in high genetic merit ani-mals. Together, the corresponding changes in lipid syn-thesis and lipid mobilization in adipose point to thepredisposition for high genetic merit cows to divertmore energy toward milk synthesis than their lower-yielding contemporaries. The Washington State Uni-versity scientists also integrated tissue-level metabo-lism with key enzymatic and endocrine control mecha-nisms. Although not the direct focus of this article, atremendous increase in knowledge of physiology andthe role of the endocrine system in partitioning use ofenergy-yielding nutrients and amino acids has beencentral to enhanced understanding of fundamental nu-trition; advances in endocrine physiology are discussedelsewhere in this issue.

The previous 25 yr has seen an explosion of knowl-edge on metabolism in the liver of dairy cows, a substan-tial portion of which has been generated using in vitroapproaches. Knowledge of changes in direction andmagnitude of various pathways of fatty acid, glucose,and amino acid metabolism has progressed substan-tially, driven largely by the need to understand adapta-tions of the liver during the transition from pregnancyto lactation as discussed earlier. During the peripartur-ient period, the liver becomes flooded with NEFA fromadipose tissue that must be oxidized, converted to ke-tone bodies, or reesterified to TG (Figure 2). Researchover the last 25 yr has clearly shown that the low rate ofsynthesis and secretion of very low density lipoproteins(VLDL) by ruminants favor accumulation of TG in thecell when TG is being actively synthesized. Groups atthe University of Wisconsin, Iowa State University,University of Illinois, Michigan State University, Cor-nell University, and Purdue University among othershave used various in vitro systems, including isolatedhepatocytes, liver slices, or tissue homogenates to quan-tify metabolic pathways as affected by substrate andmetabolite supplies, hormonal influences, and physio-logical status of the donor animal. Findings from thiscollection of research have contributed to enhanced un-derstanding of the integration of carbohydrate, fattyacid, and amino acid metabolism in healthy and dis-eased states. Baldwin’s group from the University ofCalifornia at Davis used similar approaches to thatdescribed for mammary slices to obtain estimates ofkey kinetic parameters needed to model hepatic metab-

olism. Together with whole-animal, splanchnic metabo-lism, and molecular data, tissue metabolism data ob-tained during the last 25 yr have greatly enhanced ourunderstanding of fundamental nutrition in dairy cattle.In the future, in vitro approaches can be used to addresssome of the questions raised by the multicatheteriza-tion studies, and vice versa.

PROGRESS IN MOLECULAR REGULATIONOF METABOLISM

Background and Context

During the past 25 yr there has been an importantshift in focus of nutrition research to include molecularbiology and genetics. The shift began when Paul Berg ofStanford University generated the first modified DNAmolecules in 1972. This pioneering work, for which Bergwas awarded the 1980 Nobel Prize in chemistry, com-bined the DNA of 2 organisms (human and Escherichiacoli bacteria) and created the first recombinant DNAmolecule. The ability to find the minimal sequence ofDNA that codes for a protein was made possible by thediscovery of reverse transcriptase, a retroviral enzymethat copies RNA to DNA, by Howard Temin and DavidBaltimore in 1970. Consequently, when mRNA is iso-lated from cells and reverse-transcribed, it generates aDNA strand that is complementary (cDNA) to themRNA made by that cell (or tissue). These milestoneachievements and many others resulted in the emer-gence of recombinant DNA technology and provided theprimary tools for analysis of mammalian gene struc-ture, function, and subsequent profiling tools now usedto understand the interplay between nutrition and themammalian genome.

The central dogma of molecular biology states thatDNA makes RNA makes protein. The application ofmolecular biology to understanding nutrient metabo-lism centers on identifying conditions that lead to theamplification of genetic information and synthesis ofseveral copies of RNA and ultimately the initiation andprogression of protein synthesis. The ultimate end pointof these processes in dairy cows is a coordinated changein cellular and tissue metabolism to maintain homeo-stasis or to undergo homeorhesis to support a new phys-iological state.

With few exceptions, every cell of the body containsa full set of chromosomes and identical genes yet onlya portion of these genes is expressed in each cell type.The genes that are expressed confer the unique proper-ties to each cell type. The regulation of gene expressiontherefore can be affected by specific controls at eachstep between DNA transcription and protein synthesis.The more elements there are in the pathway, the moreopportunities there are for control with different cir-

Figure 2. Schematic of metabolic relationships among adipose tissue, liver, and mammary gland during the transition period; NE =norepinephrine, Epi = epinephrine, CPT-1 = carnitine palmitoyltransferase-1, GPAT = glycerol-3-phosphate acyltransferase, TG = triglyceride,CoA = coenzyme A, VLDL = very low density lipoprotein.

cumstances. For example, control of expression of agene that encodes a key enzyme in metabolism may beexerted by binding of a nuclear protein or transcriptionfactors to promoter sequences within the 5′ region ofthe DNA that contains the genetic information for theenzyme. A classic example of this form of control isactivation of phosphoenolpyruvate carboxykinase(PEPCK) gene expression by glucagon, which involvesbinding of the cyclic-AMP responsive element bindingprotein to the PEPCK promoter. Occupation of theseDNA elements by a transcription factor then leads to anincrease in the rate of transcription of the gene throughrecruitment of other proteins and RNA polymerase.Conversely, a nuclear protein can also bind DNA andact to repress expression of gene transcription byblocking transcription initiation.

A second level of control exists in the structure of themRNA transcripts synthesized from the DNA template.These mRNA contain the protein coding informationas well as flanking nucleotide sequences. The 5′ and 3′untranslated sequences as well as the three-dimen-sional structure of the mRNA play an important rolein maintaining mRNA stability and the rate of associa-tion of the mRNA with the cell’s protein synthetic ma-chinery. For example, 5′ regions of pyruvate carboxyl-

ase (PC) mRNA contain elements that affect the associ-ation of PC mRNA with the ribosomal subunits andtherefore the rate of translation of the PC enzyme. Pro-teins that have been identified as regulatory enzymesfor key metabolic pathways, and the regulation of enzy-matic activity, are a third level of control. Considerablestudy in this area has helped to identify flux-generatingsteps for several metabolic processes in ruminants. Ap-plication of molecular biology tools serves to comple-ment rather than supersede earlier approaches. Fur-thermore, it is essential to appreciate that nutrientmetabolism is potentially controlled at all 3 levels andthat all levels of control can respond to changes in nutri-ent supply.

Application of molecular tools to understanding nu-trient metabolism has primarily used mRNA transcriptanalysis and linked the changes in mRNA to metabolicactivity, physiological state, or nutritional status. Inmost instances, the candidate transcripts are selectedbased on knowledge of the central importance of theencoded protein in controlling a particular metabolicprocess. Transcript analysis under these conditions pro-vides additional information on the origins of controlof the pathway. More recent use of whole-animal andtissue-specific gene knock-out and over-expression

models have provided additional insight to the impactof key metabolic reactions on whole animal and tissuemetabolism, but application of these technologies todate has mainly been limited to rodent models.

The majority of research on nutrient regulation ofgene expression involves use of DNA or RNA probes oramplification assays, such as PCR and real-time PCR.Recently developed array-based screening tools repre-sent an evolution of these basic approaches. Hybridiza-tion, or the process of joining 2 complementary strandsof DNA or RNA through base pairing is the core processnecessary for detection methods to quantify abundanceof a particular transcript. In a classical Northern blotanalysis of mRNA, a single-stranded DNA or RNA mol-ecule with a known nucleotide sequence is used to detecta complementary base sequence that has been immobi-lized to a solid support. When bound to its target, theprobe is then detected with a radioactive, fluorescent,enzymatic, or chemiluminescent molecule so it can bevisualized. A variation of this principle includes ribo-nuclease protection assays in which the probe and tar-get complex is formed in a solution and then separatedfor quantification of the mRNA transcript.

An important point to note is that analysis of candi-date genes relies on the nucleotide sequence informa-tion for the transcript of interest or the availabilityof a closely related cDNA from another species. Thesequence and assembly of genomes of various organ-isms has hastened the identification of genes that areexpressed in response to physiological state, nutritionalstatus, and hormonal changes.

Transcript profiling using DNA microarrays or candi-date gene analysis is a static measure of the pool sizeof individual mRNA in a cell or tissue and does notprovide any information on the dynamics of mRNA syn-thesis (transcription) or RNA degradation (RNA stabil-ity). Determining the rate of mRNA synthesis typicallyinvolves use of nuclear run-on assays that measurethe relative amount of new transcripts made from thepreviously initiated RNA polymerases and provides ameasure of the in vivo rate of transcription of DNA tomRNA. Posttranscriptional modification of RNAthrough the addition of a 5′ cap and a polyadenylation(polyA) tail protect RNA from exonucleases and aids inrecognition of the mRNA by the translational machin-ery of the cell. The rate of degradation of mRNA isinitiated by a gradual shortening of the polyA tail andremoval of the 5′ cap structure. The half-life of RNAis determined by in situ hybridization histochemistry,Northern blot analysis, or quantitative real-time PCRanalysis of RNA samples removed from cells treatedwith transcription inhibitors such as actinomycin D orα-amanitin. Levels of some key proteins (e.g., the trans-ferrin receptor) are regulated at the level of RNA stabil-

ity; however, measures of RNA degradation rate arepresently not easily attainable in vivo and thereforenutritional impacts on gene expression usually are onlydescribed for transcription rate and mRNA abundance.

Areas of Investigation and Major Milestonesin Dairy Cattle Nutrition Research

Although the application of molecular research toolsto improving our understanding of the regulation ofnutrient metabolism in dairy cattle has grown over thepast 25 yr it can still be considered in a state of infancy.Some of the discoveries during the past quarter-centurythat have used molecular biology techniques to betterunderstand the nutritional physiology of dairy cattleare described below as examples. In many cases, theimpetus for these investigations has been the knowl-edge of flux control and identification of key metabolicreactions identified during the first 75 yr of researchin dairy science. Increased application of molecular re-search tools toward Moe’s goal of “understanding ofhow animals respond to variations in amounts of keynutrients absorbed from the gut” is inevitable and willlead to the development of molecular reagents and pro-tocols that are specific to the bovine.

The availability of glucose or glucose precursors hasa profound effect on milk production and animal health.Control of glucose transporter expression in the gut andother tissues has been investigated in dairy cattle in anattempt to understand limitations in glucose absorptionwhen diets contain a high portion of cereal grains. Re-searchers from John Kennelly’s group at the Universityof Alberta found that sodium-dependent glucose trans-porter 1 (SGLT1) mRNA is found along the gastrointes-tinal tract including the rumen, omasum, duodenum,jejunum, ileum, and cecum of dairy cows. Infusion stud-ies revealed that the presence of glucose in the intestineincreases SGLT1 mRNA abundance by 2-fold, com-pared with a 60- to 90-fold increase in cotransporternumber and activity. These data highlight the impor-tance of translational and posttranslational modifica-tion as primary modes of regulation of glucose absorp-tive capacity.

Facilitated glucose transporters are proteins thatfunction in the postabsorptive exchange of glucose be-tween blood and tissues. The glucose transporter(GLUT) isoforms are membrane proteins derived froma family of closely related genes that differ in theirtissue expression. The GLUT-1 protein is considered aubiquitously expressed glucose transporter and GLUT-1 mRNA has been found in all bovine tissues examinedexcept liver. Expression of GLUT-1, -2, -3, -4, and -5in bovine follow the tissue-specific expression patternsthat have also been observed in humans, whereas ex-

pression of mRNA for GLUT-3 and GLUT-5 may beunique to bovine mammary gland.

Insulin-dependent glucose transport is mediatedthrough changes in compartmentalization of GLUT-4within cells and GLUT-4 gene expression. The fact thatGLUT-4 is not expressed in bovine mammary epithelialcells supports a lack of insulin-responsive glucose up-take by mammary tissue as confirmed using other ap-proaches. A portion of the effect of somatotropin to in-crease milk yield involves a repartitioning of glucoseaway from adipose tissue and muscle and toward mam-mary gland through changes in muscle GLUT-4 expres-sion. Although GLUT-3 and GLUT-5 have been identi-fied in bovine liver, the expression of GLUT-7, the pri-mary transporter of glucose from the hepatocyte, hasnot been confirmed. In nonruminants, GLUT-7 servesto transport glucose-6-phosphate to the endoplasmicreticulum where glucose-6-phosphatase acts to releasefree glucose into the cytoplasm. Bovine liver may offerunique opportunities to study the regulation of GLUT-7 when one considers the importance of hepatic glucone-ogenesis in lactating dairy cows and that glucokinaseactivity, the primary opposing reaction to cellular glu-cose release, is essentially absent.

A fundamental response to nutrient supply is achange in signals that regulate metabolic pathwayssuch as hormones or the direct and indirect actionsof key nutrients and metabolites to alter metabolismthrough changes in gene expression. Nutrient depriva-tion is one of the most effective experimental tools inidentifying genes that respond to nutritional status,due perhaps to the close association between nutrientsupply and hormonal status. Likewise, feed restrictionprotocols have been used to study the feedback mecha-nisms that control feed intake. The decrease duringshort-term feed deprivation in circulating concentra-tions of cholecystokinin and glucagon-like peptide 1,gastrotintestinal hormones that may play a role in feed-ing behavior, is due to a reduction in their mRNA abun-dance at the duodenum and ileum. Yves Boisclair’s re-search group and others have shown that leptin, a hor-mone secreted primarily by adipose tissue, plays a rolein energy homeostasis and several other physiologicalfunctions in dairy cattle. Leptin mRNA is expressed inseveral fat depots as well as mammary parenchyma.Circulating leptin concentrations are reduced in cattleafter 24 h of feed deprivation and in cattle during thetransition to lactation. The decrease in leptin concen-tration is associated with a decrease in leptin mRNAin adipose tissue. The response to leptin depends onthe tissue distribution and level of expression of itsreceptor in target tissues. The long-form and short-formof the leptin receptors are expressed in a tissue specificmanner in dairy cattle and expression patterns of the

2 forms likely determine the physiological actions ofleptin in those tissues, especially when coupled withthe complement of hormones and growth factors thatmodulate its activity.

As discussed earlier, considerable progress has beenachieved in understanding the physiology associatedwith the transition to lactation and a model of the un-derlying molecular mechanisms that accompany thishomeorhetic adaptation is beginning to emerge. Therecognition that fatty liver is prevalent in dairy cowsand can impair lactation performance coupled with therecognition of a reduced rate of export of TG from rumi-nant liver has prompted research on molecular pro-cesses associated with secretion of lipids as VLDL. Adecrease in expression of apolipoprotein B100 in peri-parturient dairy cows is consistent with a reduction inVLDL synthesis and suggests that liver lipid infiltra-tion may be partially regulated at a transcriptionallevel. Other studies have eliminated steps of the VLDLassembly process such as microsomal transfer proteinmRNA expression as contributors to this pathology.

Cloning and characterization of PEPCK, a key en-zyme for gluconeogenesis in dairy cattle, reveals a closeassociation between the cytosolic form of PEPCK andactivity of the enzyme in bovine liver, similar to nonru-minants. Expression of PEPCK is increased as lactationprogresses toward peak milk and is increased in re-sponse to somatotropin at the level of transcription ofthe gene. Similarly, the cloning and characterization ofPC, a key enzyme in oxaloacetate regeneration in thecell and in gluconeogenesis from lactate and alanine,indicates a link between enzyme activity and mRNAexpression in liver from transition cows. Increased PCmRNA abundance at calving supports an increased fluxof lactate and alanine carbon to glucose measured usingtransorgan balance techniques as well as increased fluxof alanine to glucose measured in liver slices (Figure3). The presence of six 5′ transcript variants for bovinePC is consistent with an additional level of regulationof PC activity through changes in the translational effi-ciency of mRNA as also observed in nonruminants.

The recognition that fatty acids are regulators of me-tabolism and act in the cell nucleus to control expressionof genes for fatty acid synthesis has been extended toinvestigations on the origins of milk fat depression indairy cows. For example, Kennelly’s group at the Uni-versity of Alberta found that the reduction in milk fatwhen fish oil is fed is linked to decreased mRNA abun-dance of acetyl CoA carboxylase, fatty acid synthase,and stearoyl-CoA desaturase (also known as ∆9-desa-turase). Bauman’s group at Cornell showed that thepotent action of trans-10, cis-12 CLA to reduce milk fatsynthesis in lactating dairy cows involves a reduction inmRNA for acetyl CoA carboxylase, fatty acid synthase,

Figure 3. Example of how data from different studies using differ-ent approaches can provide complementary information to the samemetabolic issue. These data demonstrate that contribution of alanineand lactate to glucose synthesis is greater around calving in dairycows, and that this contribution likely is regulated by the key enzymepyruvate carboxylase. The top panel shows the maximum amount ofglucose made by the liver that could be synthesized from lactateor alanine as measured by splanchnic nutrient flux techniques inmulticatheterized cows (data from Reynolds et al., 2003). The middlepanel shows the rates of conversion of radiolabeled alanine (expressedas micromoles per hour per gram of wet liver) to glucose in liver slicesobtained by biopsy (data from Overton et al., 1998). The bottom panelshows relative abundance of mRNA for pyruvate carboxylase (PC)and phosphoenolpyruvate carboxykinase (PEPCK) measured in liverobtained by biopsy (redrawn from Greenfield et al., 2000). Relativeconversion of lactate and alanine, both of which are converted topyruvate for subsequent conversion to glucose, is greater around andshortly after calving, which corresponds to greater expression of PC.

stearoyl-CoA desaturase, lipoprotein lipase, fatty acidbinding protein, and glycerol phosphate acyltransfer-ase. These changes are directly linked to a substantialreduction in lipogenic activity in explant cultures invitro. These data provide understanding of the molecu-lar basis for milk fat depression, a practical milk pro-duction problem that has been recognized for severaldecades.

The majority of advances in our understanding ofnutrient regulation of gene expression have focused oncandidate gene analysis. Microarray technology has en-abled investigation of genome-wide transcript profilesto determine the expression patterns of thousands ofgenes simultaneously. Early reports of application ofmicroarray technology for identifying differential geneexpression patterns with physiological state and nutri-tional status appear promising and considerable effortis underway currently by several research groups. Re-finements in sample handling, statistical analysis, andhierarchical clustering of expressed transcripts and de-velopment of bovine-specific metabolic maps will un-doubtedly lead to a greater understanding of the com-plex molecular basis of nutrient metabolism in supportof milk production.

FUTURE PERSPECTIVES

Considerable progress has been made toward thegoals of better understanding and prediction of ab-sorbed nutrient use envisioned by Moe and others 25yr ago. However, much remains to be learned, and itseems doubtful that available technology and mathe-matical representations alone will achieve the “HolyGrail” of predicting the subtleties of productive re-sponses (e.g., milk protein or fat yield) to changes indiet composition, environmental influences, and physio-logical state. Indeed, the marriage of measurements ofthe “omics” technologies (genomic, proteomic and meta-bolomic responses) with measurements of tissue metab-olism will be enlightening, and the identification of keyresponses to changes in diet, management, physiologi-cal state, and genetics may well lead to technologies forpredictive screening of the propensity for productivenutrient use in individual cows in a practical setting.

A shift in focus of nutrition research toward molecu-lar biology and genetics coupled with development oftools to study the response of the genome to nutritionhas led to a blended discipline known as nutritionalgenomics or “nutrigenomics.” Application of this ap-proach to dairy cattle nutrition is currently in its in-fancy and will build on the candidate gene analysis thatforms the bulk of our knowledge of molecular regulationof nutrient metabolism. There are high expectationsthat nutrigenomics, when coupled successfully with

more traditional methodologies, will provide previouslyunreachable information on the dynamics of metaboliccontrol in response to nutrient supply, will serve toclarify the causal factors of metabolic disorders, and willshape future feeding strategies to enhance productiveefficiency and animal well-being during the next 25 yr.

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