Determination of Rumen Degradability and Ruminal Effects of Three Sources of Methionine in Lactating Cows*

J. Dairy Sci. 88:223–237 © American Dairy Science Association, 2005.

Determination of Rumen Degradability and Ruminal Effects of Three Sources of Methionine in Lactating Cows* S. Noftsger, N. R. St-Pierre, and J. T. Sylvester Department of Animal Sciences, The Ohio State University, Columbus 43210

ABSTRACT Objectives were to quantify the ruminal effects and flows to the omasum of Met provided as 2-hydroxy-4(methylthio)-butanoic acid (HMB), the isopropyl ester of HMB (HMBi), and DL-Met. Eight ruminally cannulated cows were used in a replicated 4 × 4 Latin square design. Treatments were 1) no Met (control), 2) HMB at 0.10% of DM, 3) HMBi at 0.13% of DM, and 4) DLMet at 0.088% of DM. Diets were identical except for type of Met supplement and were based on corn silage and alfalfa hay at 30 and 13% of dietary DM, respectively. Samples of omasal fluid were used to determine the proportion of Met supplements passing out of the reticulorumen. Dry matter intake (20.1 kg/d) was restricted during the week of sampling to a maximum of 95% of ad libitum DMI determined during the first 2 wk of the period. Milk yields (37.7 ± 0.8 kg/d) and fat concentration (3.42 ± 0.15%) were not significantly different for control, HMB, HMBi, and DL-Met. Milk protein concentration (2.91, 2.95, 3.02, 2.96 ± 0.07%, respectively) was significantly increased by the HMBi treatment. Rumen volatile fatty acids profile and NH3 concentrations were similar across treatments. Apparent ruminal digestibility of organic matter and neutral detergent fiber were higher for the three diets supplemented with Met sources than for the control diet. In situ rate of digestibility of CP from alfalfa hay, TMR, and corn silage was affected by Met sources. Passage rates of small particles (0.071/h) and fluid (0.167/h) were not affected by treatments. Protozoal counts in the rumen and omasum were not significantly affected by Met sources. Proportion of omasal N from bacterial N was not different (0.54 ± 0.03), and bacterial N flow (305 ± 24.4 g/d) was similar across treatments. The proportion of HMB that passed into the omasum was 5.3 ± 1.5% of the amount consumed. Only a small amount

Received July 13, 2004. Accepted September 28, 2004. Corresponding author: N. R. St-Pierre; e-mail: st-pierre.8@ osu.edu. *Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript no. 18-04AS.

(2.3%) of HMBi was found as HMB in the omasum. These results indicate that little HMB escapes ruminal degradation through passage to the omasum and that the site of HMBi absorption must be preomasal. (Key words: methionine, rumen degradability, amino acid) Abbreviation key: ERD = effective ruminal digestibility, HMB = 2-hydroxy-4-(methylthio)-butanoic acid, HMBi = isopropyl-2-hydroxy-4-(methylthio)-butanoic acid, MP = metabolizable protein, NANBN = nonammonia, nonbacterial N. INTRODUCTION The US Environmental Protection Agency has issued regulations regarding N release into the environment, and more regulations will likely follow (Powers, 2003). To remain competitive and socially acceptable in the future, the dairy industry must be proactive in reducing N release into the environment. The N concentration in manure can be altered by diet manipulation (Noftsger and St-Pierre, 2003). Diets that are balanced for amino acids can potentially maintain and even improve milk yield and milk component production, while reducing the amount of N released into the environment (Noftsger and St-Pierre, 2003). 2-Hydroxy-4-(methylthio)-butanoic acid (HMB) can be used as an inexpensive source of Met (Dibner and Knight, 1984). Many studies using HMB have shown increases in milk fat yield (Patton et al., 1970b; Holter et al., 1972; Huber et al., 1984) and percentage (Lundquist et al., 1983; Huber et al., 1984), although some reported no effect (Hutjens and Schultz, 1971; Stokes et al., 1981). Most researchers found no effect of HMB on milk protein concentration (Hutjens and Schultz, 1971; Stokes et al., 1981; Hansen et al., 1991), although research in our laboratory (Noftsger and St-Pierre, 2003) showed an increase in milk protein concentration and yield that was greater than expected solely from the additional supply of metabolizable Met with Met fed in rumen-protected form (polymer coated Met) in conjunction with a rumen degradable source of Met (HMB). The degradable source of Met (HMB) may have a different

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mechanism of action, possibly through a stimulation of microbial growth. The lack of response in milk protein yield and milk yield in most trials may indicate that HMB is not escaping rumen microbial catabolism in significant concentrations to be used postruminally. Attempts to ascertain the degradability of HMB have provided widely varied answers, from 99% (Jones et al., 1988) to 50% degradable (Koenig et al., 1999). The changes in fat production and percentage may not be caused by postruminal supplementation of HMB. Patton et al. (1970a) suggested that HMB caused an increase in the protozoal biomass. Methionine is a methyl donor for the production of phosphatidylcholine. Because free choline is rapidly degraded in the rumen (Sharma and Erdman, 1988), protozoa are the primary suppliers of phosphatidylcholine, an important molecule used in the packaging of fatty acids into very low density lipoproteins and chylomicrons (Yao and Vance, 1988). The 18C and some of the 16C fatty acids in milk come from the triglycerides of chylomicrons and low density lipoproteins in the blood. Increases in protozoal passage would also provide more digestible microbial protein with a higher concentration of lysine than bacteria (Williams and Coleman, 1992). The isopropyl ester of HMB (HMBi) has been shown to have approximately 50% relative bioavailability compared with Smartamine using area under the plasma Met concentration curve over time after a pulse ruminal dose as the bioavailability indicator (Robert et al., 2001). A similar value of relative bioavailability has been obtained using a cow bioassay based on change in milk true protein concentration with Smartamine as a standard (Schwab et al., 2001). In a trial conducted in our laboratory (Sylvester et al., 2003a), HMBi increased milk and protein production and protein concentration while reducing the amount of N excreted in the manure. The hypotheses of this trial were that 1) HMB is primarily a rumen degradable source of Met, and its positive effects are primarily due to stimulation of microbial growth, predominantly protozoa in the rumen; 2) a significant portion of HMBi escapes ruminal breakdown through passage from the rumen to the lower digestive track; and 3) DL-Met is primarily rumen degradable and does not supply Met postruminally. MATERIALS AND METHODS Animals and Experimental Design Eight ruminally cannulated Holstein cows were assigned to a replicated 4 × 4 Latin square. Cows were blocked by calving date and were assigned to the experiment between 39 and 84 DIM. One cow was replaced after the first period because of health problems. There were 2 primiparous and 2 multiparous cows in each Journal of Dairy Science Vol. 88, No. 1, 2005

Table 1. Ingredient and nutrient composition of base diet (DM basis). Ingredient

Corn silage Alfalfa hay Cottonseed, whole with lint Corn grain, ground dry Soybean hulls1 Calcium soaps of fatty acids (Megalac) Soybean meal, solvent, 48% CP Blood meal, ring-dried Sodium bicarbonate Calcium carbonate Salt Dicalcium phosphate Magnesium oxide Urea Dynamate (K and Mg sulfate) Mineral and vitamin mix2 Nutrient CP, % of DM NDF, % of DM Fat, % of DM NFC, % of DM S, % of DM NEL, Mcal/kg3 RUP, % of CP intake MP, % of DM Met, g/d4 Met, % of MP Lys, g/d Lys, % of MP

Base diet (% of total ration DM) 30.00 13.00 12.00 30.50 1.85 1.00 5.50 3.00 0.75 0.50 0.40 0.38 0.25 0.20 0.10 0.57 18.4 28.8 5.8 42.1 0.21 1.70 38.0 11.6 425 1.80 158 6.80

1 Treatments [HMB (2-hydroxy-4-(methylthio)-butanoic acid), HMBi (isopropyl-2-hydroxy-4-(methylthio)-butanoic acid), and DLMet] were mixed and pelleted with the soybean hulls. The control diet contained only soybean hulls. 2 Contained 42 ppm Co, 3500 ppm Cu, 21,000 ppm Fe, 13,000 ppm Mn, 13,000 ppm Zn, 1320 ppm Io, 660 ppm Se, 3000 IU/g of vitamin A, 600 IU/g of vitamin D, and 15 IU/g of vitamin E. 3 NEL, RUP, metabolizable protein (MP), Met, and Lys were estimated using the NRC model (2001) using 20 kg/d DMI and 590 kg BW. 4 Met and Lys flows reported are digestible AA flows to the duodenum. 5 Addition of treatments may increase flow of Met (amount is dependent on the rumen undegradability of source).

replicate. The dietary treatments were 1) no methionine (control), 2) HMB at 0.10% of DM, 3) HMBi at 0.13 % of DM, and 4) DL-Met at 0.088% of DM. Methionine was supplemented on an equimolar basis across treatments, and the amount of additional Met or Met precursor on a Met basis supplied by each treatment diet was calculated to be 22 g/d at 25 kg/d DMI. Treatments were added to a base diet consisting of 57% concentrate and 43% forage, with approximately 70% of the forage DM from corn silage and 30% from alfalfa hay (Table 1). The Met concentration in the metabolizable protein (MP) of the control diet was expected to be substantially less (1.8% of MP) than the optimal for maximal use of MP for protein synthesis (2.4% of MP; NRC, 2001). Experimental periods consisted of 28 d, with d 1

DEGRADABILITY OF METHIONINE SOURCES

through 14 serving as an adjustment period, d 15 through d 20 as an adjustment period to restricted intake and 12 times/d feeding, and d 21 through d 28 was used for collection of data. Cows were housed in a conventional tie-stall barn with mattresses. They were allowed access to a concrete lot before milking, except during the 2 wk of restricted intake. Cows were milked at 0600 and 1700 h. Diets were mixed once daily as a TMR (Table 1) and fed twice daily during the adjustment period. Treatment premixes were mixed by hand into individual TMR. Orts were measured daily at 1600 h during the adjustment weeks, and the amount of feed offered was adjusted for a target of 10% orts. Starting on d 15, cows were restricted to 95% of their respective ad libitum intake determined during the prior 2 wk of adjustment and placed on automatic feeders (Ankom Technology, Macedon, NY). Cows received approximately 1/12 of their daily feed allowance every 2 h. The amount of feed offered was adjusted daily in an attempt to assure no orts during the week of sampling. The cows remained on the automatic feeders through the fourth week of each period. Care and handling of the animals was conducted as outlined in the guidelines of The Ohio State University Institutional Animal Care and Use Committee. Sampling and Laboratory Analysis Ingredient components of the TMR (hay, corn silage, whole cottonseed, and concentrate) were sampled every week. Total mixed rations were sampled daily and composited by week. Total mixed rations and component samples were analyzed using wet chemistry by DHI Forage Testing Laboratory (Ithaca, NY). Milk samples were taken at 4 consecutive milkings on d 16 through d 18 and d 23 through d 25 and analyzed by DHI Cooperative, Inc. (Columbus, OH) for milk fat and true protein by infrared spectroscopy and for MUN using a Skalar SAN Plus segmented flow analyzer (Skalar, Inc., Norcross, GA). Total milk N was calculated as (milk true protein/6.38)/0.9375 to account for milk NPN when calculating N partitioning (Mackle et al., 1999). Daily milk fat and protein yields were calculated and averaged by cow per period. Cows were weighed once weekly prior to the afternoon milking. Blood samples were collected via the coccygeal vein and arteries on d 22 of each period for free plasma AA analysis (Method A6300-AN-006; Beckman Instruments, Palo Alto, CA). Blood samples were collected mid morning and placed on ice for transport to the laboratory, where they were immediately centrifuged and the plasma removed. Blood plasma was stored at −20°C until analyzed. After thawing, samples were deproteinized using 1 mL of plasma with 100 μL of a 35% aqueous solution of sulfosalicylic acid dehydrate.

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Four solutions of physiological calibration standards were prepared prior to AA analyses using a Beckman system 6300 High Performance Amino Acid Analyzer (Beckman Instruments). Cobalt-EDTA and YbCl3 were used as markers to assess fluid flow and small particle flow, respectively, at the omasal canal (Harvatine et al., 2002). Chromic oxide was used as a total tract digestibility marker. Chromium pellets (5% chromic oxide, 95% soy hulls) were dosed at 1.1% of the daily DMI starting on d 16 and were placed in the rumen twice a day and mixed by hand. Chromium was analyzed using atomic absorption spectroscopy (Williams et al., 1962). Nitrogen-15 was used as a marker for microbial N flow. Starting on d 19, 10% enriched (15NH4)2SO4 was mixed into the rumen 3× daily along with CoEDTA for use as a microbial marker (Bowman et al., 1991). A sample of omasal fluid was taken prior to the first dose for background 15N analysis. Bacteria were separated from feed particles using a Waring blender and straining through 2 layers of cheesecloth (Noftsger et al., 2003). Bacterial and omasal samples were analyzed for 15N by The Stable Isotope Laboratory (Utah State University, Logan). The analyses were performed by continuous flow direct combustion and mass spectrometry using a Europa Scientific SL-2020 (PDZ Europa, Cheshire, England) system. Cobalt was dosed at approximately 0.5 g/d. The Co dose was mixed with 200 mL of water. Cobalt and Yb daily doses were divided into 3 equal doses placed directly into the rumen at 8-h intervals with hand mixing. The Yb-labeled feed was fed at approximately 225 g/d (asfed). Dosing began 3 d prior to first sampling (d 19). Concentrations of Co and Yb were analyzed using atomic absorption spectrophotometry. Background omasal and rumen samples for Co and Yb analysis were obtained on d 15. Digesta flow leaving the rumen was collected from the omasal canal using a system of alternating vacuum and pressure (Ahvenja¨rvi et al., 2000). Using this method, 500-mL samples were collected from a tube passed through the ruminal cannula and positioned in the omasal canal. Samples were taken 4 times/d on d 22 to 24 of the period with a 2-h shift of the sampling times after each day so that sampling was done every 2 h of the diurnal cycle. From each omasal sample, a 40-mL representative sample was diluted with an equal amount of 2% saline and refrigerated. These samples were composited by cow and period and frozen to be later analyzed by HPLC for HMB, HMBi, and free Met (Adisseo, Commentry, France). For the HMB assay, 5.0 ± 0.001 g of each sample were weighed in a centrifuge vial. After centrifugation 10 min at room temperature and 11,670 × g, the supernatant was collected. The pellet was washed once with 1 mL of mobile phase Journal of Dairy Science Vol. 88, No. 1, 2005

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(acetonitrile 8% in acidified water [pH 2.0] with H3PO4). The final pH was adjusted to 2.0 with hydroxychloric acid (1 N), and the volume was adjusted to 20.0 mL with mobile phase. The preparation was filtrated on 0.45 μm before assay. The determination of HMB concentration was determined in 20 μL of sample by HPLC; HMB was quantified in the samples by UV detection at 214 nm after separation on a Hibar prepacked RT250 to 4 LichroSorb RP18 (5 μm) column (Merck KGaA, Darnstadt, Germany) at 45°C and under isocratic conditions. The mobile phase was distributed at a flow rate of 0.8 mL/min. The determination of HMBi concentration was realized in 10 μL of sample by HPLC; HMBi was quantified in the samples by UV detection at 210 nm after separation on a HYPERSIL 150- × 4.6-mm HyPurity Elite C18 (5 μm) column under isocratic conditions. The mobile phase composed of 20% acetonitrile and 80% water acidified (pH 2.0) with H3PO4 was distributed at a flow rate of 0.9 mL/min. For Met assay, 1.5 g ± 0.01 mg of each sample were weighed in a centrifuge vial. A 0.4-mL solution of internal standards (2-amino-2deoxy-D-gluconic acid [Sigma Chemical Co., St. Louis, MO] and S-(2-aminoethyl)-L-cysteine hydrochloride [Fluka, Buchs, Switzerland]) at 1.25 mM was added. After centrifugation for 10 min at room temperature and 11,670 × g, the supernatant was collected. The pellet was washed several times with a lithium salt buffer (pH 2.2) containing 33.3 mM trilithium citrate (Merck KGaA), 35.2 mM citric acid, 0.5% thiodiethylene glycol (Sigma Chemical Co.), 0.28% hydrochloric acid, and finally octanoic acid (2 drops for 2 L of buffer); the supernatants were then pooled. The final pH was adjusted to 2.2 with hydroxychloric acid (1 N), and the volume was adjusted to 10.0 mL with the lithium salt buffer. The preparation was filtrated on 0.2 μm before assay. The determination of Met concentration was realized in 50 μL of sample by ion-exchange chromatography utilizing a Beckman 6300 amino acid analyzer (Beckman Instruments) at 570 nm after postcolumn ninhydrin derivatization. Calibration was achieved with a 50-μM mixture of acid, neutral, and basic amino acid solutions (Sigma Chemical Co.) On every other omasal fluid sample, a 40-mL aliquot was retained for protozoal counts. The 6 samples were mixed 1:1 with 50% formalin and were later composited by cow and period. Protozoa were counted using a 1mL counting chamber (Dehority, 1993). For count data, normality assumptions of residuals were tested using Proc Univariate (SAS, 1999) with the KolmogorovSmirnov test. Normality of the residuals allows statistical analysis without transformation of the data. The remaining omasal samples not preserved with formalin were composited by cow for each sampling day. The omasal samples were frozen, then later thawed and Journal of Dairy Science Vol. 88, No. 1, 2005

composited by cow and period, refrozen, and freezedried for marker and nutrient analysis. The double marker method was used to quantify digesta flow from the rumen (France and Siddons, 1986). Fecal samples for digestibility were obtained during the omasal sampling period. Fecal grab samples were taken on d 22 through d 24 with sampling times corresponding to every other omasal sample. Thus, fecal sampling was done every 4 h of the diurnal cycle over the 3-d sampling period. Samples were composited and dried in a forcedair oven at 55°C for 48 to 60 h. Chromium concentration in the feces was used to determine total fecal DM. Core samples of ruminal contents from 10 different sites in the rumen were removed at 4 different time points (3 h apart) on d 26. Contents were strained through 2 layers of cheesecloth, and pH of the fluid was measured immediately. A 50-mL aliquot of the filtered ruminal fluid was acidified with 3 mL of 6 N HCl to stop fermentation and frozen. After thawing, the acidified ruminal fluid was mixed, centrifuged at 15,000 × g at 4°C for 15 min, and then filtered through Whatman number 1 filter paper (Whatman, Clifton, NJ). The supernatant was analyzed for VFA concentrations by GLC (Pantoja et al., 1994) and for NH3N (Chaney and Marbach, 1962). Passage rates were determined on d 25 and 26 using CoEDTA for liquid and Yb-labeled feed for small particle passage. The CoEDTA- and Yb-labeled feeds were continued at the same schedule and dose as for the omasal samples until 0630 h on d 25. Rumen samples for Co analysis were strained through 2 layers of cheesecloth; a 100-mL aliquot of fluid was frozen immediately. Samples used for the estimation of liquid passage rate were obtained from 10 locations in the rumen at 0.5, 1, 2, 4, 6, 9, 12, 18, 24, and 36 h after the final dose. Whole rumen digesta samples (approximately 500 g) used for the estimation of small particle passage rate were obtained at 0.5, 2, 4, 9, 18, 24, and 36 h after the last dose. Rumen samples were immediately dried at 55°C. Rumen protozoa samples were taken 3 times/d on d 25 and 24 of the period with a 4-h shift of the sampling times after the first day so that sampling was done every 4 h of the diurnal cycle. These samples were treated similarly to the omasal protozoa samples. Any additional rumen contents were returned to the cows. Rumens were completely evacuated on d 27 at 0800 h and d 28 at 1200 h. Solids and liquids were separated using a hydraulic wine press set at 17 Newtons/cm2, weighed, sampled, and placed back into the cow within 25 min. Liquid and solid fractions were subsampled, with a representative sample reconstituted using the weight of each fraction. The control TMR, alfalfa hay, and corn silage were ground to 2 mm, and samples were placed into 10- ×

DEGRADABILITY OF METHIONINE SOURCES

20-cm polyester bags (ANKOM Technology, Macedon, NY) with a pore size of 50 ± 15 μm and suspended in the rumen. Each bag contained approximately 4 g and was duplicated at every time point. The bags were removed at 0, 2, 4, 8, 12, 24, 48, and 96 h. Incubations were done on 4 cows from the first square during each of the 4 periods. Duplicates within cow, feed, and time point were composited before analysis for NDF and protein. The omasal samples, in situ kinetics samples, and rumen samples were analyzed for NDF using amylase (Van Soest et al., 1991) and Kjeldahl N (AOAC, 1990). Statistical Analysis Data were analyzed as a replicated 4 × 4 Latin square using SAS Proc Mixed V8.1 (1999) according to the following model: Yijkl = μ + Ti + Pj:k + Sk + cl:k + eijkl

[1]

where Yijkl = μ= Ti = Pj:k = Sk = cl:k =

the dependent variable, overall mean, fixed effect of treatment i (i = 1,...,4), fixed effect of period j within square k (j = 1,...,4), fixed effect of square k (k = 1, 2), random effect of cow l within square ∼ N (0, σ2c) (l = 1,...,4), and eijkl = random residual ∼ N (0, σe2).

Mean separation was done using Fisher’s protected least significant difference. In situ data were analyzed using the NLIN procedure of SAS (SAS, 1999) according to the following model: Yijkl = (A + ADi + APj) + ACk) + εijkl

if t ≤ (L+LDi)

Yijkl = (A + ADi + APj + ACk) + (B + BDi + BPj + BCk) × (1 − EXP (− (kd + kDi + kPj + kCk) × (t − (L + LDi)))) + εijkl otherwise, subject to

where

ΣADi = 0

ΣBCk = 0

ΣAPj = 0

ΣkDi = 0

ΣACk = 0

ΣkPj = 0

ΣBDi = 0

ΣkCk = 0

ΣBPj = 0

ΣLDi = 0,

[2]

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A = estimated A pool across diets, cows, and periods; ADi = effect diet i on the A pool (i = 1, ..., 4); APj = effect of period j on the A pool (j = 1, ..., 4); ACk = effect of cow k on the A pool (k = 1, ..., 4); B = estimated B pool across diets, cows, and periods; BDi = effect of diet i on the B pool; BPj = the effect of period j on the B pool; BCk = effect of cow k on the B pool; kd = fractional degradation rate pooled across diets, cows, and periods; kDi = effect of diet i on the fractional degradation rate; kPj = effect of period j on the fractional degradation rate; kCk = effect of cow k on the fractional degradation rate; L = lag time (h) pooled across diets, cows, and periods; LDi = effect of diet i on lag time; and εijkl = residual error, approximately N (0, σe2). Model [2] is equivalent to the lag model of Orskov and McDonald (1979) but with parameter estimation explicitly accounting for the structure of the experiment (e.g., Latin square with repeated measurements within cells). The indigestible C pool is implicitly estimated as 100 minus the sum of the estimated A and B pools for each cow in each period. In its unconstrained form, model [2] is overparameterized. Constraints (or restrictions) must be imposed on the parameters to get an estimable set. This is not unique to this nonlinear model. Restrictions on discrete (class) parameters are automatically implemented by statistical software used to fit linear models (e.g., GLM procedure of SAS) or linear mixed models (e.g., MIXED procedure of SAS). Tests of the significance of each set of parameters in model [2] were conducted by fitting a full and reduced model and calculating an F statistic based on the reduction in the error sum of squares (Damon and Harvey, 1987). Standard errors of parameters and standard errors of parameter differences were calculated using the asymptotic variance-covariance matrix. Overall significance was declared at P ≤ 0.05. Initially, parameter estimates for the kinetic model of NDF in corn silage were poorly estimated because of severe collinearity among the estimates. Model [2] was reduced by removing individual lags LDi from the model (i.e., estimating a common lag across all 4 treatments). This model reduction alleviated the collinearity problem. For all estimates, mean separation was done using Fisher’s protected least significant difference, with the least significant difference statistic calculated from the asymptotic variance-covariance matrix of parameter esJournal of Dairy Science Vol. 88, No. 1, 2005

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timates. The statistical analysis described in model [2] was chosen over the traditional approach of fitting the nonlinear model of Orskov and McDonald (1979) to each cow-period subset of data followed by analyses of the parameter estimates (e.g., rate of degradation) according to the experimental design used to conduct the experiment. In this traditional approach, parameters are estimated as if each cell of the Latin square was independent of all others, ignoring that data common to a given cow or period have much more in common than data across cows and periods. This violates the assumption that the errors are independent, an assumption that underlies the method. In model [2], the effects of cows, periods, and treatments are simultaneously estimated with the parameters of the nonlinear degradation model. Thus, model [2] results in better estimates than those derived using the traditional approach. The magnitude of the improvement is currently unknown. Ultimately, model [2] should be solved as a nonlinear mixed model to account for the random nature of the cow and period effects properly and to account for the covariance of measurements made within each cell of the Latin square (repeated measures), but the computing tool to achieve this is not available yet. Effective ruminal degradabilities (ERD) were calculated from parameter estimates of model [2] for each cow-period-diet according to the following equation:

period was 92.2% of the ad libitum intake during the adjustment period. Body weights were similar between treatments (Table 2), averaging approximately 590 kg. Milk production averaged 37.7 kg/d and was not significantly different across treatments (P = 0.06), although there was a trend for the DL-Met treatment to result in lower production than the 3 other treatments. Fat concentration and yields were not significantly different among treatments. Many experiments have reported an increase in fat concentration with HMB (Patton et al., 1970b; Holter et al., 1972; Huber et al., 1984). However, the Latin square design used in this experiment was not designed for elucidating production responses, because of the short period of time cows received each diet and possible residual effects, although such effects would be balanced across treatments from the use of orthogonal Latin squares. However, responses in milk protein content to Met supplementation can generally be observed within 2 wk (Sylvester et al., 2003a). Milk protein concentration increased on the HMBi diet compared with the control and HMB diets, although the protein yield for HMBi was not different from the control. Sylvester et al. (2003a) and Schwab et al. (2001) have seen significant increases in protein concentration and yield with HMBi, but their experiments involved more cows and longer periods of supplementation. The significant response in milk protein concentration of 0.11% units is similar to what was obERD = (A + ADi + APj + ACk) + (B + BDi + BPj + BCk) served by Sylvester et al. (2003a) during the third and fourth week of supplementation (0.15% units). The non× [(kd + kDi + kPj + kCk)/(kd + kDi + kPj + kCk + kp)] significant response in milk protein production of 40 × [exp (−kp × (L + Li))] [3] g/d is also of similar magnitude to that observed by Sylvester et al. (2003a) during the third and fourth where kp = fractional rates of passage of each incubated week of supplementation. In that trial, the response in feedstuffs, calculated using NRC (2001) estimates for milk protein production did not reach statistical sigalfalfa hay (0.0434/h), corn silage (0.0557/h), and TMR nificance at P < 0.05 until the eighth week of supplementation. Somatic cell count and MUN were not signifi(0.0637/h). The resulting ERD were analyzed according to a cantly different among treatments. Latin square design as a mixed model with the fixed effects of diets and periods and the random effects of Plasma Amino Acids cows using the MIXED procedure of SAS (SAS, 1999). Least squares means of free plasma amino acid concentrations are presented in Table 3. With the exception RESULTS AND DISCUSSION of taurine, dietary treatments had no effect on free plasma amino acids, including Met concentration (TaLactation Performance ble 3). Abomasal Met infusions of 56.5 g/d (Seymour et Least squares means for production and intake are al., 1990) and duodenal Met infusions of 0, 6, 12, 18, reported in Table 2. Dry matter intake was not affected and 24 g/d (Pisulewski et al., 1996) elevate blood and by treatment. The overall DMI (20.1 kg/d) was lower plasma Met concentrations linearly. Similarly, dietary than what would be expected of Holstein cows produc- supplementation with rumen-protected Met generally ing 37.5 kg/d (NRC, 2001) because of the intake restric- is associated with higher blood or plasma Met concention. To prevent orts, some cows were restricted to <95% trations (Nichols et al., 1998; Blum et al., 1999) but not of ad libitum intake measured on d 1 through 14. The in all instances (Colin-Schoellen et al., 1995; Xu et al., actual degree of DMI restriction during the collection 1998). Sylvester et al. (2003a) reported increased Journal of Dairy Science Vol. 88, No. 1, 2005

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DEGRADABILITY OF METHIONINE SOURCES Table 2. Effect of HMB (2-hydroxy-4-(methylthio)-butanoic acid), HMBi (isopropyl-2-hydroxy-4-(methylthio)butanoic acid), and DL-Met supplementation on intake, milk production, and milk composition.1

DMI, kg/d Milk production, kg/d Gross feed efficiency6 Fat, % Fat production, kg/d Protein, % Protein production, kg/d Lactose, % Lactose production, kg/d Other solids,7 % Other solids production, kg/d MUN, mg/dL Log10 SCC per mL BW, kg

Control

HMB2

HMBi3

DL-Met

4

19.9 38.5 1.95 3.35 1.29 2.91a 1.12a,b 4.90 1.89 5.80 2.24 13.8 5.06 593

20.5 38.0 1.86 3.35 1.26 2.95a 1.12a,b 4.91 1.87 5.81 2.21 13.8 5.09 594

20.4 38.3 1.99 3.42 1.31 3.02b 1.16a 4.86 1.87 5.76 2.22 12.8 5.25 588

19.4 35.8 1.85 3.60 1.28 2.96a,b 1.07b 4.78 1.71 5.71 2.05 14.7 5.23 583

SEM

P

0.78 1.17 0.065 0.15 0.072 0.072 0.049 0.097 0.080 0.101 0.092 0.91 0.27 21.5

NS5 0.06 NS NS NS 0.01 0.05 NS 0.07 NS 0.07 NS NS NS

1 Milk data was determined during the third and fourth week of each period. Data from one cow during the fourth period (HMBi treatment) was removed because of mastitis during wk 3 of that period. 2 At 0.10% of DM. 3 At 0.13% of DM. 4 At 0.088% of DM. 5 P > 0.10. Means in a row without common superscript differ at P < 0.05. 6 Gross feed efficiency = kg of milk/kg of DMI. 7 Other solids are milk solids that are neither fat nor protein.

plasma Met concentration on diets supplemented with HMBi. Bequette et al. (1997) compared unidirectional fluxes of AA across the mammary gland to their secretion in milk protein. Based on their results, milk protein synthesis must compete with other metabolic processes within the mammary gland for the use of some AA. This competition is more acute under limiting conditions such as when the animal is in negative tissue energy balance, when DMI is reduced, and also when supply of AA is limiting. To ensure a relative steady state in the rumen, diets were fed on d 15 at a maximum of 95% of the previous 2-wk DMI, and the amount offered was further reduced if there was feed refusals on 2 consecutive d during the collection period. During these 2 wk of collection, the average cow had a tissue energy balance of −1.8 Mcal/d based on the NRC (2001) model. This negative energy balance may explain why HMBi did not raise plasma Met, contrary to what was observed by Sylvester et al. (2003a). VFA and Ammonia Rumen ammonia and VFA profiles are shown in Table 4. Ammonia in the rumen was not different (approximately 11.8 mg/dL) and was well above the 5-mg/dL minimum suggested for maximal bacterial CP synthesis (Satter and Slyter, 1974). This result indicates that any response to rumen-available Met was probably not due to provision of additional ammonia, but was specifically due to supplementation of Met. Volatile fatty acid profile was not different. Changes in microbial

growth or composition in the rumen because of a rumenavailable source of Met would be expected to cause changes in VFA profile. Some researchers (Lundquist et al., 1983; Noftsger et al., 2003) have seen changes in VFA profile associated with HMB in the diet, whereas others (Va´zquez-Anon et al., 2001) have seen no differences. Digestibility of Nutrients In vivo digestibilities of NDF and OM are reported in Table 5. Apparent OM digestibility and NDF digestibility were increased in the rumen in all diets supplemented with Met, whereas there was no difference in true OM digestibility the rumen across diets (P = 0.13) possibly because of the much larger statistical error associated with estimates of true digestibility than apparent digestibility. Total tract digestibilities of OM, DM, or N were also not affected by treatments. Digestibilities of DM (Polan et al., 1970; Hoover et al., 1999), ADF (Polan et al., 1970; Noftsger et al., 2003), crude fiber (Holter et al., 1972), and CP (Hoover et al., 1999) have been increased by HMB supplementation. Others have shown no effects on ADF digestibility (Windschitl and Stern, 1988) or NDF and hemicellulose digestibility (Windschitl and Stern, 1988; Noftsger et al., 2003). The exact mechanisms by which Met sources can alter ruminal digestion have not been clearly identified. Prevotella ruminicola 23, Butyrivirio fibrisolvens, and Selonomas ruminantium are stimulated by cysteine (Stipanuk, 2000), which can be produced from Met sources. If these Journal of Dairy Science Vol. 88, No. 1, 2005

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NOFTSGER ET AL. Table 3. Effect of HMB (2-hydroxy-4-(methylthio)-butanoic acid), HMBi (isopropyl-2-hydroxy-4-(methylthio)butanoic acid), and DL-Met supplementation of lactating dairy cow diets on free amino acid concentration in blood plasma. Treatment Control

1

HMB

HMBi2

DL-Met

3

SEM

P

(μM) Essential AA (EAA) Arg His Ile Leu Lys Met Phe Thr Trp Val Total EAA Nonessential AA (NEAA) Ala Asp Asn Gln Glu Gly Pro Ser Tyr Cit Orn Tau Total NEAA Total AA Met/EAA Lys/EAA

146 80.9 85.2 220 112 19.1 57.5 116 38.8 318 1193

135 77.4 90.7 238 97 17.0 56.4 100 38.1 342 1192

136 78.3 80.0 214 120 20.5 55.8 104 36.5 315 1160

137 84.6 96.4 257 118 21.8 62.6 117 39.4 358 1292

7.9 6.6 12.8 34.0 11.6 2.4 5.7 10.2 2.1 43.3 126

NS4 NS NS NS NS NS NS NS NS NS NS

286 31.5 18.5 99.6 197 276 98.3 115 43.6 83.6 44.8 40.2ab 1338 2531

225 27.4 18.0 89.5 192 252 87.8 104 42.4 85.5 44.1 44.9a 1216 2408

235 32.7 34.9 92.0 182 273 90.6 111 40.8 76.9 44.1 30.1c 1247 2407 (%)

285 32.0 23.0 93.6 195 282 105.6 121 52.0 74.6 48.3 35.2bc 1351 2643

30.4 3.2 17.3 11.0 11.0 21.6 9.1 8.5 6.7 8.5 4.9 1.9 76.7 184

NS NS NS NS NS NS NS NS NS NS NS 0.008 NS NS

1.56 9.46

1.43 8.30

1.78 10.20

1.74 9.38

0.12 0.42

NS 0.081

1

At 0.10% of DM. At 0.13% of DM. 3 At 0.088% of DM. 4 P > 0.10. Means in a row without common superscript differ at P < 0.05. a,b,c Means in a same row with a different subscript are significantly different at P < 0.05. 2

organisms are provided with supplemental Met sources, causing an increase in overall numbers of fibrolytic organisms, fiber digestibility could possibly be improved. Additionally, supplemental dietary Met sources could spare Met precursors for more efficient protein synthesis or by shifting bacterial species (Noftsger et al., 2003) Digestibility rates and pool sizes of NDF and protein determined using in situ kinetics are reported in Table 6. Average ERD of NDF was 19.7, 23.1, and 16.2% for corn silage, alfalfa hay, and TMR, respectively. Rates of digestion of NDF were fastest for the HMBi treatment of alfalfa hay and TMR, with a longer lag time. The combination of faster rate and longer lag time for HMBi made the calculated effective rumen digestibility similar for all treatments. Effective ruminal NDF digestibility of corn silage was greater in the control and Journal of Dairy Science Vol. 88, No. 1, 2005

HMB treatments than in the HMBi and DL-Met treatments. These treatments also had a larger pool of digestible NDF (B pool). In situ estimates of NDF ruminal digestibility of the TMR was considerably less than in vivo estimates (16.4 vs. 39.5%, respectively). In addition, treatments had no effect on in situ NDF digestibility of the TMR (Table 6), whereas in vivo NDF digestibility was significantly increased by 2.2 to 3.6% by Met sources (Table 5). In situ estimates of ruminal NDF digestibility are commonly less than those found in vivo. Torrent et al. (1994) found that NDF digestibilities of identical samples of alfalfa hay, brewers grains, and beet pulp were lower when estimated using polyester bags in steers than when estimated using markers in sheep. Firkins et al. (1998) reported that in situ procedures could underestimate digestibility because of

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DEGRADABILITY OF METHIONINE SOURCES Table 4. Least squares means for rumen pH, volatile fatty acids, and ammonia in cows fed diets that vary in source of Met.1

PH Rumen NH3, mg/dL Total VFA, mM Concentration of VFA, mol/100 mol Acetate (A) Propionate (P) Butyrate Valerate Isobutyrate Isovalerate A:P

Control

HMB2

HMBi

DL-Met

SEM

P

5.96 12.7 120.7

5.95 12.1 121.0

5.91 11.1 121.4

5.94 11.1 118.0

0.046 1.05 2.6

NS3 NS NS

64.0 22.4 10.3 1.14 0.84 1.31 2.88

62.0 24.6 10.2 1.19 0.81 1.16 2.55

62.8 23.4 10.6 1.17 0.81 1.22 2.72

62.8 23.8 10.2 1.17 0.74 1.24 2.70

0.77 0.96 0.34 0.039 0.036 0.085 0.139

NS NS NS NS NS NS NS

1

Averaged across 4 sampling time points, 3 h apart. HMB = 2-Hydroxy-4-(methylthio)-butanoic acid at 0.10% of DM; HMBi = isopropyl HMB at 0.13% of DM. DL-Met at 0.088% of DM. 3 P > 0.10. 2

lower pH inside the bags than in the rumen contents or overestimate digestibility caused by particle efflux. In situ rate of CP digestibility and estimated ERD was highest for the DL-Met treatment in the TMR because of a greater rate of CP degradation. This increase in CP degradability caused by DL-Met was not observed with the alfalfa hay and corn silage substrate. The ERD of alfalfa hay CP was highest for HMB and lowest for HMBi, again because of differences in rates of degradation (Table 6). Treatments had no effect on the ERD of CP in corn silage. Hoover et al. (1999) reported a quadratic effect of HMB on ruminal CP digestibility, with HMB increasing digestibility at 0.11% of the diet DM, but not at 0.22%. Isopropyl-HMB, assuming a 50% ruminal breakdown (Schwab et al., 2001), would provide ruminally available HMB at approximately 0.05% of the diet DM, whereas the HMB treatment provided HMB at 0.10% of diet DM in this experiment and would be more similar to the intermediate treatment of Hoover et al. (1999). In situ ERD of the CP in TMR, alfalfa hay and corn silage measured using cows fed the control diet were lower than values calculated using NRC

(2002) (in situ: 58.0, 73.3, and 63.0% and NRC: 62.2, 83.3, and 65.2% for TMR, hay and corn silage, respectively). Differences in alfalfa hay ERD are primarily due to differences in the size of the A pool (in situ: 36.2%; NRC: 44.4%). Differences in corn silage ERD are primarily due to differences in C pools (in situ: 11.7%; NRC: 18.5%). More importantly, the NRC (2001) model does not include a mechanism to directly account for the effect of Met supplementation on ruminal CP degradability. Rumen Pool and Passage Rates Rumen pool and passage rate results are shown in Table 7. Rumen mass of wet digesta and DM were not different among treatments and averaged 70.4 and 10.2 kg, respectively. There was a trend (P = 0.09) for the control diet to have a higher concentration of NDF in the ruminal DM content than the other 3 diets, possibly because of increased NDF digestibility in the Met-supplemented diets. Passage rates of liquid and small particles were not affected by treatment and averaged

Table 5. Digestibility of nutrients for diets that vary in source and availability of Met. Control

HMB1

HMBi

DL-Met

SEM

P

(%) Rumen OM Apparent OM True NDF Total tract OM DM

43.8a 53.3 37.2a

51.6b 59.7 40.7b

48.3b 56.5 39.4b

50.8b 55.5 40.8b

1.38 3.17 1.04

<0.0012 NS <0.001

64.6 66.6

65.5 66.1

64.3 67.3

64.8 66.9

1.59 1.54

NS NS

1 HMB = 2-Hydroxy-4-(methylthio)-butanoic acid at 0.10% of DM, and HMBi = isopropyl HMB at 0.13% of DM. DL-Met at 0.088% of DM. 2 P > 0.10. Means in a row without common superscript differ at P < 0.05.

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NOFTSGER ET AL. Table 6. Digestibility of CP and NDF in TMR, alfalfa hay, and corn silage using polyester bags removed at 8 time points for diets that vary in source of Met. Control

HMB1

HMBi

DL-Met

SED2

P3

TMR NDF A,4 % B, % C, % Rate /h Lag, h ERD,5 % CP A, % B, % C, % Rate, /h Lag, h ERD, %

3.9 61.0a 35.2ab 0.023a 3.9ab 15.7

1.3 67.0ab 31.7ab 0.028ab 3.2ab 15.2

2.7 58.6a 38.7b 0.038b 6.3b 16.5

1.9 80.2b 18.0a 0.022a 1.1a 17.5

2.6 9.0 9.0 0.006 2.2 1.4

NS 0.04 0.04 0.05 0.05 NS

37.1 47.4 15.5 0.053a 0 58.0a

36.9 45.8 17.4 0.069ab 1.5 58.4a

34.7 48.7 16.6 0.068ab 0 59.3ab

35.9 46.3 17.8 0.076b 0 61.2b

3.0 4.1 4.1 0.014 0.6 0.83

NS NS NS 0.05 NS 0.03

0.6 47.9 51.5b 0.049a 1.6a 23.4

2.1 3.4 3.4 0.008 1.3 1.76

NS NS 0.04 <0.005 0.05 NS

Alfalfa hay NDF A, % B, % C, % Rate, /h Lag, h ERD, % CP A, % B, % C, % Rate, /h Lag, h ERD,% NDF A, % B, % C, % Rate, /h Lag, h ERD, % CP A, % B, % C, % /h Lag, h ERD, %

1.0 45.9 53.0b 0.047a 0.8a 23.6

3.5 52.0 44.5a 0.039a 2.7a 23.1

1.6 46.3 52.1b 0.067b 5.5b 22.2

36.2 53.8 10.0 0.100ab 0 73.3b

31.1 32.5 58.4 57.2 10.5 10.4 0.133b 0.090a 0 0 75.0c 70.5a Corn silage

32.8 57.3 9.9 0.105ab 0 73.1b

3.0 3.7 3.7 0.017 — 0.70

NS NS NS 0.05 NS <0.005

2.2 89.8b 8.0a 0.012 0.0 22.4b

2.1 96.5b 1.4a 0.010 0.0 23.7b

1.2 67.4a 31.4b 0.025 0.0 16.0a

0.1 75.6a 24.3b 0.018 0.0 16.7a

0.8 3.8 3.9 0.0021 0.0 2.0

NS 0.049 0.047 NS NA6 <0.001

54.4 33.9 11.7a 0.049a 9.2b 63.0

51.5 34.5 14.0a 0.041a 0.0a 63.7

53.2 33.9 12.9a 0.036a 3.8b 63.4

52.8 26.2 21.0b 0.075b 7.7b 63.0

2.0 4.3 4.3 0.014 2.2 1.6

NS NS 0.03 0.05 0.05 NS

1 HMB = 2-Hydroxy-4-(methylthio)-butanoic acid at 0.10% of DM and HMBi = isopropyl HMB at 0.13% of DM. DL-Met at 0.088% of DM. 2 SED = Standard error of the differences. 3 P > 0.10. Means in a row without common superscript differ at P < 0.05. 4 A, B, and C are kinetic pools. The A pool is the fraction of the constituent (NDF or CP) that is instantaneously digestible, the B pool is the potentially digestible fraction, and the C pool is the indigestible fraction. 5 ERD = Effective ruminal digestibility estimated as: ERD = A + B [kd/kd + kp] × [exp (−kpL)]. Rate of passage (kp) was calculated using NRC (2001) estimates for concentrates (0.0754/h), alfalfa hay (0.0434/h), corn silage (0.0557/h), and TMR (0.0637/h). 6 NA = Not available; lag model could not be uniquely solved because of collinearity among parameters.

0.157 and 0.071/h, respectively. The rumen pH was also not different across diets and averaged 5.94. This pH was slightly lower than expected, as steady-state feeding should prevent most of the diurnal variation normally seen in pH (Dehority, 2003). Journal of Dairy Science Vol. 88, No. 1, 2005

Counts of rumen and omasal protozoa were not different across treatments (Table 7). The omasal protozoa showed a nonsignificant numerical increase with the HMB diet. Current methods for counting protozoa have high measurement errors. Consequently, treatments

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DEGRADABILITY OF METHIONINE SOURCES Table 7. Least squares means for rumen pool measurements and passage rates in cows fed diets that vary in source of Met.

Rumen mass, kg Wet digesta3 DM Rumen digesta DM, % NDF, % of DM N, % of DM Ash, % of DM Ruminal liquid volume,4 L Liquid passage rate, /h Small particle passage rate, /h Rumen protozoa,5 /mL Omasal protozoa, /mL

Control

HMB1

HMBi

DL-Met

SEM

P

71.5 10.45

69.7 10.31

69.7 9.66

70.8 10.26

2.90 0.60

NS2 NS

14.49 56.14 3.07 9.07 61.0 0.143 0.075 1.36 6.11

15.01 54.81 3.04 9.08 59.3 0.172 0.069 1.29 6.51

13.79 53.14 3.06 9.55 60.0 0.142 0.060 1.49 6.52

14.02 54.74 3.09 9.27 60.8 0.172 0.079 1.46 5.73

0.45 0.85 0.052 0.367 2.76 0.028 0.006 0.159 0.790

0.099 0.090 NS NS NS NS NS NS NS

1 HMB = 2-Hydroxy-4-(methylthio)-butanoic acid at 0.10% of DM, and HMBi = isopropyl HMB at 0.13% of DM. DL-Met at 0.088% of DM. 2 P > 0.10. Means in a row without common superscript differ at P < 0.05. 3 Total pool sizes of wet digesta were determined by the average of 2 d of rumen evacuations. 4 Ruminal liquid volume = wet digesta − (wet digesta × 105°C DM). 5 Protozoa counts are expressed as 106/mL for rumen and 105/mL for omasal measurements.

must result in large differences in protozoal counts to be significantly different. Nitrogen Partitioning Nitrogen partitioning is reported in Table 8. Milk N production was significantly higher with the control and HMBi diet than with the DL-Met treatment; the HMB treatment was intermediate. On average, 31.3% of intake N appeared as milk N. This agrees with N efficiencies observed in other trials (Noftsger and StPierre, 2003; Sylvester et al., 2003a). Based on milk urea N and BW, the equation of Kauffman and StPierre (2001) predicted a mean urinary N excretion of 226 g/d across treatments. Using predicted UN excretion and fecal excretion of N based on Cr flow, environmental N load (kg of N excreted/kg of N in milk) was not different across treatments. Our laboratory has reported an improvement of this ratio when the supply of Lys and Met was increased in combination with increased digestibility of supplemental RUP (Noftsger and St-Pierre, 2003). This improvement was due to a reduction in dietary CP with a concomitant decrease in urinary N and increase in milk N. Retained N averaged −17 g and was not different between treatments. With cows past their second month in lactation and fed ad libitum, retained N should be approximately zero. In this experiment, intake of cows during the sampling period (d 14 to 28) was restricted to 90 to 95% of DMI measured during the adjustment period (d 1 to 14), possibly resulting in small, negative estimated retained N.

Nitrogen Flow to the Omasum Flow and partitioning of N to the omasum are reported in Table 9. Flows of total N, ammonia N, bacterial N, and nonammonia, nonbacterial N (NANBN) were not different among treatments. The proportion of N intake flowing to the omasum as NANBN was not affected by treatments and averaged 40.6%. This in vivo estimate of intake N escaping ruminal degradation is very close to the average value of 40.8% determined in situ and supports the use of this latter method to estimate in vivo ruminal protein degradability. The proportion of omasal N from bacterial N was not different and averaged 0.54. Some research has shown an increase in microbial protein production with supplementation of HMB (Hoover et al., 1999; Va´zquez-Anon et al., 2001), whereas others saw none (Windschitl and Stern, 1988). Using continuous culture fermenters, we did not find any effect of HMB or DL-Met on bacterial protein production but found a larger proportion of bacterial N from NH3 N for diets supplemented with HMB at either 0.055 or 0.11% of DM (Noftsger et al., 2003). As pointed out by Koenig et al. (2002), the degree to which the end products of microbial HMB use or degradation are limiting in the rumen should ultimately determine whether HMB supplementation results in measurable improvement in microbial protein synthesis. Rumen Passage of Met Supplements Extents of passage of HMB, HMBi, and Met to the omasum are reported in Table 10. Passage of the Met sources was determined using fluid passage rates deterJournal of Dairy Science Vol. 88, No. 1, 2005

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NOFTSGER ET AL. Table 8. Nitrogen partitioning and efficiency with diets that vary in source of Met.

N intake, g/d Fecal N, g/d Estimated urinary N,3 g/d Milk N production, g/d Estimated retained N, g/d Gross N efficiency4 Environmental N load5 Total tract N digestibility, %

Control

HMB1

HMBi

DL-Met

SEM

P

585 196 225 188a −23 32.1 2.24 66.5

614 200 229 187a,b −1 30.4 2.29 67.4

621 219 223 194a −13 31.5 2.28 64.7

571 200 225 178b −32 31.0 2.39 65.0

23.6 14.9 11.7 8.23 14.0 0.95 0.10 2.25

NS2 NS NS 0.05 NS NS NS NS

1 HMB = 2-Hydroxy-4-(methylthio)-butanoic acid at 0.10% of DM and HMBi = isopropyl HMB at 0.13% of DM. DL-Met at 0.088% of DM. 2 P > 0.10. Means in a row without common superscript differ at P < 0.05. 3 N = 0.0259 × BW (kg) × milk urea N (mg/dL) (Kauffman and St-Pierre, 2001). 4 Calculated as milk N/N intake × 100. 5 Calculated as kg of N excreted/kg of N in milk; environmental N load calculation assumes zero N balance.

mined by using CoEDTA. Because HMB is readily soluble in aqueous media (Koenig et al., 2002), the assumption is that HMB flows with the fluid phase. The degradability was determined using actual grams of HMB provided by the dose. Approximately 85% of the dose in the HMBi treatment is HMB. Only a small percentage of the HMB and HMBi ingested appeared as HMB in the omasal fluid, and HMBi concentrations were below detection level. This supports the observation made by several researchers who have found little rumen escape of HMB supplements in lambs (Papas et al., 1974) and cows (Jones et al., 1988). Others have found higher values of undegradability, but these experiments are usually carried out with in vitro systems (Patterson and Kung, 1988), pulse dosing of HMB (Koenig et al., 1999, 2002), or both (Va´zquez-Anon et al., 2001). These may not adequately represent the in vivo situation. Continuous culture methods are experimental models of the rumen, but most do not contain either a rumen mat or protozoa. Protozoa must sequester themselves along the rumen wall or in the mat to remain in the rumen long enough to divide. Passage rates can vary by cow, stage of lactation, and DMI. Pulse dosing raises ruminal concentrations far above that ob-

served when cows are fed a diet at the suggested dose of 0.10 to 0.11% of DM. In the experiment of Koenig et al. (2002), rumen HMB concentration following the lowest HMB pulse dose of 25 g took 6 h to return to the average steady-state concentration observed in our experiment (23 μg/mL, data not shown). These high concentrations following pulse dosing may saturate microbial enzymatic pathways or transport mechanisms across the microbial membranes. Rumen undegradability of HMBi has been determined previously to be approximately 50% using blood (Robert et al., 2001) and milk true protein changes (Schwab et al., 2001) as indicators of bioavailability to the cow. With HMBi, approximately one-half of the HMBi is absorbed through the rumen wall, where the isopropanol is removed and most of the HMB is released into the bloodstream (Robert et al., 2001). The remaining 50% is hydrolyzed in the rumen to HMB and isopropanol (Robert et al., 2002). This remaining rumen-available HMB would behave similarly to unmodified HMB. Because the doses of HMB and HMBi contained equivalent amounts of Met precursor, 50% degradability would leave an amount of HMB in the rumen equivalent to one-half the dose of the unmodified

Table 9. Flow and partitioning of N to the omasum with diets that vary in source of Met.

Omasal N, g/d Ammonia N Microbial N Nonbacterial NAN3 Nonbacterial NAN, % of N intake Proportion of omasal N from bacteria

Control

HMB1

HMBi

DL-Met

SEM

P

556 12.3 303 241 41.2 0.545

565 14.1 297 254 41.3 0.525

596 13.2 331 252 40.6 0.555

531 17.7 289 224 39.2 0.545

34.9 1.4 24.4 35.1 4.0 0.026

NS2 NS NS NS NS NS

1 HMB = 2-Hydroxy-4-(methylthio)-butanoic acid at 0.10% of DM and HMBi = isopropyl HMB at 0.13% of DM. DL-Met at 0.088% of DM. 2 P > 0.10. 3 NAN = Nonammonia N.

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DEGRADABILITY OF METHIONINE SOURCES Table 10. Percentage of HMB and HMBi dose appearing as HMB in the omasal fluid and passage of free Met to the omasum. NA = not applicable. Control 2

Percentage of dose Passage of free Met, g/d

NA 5.2

HMB 5.33 4.9

a

HMBi b

2.33 5.5

DL-Met

SEM

P

NA 4.9

1.51 0.32

0.023 NS

1 HMB = 2-Hydroxy-4-(methylthio)-butanoic acid at 0.10% of DM and HMBi = isopropyl HMB at 0.13% of DM. DL-Met at 0.088% of DM. 2 Liquid HMBi contains 90% HMBi monomer, which is made up of 78% HMB, amounting to 90% × 78% = 70% HMB in the liquid fed HMBi. Percentage of dose is based on actual intake of HMB. 3 P > 0.10. Means in a row without common superscript differ at P < 0.05.

HMB treatment. Therefore, passage of HMB to the omasum of cows on the HMBi treatment would be expected at approximately one-half the level found on the HMB treatment. This is supported by our results, where only 2.3% of the HMB from HMBi passed, whereas 5.3% of the HMB from the HMB treatment passed out through the omasum. Escape of free Met was small and similar for all treatments (5.1 g/d), indicating that DL-Met did not pass out of the rumen in significant amounts. In fact, the passage of free Met on the DL-Met treatment was numerically less than the control. These data do not support the hypothesis that the supplementation of diets with unprotected DL-Met results in an additional supply of Met postruminally (Velle et al., 1997; Volden et al., 1998). Also, our omasal flow data do not support the hypothesis that a large portion of dietary HMB escapes ruminal breakdown because of its association with the ruminal liquid fraction, which has a relatively high rate of passage in conjunction with a relatively slow rate of ruminal degradation (Koenig et al., 1999, 2002). Similarly, the same mechanism (escape with the fluid phase to the omasum) cannot be responsible for the mode of action of HMBi. Our results do not rule out the possibility of HMB being directly absorbed through the rumen wall. McCollum et al. (2000) found, using an in vitro system, that both the omasal and ruminal epithelia have the capacity to absorb HMB, although the capacity per unit of dry tissue was far less for the ruminal epithelium. The proportion of HMB escaping ruminal breakdown through ruminal wall absorption under normal, in vivo conditions is unknown. In contrast, data from Robert et al. (2001) suggest that this mechanism results in a significant proportion of HMBi escaping ruminal degradation. CONCLUSIONS The proportion of ingested HMB escaping the rumen was only 5% based on the amount flowing into the omasum. Rumen digestibility of OM (apparent) and NDF were improved by all 3 Met sources, indicating a ruminal effect in all 3 instances. The differences in degrada-

bility of HMB and HBMi were evident in the changes in milk protein concentration and production observed with HMBi, but not with HMB. The ruminal effects of HMB and HMBi appeared as changes in NDF digestibility, indicating some changes in the rumen microbial ecosystem. The passage of HMBi as HMB into the omasum further supports prior estimates of 50% rumen undegradability. No measurable amounts of HMBi appeared in the omasum. None of the Met sources had an effect on rumen or omasal protozoa population. With more precise molecular methods for protozoal enumeration, it may be possible to quantify more precisely protozoal population and, hence, the potential effects of HMB on protozoa (Sylvester et al., 2003b). The high rumen degradability of DL-Met is confirmed by the absence of change in milk composition and of change in free Met flow in the omasum with the DL-Met-supplemented diet. ACKNOWLEDGMENTS The authors thank Adisseo for its generous financial support and Brian Sloan and Jean-Claude Robert for their advice on the research protocol. Our appreciation is extended to Venture Milling for its generous donation of the blood meal used in this experiment. We thank the farm crew at the Waterman Dairy Center for their help with feeding and milking of the animals as well as Emily Oelker, Chad Knueve, and Pam Bucci for helping with omasal sampling; Sanjay Karnati for technical assistance; and Jeff Firkins for helpful comments and suggestions on an earlier version of this manuscript. REFERENCES Ahvenja¨rvi, S., A. Vanhatalo, P. Huhtanen, and T. Varvikko. 2000. Determination of reticulo-rumen and whole-stomach digestion in lactating cows by omasal canal or duodenal sampling. Br. J. Nutr. 83:67–77. Association of Official Analytical Chemists. 1990. Official Methods of Analysis. Vol. I. 15th ed. AOAC, Arlington, VA. Bequette, B. J., F. R. C. Backwell, A. G. Calder, J. A. Metcalf, D. E. Beever, J. C. MacRae, and G. E. Lobley. 1997. Application of a U-3C-labeled amino acid tracer in lactating dairy goats for simultaneous measurements of the flux of amino acids in plasma and the partition of amino acids to the mammary gland. J. Dairy Sci. 80:2842–2853. Journal of Dairy Science Vol. 88, No. 1, 2005

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