Lymphocyte Proliferation Response of Lactating Dairy Cows Fed ...

Lymphocyte Proliferation Response of Lactating Dairy Cows Fed Varying Concentrations of Rumen-Protected Methionine K. J. SODER1 and L. A. HOLDEN2 Department of Dairy and Animal Science, The Pennsylvania State University, University Park 16802

ABSTRACT

INTRODUCTION

Six midlactation Holstein cows were used in a replicated 3 × 3 Latin square design to characterize the influence of dietary concentration of Mepron威85 (Degussa Corp., Allendale, NJ) on isolated mononuclear cell composition and lymphocyte proliferation. Cows were fed a common total mixed ration containing corn silage, legume silage, chopped legume hay, and a grain and mineral pellet that was top-dressed with one of three treatments. Treatments consisted of 1) 0 g/d of Mepron威85, 2) 15 g/d of Mepron威85 (11 g of rumenprotected Met), or 3) 30 g/d of Mepron威85 (22 g of rumen-protected Met). Cows were housed in a tie-stall barn, had continuous access to fresh water, and were fed once daily at 0900 h for ad libitum intake. Dry matter intake (DMI), orts, and milk yields were recorded daily, and weekly milk samples were collected for analyses of fat, protein, SCC, and milk urea nitrogen. Blood and milk samples were collected before the beginning of the experiment and during wk 2 and 4 of each 28-d treatment period. Blood was analyzed for serum methionine, lymphocyte proliferation, and phenotypic composition of isolated mononuclear cells. Milk samples were analyzed for phenotypic composition of isolated mononuclear cells. Least square means for DMI, milk yield, milk composition, and phenotypic mononuclear cell composition of blood and milk were not affected by treatment. Proliferative ability of peripheral blood T lymphocytes increased for cows consuming 30 g/d of Mepron威85. (Key words: methionine, proliferation, lymphocyte)

Methionine has been identified as one of the firstlimiting amino acids in dairy rations (31). Research has demonstrated that milk protein is the most sensitive of the production responses to alterations in supply of Met (5, 10), although increases in FCM (22), DMI (24), and BW (24) have also been observed with Met supplementation. In addition to production responses in dairy cows, considerable evidence has been generated that may substantiate an important role for Met in immune function. Human lymphocytes have been shown to be dependent upon Met for growth; researchers (12) have reported that peripheral lymphocytes from healthy donors could not be cultured in medium deficient in exogenous Met. Similar observations have been reported in virus-transformed and tumor-altered lymphocyte cells (6, 13, 35). Limited research in some species further suggests that immune function is dependent on adequate Met availability. Weanling rats whose dams were deprived of Met and choline during both gestation and lactation exhibited increased susceptibility to Salmonella typhimurium and decreased lymphocyte stimulation to ovine red blood cells (18, 39). It was not possible in these studies to distinguish between the effects of Met and choline, but other studies (7, 37) showed that choline deficiency did not affect immune responses of chickens. Supplemental Met has been shown to increase antibody concentrations in chicks (37). Although this mechanism is not yet clear, the authors (37) speculated that additional Met may be important in thymus-derived T helper lymphocyte function. Methionine may stimulate the immune system of dairy cattle, as suggested by work with other species (7, 18, 37, 39). This stimulatory effect may have an economic impact on incidence of disease, particularly mastitis. Mastitis is the most costly disease of the dairy industry with economic losses of approximately $2 billion annually in the US (20). The majority of dairy cows in the US will have at least one incidence of mastitis (8), which will cost the producer an average of $200/ cow per year per case (19). Rumen-protected Met may provide multiple benefits to the dairy industry by im-

Abbreviation key: ConA = concanavalin A, IL-2 = interleukin-2, ME15 = 15 g/d of Mepron威85, ME30 = 30 g/d of Mepron威85, PAE = 1× PBS + 20% acid citrate dextrose + 20 mM EDTA, PBMC = peripheral blood mononuclear cells, PHA = phytohemagglutinin, PWM = pokeweed mitogen.

Received January 8, 1999. Accepted April 26, 1999. 1 1Current address: USDA-ARS, Pasture Systems and Watershed Management Research Laboratory, University Park, PA 16802. 2 Reprint requests: 324 Henning Building, University Park, PA 16802. 1999 J Dairy Sci 82:1935–1942

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proving milk yield and composition and stimulating immune function, thereby improving disease resistance. However, very limited research has been completed that evaluates the effects of Met on the immune status of the dairy cow. Preliminary research (Colin Peel, Degussa Corp., Allendale, NJ, personal communication, 1998) has found that rumen-protected Met may have the potential to decrease SCC of dairy cows. In addition, no research is available that evaluates the effects of varied concentrations of dietary Met on lymphocyte proliferation of the dairy cow, an important component of immune response. Therefore, the objective of this experiment was to determine the effect of dietary concentration of Mepron威85 (Degussa Corp.) on milk and blood mononuclear cell population and peripheral blood lymphocyte proliferation of lactating dairy cows. MATERIALS AND METHODS

TABLE 1. Ingredient composition of TMR on a DM basis. Ingredient

DM

Corn silage Alfalfa hay Alfalfa haylage Grass haylage Ground corn Canola meal Dark distillers corn Wheat middlings Cooked soybeans Barley ProBase威,1 Limestone Sodium bicarbonate Salt Sodium bentonite Magnesium oxide Dicalcium phosphate Penn State Trace Mineral Premix #4威,2 Se premix, 0.06% Se Vitamins A, D, and E3

(%) 28.32 8.75 8.26 3.76 16.35 9.95 7.52 4.12 4.98 3.68 1.28 1.06 0.66 0.45 0.41 0.20 0.07 0.05 0.04 0.09

1

Agway, Inc. (Syracuse, NY); bypass protein blend. Agway, Inc.; contained 25.3% Ca, 5.8% S, 5455 ppm of Cu, 20,202 ppm of Fe, 0.1 ppm of I, 17,172 ppm of Mn, and 54,545 ppm of Zn. 3 Contained 1.36 million IU/kg of vitamin A, 454,545 IU/kg of vitamin D, and 1363 IU/kg of vitamin E. 2

Cows and Dietary Treatment Six multiparous Holstein cows were used in a replicated 3 × 3 Latin square design to evaluate the effects of Mepron威85 on isolated mononuclear cell composition and lymphocyte proliferation. Cows were selected based upon lactation number, milk yield, stage of lactation, SCC, and absence of mastitis-causing organisms. Samples from cows were cultured for mastitis-causing organisms at freshening and again 1 wk before beginning the experiment; cows were blocked based upon pretrial SCC, resulting in a high and low SCC square. Cows were 100 DIM (±20 d) at the beginning of the experiment. Cows were housed at The Pennsylvania State University dairy production research center (University Park) in individual tie stalls fitted with rubber mats and bedded with sand; cows had continuous access to fresh water. The experiment consisted of 3 treatment periods of 28-d each. Cows received each of three treatments arranged in a replicated 3 × 3 Latin square design. Cows were fed a common TMR containing corn silage, legume silage, chopped legume hay, and a grain and mineral pellet (Table 1). Diets were formulated to meet or exceed NRC (17) recommendations. Cows were fed once daily at 0900 h for ad libitum intake. Treatments were topdressed and hand-mixed in the TMR at feeding time and consisted of 1) 0 g/d of Mepron威85 (control), 2) 15 g/d of Mepron威85 (ME15), or 3) 30 g/d of Mepron威85 (ME30). These feeding levels provided approximately 0, 11, or 22 g, respectively, of supplemental Met postruminally for absorption (Mepron威85 Dairy Ration Evaluator, Degussa Corp.). Journal of Dairy Science Vol. 82, No. 9, 1999

Experimental Measures Daily intake and orts were recorded throughout the experiment. Cows were milked at 0500, 1300, and 1900 h. Milk yield was recorded daily at each milking. Milk samples were taken once weekly at consecutive a.m. and p.m. (0500 and 1900 h) milkings, preserved in 2bromo-2-nitropropane-1,3 diol, and analyzed for fat, protein, SCC, and milk urea nitrogen by the Pennsylvania DHIA testing laboratory (infrared analysis; Foss 605B Milko-Scan; Foss Electric, Hillerød, Denmark). The TMR and orts samples were collected daily and composited weekly by treatment. Milk samples (1 L) were collected on 2 d immediately before the start of the experiment (d −3 and −1) and twice during wk 2 (d 11 and 13) and wk 4 (d 26 and 28) of each treatment period during the 0500 h milking. The phenotypes of isolated mononuclear cell populations from the samples were characterized via flow cytometry. Means were pooled by week (d −3 and −1, d 11 and 13, and d 26 and 28) to obtain one mean value for each collection week (two values per treatment period plus one value for pretreatment collection; n = 7). Blood was collected via jugular venipuncture immediately before the beginning of the trial (d −2), and during wk 2 (d 10) and wk 4 (d 27) of each treatment period immediately following the 0500 h milking. Blood samples were analyzed via flow cytometry for lymphocyte proliferation and serum Met concentration (n = 7). Fifteen milliliters of blood was

METHIONINE AND LYMPHOCYTE PROLIFERATION

collected in plain Vacutainer tubes, allowed to clot at room temperature and centrifuged at 4000 × g for 20 min, and serum was collected and frozen at −20°C. The frozen serum was later shipped on dry ice for serum Met analysis (liquid chromatography; Olson Biochemistry Labs, Brookings, SD). An additional 250 ml of blood was collected in sterile glass bottles containing 25 ml of EDTA for lymphocyte proliferation and flow cytometry analyses. Sample Analyses The TMR samples were analyzed for DM, CP, ADF, NDF, (3) soluble protein (38), mineral composition (near-infrared spectroscopy; Skyview Laboratory, Inc., Jennerstown, PA), and amino acid composition [Degussa Corp.; (3)]. The TMR analyses did not include Met supplementation. Cell preparation. Peripheral blood mononuclear cells (PBMC) were isolated from citrated venous blood samples as previously described (33). The PBMC were then purified using a Ficoll-Paque density gradient (1.077 g/ml; Pharmacia, Piscataway, NJ). Any red blood cell contamination of the mononuclear cell pellet was eliminated by water lysis. After mononuclear cell purification, the PBMC were washed three times in Hank’s Balanced Salt Solution (Sigma, St. Louis, MO). Milk aliquots (1 L) were collected in Nalgene containers for transportation to the laboratory. The milk was immediately mixed with an equal volume of buffer containing 1× PBS, pH 7.2; 20% acid citrate dextrose (10% solution); and 20 mM EDTA (PAE). The milk and PAE buffer were centrifuged at 250 × g for 30 min at 15°C. Cell pellets were washed three times in PAE buffer, diluted with PAE buffer in 50-ml centrifuge tubes, and separated by a Percoll density gradient (1.086 g/ml; Pharmacia); centrifugation was as previously described (25). Flow cytometric analysis. Flow cytometry, as previously described (33), was performed to characterize the phenotype of isolated mononuclear cell populations from milk and peripheral blood. Briefly, isolated mononuclear cells were resuspended to 1 × 107 cells/ml in PBS supplemented with 2% fetal bovine serum (Hyclone, Logan, UT). Cells (50 µl) were incubated for 30 min at 4°C with 50 µl of monoclonal antibodies (Table 2). Background immunofluorescence caused by nonspecific labeling of Fc receptor was determined by incubating 50 µl of cells in 50 µl of PBS supplemented with 2% fetal bovine serum. The cells were then washed and incubated with 100 µl of fluorescein isothiocyanate conjugated goat F(ab′)2 fragment against mouse IgG (diluted 1:2000; USB, Cleveland, OH) with a previously described staining procedure (9). Analyses were per-

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formed with a counter (EPICS PS 753; Coulter Electronics, Miami, FL). Data from 10,000 cells were collected, and two-parameter analysis of forward angle versus 90° light scatter was used to identify the lymphocyte population for fluorescence analysis. Immunofluorescence histograms were expressed as the percentage of cells positive for staining. Lymphocyte proliferation analysis. The ability of isolated PBMC to proliferate in response to various mitogenic stimuli was examined. The PBMC were resuspended (2 × 106 cells/ml) in proliferation medium containing RPMI 1640 (Sigma), 10% fetal bovine serum, and 0.01 mM 2-mercaptoethanol (Fisher, Pittsburgh, PA). One hundred microliters of resuspended cells was added to a round bottom, 96-well plate. Triplicate sets of samples were stimulated with 100 µl of concanavalin A (ConA; 0.5 µg/well; Sigma), phytohemagglutinin (PHA; 1.0 µg/well; Sigma), pokeweed mitogen (PWM; 0.1 µg/well; Sigma); or recombinant human interleukin2 (IL-2; 12.5 U/well; Boehringer Mannheim, Indianapolis, IN). All treatments were diluted in proliferation medium. A triplicate set of wells containing only 100 µl of cells and 100 µl of proliferation medium served as controls. After incubation for 48 h at 37°C, 0.4 µCi of 3 HTdr (Amersham, Arlington Heights, IL; 20 µl/well; stock was diluted in proliferation medium) was added to all wells. After an additional 18 h of incubation at 37°C, the cells were harvested with a semi-automatic cell harvester (Skatron, Sterling, VA), and 3HTdr incorporation into cellular DNA was measured with a liquid scintillation counter (Beckman LS 60001C; Beckman Instruments, Inc., Columbia, MD). Results were expressed as increased counts per minute divided by the counts per minute of the control. Statistical Analyses The DMI, milk yield, and milk composition data were analyzed by ANOVA using the GLM procedure of SAS威 (30). The model Y = square + square(cow) + square(period) + treatment + error was used for the analyses where square = high or low SCC square (n = 2), and period = treatment period (n = 3). The repeated measures data (serum Met, lymphocyte proliferation, and flow cytometry) were analyzed using the MIXED procedure of SAS威 (30). The statistical model Y = square + treatment + treatment(cow) + time + time × treatment + error was used for the analyses where square = high or low SCC square (n = 2), and time = the pooled mean values for each collection week (n = 7). For each analyzed variable, cow nested within treatment was subjected to three covariance structures: unstructured, autoregressive order 1, and compound symmetry. The covariance structure resulting in the Journal of Dairy Science Vol. 82, No. 9, 1999

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SODER AND HOLDEN TABLE 2. Monoclonal antibodies specific to bovine mononuclear cell differentiation molecules used to define the composition of mononuclear cell populations from peripheral blood mononuclear cells and milk.1 Molecule

Cell type2

Mab3

Dilution4

CD2 CD4 CD8 γδ MG B Cell IL-2r MHC II

αβ T lymphocyte Natural killer cells T helper T cytotoxic γδ T lymphocyte Monocyte, granulocyte B lymphocyte Interleukin-2 receptor Major histocompatibility complex class II

BAQ95A CACT83B CACT80C B7A1 DH59B BAQ44A CACT116A TH14B

1:100 1:160 1:400 1:100 1:100 1:100 1:200 1:200

1

Monoclonal antibodies were raised in mice that were specific for bovine mononuclear cell surface antigens, Veterinary Medicine Research and Development, Inc. (Pullman, WA). 2 Cells expressing the molecule. 3 Mab = Monoclonal antibodies that specifically react with the mononuclear cell differentiation antigen. 4 All Mab were originally at a stock concentration of 1 mg/ml and then diluted with PBS.

largest Akaike’s Information Criterion and Schwarz Bayesian Criterion was used (15). All means presented are least squares means. Significant differences among treatments were declared at P < 0.05. RESULTS AND DISCUSSION Ingredient and nutrient composition of the TMR are presented in Tables 1 and 3. The nutrient content of the diet met or exceeded NRC (17) requirements for cows at the production levels observed in this experiment. The basal diet plus Mepron威85 (0, 15, 30 g/d) provided 100, 118, or 132% of Met requirements, respectively (Mepron威85 Dairy Ration Evaluator). The basal diet provided 108% of the lysine requirement for all treatments (Mepron威85 Dairy Ration Evaluator). The supplemental Met concentrations in the current experiment were similar to other studies evaluating production responses (4, 5, 10, 11, 23), which ranged from 0 to 60 g of supplemental Met (as fed). Armentano et al. (1), however, fed rumen-protected Met at 85 and 100% of NRC (17) recommended amounts and reported inTABLE 3. Mean chemical composition of TMR on a DM basis. Item 2

CP, % Soluble protein,2 % NEL3 Mcal/kg ADF, % NDF, % Ca, % P, % Mg, % K, % Met, g/100 g of DM2 Lys, g/100 g DM

X1

SD

18.17 32.03 1.67 20.00 32.60 0.94 0.47 0.31 1.39 0.28 0.68

0.64 2.79 0.04 1.21 1.47 0.05 0.01 0.07 0.21 0.03 0.07

n = 6. Basal TMR only; did not include supplemental Met. 3 Estimated according to NRC (17). 1 2

Journal of Dairy Science Vol. 82, No. 9, 1999

creased milk protein content with increasing dietary Met. Lymphocyte Proliferation and Isolated Mononuclear Cell Phenotypic Characterization The primary objective of this study was to determine the optimal dose of Mepron威85 necessary to stimulate lymphocyte proliferation in the dairy cow. The lymphocyte proliferation responses of PBMC are summarized in Table 4. Cows on the ME30 treatment had higher (P < 0.05) lymphocyte proliferation in response to ConA (P = 0.04) and PHA (P = 0.02) than to the control or ME15 treatments. Lymphocyte proliferation responses to PWM and IL-2 were similar for all treatments, averaging 116.33 and 59.55 cpm × 103, respectively. Phenotypic composition of mononuclear cell populations in PBMC and milk were not affected by treatment (Tables 5 and 6) and were similar to values reported by Park et al. (25), who monitored mononuclear cell populations in blood and mammary gland secretions throughout various stages of lactation. The mitogens ConA and PHA activate T lymphocytes; PWM activates both T lymphocytes and B lymphocytes, and IL-2 activates any cell containing IL-2 receptors (14). The results of this experiment suggest that peripheral T lymphocyte activity was enhanced by Mepron威85, as shown by the increased proliferative responses to ConA and PHA (14, 34). The PWM proliferative response tended to increase as supplemental Mepron威85 increased, further suggesting increased lymphocyte activity. The IL-2 proliferation was not affected by treatment. No shifts in phenotypic composition of mononuclear cell populations in PBMC or milk were observed with supplemental Mepron威85 feeding. Lack of shifts in PBMC and milk mononuclear cell populations may not be unusual because there was not a mastitic challenge that would cause a recruitment of

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METHIONINE AND LYMPHOCYTE PROLIFERATION TABLE 4. Proliferation of isolated mononuclear cells from the peripheral blood to various mitogens in cows fed 0 g/d (control), 15 g/d (ME15), or 30 g/d (ME30) of Mepron威85.a Treatment Mitogen (cpm × 10 ) 2

3

3

Control

ME15

a

ConA PHA PWM IL-2

ME30 a

128.48 90.69a 109.59 62.76

b

126.97 100.39a 113.68 61.61

140.39 130.90b 125.76 54.28

SEM

P

3.97 8.95 6.83 3.65

0.04 0.02 0.27 0.24

Means within the same row with different superscripts differ (P < 0.05). Degussa, Corp., Allendale, NJ. 2 ConA = concanavalin A, PHA = phytohemagglutinin, PWM = pokeweed mitogen, and IL-2 = interleukin a,b 1

2. 3 Proliferation expressed as increased counts per minute over control cultures; values are least squares means; n = 7.

mononuclear cells to the mammary gland (34). Therefore, it appeared that feeding Mepron威85 increased the proliferative ability of T lymphocytes, as suggested in other species (6, 12, 13, 37). Whether cows showing a higher lymphocyte proliferation response would be more resistant to infection could not be concluded from this experiment. In addition, it is not known what effects Mepron威85 may have on lymphocyte proliferation during periods of immunosuppression, such as the periparturient period. Certain normal physiological events associated with parturition may suppress lymphocyte function (such as increased estrogen and cortisol concentrations), which, in turn, may increase susceptibility to infection (33). Further studies are necessary not only to clarify the relationship between lymphocyte function and susceptibility to infection but also to further investigate the possibility of stimulating the immune system in response to increased dietary Met concentrations.

for cows on the ME30 treatment, intermediate for cows on the ME15 treatment, and lowest for control cows. These data provide evidence that the Met was protected from ruminal degradation and was absorbed into the blood (24). These serum values agree with other studies (21, 26, 28) that also reported linear increases in serum Met with increasing dietary Met. Pisulewski et al. (26) theorized that a linear increase in serum Met across all treatments suggested that serum Met might not serve as an accurate criterion for Met adequacy in the diet. This theory is supported by other researchers (23, 24) who also observed the absence of any inflection point in the Met response curve to increasing doses of ruminally protected Met in dairy cows. This data is in contrast to data for growing ruminants in which plasma-free amino acids may serve as a criterion of amino acid adequacy (36). DMI, Milk Yield, and Composition A secondary objective of this study was to monitor production parameters in response to varying concentrations of supplemental Mepron威85. Mean DMI, daily milk yields, and 4% FCM were not affected by treatment

Serum Met As expected, serum Met was significantly (P < 0.001) affected by treatment (Table 7). Serum Met was highest

TABLE 5. Isolated mononuclear cell populations from peripheral blood as determined by flow cytometric analysis with cell-type specific antibody markers in cows fed 0 g/d (control), 15 g/d (ME15), or 30 g/d (ME30) of Mepron威85.1 Monoclonal antibody2 Treatment

CD2

CD4

CD8

Control ME15 ME30

35.3 33.1 34.5

22.5 20.3 23.8

7.8 7.7 7.4

γδ

MG

B

IL-2r

MHCII

35.3 33.1 34.5

22.6 20.4 23.9

(% of cells positive for staining)

SEM3

1.53

1.89

0.53

10.0 9.8 8.2 2.62

22.9 23.8 20.9 1.63

35.8 34.4 37.2 2.62

1.55

1.90

1

Degussa, Corp., Allendale, NJ. MG = monocyte or granulocyte, B = B lymphocyte, IL-2r = interleukin-2 receptor, and MHCII = major histocompatibility complex class II. 3 n = 7. 2

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SODER AND HOLDEN TABLE 6. Isolated mononuclear cell populations from bovine milk as determined by flow cytometric analysis with cell-type specific antibody markers in cows fed 0 g/d (control), 15 g/d (ME15), or 30 g/d (ME30) of Mepron威85.1 Monoclonal antibody2 Treatment

CD2

CD4

CD8

Control ME15 ME30 SEM3

37.35 36.70 37.51

13.18 13.22 14.28

(% of cells positive for staining) 16.31 2.75 43.1 4.96 16.69 2.68 53.5 4.40 17.64 2.39 42.1 6.07

3.95

2.69

γδ

2.85

0.51

MG

B

11.2

1.04

IL-2r

MHCII

1.20 1.03 1.44

13.6 12.5 13.6

0.68

4.10

1

Degussa, Corp., Allendale, NJ. MG = monocyte or granulocyte, B = B lymphocyte, IL-2r = interleukin-2 receptor, and MHCII = major histocompatibility complex class II. 3 n = 7. 2

and averaged 24.67, 45.84, and 38.27 kg/d, respectively (Table 7). Percentages of milk protein and milk fat were not affected by treatment and averaged 3.13% and 2.97%, respectively. Treatment also had no effect on protein and fat yields, averaging 1.42 and 1.34 kg/d, respectively. Milk urea nitrogen was not affected by treatment, averaging 14.20 mg/dl across all treatments. Mean SCC was significantly affected (P = 0.03) by treatment. Cows on the ME15 treatment had higher SCC than did control or ME30 cows. The elevated SCC for the ME15 treatment might have been due to one cow that contracted mastitis at the end of the trial while on the ME15 treatment. The SCC was significantly affected by square (data not shown). Cows in square 1 (high SCC) had greater (P = 0.03) SCC than cows in square 2, averaging 288,321 and 40,912, respectively. Two cows on the trial (both in square 1) had high SCC (>100,000) throughout the trial, even though milk sam-

ples cultured for mastitis-causing organisms were repeatedly negative throughout the trial. Total crude protein and Lys intakes (basal diet plus treatment) were not affected by treatment. As expected, total Met intake (basal diet plus treatment) increased linearly (P < 0.001) with increasing Mepron威85 supplementation. In agreement with the findings in the present study, other researchers reported no effect of rumen-protected Met on DMI (1, 2, 22, 24, 26, 29), milk yield (10, 11, 22, 24, 27), or milk composition (22, 24, 27). Lack of milk yield and composition response may indicate that dietary Met status was not first-limiting for milk yield or composition (5, 31, 32). Additionally, early lactation cows tend to show a greater response to rumen-protected Met than do midlactation cows because of greater nutrient demands during early lactation (10, 16, 31). Although, a few studies have shown increased milk yield in midlactation cows supplemented with rumen-

TABLE 7. Mean serum Met, DMI, nutrient intake, and milk yield and composition in cows fed 0 g/d (control), 15 g/d (ME15), or 30 g/d (ME30) Mepron威85.1 Treatment Item

Control

ME15

ME30

SEM

P

Serum Met, µmol/dl DMI, kg/d CP intake, kg/d Met intake, g/d Lys intake, g/d

a

2.63 24.99 4.54 69.97a 169.94

b

3.19 24.22 4.41 82.84b 164.75

c

3.84 24.79 4.52 99.41c 168.58

0.10 0.40 0.07 1.11 2.70