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The objective was to determine the effects of abomasal infusion of linseed oil on liver triglyceride (TG) accumulation and adipose tissue lipolysis during an experimental protocol for induction of fatty liver. Eight nonpregnant, nonlactating Holstein cows were randomly assigned to treatments in a replicated 4 x 4 Latin square design. Treatments were abomasal infusion of water (W), tallow (T), linseed oil (LO), or half linseed oil and half tallow (LOT) at a rate of 0.56 g/kg of body weight per day. Each experimental period consisted of a 4-d fast concurrent with administration of treatments into the abomasum in 6 equal doses per day (every 4 h). Cows were fed ad libitum for 24 d between periods of fasting and lipid infusion. Infusion of linseed oil (LO and LOT) increased alpha-linolenic acid (C18:3n-3) content in serum (12.2, 10.4, 4.2, and 4.6 g/100 g of fatty acids for LO, LOT, T, and W, respectively), but not in the nonesterified fatty acid (NEFA) fraction of plasma. Treatments had no effect on plasma NEFA concentrations. Abomasal infusion of lipid increased in vitro stimulated lipolysis in subcutaneous adipose tissue, compared with W (4,294, 3,809, 4,231, and 3,293 nmol of glycerol released x g(-1) tissue x 2 h(-1) for LO, LOT, T, and W, respectively), but there was no difference between fat sources. Hepatic TG accumulation over 4-d fast was 2.52, 2.60, 2.64, and 2.09 +/- 0.75 microg of TG/microg of DNA for W, LO, LOT, and T, respectively, which did not differ. Abomasal infusion of LO did not reduce liver TG accumulation, plasma NEFA concentration, or alter in vitro adipose tissue lipolysis when compared with T. These results contrast with a previous study involving i.v. infusion of lipid emulsion derived from LO. Discrepancies might be explained by the use of different administration routes and a relatively modest induction of liver TG accumulation in the current experiment.

Two experiments were conducted to evaluate if supplementing rumen-protected choline (RPC; Reashure, Balchem Encapsulates, Slate Hill, NY) could prevent or alleviate fatty liver in dairy cattle. The first experiment evaluated the effect of supplementing RPC on hepatic triacylglycerol (TAG) accumulation during fatty liver induction. Twenty-four dry cows between 45 to 60 d prepartum were paired by body weight (BW) and body condition score (BCS) and randomly assigned to control or supplementation with 15 g of choline as RPC/d. From d 0 to 6, before treatment application, all cows were fed 1.4 kg/d of concentrate and forage ad libitum. Samples of blood and liver, obtained during the pretreatment period, were used for covariate adjustment of blood metabolites and liver composition data. During fatty liver induction (d 7 to 17), cows were fed 1.4 kg/d of concentrate with or without supplementation with RPC, and forage intake was restricted, so cows consumed 30% of the total energy requirements for pregnancy and maintenance. Supplementation with RPC during fatty liver induction did not affect plasma glucose and plasma beta-hydroxybutyrate (BHBA) concentration but did decrease plasma nonesterified fatty acid (NEFA; 703 vs. 562 microEq/L, SE = 40) and liver TAG accumulation (16.7 vs. 9.3 microg/microg of DNA, SE = 2.0). In the second experiment, we evaluated the effect of supplementing RPC on the clearance of liver TAG when cows were fed ad libitum after the induction of fatty liver by feed restriction. Twenty-eight cows between 45 and 60 d prepartum were paired according to BCS and BW and assigned to treatments. Fatty liver was induced by feeding 1.4 kg/d of concentrate (without RPC) and restricting forage intake, so cows consumed 30% of maintenance and pregnancy energy requirements for 10 d. From d 11 to 16, after feed restriction, cows were fed forage ad libitum and 1.4 kg/d of concentrate with or without RPC. Treatments were not applied during fatty liver induction; however, following feed restriction, liver for cows assigned to control and RPC treatments contained 6.8 and 12.7 microg of TAG/microg of DNA, respectively. Measurements obtained before treatment served as covariates for statistical analysis. During the depletion phase, plasma glucose, BHBA, and NEFA were not affected by treatment. Liver TAG, expressed as covariate adjusted means, was 6.0 and 4.9 microg/microg of DNA (SE = 0.4) on d 13, and 5.0 and 1.5 microg/microg of DNA (SE = 0.9) on d 16 for control and RPC, respectively. Rumen-protected choline can prevent and possibly alleviate fatty liver induced by feed restriction.

We conducted three experiments to determine the effects of nutritional and hormonal status on microsomal triglyceride transfer protein (MTP) activity and mass. In experiment 1, 18 nonlactating Holstein cows, 75 d before expected calving date, in their second gestation or greater were monitored from d 75 to 55 prepartum. Cows were fed a control diet from d 75 to 62 prepartum for covariable measurements. From d 61 to 55 prepartum, six cows continued to receive the control diet, six cows were restricted to 2.3 kg of grass hay/d, and six cows were fed the control diet plus 1.8 kg of concentrate/d and 500 ml of propylene glycol given 2 times/d as an oral drench. Plasma glucose and serum insulin concentrations were highest in cows that received propylene glycol and lowest in feed restricted cows. Plasma nonesterified fatty acids (NEFA) and liver triglyceride (TG) concentrations were highest in feed restricted cows and not different between cows that received the control diet and cows that received propylene glycol. Hepatic MTP activity and mass were not affected by treatment in experiment 1. In experiment 2, bovine hepatocytes isolated from the caudate process of five preruminating Holstein bull calves were incubated with either 0, 0.5, 1.0, or 2.0 mM NEFA for 48 h. Intracellular TG increased linearly as NEFA concentration in the media increased. Concentration of NEFA in the incubation media had no effect on MTP activity or mass. There was a quadratic effect of concentration of NEFA in the incubation media on MTP mRNA. In experiment 3, bovine hepatocytes isolated from the caudate process of five preruminating Holstein bull calves were incubated with 2 mM [1-14C]oleate for 24 h to accumulate TG, followed by a 36-h period of TG depletion, during which hepatocytes were incubated with no hormone, 10 nM insulin, or 10 nM glucagon. There was no effect of insulin or glucagon on intracellular TG, MTP activity or mass. Cells incubated with no hormone had higher levels of MTP mRNA compared to cells incubated with insulin or glucagon during the depletion period. Results suggest that hepatic MTP mRNA may be affected by TG accumulation, insulin, and glucagon in vitro. However, hepatic MTP activity and mass are not affected by nutritional status of nonlactating dairy cows, TG accumulation in vitro, or insulin and glucagon in vitro.

We determined the relationship between microsomal triglyceride transfer protein (MTP) (activity, mass, and mRNA) and liver triglyceride concentration in 16 dairy cows (13 multiparous and three primiparous) from 27 d before expected calving (d -27) to 35 d postpartum (d 35), the time period when fatty liver is most likely to develop. In addition, dry matter intake, plasma nonesterified fatty acids (NEFA), and plasma glucose were monitored. There were no significant parity x time interactions. Dry matter intake, plasma NEFA, plasma glucose, and liver triglyceride were significantly affected by day of sampling. Dry matter intake was 10.7, 8.0, and 19.5 kg/d on d -27, 2, and 35, respectively. Plasma NEFA concentration was higher on d 2 (1113 microEq/L) compared with d -27 (201 microEq/L) and 35 (358 microEq/L). Plasma glucose concentration was 63.3, 54.3, and 57.8 mg/dl on d -27, 2, and 35, respectively. Hepatic triglyceride (TG) concentration increased from 1.8 to 11.8% liver TG (DM basis) on d -27 and 2, respectively. There was no difference between hepatic triglyceride concentration on d 2 and 35. There was a significant effect of day of sampling on hepatic MTP activity and mRNA. Hepatic MTP activity decreased from 2.08 to 1.79 nmole triolein transferred/ h per mg of microsomal protein on d -27 and 2, respectively, and increased from 1.79 to 2.17 nmole triolein transferred/h per mg of microsomal protein on d 2 and 35, respectively. Hepatic MTP mRNA increased from d -27 to 2 and remained elevated from d 2 to 35. There was no effect of day of sampling on MTP mass. There were no significant correlations between hepatic MTP activity, mass, or mRNA with either liver TG or plasma NEFA on any of the sampling days. The cause of a decrease in hepatic MTP activity and increase in mRNA on d 2 is unknown. However, the lack of correlation between MTP activity, mass, or mRNA with either liver TG or plasma NEFA on d 2 postpartum suggests that MTP probably does not play a role in the etiology of fatty liver that occurs in dairy cows at calving.

Two experiments were conducted to test the following two hypotheses: 1) fatty liver could hamper hepatic conversion of ammonia to urea and increase circulating ammonia or Gln% [Gln% = Gln x 100/(Gln + Glu)] in cows around parturition; 2) decreased ureagenesis might cause alkalosis and in turn reduce blood Ca. In the first experiment, 14 Holstein cows were monitored from 27 d prepartum to 35 d postpartum. There was a rise in circulating ammonia and Gln% at calving, suggesting an increase in ammonia passing to and through the liver. Stepwise regression analysis revealed the following relationship for plasma samples at 22 h and liver triglyceride at 2 d postpartum: ammonia (microM) = 32.1+/-0.89 triglyceride (% DM), Gln% = 71.2 + 0.23 triglyceride (% DM) + 1.31 urea (mM). The positive correlation between liver triglyceride and plasma ammonia and Gln% suggests that hepatic triglyceride accumulation might inhibit ureagenesis, thereby increasing ammonia concentration at the perivenous hepatocytes where Gln synthesis occurs and increasing ammonia concentration in blood leaving the liver. In the second experiment, 28 rats were used to determine whether hepatic triglyceride accumulation, induced by choline deficiency, affects urinary ammonia N and blood pH homeostasis. There was a trend for a positive correlation between urinary ammonia N and liver triglyceride. No correlation between liver triglyceride and blood pH, bicarbonate, pCO2 or plasma Ca was found. In conclusion, hepatic triglyceride accumulation may inhibit ureagenesis and result in increased circulating ammonia, Gln% and urinary ammonia N in vivo. Hepatic triglyceride accumulation did not affect blood pH homeostasis.

Two trials were conducted to examine the effects of supplemental methionine, provided as methionine hydroxy analog 13 g/d), or fat (454 g of calcium salts of long-chain fatty acids/d) on hepatic triglyceride concentration. In the first experiment, methionine hydroxy analog or fat was fed during feed restriction to determine if hepatic triglyceride accumulation is affected. The objective of the second experiment was to determine if feeding fat or methionine hydroxy analog influences the rate of triglyceride depletion from the liver of cows in positive energy balance following the induction of fatty liver by feed restriction. In experiment 1, feeding methionine hydroxy analog decreased plasma glucose, increased plasma nonesterified fatty acids, and had no effect on liver triglyceride. Feeding fat increased plasma nonesterified fatty acids and increased hepatic triglyceride during the 10-d feed restriction period. In experiment 2, feeding fat decreased the rate of triglyceride depletion from liver when cows were allowed to resume ad libitum consumption of feed; methionine hydroxy analog had no effect. Results of these studies indicate that feeding supplemental fat or methionine hydroxy analog at levels tested does not prevent or alleviate fatty liver induced by feed restriction.

Plasma NEFA concentrations increase prior to and at parturition, resulting in increased fatty acid uptake by the liver, fatty acid esterification, and triglyceride storage. Liver triglyceride concentration increases four- to fivefold between d 17 prior to calving and d 1 following calving. Increases in liver triglyceride following calving do not appear to be dramatic. Severity of fatty liver 1 d postpartum is correlated negatively with feed intake 1 d prepartum. Export of newly synthesized triglyceride as very low density lipoprotein occurs slowly in ruminants and is a major factor in the development of fatty liver. Nutritional strategies to minimize the elevation in plasma NEFA prior to calving results in lower liver triglyceride at calving. Fatty liver probably precedes clinical spontaneous ketosis. Liver triglyceride to glycogen ratio may be used to predict susceptibility of cows to ketosis. Consequently, strategies to reduce liver triglyceride at calving may decrease incidence of ketosis. Research to determine methods to reduce fatty acid delivery to the liver or to enhance hepatic export of very low density lipoprotein near calving is warranted. Identification of the cause for the slow rate of assembly and secretion of hepatic very low density lipoprotein in ruminants will be required to assess the feasibility of increasing export of very low density lipoprotein.

Plasma glucose concentration during late gestation was thought to be important for the development of fatty liver near parturition. Thirteen multiparous cows were given a 1-L oral drench of propylene glycol once daily beginning 10 +/- 3.6 d prepartum until parturition. Eleven control cows received a 1-L water drench. Plasma glucose increased following propylene glycol administration. Plasma NEFA concentration was 403 and 234 microM, and plasma insulin concentrations were .354 and .679 ng/ml, for control cows and cows treated with propylene glycol measured from 1 to 7 d prepartum. Plasma NEFA tended to be lower in cows treated with propylene glycol from 1 to 21 d postpartum. Prepartum propylene glycol administration reduced hepatic triglyceride accumulation by 32 and 42% at 1 and 21 d postpartum, respectively. Prepartum plasma BHBA was reduced during propylene glycol administration. Prepartum plasma glucose, NEFA, BHBA, and insulin were strongly correlated with liver triglyceride at 1 d postpartum (r = -.49, .45, .36, and -.49, respectively). Pre- and postpartum DMI were not affected by treatment. Milk production and composition measured through 21 d postpartum were not different between groups.

Two trials were conducted to determine if estrogen contributes to development of fatty liver in dairy cattle. During trial 1, eight late lactation, nonpregnant cows were assigned to 0 or 15 mg estradiol-17 beta benzoate/d treatment. Days 1 to 3 of the trial were for baseline measurements, and treatments were given from d 4 to 21; on d 20 and 21 animals were fasted. Short-term feed deprivation resulted in increased plasma FFA concentrations and rapid accumulation of triglyceride into liver tissue obtained by biopsy. During starvation, plasma FFA concentration and liver triglyceride content were lower for cows receiving the estradiol-17 beta treatment relative to cows receiving control treatment. Very low density lipoprotein concentration in blood increased dramatically in three of four animals during estradiol-17 beta administration. Because of the decrease in milk production during estradiol-17 beta treatment, it was not known whether this represented a decrease in very low density lipoprotein clearance from blood or reflected a lipotropic response to estradiol-17 beta. Therefore, a second trial was conducted employing nonlactating cows, and control and estradiol-17 beta-treated animals were pair fed. The trial was 33 d with d 1 to 3 for baseline measurements, and treatments were administered from d 4 to 33. All animals were starved from d 19 to 23. Estradiol-17 beta increased hepatic lipid and triglyceride accumulation and plasma very low density lipoprotein concentration during starvation. Plasma FFA concentration was also increased by estradiol-17 beta during this time; therefore, a direct or indirect effect of estrogen on hepatic lipid metabolism could not be delineated.

Previous research has implicated linseed oil in altering lipid metabolism of dairy cattle when infused intravenously as lipid emulsion. The objective was to determine the effects of abomasal linseed oil (LO) infusion on liver triglyceride (TG) accumulation and subcutaneous adipose tissue lipolysis during fatty liver induction. Eight nonpregnant, nonlactating Holstein cows were randomly assigned to treatments in a replicated 4 x 4 Latin square design. Treatments included abomasal infusion of water (W), tallow (T), LO, or half tallow plus half LO (LOT) at a rate of 0.56 g/kg BW per day for 4 d. Daily treatments were administered directly into the abomasum, divided into 6 doses and administered every 4 h. Infusion of treatments was concurrent with a 4 d fast. Cows were fed ad libitum for 24 d between periods of fasting and lipid infusion. T contained 43% C18:1, 24% C16:0 and 14% C18:0. LO contained 51% C18:3, and 17% C18:2. For most parameters, contrasts were: W vs. LO + LOT + T (effect of lipid), LO vs. T (linear effect of LO), and LOT vs. LO + T (nonlinear dose effect of LO). For measurement of blood fatty acid composition, differences among treatment means were determined using the PDIFF procedure of PROC MIXED. Infusion of linseed oil (LO or LOT) increased α-linolenic acid (g/100 g fatty acid) in serum (P < 0.05) but not in the nonesterified fatty acid (NEFA) fraction of plasma. Treatments had no effect on plasma NEFA concentrations. Infusion of LO increased total serum fatty acid concentration (nonlinear dose effect; P = 0.06). Abomasal infusion of lipid significantly increased isoproterenol stimulated lipolysis by subcutaneous adipose tissue explants in vitro. Liver TG content increased 4.8, 4.9, 2.3 and 2.3 μg TG/ μg of DNA during the fast for W, LO, LOT and T, respectively; infusion of lipid decreased liver TG accumulation (P = 0.06) when compared to W, but this was due to T (LO vs. T; P = 0.03). Abomasal LO infusion failed to reduce liver TG accumulation, plasma NEFA concentration or alter subcutaneous adipose tissue lipolysis. [ABSTRACT FROM AUTHOR]

The objective was to determine the effects of intravenous infusion of triacylglycerol (TAG) emulsions derived from different lipid sources on energy metabolism during a 4-d fast. Six nonpregnant, nonlactating multiparous Holstein cows were randomly assigned to treatments in a replicated 3 x 3 Latin Square design. Treatments included intravenous infusion of tallow, linseed oil, or fish oil emulsions at a rate of 0.54 g of TAG/kg of body weight per day; infusions were concurrent with a 4-d fast. The emulsions were administered for 20 to 30 min every 4 h throughout the 4-d fast. Cows were fed ad libitum for 24 d between the fast/infusion periods. Infusion of tallow, linseed oil, or fish oil emulsions increased plasma concentrations of palmitic acid, linolenic acid, and eicosapentaenoic and docosahexaenoic acids, respectively. Infusion of linseed oil emulsion decreased plasma TAG concentrations compared with tallow and fish oil treatments, which were similar. Infusion of the tallow emulsion resulted in the highest concentrations of plasma nonesterified fatty acid (NEFA), insulin, and glucose, whereas the infusion derived from linseed oil had the lowest NEFA and β-hydroxybutyric acid concentrations. The different TAG emulsions had no effect on total or peroxisomal oxidation of [1-14C]oleic acid in liver homogenates. Liver TAG content increased 12.0, 7.8, and 14.1 µ/µg of DNA during the fast for tallow, linseed oil, and fish oil treatments, respectively; linseed oil was different from fish oil and tended to be different from tallow. [ABSTRACT FROM AUTHOR]