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Feeding 2-Hydroxy-4-(Methylthio)-Butanoic Acid to Periparturient Dairy Cows Improves Milk Production but not Hepatic Metabolism^*

¹ Department of Animal Science, Cornell University, Ithaca, NY 14853
² Novus International, Inc., St. Louis, MO 63141

Corresponding author: T. R. Overton; e-mail: tro2{at}cornell.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Forty-eight Holstein cows, entering second or later lactation, were utilized to determine the effects of 2-hydroxy-4-(methylthio)-butanoic acid (HMB) on milk production, hepatic lipid metabolism, and gluconeogenesis during the periparturient period. Cows were fed one of 3 diets as TMR starting 21 d before expected calving. These diets contained 0 (the basal diet), 0.09 (+HMB), or 0.18 (++HMB)% HMB. From parturition to 84 DIM, cows were fed diets that contained 0, 0.13, or 0.20% HMB. Prepartum and postpartum dry matter intakes were similar among cows fed the basal diet, +HMB and ++HMB. There was a quadratic effect on milk yield such that cows fed +HMB had the greatest milk yield; yields of milk by cows fed the basal diet and ++HMB were similar. This led to trends for increased yields of 3.5% fat-corrected milk and total solids when cows were fed +HMB. Percentages of fat, protein, and total solids in milk were not affected by treatment. Despite differences in milk yield, calculated energy balance was not affected by treatment. Plasma concentrations of NEFA, ß-hydroxybutyrate, and glucose were not different among treatments. Liver triglyceride content was similar among treatments on d 1 postpartum and was increased for cows consuming +HMB on d 21 postpartum compared with the other dietary treatments. Capacities for metabolism of [1-¹⁴C]palmitate by liver slices in vitro were not affected by treatment; however, conversion of [1-¹⁴C]propionate to CO₂ and glucose decreased as the amount of HMB consumed by cows increased on d 21 postpartum. Cows consuming +HMB had greater days to first ovulation compared with cows consuming the basal diet and ++HMB as measured by plasma progesterone concentrations. These data suggest that adding HMB to low Met diets to achieve a predicted Met supply of approximately 2.3% of metabolizable protein supply is beneficial for increasing milk production but does not appear to benefit hepatic energy metabolism during early lactation.

Key Words: dairy cow • methionine • gluconeogenesis • hepatic lipidosis

Abbreviation key: HMB = 2-hydroxy-4-(methylthio)-butanoic acid, TG = triglyceride, VLDL = very low density lipoproteins

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Requirements for energy, glucose, and amino acids increase dramatically during the periparturient period to support the onset of milk production. This requires remarkable coordination of metabolism to meet the demands of lactation (Bell, 1995). Energy requirements are met, in part, by rapid mobilization of fatty acids from adipose tissue, resulting in increased circulating concentrations of NEFA. Although these NEFA can be used by other tissues for energy and for milk fat, the liver extracts NEFA in proportion to its concentration in the bloodstream (Emery, 1992; Reynolds et al., 2003), resulting in hepatic lipidosis because the capacity of ruminant liver for fatty acid oxidation and export as very low density lipoproteins (VLDL) is low relative to fatty acid uptake (Grummer, 1993). The resulting triglyceride (TG) accumulation in the liver has been suggested to reduce the capacity of the liver to detoxify ammonia to urea (Strang et al., 1998), potentially leading to decreased capacity for gluconeogenesis from propionate (Overton et al., 1999). The corollary is that strategies to decrease the accumulation of TG in liver of periparturient dairy cows may alleviate the potential inhibition of these important metabolic processes.

Given the potentially detrimental metabolic effects of accumulation of TG in liver during the periparturient period, several nutrients have been identified as potentially limiting for hepatic disposal of fatty acids either through oxidation or export (Grummer, 1993; Bauchart et al., 1998). Research conducted in our laboratory (Piepenbrink and Overton, 2003) has demonstrated that supplementation of choline to dairy cows during the periparturient period resulted in coordinated changes in fatty acid and carbohydrate metabolism consistent with alleviation of the impairment of TG accumulation on hepatic metabolism. Phosphatidylcholine plays an important role in VLDL assembly and stability of the lipoprotein particle (Vermeulen et al., 1997). Although we cannot determine whether the effects on fatty acid metabolism reported in our previous experiment (Piepenbrink and Overton, 2003) are related to VLDL synthesis and secretion, Yao and Vance (1988) reported that hepatocytes isolated from rats fed a choline-free diet and supplemented in vitro with either choline or Met increased their secretion of phosphatidylcholine and export of TG as VLDL.

Methionine is another potentially limiting nutrient for hepatic metabolism of fatty acids during the periparturient period. Although most commonly noted for its role as one of the two most limiting AA for production of milk and milk protein (Schwab, 1994), suggestions that Met as DL-Met or Met hydroxy analog may have a role in hepatic lipid metabolism and ketosis have existed for more than 30 yr (McCarthy et al., 1968; Waterman and Schultz, 1972). Methionine is required for synthesis of carnitine (Combs, 1992), which is required for the transport of long-chain fatty acids across mitochondrial and peroxisomal membranes, allowing the fatty acids to undergo oxidative processes (Zammit, 1999). Emmanuel and Kennelly (1984) found that 28% of radioactivity from L-[methyl-¹⁴C]Met was recovered as choline in lactating goats, indicating that choline biosynthesis is an important fate of Met fed to ruminants. Furthermore, Met is required for synthesis of apolipoprotein B100 (apoB100; Grummer, 1993), which is essential for synthesis and secretion of VLDL particles. Despite these possible roles, few data exist that describe the effects of Met on hepatic metabolism in ruminants. In an experiment with limited replication, Durand et al. (1992) demonstrated that infusing Met and Lys into the portal vein of early-lactation cows caused the hepatic balance of VLDL to be positive, whereas the balance of VLDL was negative during both the preinfusion and postinfusion periods in these cows. In a subsequent experiment conducted during the first 4 wk of lactation, cows fed ruminally protected Met had similar concentrations of TG and apoB100 in liver compared with controls, whereas those fed both ruminally protected Met and Lys had lower concentrations of TG and higher concentrations of apoB100 in liver compared with controls (Bauchart et al., 1998). Bertics and Grummer (1999) fed Met hydroxy analog to cows that were subjected to fatty liver induction by feed restriction and found that cows fed Met hydroxy analog had higher plasma concentrations of NEFA, and neither accumulation nor depletion of TG within the liver were affected by feeding Met hydroxy analog. Collectively, these data do not allow evaluation of the role of Met in hepatic fatty acid metabolism independent of Lys supply.

We hypothesized that hepatic fatty acid metabolism and cow performance might be sensitive to Met supply during the periparturient period and early lactation. Therefore, the objective of this experiment was to determine the effects of increasing Met supply on hepatic metabolism, key metabolites in blood, and cow performance by feeding increasing amounts of a Met analog (DL-2-hydroxy-4-methylthiobutanoic acid; HMB) to periparturient cows fed diets formulated to be deficient in Met and adequate in Lys.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The Cornell University Institutional Animal Care and Use Committee approved all procedures involving animals. Forty-eight Holstein dairy cows, entering second or greater lactation, were moved into individual tie stalls approximately 28 d before expected parturition and fed for ad libitum intake a common prepartum diet (Basal, Table 1) once daily as TMR. On d 21 before expected parturition, cows were assigned to one of 3 diets containing increasing amounts of HMB (0, 0.09, and 0.18% of DM for the basal diet, +HMB, and ++HMB, respectively; Alimet feed supplement, Novus International, St. Louis, MO) in a completely randomized design. After parturition, cows were fed postpartum diets (Table 1) containing HMB at 0, 0.13, and 0.20% of DM (for the basal diet, +HMB, and ++HMB, respectively) until 84 d of lactation. Cows assigned to each of the 3 prepartum treatments were maintained on their respective treatments during the postpartum period. Pre- and postpartum basal diets were formulated using the CPM Dairy (version 1.0) ration evaluator and were found to be deficient in Met and adequate in Lys as described by NRC (2001). Additions of HMB were made to the basal diet to achieve projected Met concentrations in metabolizable protein of 2.34 and 2.70% for prepartum diets and 2.36 and 2.63% for postpartum diets (+HMB and ++HMB, respectively; Table 2). The +HMB diets were formulated to bring Met concentrations close to NRC (2001) requirements for cows in established lactation, and the ++HMB diet was formulated to provide Met in excess of the recommendations, given the potential additional requirement for Met during hepatic metabolism of fatty acids. The source of HMB used in this experiment was estimated to have a ruminal escape value of 40% when given as a single bolus to ruminants (Koenig et al., 2002).

Cows were milked 3x per day at approximately 8-h intervals, and milk weights were recorded using in-line meters (DeLaval, Kansas City, MO). Milk was sampled from each milking during one 24-h period each week and composited. The composite samples were stored at 4°C with a preservative (bronopol tablet; D & F Control System, San Ramon, CA) until analyzed for fat, protein, and lactose using infrared analysis (AOAC 2000: method 972.160), and SCC by an optical fluorescent method (AOAC 2000: method 978.26) (Dairy One Cooperative Inc.). The calibration reference methods for the infrared milk analysis were as follows: fat by modified Mojonnier ether extraction (AOAC 2000: method 989.05), Kjeldahl true protein (AOAC 2000: method 991.22), and lactose by difference of oven drying total solids (AOAC 2000: method 990.20) minus the sum of modified Mojonnier ether extraction, Kjeldahl true protein (AOAC 2000: method 991.22), estimated nonprotein nitrogen (0.19% on a protein basis), and estimated ash ([true protein x 0.596] +0.5379). Body weights of cows were measured weekly. Two individuals assigned BCS to cows weekly (1 = thin, 5 = fat; Wildman et al., 1982).

Blood samples were collected by venipuncture of the coccygeal vein/artery using heparinized Vacutainer tubes (Becton Dickinson, Franklin Lanes, NJ). Samples were obtained on 1 d during the 7 d before assignment to treatment, and then every other day from approximately 21 d prior to expected parturition to 60 d after parturition. Sampling time (approximately 0900 h) corresponded to the time after orts were removed, after lactating cows returned from the parlor, and prior to feeding. Blood samples were placed on ice immediately following collection. Plasma was harvested after centrifugation of the blood at 2060 x g for 15 min at 5°C. Plasma was stored at -20°C until subsequent analysis for glucose by enzymatic analysis (glucose oxidase) using a commercial kit (kit 510-A; Sigma Chemical). Plasma concentrations of NEFA were analyzed by enzymatic analysis (NEFA-C; WAKO Pure Chemical Industries, Osaka, Japan) using modifications described by McCutcheon and Bauman (1986) and Sechen et al. (1990). Plasma concentrations of BHBA were quantified (BHBA dehydrogenase) using a commercial kit (kit 310-UV; Sigma Chemical). All spectrophotometric measurements were conducted using a Versa_max tunable microplate reader (Molecular Devices, Sunnyvale, CA). Inter-assay variation was maintained at <5%. Only samples obtained through 30 d of lactation were analyzed for metabolites. Postpartum plasma samples were analyzed for progesterone by radioimmunoassay (Elrod and Butler, 1993). The assay limit of detection was 0.2 ng/mL. Inter- and intra-assay coefficients of variation were 11.9 and 15.7%, respectively.

Liver samples were obtained by percutaneous trochar biopsy (Veenhuizen et al., 1991) from each cow on 1 d during the week prior to assignment to treatment, 1 d after calving, and again 21 d after calving. Total liver removed was approximately 3 g for each biopsy. Liver was blotted to remove excess blood and connective tissue. A portion of the liver was frozen in liquid nitrogen for subsequent analysis for content of TG and glycogen (described below). The remaining liver was transported to the laboratory in ice-cold PBS (0.2 mM; pH 7.4) and used immediately to determine hepatic capacities for fatty acid metabolism and gluconeogenesis using an in vitro metabolic incubation system (described below).

Liver Composition
The concentration of glycogen in liver was determined using a procedure that has been previously described (Lo et al., 1970). Liver TG content was determined using a modification of the procedure described by Rukkwamsuk et al. (1999a). Liver (between 35 and 50 mg) was weighed into disposable tubes, and 0.5 mL of 20% KOH (wt/vol) was added. Liver was allowed to digest overnight. Vortexing the tubes after 1 h improved consistency of digestion by ensuring complete submersion of the liver sample. The next day, samples were neutralized with 0.5 mL of a 10% H₂SO₄ (vol/vol), followed by addition of 0.5 mL of 5% CHAPS (3-[(3-c h o l a m i d o p r o p y l) d i m e t h y l a m m o n i o ] - 1 - p r o p a n e -sulfonate; wt/vol) to emulsify lipids. For analysis of the glycerol in the sample, the Infinity Triglyceride Reagent (Sigma Chemical, St. Louis, MO) was used. Glycerol standards (Sigma #339-11) were diluted with water to obtain a standard curve from 0 to 500 mg/dL of triolein-equivalent glycerol. For the analysis, 96-well plates were used. Water was used as a blank because the absorbance of the KOH, H₂ SO₄, CHAPS mixture was not different from that of water. Prior to use, standards and samples were vortexed. To each well was added 2.5 µL of water, standard, or sample solution (in triplicate). Then 250 µL of the Infinity Triglyceride Reagent was added. Plates were mixed for 12 s and then incubated for 7 min at 37°C before being read at 660 nm in a microplate reader (Versamax; Molecular Devices Corp., Sunnyvale, CA). Triglyceride concentration was then calculated based on initial wet tissue weight and volume dilution.

Liver Incubations
Upon return to the laboratory (usually within 1 h of biopsy), liver was sliced using a Krumdieck Tissue Slicer (Alabama Research and Development, Munford, AL) filled with ice-cold PBS. Slices of tissue (54 ± 2 mg) were then weighed into flasks. For conversion of [1-¹⁴C] propionate to CO₂ and glucose, incubation procedures, previously described (Piepenbrink and Overton, 2003) were used, except that propionate incubations were terminated with 0.5 mL 25% TCA (wt/vol) instead of the H₂SO₄.

Conversion of [1-¹⁴C] palmitate to CO₂ was measured as previously described (Piepenbrink and Overton, 2003) except that these flasks were covered with rubber septa without a hanging well for the duration of the incubation period (120 min). At either 0 or 120 min, liver was removed from the medium using forceps to terminate the incubations. The liver was rinsed in 1 mL of 3% BSA in KRB (at 37°C, wt/vol), followed by a rinse in 1 mL of saline (0.9% NaCl). Rinses were added to the incubation flasks, and then a septa containing folded filter paper in the hanging well was placed on top. At this time, 1 mL of a 40% perchloric acid (wt/vol) was injected into the media and 0.1 mL of 30% NaOH (wt/vol) was injected onto the filter paper to collect CO₂. The flasks were placed in an ice bath for 1 h to ensure complete evolution of CO₂ and to limit any further metabolism.

Determination of stored esterified products.
After liver slices were rinsed, they were transferred into 16- x 125-mm glass extraction tubes that contained 5 mL of a 3:2 mixture (wt/vol) hexane:isopropanol (Hara and Radin, 1978). Tubes were then capped and placed on a wrist-action shaker overnight. The next day, tubes were centrifuged at 1800 x g for 5 min. The solvent solution was poured into disposable tubes, and another 2 mL of the hexane:isopropanol mixture was added to the tubes containing the liver tissue. Tubes were capped and vortexed for 30 s. The initial 5 mL of solvent was returned to the extraction tube, and 2 mL of a 6.7% NaSO₄ (wt/vol) solution was added to cause phase separation, as well as removal of nonlipid components from the hexane layer. The tubes were vortexed and then centrifuged at 1800 x g for 5 min. The hexane layer was removed to scintillation vials and placed in an evaporator at 37°C for a minimum of 4 h. After evaporation, 10 mL of scintillation cocktail (Scintisafe Econo 2; Fisher Scientific, Pittsburgh, PA) was added to each vial, and radioactivity was measured using liquid scintillation spectroscopy (model 2200 CA Scintillation Counter, Packard Instruments Company).

Determination of conversion of [1-¹⁴C] propionate to glucose.
Flask contents were neutralized after CO₂ collection with a saturated NaOH solution by drop-wise addition and the supernatant frozen at -20°C until isolation of glucose. To isolate glucose, the modified batch procedure of Azain et al. (1999) was used. Because of the added cation/anion load on the resins from using TCA and NaOH instead of the H₂SO₄ and Ba(OH)₂ solutions, it was found that the amount of resin added needed to be increased to give a more consistent recovery of glucose. For this, 2.2 mL of 50% (wt/vol) slurry of the anion exchange resin (AG 1-X8 resin, acetate form; Bio-Rad, Hercules, CA) in water was added in the first step. Tubes were capped and vortexed vigorously for 30 s. This was repeated 2 more times, allowing resin to settle momentarily between mixing bouts. Afterward, 2.0 mL of the cation exchange resin (AG 50W-8 resin, hydrogen form: Bio-Rad) was added. Again, the tubes were capped and vortexed as above. Tubes were then centrifuged at 1390 x g for 15 min. The resins then formed a semi-solid pellet that allowed the aqueous layer to be poured into scintillation vials. The vials were then placed into a 37°C water bath, and a steady stream of air was used to evaporate the water. The next day, 1 mL of water was added to reconstitute the glucose. Ten milliliters of scintillation cocktail (Scintisafe Econo 2) was added to each vial, and radioactivity was measured using dual label liquid scintillation spectroscopy (model 2200 CA, Packard Instrument Company). By counting an equal volume of the internal standard ([³H]L-1-glucose) that was added to each flask prior to neutralization, correction of aqueous volume trapped in the resin and losses during processing of the flasks following incubations could be determined. Recovery of the internal standard averaged 38 ± 10%.

Statistical Analysis
Two cows fed ++HMB developed mastitis due to Staphylococcus aureus infection and were removed from the study at approximately 3 wk postpartum. Their remaining data were treated as missing values. Before statistical analysis, daily measurements (DMI, milk production) were reduced to weekly means. To estimate energy balance, the NRC (2001) equations were used. For in vitro incubations, triplicate values for blanks and live flasks were averaged, and blanks subtracted from values obtained from live flasks. Rates of substrate conversion were calculated based on the known amount of radioactivity added to each flask that was converted to end products over the 2-h incubation period.

Prepartum and postpartum data were analyzed separately because treatments were equally spaced during the prepartum period and unequally spaced during the postpartum period. Performance and blood metabolite data were subjected to repeated measures ANOVA using the MIXED procedure of SAS (SAS Users Guide, 2001). Pretreatment values for all measurements, except milk yield and composition, were utilized during analysis of covariance. In the case of energy balance, pretreatment BW was used in analysis of covariance. Week or day was used in the repeated statement with cow within treatment as the error term and the autoregressive covariate structure (Littell et al., 1998). Degrees of freedom were estimated using the Satterthwaite specification. Single degree of freedom orthogonal contrasts were used to test the linear and quadratic effects of HMB supply; the models used to analyze postpartum data employed adjusted coefficients due to the uneven spacing of treatments. Liver data collected on d 1 and 21 postpartum were subjected to ANOVA using the MIXED procedure of SAS (SAS User’s Guide, 2001); the orthogonal contrasts used to analyze the d 1 liver data were based on equally spaced treatments, and those used to analyze the d 21 liver data were based on unequally spaced treatments, as data collected on d 1 postpartum are reflective of treatments applied during the prepartum period.

For determining reproductive status as affected by treatment, concentrations of plasma progesterone were used to determine days to first ovulation. Ovulation was assumed to occur when progesterone values increased by more than 2 ng for more than 2 consecutive samples. For cows that did not have an increase in progesterone before blood sampling ended, a value of 60 d was used. The data for days to first ovulation were subjected to ANOVA using the MIXED procedure of SAS (SAS Users Guide, 2001). Least squares means are reported with the highest standard error for each variable. Significance was declared at P 0.05 and trends at 0.05 < P 0.15. Only significant treatment x time interactions will be discussed.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Nutrient composition of the pre- and postpartum diets fed in this study is presented in Table 2, and the nutrient composition of individual feeds and protein mixes is presented in Tables 3 and 4, respectively. Both prepartum and postpartum diets were slightly lower than expected in CP and net energy content. Part of this discrepancy was due to variability in the content of CP and energy in forages over the course of the study. Blood meal and protected soybean meal were included in the diets to maintain a high level of Lys in the diets. As predicted by CPM Dairy, Lys accounted for more than 7.15% of metabolizable protein supply for all 6 diets.

View larger version (14K): [in this window] [in a new window]   Figure 1. Milk yield of cows consuming 3 levels of 2-hydroxy-4-methylthiobutanoic acid (HMB) from 21 d prepartum through 84 d postpartum. There was a significant quadratic effect (P = 0.05, SEM = 1.00) and a tendency for a treatment by time effect (P < 0.15).

Energy balance was not significantly different among cows fed the 3 treatments during either the prepartum or postpartum periods (P > 0.15; Figure 2). The lack of differences among treatments probably resulted from the slight, nonsignificant increase in feed intake (Table 5) as cows consumed greater amounts of HMB and small, nonsignificant, differences in milk composition (Table 5), even though cows consuming +HMB had greater milk production than cows consuming ++HMB or the basal diet.

View larger version (17K): [in this window] [in a new window]   Figure 2. Calculated energy balance of cows consuming three levels of 2-hydroxy-4-methylthiobutanoic acid (HMB) from 21 d prepartum through 84 d postpartum. Body weight measured prior to treatment assignment was used in analysis of covariance. There were no differences in energy balance between cows on the three treatments (P > 0.15, SEM = 1.27).

View larger version (16K): [in this window] [in a new window]   Figure 3. Body weights of cows consuming 3 levels of 2-hydroxy-4-methylthiobutanoic acid (HMB) from 21 d prepartum through 84 d postpartum. Weights obtained before assignment to treatment at approximately 28 d before the expected calving date of each cow were used during analysis of covariance. Effects of treatment and the interaction of treatment and time were not significant (P > 0.15; SEM = 7.8).

View larger version (16K): [in this window] [in a new window]   Figure 4. Body condition scores of cows consuming three levels of 2-hydroxy-4-methylthiobutanoic acid (HMB) from 21 d prepartum through 84 d postpartum. Body condition scores obtained before assignment to treatment at approximately 28 d before the expected calving date of each cow were used during analysis of covariance. Effects of treatment and the interaction of treatment and time were not significant (P > 0.15; SEM = 0.08).

View larger version (20K): [in this window] [in a new window]   Figure 5. Plasma concentrations of nonesterified fatty acids (NEFA) from cows consuming three levels of 2-hydroxy-4-methylthiobutanoic acid (HMB) from 21 d prepartum through 84 d postpartum. Plasma samples obtained before assignment to treatment were used during analysis of covariance. The interaction of treatment and time was not significant (P > 0.15; SEM = 78.1).

Concentrations of TG in liver were not affected (P > 0.15) by treatment on d 1 postpartum; however, cows fed +HMB tended (P < 0.15) to have increased concentrations of liver TG on d 21 postpartum (Table 8). Concentrations of glycogen tended (P < 0.08) to be affected quadratically on d 1 postpartum, such that cows fed +HMB had the lowest concentration of liver glycogen (Table 8). Concentrations of liver glycogen and 21 d postpartum tended (P < 0.10) to be decreased linearly by feeding increasing amounts of HMB (Table 8).

Bertics and Grummer (1999) concluded that there were no effects on liver TG of feeding Met hydroxy analog to nonlactating cows that were induced to develop fatty liver by feed restriction. The analog had no effect on the amount of TG in the liver or the amount of TG that was depleted from the liver 3 and 6 d after termination of feed restriction. Liver lipid content at 1 wk postpartum was not different due to feeding rumen-protected Met and Lys in one study (Socha, 1994), although other researchers (Bauchart et al., 1998) reported that the content of liver TG decreased only when both rumen-protected Met and Lys were fed to cows and not when rumen-protected Met was fed alone.

The capacity of liver slices to convert [1-¹⁴C]palmitate to CO₂ was not different (P > 0.15) among cows fed the 3 dietary treatments on either d 1 or 21 postpartum (Table 9). Likewise, the feeding of HMB did not affect the capacity of liver slices to incorporate [1-¹⁴C]palmitate into esterified products stored intracellularly on d 1 postpartum (Table 9). On d 21 postpartum, the capacity of liver slices to incorporate [1-¹⁴C]palmitate into esterified products stored intracellularly was affected quadratically (P < 0.05) by treatment, such that cows fed ++HMB had the greatest capacity to convert [1-¹⁴C]palmitate to stored esterified products. Because conversion to CO₂ was not affected by treatment, we infer that in vivo rate of oxidation of NEFA by liver probably was not affected by dietary supply of HMB. In vitro capacity for [1-¹⁴C]palmitate esterification and storage was modestly affected by HMB supplementation but only on d 21 postpartum. In the study of Pullen et al. (1989), Met hydroxy analog had no effect on the incorporation of NEFA into plasma TG, indicating that the analog probably did not alter hepatic lipoprotein synthesis. Given that neither liver TG nor in vitro capacities for metabolism of [1-¹⁴C]palmitate by liver slices on d 1 were affected by Met supplied as HMB, and effects on metabolism of [1-¹⁴ C]palmitate by liver slices on d 21 postpartum were modest at best, these data suggest collectively that hepatic metabolism of fatty acids is not sensitive to Met supplied as HMB.

Despite the apparent decrease in hepatic capacity for propionate metabolism as the amount of HMB fed to cows increased, cow performance relative to the controls was not impaired (Table 5), and cows fed +HMB that produced the most milk and most lactose also had the greatest concentrations of plasma glucose during the postpartum period (Table 7). In this study, we measured maximal capacity for gluconeogenesis from supra-physiological concentrations of propionate rather than total glucose production by liver. Danfær et al. (1995) demonstrated that infusing ruminants with either propionate or AA could change the substrate utilized for gluconeogenesis without altering total hepatic glucose production. Even if feeding HMB directly or indirectly altered the capacity of liver to metabolize propionate, it is likely that overall capacity of liver to produce glucose from all glucogenic substrates was not affected, at least for cows fed +HMB.

There was a quadratic effect (P < 0.04) for days to first ovulation as determined by concentrations of progesterone in plasma. Average days to first ovulation were 43 for cows consuming +HMB, whereas days to first ovulation were 28 and 34 for the basal diet and ++HMB, respectively (SEM = 4). Rukkwamsuk et al. (1999b) observed a correlation between concentrations of liver TG at 2 wk after calving with days to first ovulation. In this study, the longest number of days to first ovulation occurred in cows that were consuming +HMB and also produced the most milk. These cows also had higher concentrations of liver TG at 21 d postpartum. Table 10 was compiled using reported disorders recorded in individual cow health records. Cases of each disorder appear to be relatively evenly distributed across the 3 treatments.

Supplementation of both Lys and Met to dairy cows, beginning prior to parturition and continuing through early lactation, resulted in increased energy-corrected milk yield (Socha et al., 1994) and milk protein yield (Carson et al., 1998; Socha et al., 1994); however, effects on both milk protein yield and milk fat yield to Lys and Met were variable and dependent on AA status of the prepartum diet (Carson et al., 1998). Clearly, research is warranted to investigate further these interactions of peripartal diet and supply of Met and Lys on milk yield and milk component synthesis.

We hypothesized that Met supplied as HMB would improve hepatic capacity to metabolize NEFA such that the rate of accumulation of NEFA as liver TG would decrease and production responses potentially would follow. We optimized milk yield in this experiment when Met was supplied as HMB at approximately 2.3% of metabolizable protein supply during the periparturient period and early lactation when Lys was considered to be adequate, which concurs substantially with published recommendations for cows during established lactation (NRC, 2001). However, given the substantial lack of effect of Met as HMB on hepatic fatty acid metabolism, the mechanism for the increased milk yield does not appear to be related to improved energy metabolism in liver.

In addition to supplying postruminal Met, numerous effects of HMB on ruminal fermentation have been reported, ranging from increased gas production (Robert et al., 1998), CP digestion, nonammonia N flow, microbial protein efficiency (Sloan et al., 2000), bacterial protein synthesis (Vázquez-Añón et al., 2001), decreased ruminal free fatty acids and increased polar lipids (Patton et al., 1970), increased acetate/propionate ratios (Ellis et al., 1986), and increased fiber, protein, and ether extract digestibility (Polan et al., 1970). Although the mechanisms by which HMB stimulates ruminal fermentation are not well defined, effects appear to be dependent on dose of HMB. Using in vitro systems, when HMB was supplied at high doses (0.45, 0.22, and 1.45% of DM), ruminal effects were nullified (Robert et al., 1998; Sloan et al., 2000; Vázquez-Añón et al., 2001). The dual effect of HMB on stimulating ruminal fermentation and supplying postruminal Met might explain the milk yield response measured in this experiment. The increased milk yield by cows consuming +HMB might be caused by the combination of adequate supply of Met derived from HMB to the cow and the direct effect of HMB on ruminal fermentation. In cows consuming ++HMB (0.20% of DM), the effect of HMB on ruminal fermentation may have been negated. Alternatively, any benefits of increased postruminal Met derived from HMB may have been limited by postruminal Lys supply (Rulquin et al., 1993; NRC, 2001). However, in this study we did not collect data that would have allowed us to measure the postruminal supplies of Met and Lys or to find that ruminal fermentation was indeed altered by treatment.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Milk yield increased in a quadratic fashion in response to supplementation with HMB during the periparturient period. However, the lack of meaningful effects of HMB supply on metabolic parameters and long-chain fatty acid metabolism indicate that the mechanism was not related to alterations in hepatic long-chain fatty acid metabolism in periparturient cows as hypothesized. Future research should seek to determine the mechanism underpinning the increase in milk yield when moderate amounts of HMB are included in the diet.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The authors thank Papillon Agricultural Products, Easton, MD, for donating the blood meal and blending the protein mixes used in this experiment, West Central Soy, Ralston, IA, for donation of the SoyPlus used in this experiment, and Bioproducts Inc., Fairlawn, OH, for donation of the EnerG II used in this experiment. Larry Chase, Bill Chalupa, and Charlie Sniffen also are gratefully acknowledged for their input on formulation of the experimental diets fed during this experiment. The authors also thank Mary Partridge, Tom Muscato, and the staff of the Cornell Teaching and Research Center for their assistance with experiment management and daily care of the animals. Special appreciation also is extended to Ramona Slepetis, Christina Sheehan, Kristen Vyhnal, and Lisa Ruzzi for their assistance with sample collection and analysis, and liver incubations.

	FOOTNOTES

 
^* Supported, in part, by Novus International, Inc., St. Louis, Missouri, and in part, by the Cornell University Agricultural Experiment Station federal formula funds, Project No. 127453, received from Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.

Received for publication November 20, 2002. Accepted for publication August 18, 2003.

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