Nutritional Management of Ketosis and Fatty Liver in Dairy Cows

Nutritional Management of Ketosis and Fatty Liver in Dairy Cows José E.P. Santos and Leandro F. Greco Department of Animal Sciences University of Florida Gainesville-FL Clinical and Subclinical Ketosis Subclinical Excess of ketones in the absence of clinical signs of disease (β-oh-butyrate > 14 mg/dl) Clinical Elevated blood concentration of ketone bodies (> 27 mg/dl) in association with hypoglycemia (Glucose < 45 mg/dl) with clinical signs of disease Anorexia, rumen atony, dry feces, decreased milk yield, nervous ketosis Lactational Incidence and Prevalence 30 Prevalent metabolic 25 disorder in high producing di dairy cows 20 Lactational incidence in first 9 wks 43% (BHBA > 15 mg/dl) % 15 10 5 0 0 2 4 6 8 10 wk postpartum Duffield et al. (2002) 1

Ketone bodies CO 2 O CH 3 -C-CH 3 O Acetone CH 3 -C-CH 2 -COO - Acetoacetate NADH + H NAD + 3-OH-butyrate dehydrogenase OH CH 3 -C-CH 2 -COO - β-oh-butyrate * β-oh-butyrate is incorrectly classified as a ketone body Ketone bodies (β-oh-butyrate) Normally produced during β-oxidation of butyrate by rumen wall Supply energy Adipose tissue synthesis Milk fat synthesis Brain during starvation Integral part of normal ruminant intermediary metabolism Synthesis and Utilization of Ketones Liver Blood Extra-hepatic tissues Acyl-CoA A FFA Acetyl-CoA Glucose Ketones Urine Milk Ketones Glucose Acyl-CoA Acetyl-CoA TCA Lungs Ketones TCA 2

Causes Inadequate energy intake Increased triacylglycerol mobilization Excessive incomplete hepatic FA oxidation Dry Matter Intake and Plasma NEFA 66 DMI NEFA 1000 lb/d DMI, 44 33 22 800 600 400 NEFA A, um 11-20 -10 0 10 20 30 Day relative to calving 200 Bertics et al. (1992) Fatty Liver or Hepatic Lipidosis Increased accumulation of triglycerides in the liver Diagnosed by liver biopsy No specific blood test 3

Classification of types of fatty liver % Triglyceride Category by wet tissue weight Normal healthy <1 Mild fatty liver < 5 Moderate fatty liver 5-10 Severe fatty liver >10 Gaal et al., 1983 Severe form is associated with serious effects on health and production Poor Transition Can Result in Metabolic Disorders Fatty liver Drackley (2004) Concentrations of Triglyceride and Glycogen in Hepatic Tissue of Transition Dairy Cows t (% wet wt) Componen 5 4 3 2 1 0 Triglyceride Glycogen -40-30 -20-10 0 10 20 30 40 50 60 70 Days relative to calving Data from Underwood (2003) 4

How prevalent is fatty liver? Moderate 20%, 33%, 33%, 40%, 45%, 48%, 53%, 65% Severe 5%, 5%, 11%, 14%, 15%, 15%, 15%, 20%, 24% May affect over half to two-thirds of lactating cows! Incidences reported from 9 studies around the world Summary from Drackley (2006) Fatty Liver Can Happen in Days Cow with NEFA concentration in plasma of 1.0 mm in the day of calving Assuming that palmitic acid (C 16 H 32 O 2 ) is the main NEFA in blood : 1 mm = 256 mg/l Removal of NEFA depends upon its concentration in plasma and the blood flow to the liver Hepatic blood flow of 1,000 L/h with 256 mg of NEFA/L = 256 x 1000 x 24 h = 6.1 kg of NEFA flowing through the liver in 24 h 20 to 30% extraction efficiency 1.2 to 1.8 kg of FA extracted by the liver in a single day for a cow with plasma NEFA of 1.0 mm Bovine liver : 8 to 10 kg or 3 to 4 kg of dry weight Liver triacylglycerol can increase in 5 to 10% units in a single day Therefore, hepatic lipidosis can occur in 1 to 2 days 5

Liver TG, % (DM basis) 30 25 20 15 10 5 0 7.1 Control Force Fed 22.8 15.8-17 -1 Day Relative to Calving Bertics et al. (1992) BHBA and Immune Function 21 Grinberg et al. (2006 ) Infect. Immun. 76: 2802 2807 Transition Period High energy requirements for milk synthesis Coordinated adaptations Increased sensitivity of adipose tissue Insulin resistance High blood GH Increased adrenergic receptors on the adipocyte cell membrane Decreased hepatic glycogen and increased lipid content Glycogenolysis associated with influx of NEFA High energy demands associated with low energy intake in presence of hormonal shifts hepatic lipidosis 6

Adipose (Fat) Tissue Healthy well-fed cow Liver NE, Epi NEFA Insulin NEFA NEFA TG CO 2 Propionate Mitochondria Milk Fat Glucose TG Mammary Gland VLDL TG Modified from Drackley, 1999 Adipose (Fat) Tissue Cow in negative energy balance Liver NE, Epi NEFA Insulin NEFA NEFA TG Propionate Ketone Bodies Mitochondria Milk Fat Glucose TG Mammary Gland VLDL TG Modified from Drackley, 1999 Hormones Trigger Mobilization of Stored Triacylglycerols [glucose] Epinephrine and glucagon Lipid droplets in adipocytes is coated with perilipins, preventing lipid mobilization. 1. When [glucose] is low in blood, it triggers the release of glucagon, which binds its receptor in the adipocyte membrane stimulates adenylyl cyclase, via a G protein, to produce camp. 2. This activates PKA 3. PKA phosphorylates the HSL 4. PKA phosphorylates perilipin molecules on the surface of the lipidid droplet. This allows HSL access to the surface of the lipid droplet 5. HSL hydrolyzes triacylglycerols to FFA. 6. FAs leave the adipocyte, bind serum albumin in the blood, and are carried in the blood; 7. Enter tissues via a specific fatty acid transporter. 8. In tissues (liver, muscle), FA are oxidized to CO 2, and the energy of oxidation is conserved in ATP 7

Working Model for Lipolysis in Dairy Cattle Koltes and Spurlock (2011) J. Dairy Sci. 94: 1839-1848 Hepatic FA Metabolism Ruminant liver Little hepatic lipase activity (LPL) Low capacity to export VLDL Oxidation of FA: main route for FA disposal Ketogenesis becomes extremely important Mitochondria and peroxisomes Regulation of ketogenesis 3 levels Adipose tissue level: lipolysis FA entry into the mitochondria (inner membrane is not permeable to acyl CoA) Acetyl CoA can undergo ketogenesis or complete FA oxidation: energy expenditures of the liver 8

Adipocyte containing TAG Fatty Acid Fatty Acid Hydrolysis of the ester bond Fatty Acid ll membrane Ce Insulin Albumin Free Fatty Aci id HSL Glycerol + 3 Fatty Acids + Glucagon and cathecholamines Fatty Acids Are Activated and Transported into Mitochondria (size dependent) FA 12C cross the mitochondrial membrane without transporter protein FA 14C use carnitine shuttle to enter mitochondria: carnitine acyltransferase is inhibited by [malonyl-coa] FA is transiently attached to carnitine to form Fatty Acyl-carnitine catalyzed by CAT I in the outer mitochondrial membrane. Fatty acyl group is transferred from carnitine to intramitochondrial coenzyme A by CAT II. B-oxidation then proceeds Ketogenesis Hormone regulation Insulin/glucagon NEFA HSL Adipose Tissue Hepatocyte NEFA Malonyl CoA AcCoA carboxylase Acetyl CoA Fed state - Low E state Carnitine transport Fed state β-oxidation Acyl-CoA Acetyl-CoA Aceto Acetate Esterification i - TAG VLDL E + CO 2 Β-OH-butyrate dehydrogenase CO 2 Acetone β-oh-butyrate 9

Why Ketogenesis vs Complete Oxidation? ATP synthesis is reduced (liver does not need to increase its energy and O 2 expenditure) Ketonescanbeexported from the liver and used as energy source by other tissues (muscle, brain, mammary gland, etc) Ketones are water-soluble (no need for albumin to transport). Albumin is low early postpartum Possible lack of OAA in the mitochondria (not demonstrated in ruminants) gluconeogenic enzymes are upregulated in periods of low energy supply catalase Heat To cytosol then to mitochondria When FFA Uptake is Extensive, Hepatocyte Increases Peroxissomal β-oxidation Peroxissomal β-oxidation allows the tissue to dispose of excess of FA Generates less ATP per mol of FA oxidized First oxidative step, electrons pass directly to O 2, generating H 2 O 2, catalase (heat) and the NADH formed in the second oxidative step are exported to the cytosol, eventually entering mitochondria. Very long chain FA stimulates peroxissomal oxidation. High dietary fats induces the synthesis of the enzymes of peroxisomal oxidation in the liver. Liver peroxisomes do not contain the enzymes of the citric acid cycle long-chain or branched FA are catabolized to shorter-chain products (hexanoyl-coa), which are exported to mitochondria and completely oxidized. Insulin Affects Tissues Differently Muscle Uptake of glucose and immediate use (exercise) or storage as glycogen (exercising muscles can also take up glucose without insulin) Liver Liver Uptake of glucose and storage as glycogen Inhibits glycogen phosphorylase Activates glycogen synthase Inhibits gluconeogenesis Promotes re-esterification of FA to TAG (some excess glucose conversion to fatty acids in ruminants) Adipose Tissue Promotes glucose uptake and conversion to glycerol for fat production 10

Insulin Control Gastrointestinal hormones Amino acids Pancreas Insulin Beta cells Blood glucose Most Cells Protein synthesis Muscle Glucose uptake Glycogen synthesis Adipose Glucose uptake Glycerol production Triglyceride breakdown Triglyceride synthesis Liver Glucose uptake Glycogen synthesis Fatty acid synthesis Glucose synthesis amino acids triglycerides glucose Brain No effect Feedback Effects of Glucagon Prevents hypoglycemia Stimulates glycogenolysis Stimulates gluconeogenesis Increases with exercise/stress independent of blood glucose Exerts effects through camp second messenger system (phosphorylation of target enzymes) Glucagon Control Exercise Amino acids Pancreas Alpha cells Epinephrine (stress) Adipose Triglyceride breakdown Triglyceride storage Liver Glycogen breakdown Glucose synthesis Glucose release Fatty acids Blood glucose Brain No effect Feedback 11

Hepatic lipidosis Multiparous cows > Primiparous Greater TAG infiltration into the liver in multiparous Althoughketosismight g not follow the same pattern Decreased ureagenesis: TAG infiltration decreases ureagenesis independent of other metabolic changes during transition Decreased gluconeogenesis in some, but not all studies Approaches to Minimize Ketosis and TAG Accumulation in the Liver Reduce TAG mobilization Diets that promote propionate synthesis Gluconeogenic precursors Increase FA oxidation by the liver Carnitine and up regulation of peroxissomal oxidation Increase VLDL export by the liver Choline Shorten the Dry Period: Attempting to Put the Cow in a More Favorable Energy Status 12

1. Control (n=21) 56 d dry period 28 d low energy diet, 28 d mod. energy diet 2. Shortened Dry Period (n=23) 28 d dry period High energy diet 3. Continuously Milked (n=21) 0 d dry period High energy diet (Rastani et al., 2005) Energy Balance Energy Balance (Mcal/d) Prepartum 15 10 5-5 -10-15 Postpartum 0-10 -8-6 -4-2 0 2 4 6 8 10 56 d 28 d 0 d Week Relative to Calving Rastani et al., 2005 Liver TG g DNA Liver TG, ug/ug 30 25 20 15 10 5 0 56 28 0 Tx*day: P = 0.06 28 vs. 0: P < 0.02-30 1 35 Day Relative to Calving Rastani et al., 2005 13

Effect of Propylene Glycol on Liver Lipids and TG Studer et al. (1993) Studer et al. (1993) % (DM basis) 33 30 27 24 21 18 15 12 d +1 d +21 % (DM basis) 17 14 11 8 5 2 d +1 d +21 Control PG 1L/d Control PG 1L/d MONENSIN Active against Gram + microorganisms (in excessive concentrations can also inhibit Gram -) Diverges C and H partition in the rumen Reduces methanogenesis Increases propionate production at the expense of C 2 and C 4 Reduces CO 2 and CH 4 production Modifies rumen ph Alters pattern of DM intake and concentrations of lactate Alters rumen N metabolism Reduces proteolysis and deamination (reduces rumen N-NH 3 ) BHBA, mg/dl 11 P < 0.001 10 9 8 7 6 Control Monensin 5 4 14 7 14 50 600 500 P < 0.04 400 Arieli et al. (2008) J. Dairy Sci. NEFA, µeq/l 300 200 100 Control Monensin 0 14 7 14 50 14

Impact of Monensin CRC on Hepatic Composition (Triacylglycerol and Glycogen) TG % 20 18 16 14 12 10 8 Monensin Placebo P = 0.12, % Glycogen 1.5 1.2 0.9 0.6 Monensin Placebo Monensin effect: P = 0.02 6 4 0.3 2 0 24 hours 3 weeks 0.0 24 h 3 weeks Time relative to calving Time relative to calving Zahra et al. (2004) J. Dairy Sci. Study Cow Health Ketosis Risk ratio (95% CI) % Weight Wilson_A (1992) 0.98 (0.06,15.07) 0.4 MH Relative Risk = 0.75 (0.63, 0.90) Wilson_B (1992) 0.32 (0.01,7.58) 0.6 Beckett (1998) 0.96 (0.06,15.29) 0.4 Duffield (1998) 0.50 (0.17,1.46) 3.9 Vallimont_A (2001) 0.65 (0.18,2.38) 1.7 Vallimont_B (2001) 0.36 (0.04,3.03) 1.1 Duffield (2002) 0.64 (0.31,1.30) 7.5 Green_A (2004) 0.74 (0.48,1.15) 15.9 Green_B (2004) 0.71 (0.45,1.10) 15.8 Green_C (2004) 0.86 (0.56,1.30) 15.8 Green_D (2004) 0.45 (0.24,0.86) 10.6 Green_E (2004) 1.13 (0.70,1.82) 10.4 Green_F (2004) 0.80 (0.48,1.35) 10.8 Zahra_A (2006) 0.36 (0.08,1.67) 2.3 Zahra_B (2006) 0.96 (0.30,3.07) 2.0 Petersson-Wolfe_A (2007) 2.00 (0.19,21.28) 0.4 Petersson-Wolfe_B (2007) 1.96 (0.18,20.83) 0.4 Overall (95% CI) 0.75 (0.63,0.90).013325 1 75.0479 Risk ratio Duffield et al. (2008) J. Dairy Sci. 91:2328 2341 Ruminally Protected Amino Acids (Methionine) AA can be used as gluconeogenic precursors Might enhance oxidation of fatty acids by the hepatic tissue Might enhance VLDL synthesis and secretion (phospholipid synthesis and/or apolipoprotein synthesis) Might reduce ketogenesis Supply limiting amino acids for milk and milk protein synthesis 15

Effect of Supplemental Methionine Analog (Hydroxy Methyl Butanoic Acid) on Hepatic Metabolism Item Treatment Hepatic TG, mg % NEFA, meq/l Glucose, mg/dl Reference Control 23.0 0.270 61.2 Bertics and Grummer, 1998 13 g Met 20.0 0.346 59.4 Control 12.7 0.820 58.0** Bertics and Grummer, 1997 13 g Met 15.4 1.076** 50.3 Effect of CP/RUP on Hepatic TG of Multiparous Transition Cows Treatment (Diet CP, %) Hepatic TG, % 11.7 (26.5 % RUP) 15.5 (43.2% RUP) 20.6 (51.5% RUP) Prepartum 0.55 0.75 1.04 Postpartum 5.6 7.0 7.0 Huyler et al. (1999) Rumen-Protected Choline Choline is necessary for synthesis of the main phospholipids Phosphatidil choline (lecithin) Lisophosphatidil choline Lecithin is required for synthesis and secretion of hepatic VLDL and intestinal chylomicrons Deficiency can result in fatty liver 16

Impact of Rumen-Protected Choline on Fatty Liver Treatament Fatty liver Control Choline Induction NEFA, µeq/l 703 562* Hepatic TG µg/µg DNA 16.7 9.3* Recovery Day 3, % of previous 60.4 52.2 Day 6, % of previous 48.5 29.9 * P < 0.05 Cooke et al. (2007) J. Dairy Sci. 90:2413-2418 Effect of RPC on clinical ketosis of Dairy Cows P = 0.001 30 Control RPC 25 20 P = 0.01 15 10 P = 0.05 5 0 Ketonuria Clinical Relapses Lima et al. Vet. J. (2012) in press doi.org/10.1016/j.tvjl.2011.09.019 Effect of Feeding RPC on Hepatic Composition in Dairy Cows at 9 DIM Treatment Control RPC P Primiparous Multiparous Primiparous Multiparous TRT PAR TRT x PAR DM, % 63.9±0.04 45.5±0.05 62.6±0.04 57.7±0.05 0.23 0.01 0.13 Glycogen As is, % 0.95±0.20 0.92±.23 1.15±0.22 1.13±0.25 0.36 0.91 0.99 DM, % 1.70±0.48 2.32±0.54 1.96±0.52 2.44±0.60 0.72 0.31 0.89 Triacyglycerol As is, % 4.99±1.56 6.74±1.76 3.33±1.68 4.83±1.94 0.31 0.36 0.94 DM, % 8.10±.29 12.69±2.59 4.87±2.48 7.17±2.86 0.10 0.19 0.66 Hepatic lipidosis, 1 % 28.6 (4/14) 54.6 (6/11) 8.3 (1/12) 22.2 (2/9) 0.05 0.12 0.65 1 Hepatic triacylglycerol > 5% of wet tissue (Gaal et al., 1983). Lima et al. Vet. J. (2012) in press doi.org/10.1016/j.tvjl.2011.09.019 17

Plasma Choline (mg/dl) on d 1 Postpartum and Hepatic Triacylglycerol (Log 10 of % of wet weight) on d 9 Postpartum, Control (, ) and RPC (, ) Linear relationship (P < 0.001) Control, Log 10 of hepatic triacylglycerol = 1.541 0.01133 x plasma choline (mg/dl), r 2 = 0.28 RPC, Log 10 of hepatic triacylglycerol = 1.599 0.009576 x plasma choline (mg/dl), r 2 = 0.64. Controlling caloric intake prepartum Overfed Cows Consumed 56% more Calories Prepartum than Required based on NRC NEL Intak ke (Mcal/d) 45 40 35 156% of requirement 30 25 20 15 No effect on intake postpartum 10 Overfed Limit-fed 5 81% of reqt. 0-10 -8-6 -4-2 0 2 4 6 8 10 12 14 16 Weeks relative to calving Douglas et al. J. Dairy Sci. (2006) 18

Overfeeding During the Dry Period Increases Hepatic TG After Calving 8 ride (%) Triglyce 6 4 2 Overfed Limit-fed 0-75 -50-25 0 25 50 75 Days from parturition Douglas et Douglas al. J. Dairy et al. Sci. (2006) (2006) Prepartum caloric intake of cows fed diets with differing Forage/NE L content Prepartum caloric intake, Mcal/d Reference Low Energy High Energy P Douglas et al. (2007) 11.6 16.2 0.001 Douglas et al. (2006) 11.5 22.0 0.001 Rabelo et al. (2003; 2005) 17.77 21.5 0.001001 Doepel et al. (2002) 17.3 20.6 0.05 Hayirli et al. (2011) 15.1 20.5 0.001 Janovick and Drackley (2011) 14.3 19.1 0.01 Kanjanapruthipong et al. (2010) 14.7 18.8 0.01 Average 14.6 19.8 Effect of prepartum caloric intake on milk yield Milk Yield, kg/d Reference Low Energy High Energy P Douglas et al. (2007) 34.4 39.9 0.12 Douglas et al. (2006) 42.4 40.4 0.20 Rabelo et al. (2003; 2005) 38.0 37.3 0.67 Doepel et al. (2002) 34.8 37.2 0.22 Hayirli et al. (2011) 34.8 34.4 0.70 Janovick and Drackley (2011) 29.9 33.2 0.10 Kanjanapruthipong et al. (2010) 26.3 29.0 0.03 Average 34.4 35.9 19

Effect of prepartum caloric intake on FCM yield Milk Yield, kg/d Reference Low Energy High Energy P Douglas et al. (2007) 35.6 37.9 NS Douglas et al. (2006) 40.8 39.8 NS Rabelo et al. (2003; 2005) 38.5 40.4 0.59 Doepel et al. (2002) 39.1 40.3 NS Hayirli et al. (2011) 33.7 35.2 0.27 Janovick and Drackley (2011) 40.5 46.1 0.09 Kanjanapruthipong et al. (2010) 26.1 28.4 0.04 Average 36.3 38.3 Effect of prepartum caloric intake on BHBA postpartum BHBA, mg/dl Reference Low Energy High Energy P Douglas et al. (2007) 5.2 4.7 0.56 Douglas et al. (2006) 4.8 6.0 0.05 Rabelo et al. (2003; 2005) 5.4 5.0 50 0.45 Doepel et al. (2002) ~10 ~10 NS Hayirli et al. (2011) 11.6 11.4 0.96 Janovick and Drackley (2011) 4.5 6.6 0.01 Kanjanapruthipong et al. (2010) 6.1 3.8 0.04 Average 6.8 6.8 Far Off Cows Item Multiparous Primiparous BW, kg 650 570 BW 0.75, kg 129 117 Net energy, Mcal/day Maintenance 10.3 9.3 Growth 0.5 1.0 Conceptus growth 4.0 4.0 Weight gain at 0.2 kg/day 1.0 1.0 Total, Mcal/day 15.8 15.3 Metabolizable protein, g/day Maintenance 570 560 Growth 240 240 Conceptus 30 80 Total, g/day 840 880 DM intake, kg/day 13 11 Diet NE L, Mcal/kg DM 1.22 1.39 MP required, g/kg DM 64.6 80.0 Diet CP, % DM 10.8 13.3 20

Far Off Dry Cows Dry-off until ~ 255 d of gestation Ration considerations NE L ~ 1.30 to 1.35 Mcal/kg for maintenance of BCS (15 to 17 Mcal/d) Do not overfeed calories (dietary NDF ~ 50 to 55% to limit caloric density and feed intake) Supply sufficient metabolizable protein (850 to 900 g/day) Evidence of increased insulin resistance during the immediate peripartum period with overfeeding during faroff period (Dann et al., 2003) Trying to regain BCS can be a problem during this period of lactation 14 Effect of Fatness on DMI kg / day 12 10 8 6 Thin Cows Fat Cows -21-18 -15-12 -9-6 -3 0 Days From Calving Adapted from Hayirili, 1998 Correlations- Rabelo et al.(2003) Liver TG, d1 r P < Energy intake, final wk PP.05.70 Energy balance, final wk PP.09.47 Energy intake change, wk -4 vs d-1.45.0004 Energy balance change, wk 4 vs d-1.45.0004 Plasma NEFA change, final wk.46.0007 21

Summary Avoid obese cows at dry off reproductive and dietary management during lactation Do not overfeed dry cows during the early dry period Maximize DM intake during the last 3 weeks of gestation and first 3 of lactation, but minimize drop in intake prepartum Close up period Group cows and heifers separately Close up diets with moderate NFC (30 to 35%) and NDF (38 to 45%) Add monensin at 300 mg prepartum and 24 ppm postpartum in the first 3 weeks of lactation Use additives if necessary (300 ml of PPG and 15 g/d of rumen-protected choline) Watch acidogenic salts in close up rations (urine ph & [Cl - ] in the diet) Minimize early postpartum health problems Catch and treat ill animals early (effective fresh cow protocols) 22