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Ruminal Degradability, Intestinal Disappearance, and Plasma Methionine Response of Rumen-Protected Methionine in Dairy Cows
J. Dairy Sci. 84:1480–1487 American Dairy Science Association, 2001.
Ruminal Degradability, Intestinal Disappearance, and Plasma Methionine Response of Rumen-Protected Methionine in Dairy Cows1 K. M. Koenig and L. M. Rode2 Agriculture and Agri-Food Canada, Research Branch Lethbridge, AB, Canada T1J 4B1
ABSTRACT Bioavailability of Met from a rumen-protected Met product was evaluated in two experiments using three ruminally and duodenally cannulated lactating (experiment 1) and nonlactating (experiment 2) dairy cows. In the first experiment, the ruminal in situ and mobile bag technique was used to assess ruminal degradability and intestinal disappearance of Met from the protected Met product. Effective ruminal degradability of Met at a ruminal outflow rate of 0.11/h was 21.7%. Combining effective ruminal degradability with intestinal digestibility yielded an estimate of Met availability of 25%. In the second experiment, designed as a 3 × 3 Latin square, Met availability was assessed by determining the response of plasma Met to supplementation of the protected Met product relative to that of duodenally administered Met. The periods were 1 wk with cows fed a meal containing 0, 20, or 63 g of protected Met on d 1 and infused intraduodenally with 10.7 g of Met on d 4. Blood was collected at various times relative to the time of oral dosing and the commencement of the duodenal infusion. Plasma Met response measured as area under the curve increased linearly with increasing protected Met. The response of plasma Met increased by 33 and 65.5% of the control values for 20 and 63 g of protected Met, respectively. Intestinal bioavailability of Met in the protected Met product ranged from 22 to 34%. (Key words: rumen-protected methionine, intestinal availability, dairy cow) Abbreviation key: AUC = area under the curve, ERD = effective ruminal degradability, RPAA = rumen-protected AA, RPMet = rumen-protected Met. INTRODUCTION Protein is one of the major limiting nutrients in the diets of lactating dairy cows. This is particularly the Received August 25, 2000. Accepted January 4, 2001. Corresponding author: K. M. Koenig; e-mail: [email protected]. 1 Contribution Number 3870043 of the Lethbridge Research Centre. 2 Current address: Biovance Technologies, Inc., 3303 Beauvais Place S., Lethbridge, Alberta, T1K 3J5.
case for cows in early lactation when DMI is relatively low and protein requirement is high. Feeding a diet containing more protein is not a satisfactory solution because the breakdown of dietary protein in the rumen is one of the most inefficient processes in ruminant nutrition and will lead to the excretion of excess nitrogen. In typical North American dairy rations, only 25 to 35% of the feed protein reaches the small intestine for absorption. In an attempt to overcome this inefficiency, dietary protein sources that are considered to be good sources of bypass or rumen undegradable intake protein have been used. An alternate strategy is to feed rumen-protected AA (RPAA) so that any AA imbalances are corrected and overall utilization of dietary protein is improved. Dairy diets formulated for high milk production are often deficient in Met or Lys (Rulquin et al., 1993; Schwab et al., 1992) and possibly several additional AA. Protein supplements such as fish meal, which is relatively high in rumen undegradable Met and Lys, and blood meal, which is moderate in rumen undegradable Met but high in Lys, also contribute to additional alpha-amino N beyond the animal’s requirement. This extra N must be excreted as urea at an additional energy cost to the animal. If AA can be protected from ruminal breakdown and be absorbed in the intestine, then the total amount of protein absorbed by the dairy cow will be utilized more efficiently. This will result in a potential reduction in the protein levels required in dairy cow diets. Rumen-protected AA products must be resistant to degradation until they escape the rumen to the small intestine. In the small intestine, they must then be available for absorption to be of value to the animal. While several RPAA products are commercially available, the efficacy of many of these products is not well established. The objective of this study was to quantitate the bioavailability of a rumen-protected methionine (RPMet) product based on 1) in situ ruminal degradability and intestinal disappearance, and 2) the response of blood plasma Met concentrations to RPMet and duodenally administered Met.
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BIOAVAILABILITY OF PROTECTED METHIONINE Table 1. Ingredient composition of TMR. Item
%, DM basis
Barley silage Alfalfa cubes Barley grain (medium roll) Beet pulp Blood meal Soybean meal Canola meal Dried molasses NaHCO3 CaHPO4 Mineral premix1 Vitamin premix2 Flavor3 Liquid molasses Canola oil
29.80 20.11 29.07 7.26 6.25 2.23 2.17 0.43 0.89 0.57 1.18 0.015 0.009 0.0009 0.0020
1 Supplies per kilogram of TMR: 1.0% NaCl, 92 mg/kg of Zn, 87 mg/ kg of Mn, 24 mg/kg of Cu, 1.2 mg/kg of I, 0.48 mg/kg Se, and 0.37 mg/kg Co. 2 Supplies per kilogram of TMR: 1500 IU of vitamin A, 150 IU of vitamin D, and 1.5 IU of vitamin E. 3 Cattle feeding flavor (Alltech, Inc., Guelph ON, Canada).
MATERIALS AND METHODS Experiment 1 Animals and diet. Three lactating Holstein cows (97 ± 30 DIM and 629 ± 16 kg BW; X ± SD) with ruminal (Bar Diamond, Parma, ID) and duodenal T-type cannulas (placed proximal to the common bile and pancreatic duct approximately 10 cm distal to the pylorus) were used to evaluate ruminal degradation and intestinal disappearance of Met from an RPMet product. The cows were housed in individual tie stalls on mattresses bedded with wood shavings and were milked twice daily in their stalls at 0700 and 1700 h. The cows were offered a TMR (Table 1) twice daily at 0630 and 1530 h, at a level that permitted approximately 10% orts. Feed offered and orts were recorded daily. The TMR and barley silage were sampled weekly, and orts were collected daily and composited weekly to calculate DMI. The DM content of the feed and orts was determined by drying in a forced-air oven at 55°C for 48 h. The cows were adapted to the diet for 28 d before commencing three consecutive 14-d periods. Measurements and samples were obtained from d 4 to 14 of each period. During the experiment, the cows averaged 26 ± 3.1 kg/d of DMI and 24.6 ± 5.53 kg/d of milk. Between h 0900 and 1100 the cows were released outside to an exercise pen, except when measurements were required during this time. The cows were cared for according to the guidelines of the Canadian Council on Animal Care (1993). In situ rumen and mobile bag procedure. Bags measuring 3.0 × 3.5 cm were constructed from polyester
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monofilament fabric (PeCAP, 51 micron, Tetko, Inc., Elmford, NY). Forty polyester bags containing 1.0 g of Mepron M85 (85% minimum D,L-Met content, Degussa Corporation, Allendale, NJ) were heat sealed and distributed among five mesh retaining bags (eight polyester bags per mesh bag) and incubated in the rumen to determine DM and Met degradation. The mesh bags with 3- × 5-mm pores permitted ruminal fluid to percolate freely and contained a weight to control the position of the bags within the ruminal contents. One mesh bag, containing eight polyester bags, was removed from the rumen after 3, 6, 12, 24 and 96 h of incubation in the rumen. Upon removal, the polyester bags were washed under cold running tap water until there was no color visible in the rinse water. Four of the bags (mobile bags) were placed one at a time at 15-min intervals into 200 ml of pepsin-HCl solution (2 g of pepsin A (Sigma Chemical Co., St. Louis, MO) per liter of 0.01 N HCl) for 1 h at 39°C to simulate digestion within the abomasum. Bags that were not placed in the pepsin-HCl solution immediately after rumen withdrawal were stored at 4°C. Following the 1-h pepsin-HCl incubation, the bags were introduced at 15-min intervals, through the neck of the duodenal cannula and into the duodenum. Within 12 to 24 h after placement into the duodenum, the mobile bags were recovered from the feces and washed under cold running tap water as described previously for the bags removed from the rumen. Zero-hour bags were incubated in 600 ml of artificial rumen fluid prepared according to the method of Goering and Van Soest (1970), but excluding the microminerals, for 30 min at 39°C. After incubation, four 0-h bags were washed and incubated in pepsin-HCl and then placed in the duodenum and recovered from the feces as previously described for the other time points. The polyester bags containing the residue from rumen and mobile bags were oven-dried at 55°C for 48 h and then weighed to determine DM disappearance. The rumen and mobile bag procedure was repeated in each cow for a second and third period. Rumen and mobile bag residues were ground to a fine powder using a mortar and pestle, mixed with 0.1 N HCl to solubilize the Met, diluted with 0.1 N HCl, and filtered through a 0.45-micron syringe-tip filter (ColeParmer, Chicago, IL). Methionine was quantified by HPLC based on precolumn derivatization with phenylisothiocyanate (Cohen et al., 1989). The methionine content of the RPMet as assessed from the extraction and analysis of triplicate samples of 1.0 g was 87.1% and was marginally higher than the product specification of 85% minimum. The amount of RPMet (DM basis) that was initially placed in the polyester bags before incubation was multiplied by the measured Met concentration (87.1%) to calculate the Met content. Journal of Dairy Science Vol. 84, No. 6, 2001
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Ruminal DM and Met disappearance at each individual incubation time were calculated as the difference between the DM and Met content of the initial samples and the residues remaining after incubation in the rumen and expressed as a percentage of the DM and Met content of the initial sample, respectively. Kinetic parameters of DM and Met disappearance in the rumen were estimated using an iterative least squares method of the nonlinear regression procedure of SAS (1988) by fitting the following model (Orskov and McDonald, 1979) to the means of quadruplicate values for each cow in each period: y = a + b(1 − e−ct ), where y is the percentage of DM or Met that disappears at time t (%), a is the fraction of DM or Met rapidly solubilized (%), b is the fraction of DM or Met potentially degradable (%), c is the fractional rate constant for the disappearance of fraction b (/h), and t is the time of incubation (h). The potentially degradable fraction (%) was calculated as (a + b). Effective ruminal degradability (ERD) was calculated from the equation: ERD = a + (b × c)/(c + k), where k is the fractional passage rate from the rumen and constants a, b, and c are as defined previously. Effective ruminal degradabilities of DM and Met were estimated for hypothetical ruminal passage rates of 0.05, 0.08, and 0.11/h. Total tract DM and Met disappearance of RPMet at each individual incubation time were calculated as the difference between the DM and Met content of the initial sample and the residue remaining after recovery of the mobile bag in the feces and expressed as a percentage of DM and Met content of the initial sample, respectively. Dry matter and Met disappearance of RPMet in the intestinal tract was calculated as the difference between the average DM and Met disappearance at each time point in the rumen-incubated bags and the mobile bags for each cow, in each period, at each incubation time. Intestinal DM and Met disappearance of RPMet was also expressed as a percentage of the RPMet entering the intestine. An estimate of bioavailability (%) was calculated by combining effective ruminal degradability and intestinal digestibility as follows: (100 − ERD) × fractional intestinal digestibility. Experiment 2 Animals and diet. Three nonlactating Holstein dairy cows (750 ± 89.0 kg of BW, 11.9 ± 0.7 kg of DMI; Journal of Dairy Science Vol. 84, No. 6, 2001
x¯ ± SD) with ruminal and duodenal cannula were used to assess the gastrointestinal availability of RPMet by comparing the response of plasma Met to an oral dose of RPMet and duodenally administered D,L-Met. The experiment consisted of three periods of 6 d (d 1 to 3 for oral dosing and sampling and d 4 to 6 for duodenal dosing and sampling). One day of rest separated the periods. Cows were weighed 1 d before the beginning of the first period and at the end of each period at the same time each day to determine the mean BW for each cow. Cows were housed and cared for as described for the previous experiment. Two weeks before the experiment, the cows began receiving the experimental diet which consisted (DM basis) of 3.3 kg of barley silage, 2.9 kg of grass hay, and 3.6 kg of barley grain concentrate daily (Table 2). One half of the daily allotment of barley silage was offered at 0600 h and the other half along with the grass hay was offered at 1530 h. The concentrate was fed at 1430 h. Samples of the barley silage, hay, and concentrate were collected weekly and composited. Feed refused was weighed daily and sampled, and the samples were composited for each period. Feed and orts samples were dried at 55°C in an oven for 48 h. Feed intake was determined as the difference in the DM of feed offered and refused. Dosing of RPMet and D,L-Met. In each of the three periods, the cows received one of three oral doses: 0 (control), 20, and 63 g of RPMet (Mepron M85); equivalent to 0, 17.4 and 54.9 g of D,L-Met (based on 87.1% Met). Coarsely ground barley grain served as the carrier for the dose. Cows began receiving 0.5 kg of the grain carrier at 0600 h, 4 d before the beginning of the first period and continued to receive it throughout the experiment. On the first day of each period, the oral dose of RPMet was mixed with the barley grain. On this day, the 0600 h feed allotment was withheld from cows until the dose mixed with the barley grain was administered. The cows were expected to consume the dose within 10 Table 2. Ingredient composition of the diet of experiment 2. Item
%, DM basis
Barley silage Grass hay Barley grain (medium roll) Canola meal Canola oil Liquid molasses Mineral premix1 Vitamin premix2
33.55 29.65 30.51 3.98 0.17 0.42 1.7 0.02
1 Supplies per kilogram of concentrate: 4.1% NaCl, 360 mg/kg of Zn, 340 mg/kg of Mn, 93 mg/kg of Cu, 4.7 mg/kg of I, 1.9 mg/kg of Se, and 1.4 mg/kg of Co. 2 Supplies per kilogram of concentrate: 54,500 IU of vitamin A, 5450 IU of vitamin D, and 54 IU of vitamin E.
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min. If, however, the dose was not consumed within 30 min, the dose that remained was placed into the rumen under the mat layer. Blood was collected via jugular venipuncture into sterile heparinized Vacutainer tubes (Vacutainer Systems, Becton Dickinson, Rutherford, NJ) at 0, 1, 3, 6, 9, 12, 24, and 48 h relative to the time of oral dosing. Seventy-two hours after oral dosing, each cow received Met by infusion into the duodenum. An indwelling catheter was placed in the jugular vein of the cows 10 to 15 h before infusion. To simulate the rate of Met delivery to the intestine from orally administered RPMet, D,L-Met was continuously infused at an exponential rate of decline in concentration into the lumen of the duodenum over a 12-h period beginning at 0600 h on d 4 of each period. The D,L-Met (BDH Chemicals Ltd., Poole, London), 13.7 g, was dissolved in 400 ml of 0.1 N HCl. Once dissolved, the volume of the Met infusate was adjusted to 450 ml with 0.2 M NaOH to yield a pH of 2.8. A volume of approximately 450 ml was maintained in the Met infusate reservoir by a constant inflow of 0.0018 N HCl, pH 2.8, at a rate equivalent to the outflow that entered the intestine. After 12 h, the volume of fluid remaining in the infusate reservoir was measured and analyzed for Met to determine the exact quantity of Met administered to the duodenum. Blood was collected via the catheter into heparinized tubes for 0, 1, 2, 3, 4.5, 6, 7.5, 9, 10.5, 12, and 24 h relative to the commencement of the duodenal Met infusion. At 48 h, blood was collected by jugular venipuncture. Plasma was separated from cells by centrifuging at 3000 × g for 15 min and collecting the supernatant. The plasma supernatant was deproteinized by adding an equal volume of acetonitrile and centrifuging at 3000 × g. The protein-free supernatant was collected and stored at −70°C until analyzed for Met. Samples of deproteinized plasma and the remaining infusate were analyzed for Met by HPLC as described previously. Calculations and statistical analysis. The response to the various oral doses of RPMet and the duodenal administration of D,L-Met was determined as the area under the curve (AUC) for plasma Met concentration over time by the trapezoidal method using SAS (1988). Data for AUC was analyzed using the GLM procedure of SAS (1988) to account for the effects of cow, period, and dose. Linear and quadratic effects were tested using orthogonal contrasts. Differences were considered significant at P < 0.05, and trends were discussed at 0.05 < P < 0.15. The response of plasma Met to 20 and 63 g of RPMet and the duodenally infused Met was expressed as a percentage of the AUC above the baseline response at 0 g of RPMet:
Figure 1. In situ ruminal DM (䊐) and Met (•) disappearance from rumen-protected methionine. Each time point represents the mean of three cows, where the value for each cow was calculated as the mean of three periods, each of which were estimated from the incubation of four bags. The vertical bars represent the SEM. Where the vertical bars are not visible, the SEM is smaller than the size of the symbol representing the mean.
100 × [(AUC20 or 63 RPMet or Met − AUC0 g RPMet)/AUC0 g RPMet]. The amount of absorbable Met delivered to the intestine was then calculated from the ratio of the plasma response of Met (percentage above the baseline response) for the RPMet and the duodenally infused Met multiplied by the amount of Met infused into the lumen of the duodenum: (AUC20 or 63 RPMet % above baseline/AUCMet % above baseline) × g of Met infused into the duodenum. RESULTS AND DISCUSSION In Situ Ruminal Digestion Kinetics One cow was removed from the first experiment before completion of the third period and, therefore, her data for this period were not included in the study. In situ ruminal DM and Met disappearance curves are presented in Figure 1 and the ruminal digestion kinetics in Table 3. The ruminal fractional degradation rate (parameter ‘c’) was 0.0224 and 0.0225/h for DM and Met, respectively. The low fractional degradation rate is indicative of a stable RPMet product. Potential degradability was 93.6% for DM and 95.7% for Met. These values are consistent with a product that is potentially high in digestibility and bioavailability. Effective ruminal degradability, and ruminal escape or bypass, are a function of the ruminal outflow rate. At a ruminal outflow rate of 0.05/h, ERD was calculated as 29.5% for DM and 34.0% for Met. At a ruminal outflow rate of Journal of Dairy Science Vol. 84, No. 6, 2001
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0.08/h, ERD was 21.3 and 26.1% for DM and Met, respectively. The ruminal retention time of the RPMet product is reported to be about 6 h (Greissinger et al., 1994), corresponding to a fractional turnover rate of approximately 0.11. At a ruminal outflow rate of 0.11/ h, ERD was calculated as 16.8% for DM and 21.7% for Met. For a RPMet product, particle size and specific gravity (density) are critical factors affecting ruminal outflow rate. If particles are too dense, they will sink in the rumen, and if density is too low, then particles will float. In either case, ruminal outflow rate will decrease to the range of 0.08 to 0.05/h or lower, significantly increasing the effective degradability of the product. Ruminal, Intestinal, and Total Tract Disappearance Ruminal and total tract disappearance of DM and Met (Table 4) were similar, which is consistent with the high Met content of RPMet (85% minimum D,LMet). Ruminal disappearance of Met was marginally higher than ruminal DM disappearance. Because small flaws or cracks in the RPMet coating would allow for solubilization of the Met and since the coating material appears to be of low digestibility, these results are to be expected. The extent of ruminal RPMet disappearance observed in the present study is in good agreement with the results of Overton et al. (1996), but slightly lower than the results of Berthiaume et al. (2000). Total tract DM and Met disappearance of RPMet ranged from 39 to 50% for all ruminal incubation times, except for 24 and 96 h, where total tract disappearance was higher and ranged from 55 to 90%. Higher total tract disappearance values for these time points reflected the higher ruminal disappearance observed for these times. Intestinal tract disappearance, measured as total tract minus ruminal disappearance, declined Table 3. In situ ruminal digestion kinetics and effective degradability of rumen-protected methionine. Parameter1
DM
SEM
Met
SEM
a, % b, % c, /h a+b ERD, k = 0.05/h k = 0.08/h k = 0.11/h
1.45 92.17 0.0224 93.63 29.45 21.28 16.81
0.43 1.34 0.0031 1.12 2.13 1.65 1.30
6.77 88.97 0.0225 95.73 33.98 26.06 21.71
0.70 1.61 0.0034 2.01 2.57 2.11 1.77
1 Parameters were calculated from the fitted equation y = a + b(1−e− ), where y = percentage of DM or Met disappearance from the polyester bag at time t, a = soluble fraction (%), b = slowly degradable fraction (%), c = fractional rate constant at which b is degraded (/h), and t = time of incubation (h). The potentially degradable fraction (%) was calculated as (a + b). Effective ruminal degradability (ERD) was calculated using the equation a + bc/(c + k), where k = 0.05, 0.08, and 0.11/h. ct
Journal of Dairy Science Vol. 84, No. 6, 2001
Table 4. Dry matter and methionine disappearance from rumenprotected methionine in the rumen, intestine and total tract of dairy cows.1 Hours of ruminal incubation
DM Rumen2, % SEM Intestine,3 % SEM Intestine,4 % SEM Total tract,5 % SEM Methionine Rumen, % SEM Intestine,3 % SEM Intestine,4 % SEM Total tract, % SEM
0
3
6
12
24
96
2.7 0.1 37.9 3.9 39.2 4.2 40.5 4.0
6.5 0.2 33.6 1.9 36.1 2.2 40.1 1.8
12.5 0.9 26.6 1.4 30.7 2.0 39.1 2.0
21.4 0.9 24.4 1.5 31.1 1.9 45.7 0.7
39.1 1.5 16.7 2.4 27.7 4.2 55.8 2.5
82.1 0.2 3.8 0.3 20.2 2.6 85.9 0.2
8.6 0.4 35.9 3.8 39.5 4.4 44.5 4.0
14.7 1.9 28.7 1.3 34.7 2.3 43.4 2.3
17.8 0.8 24.4 1.5 29.6 1.8 42.2 0.8
25.0 1.3 25.0 1.3 33.4 1.4 50.1 1.1
42.9 1.3 15.8 2.0 28.1 3.7 58.8 1.7
85.5 1.3 4.0 1.3 26.1 6.6 89.6 0.3
1 n = 3. Observations were obtained from three cows, replicated for three periods with four bags per incubation time. 2 Ruminal disappearance (%) = [(initial − residue remaining after ruminal incubation)/initial] × 100%. 3 Intestinal tract disappearance (%) = total tract disappearance (%) − ruminal disappearance (%). 4 Intestinal tract disappearance as a proportion of the ruminally protected methionine leaving the rumen (%) = [(total tract disappearance (%) − ruminal disappearance (%)/(100 − ruminal disappearance (%))] × 100%. 5 Total tract disappearance (%) = [(initial − residue remaining after recovery of mobile bag from feces)/initial] × 100%.
steadily as ruminal incubation time increased. When intestinal disappearance was expressed as a percentage of the RPMet entering the intestine, Met disappearance ranged from 26.1 to 39.5% with an average value of 31.9%. The length of ruminal incubation did not appear to affect digestibility of Met entering the intestine. Combining ERD and intestinal digestibility will yield an estimate of bioavailability. At a ruminal outflow rate of 0.08/h, Met bioavailability was estimated to be 23.6% ([100 − ERD] × intestinal digestibility). At 0.11/h, Met bioavailability was 25%. Generally, the better the ruminal protection, the lower the intestinal availability for encapsulated RPAA. Therefore, there is often a tradeoff between ruminal resistance and intestinal availability. The exception to this would be with products with pHsensitive polymer coatings (Blum et al., 1999). The samples size to surface area used in the mobile bag technique is generally larger (de Boer et al., 1987; Sauer et al., 1983; Varvikko and Vanhatalo, 1990) than that used for the ruminal in situ technique. A smaller bag is favored for the mobile bag technique to avoid disruption of the flow of digesta through the intestine. As the sample size to surface area increases, however,
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feed particles within the bags can become compacted, restricting solubilization and access of digestive enzymes to the feed particles, which will thereby underestimate in situ digestibility. Underestimation of in situ ruminal digestibility is apparent particularly at the early time points of the ruminal incubation (Nocek, 1988). The larger sample size to surface area may, however, have less of an effect on intestinal digestibility, due to the length of time the bags remain in the intestine before the measurement of substrate disappearance. The mobile bag technique has compared favorably with true protein digestibility measured conventionally in swine (Sauer et al., 1983) and cattle (de Boer et al., 1987). Although, in a recent study, the mobile nylon bag technique was reported to underestimate the bioavailability of Met from RPMet (Berthiaume et al., 2000). The mobile nylon bag technique used in this evaluation does not include any preintestinal loss of Met due to mastication. Mastication losses are difficult to measure, but they are a function of particle size and density. Estimates for RPAA with physical characteristics similar to the product used herein suggest that mastication and the preintestinal losses associated with mastication may be 10 to 15% of the oral dose (unpublished). However, without direct measurements for these mastication losses, it would be inappropriate to include the values in calculation of bioavailability. Intestinal disappearance was 25 to 36% for ruminal incubation times of 0 to 12 h in the present study, compared with values of 52 to 63% in the study of Overton et al. (1996) and 48% (for 4.5 h of incubation) in the study of Berthiaume et al. (2000). There were some methodological differences between the studies with regards to the sample size to surface area of the polyester bags, and the length of the time mobile bags were incubated in pepsin-HCl before insertion into the duodenum. The similarity between the present study and that of Overton et al. (1996) for ruminal degradability of RPMet suggests that access of digestive fluids was probably not a contributing factor to the differences observed for intestinal digestibility. Incubation of mobile bags in pepsin HCl for 1 or 2.5 h would have had little effect on the extent of intestinal degradation of RPMet and release of Met due to the acid-insensitive coating. Residence time within the intestinal tract could, however, be expected to affect the extent of Met release from the RPMet product. The incubation of the material within the rumen indicated that the extent of Met degradation increased with increasing time of exposure to digestive fluids. Higher DMI by cows in the present study, if coupled with a faster rate of passage through the lower tract, may have reduced intestinal disappearance and hence bioavailability of RPMet rela-
Figure 2. Response of plasma methionine to a 0 (•), 20 (䊏), and 63 (▲) g oral dose of rumen-protected methionine and the intraduodenal infusion of 10.7 g of D,L-methionine (䊐) in nonlactating dairy cows. The vertical bars represent the SEM (n = 3).
tive to the other two studies (Berthiaume et al., 2000; Overton et al., 1996). Response of Plasma Methionine to RPMet The response of plasma Met to a 20 and 63 g oral dose of RPMet was evaluated in the second study, relative to the direct administration of 10.7 ± 0.2 g of Met to the duodenum over a 12-h infusion. Twenty grams of RPMet was investigated, as 10 to 20 g is the dose recommended by the manufacturer of the RPMet product to meet the requirements of high producing dairy cattle. The cows readily consumed the smaller, 20-g dose of RPMet and the carrier, but two of the three cows refused to consume the higher dose of RPMet (63 g) within the 30 min permitted, and, therefore, the dose and carrier remaining was placed directly into the rumen through the rumen cannula. In other studies in which cows were fed similar amounts of RPMet mixed or top-dressed on rations, no negative effects on DMI have been reported (Blum et al., 1999; Papas et al., 1984b; Rogers et al., 1987). Plasma Met concentration peaked between 9 to 12 h after oral dosing of RPMet, and 3 h after initiation of the duodenal infusion of Met (Figure 2). The delay in reaching peak concentration following oral dosing of RPMet is due in part to the residence time in the rumen (6 to 9 h), and the time required for absorption and accumulation of Met in the plasma pool. Plasma Met concentrations had returned to baseline levels at 24 h after receiving the oral dose of RPMet and the duodenal infusion of Met, and, therefore, the response measured as AUC for plasma Met was determined from 0 to 24 h (Figure 2). Plasma Met response Journal of Dairy Science Vol. 84, No. 6, 2001
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(AUC) tended (P = 0.125) to increase linearly with increasing levels of RPMet (data not presented). The increase in plasma Met above the control level (0 g of RPMet) reached 32.5 and 65.5% for cows receiving 20 and 63 g of RPMet, respectively. As expected, plasma Met responded similarly (P > 0.05) to the duodenal infusion of Met when administered 24 h after the return to baseline of the plasma Met from the three oral treatment levels of RPMet. The increase in plasma Met response following duodenal administration of Met, ranged from 51 to 71% above the baseline response (plasma Met response to oral supplementation of 0 g RPMet), with an average response of 59.2%. To estimate the amount of Met delivered to the intestine by the RPMet product, the response (as a percentage above the baseline response at 0 g of RPMet) of plasma Met to oral RPMet supplementation was expressed as a proportion of the average response due to the duodenal administration of Met, and then multiplied by the amount of Met administered to the duodenum. Thus, 20 g of RPMet provided approximately 5.9 g of absorbable Met (32.5%/59.2% × 10.7 g of Met), and 63 g of RPMet delivered 11.8 g of absorbable Met (65.5%/59.2% × 10.7 g Met) to the intestine. An estimate of Met bioavailability was then calculated by expressing the quantity of absorbable Met delivered to the intestine as a percentage of the Met content of the oral dose of RPMet. Thus, the bioavailability of Met in RPMet, based on the response of plasma Met ranged from 22 to 34% [100% × 11.8 g of Met absorbed/(63 g of RPMet × 87.1% Met) and 100% × 5.9 g of Met absorbed/(20 g of RPMet × 87.1% Met)], and averaged 28%. The above estimate of RPMet bioavailability was based on a single-point calibration for the response of plasma Met to duodenally delivered Met. The variation of the response of plasma Met to a single level of duodenal Met was reduced for each animal at the expense of measuring the response to several levels with greater variation. The assumption was made that the plasma Met response to increasing duodenal Met is linear up to the amount of Met that was investigated. As previously discussed, a linear relationship was obtained for the response of plasma Met to increasing RPMet. The amount of Met delivered to the intestine (determined by the amount of Met remaining in the infusate reservoir after 12 h of administration) was less than the amount that was originally predicted to be delivered. As a result, the plasma Met response to the higher dose of RPMet (11.8 g of absorbable Met) was beyond the range of response to the Met administered duodenally (10.7 g of absorbable Met), but only marginally. The estimate of Met bioavailability based on plasma Met response may also be prone to sampling error if the Journal of Dairy Science Vol. 84, No. 6, 2001
peak plasma Met response after oral administration occurred between the times of plasma sampling. Missing the peak plasma Met response could result in the underestimation of the calculated AUC and hence the estimate of Met bioavailability. Amino acid concentrations in plasma or serum provide a qualitative measure of the postruminal delivery of essential AA from RPMet products (Blum et al., 1999; Papas et al., 1984a, 1984b). A quantitative measure was obtained in the present experiment by relating the plasma Met response of RPMet to the response of a known quantity of Met delivered directly to the intestine, the major site of AA absorption. The quantitative estimation of Met bioavailability required that a linear or other definable relationship exist between the plasma Met concentration and the amount of Met delivered postruminally. Identification of an inflection point or break point in a curve for the response of plasma AA concentrations to postruminally delivered AA has been used to identify the requirements of essential AA (Bergen, 1979). When the provision of the essential AA is below the requirement, the AA concentration in plasma may either increase marginally or not at all, but once the requirement for the AA has been met, plasma concentration increases more rapidly (Bergen, 1979; Broderick et al., 1974). The Met requirements of the nonlactating cows used in the present study were considered adequately met by the experimental diet (Cornell-PennMiner Dairy, version 1), and thus, the response of plasma Met tended to increase linearly (P = 0.125) with the postruminal supply of Met. Linear responses in plasma Met to incremental amounts of RPMet (Papas et al., 1984a; Piepenbrink et al., 1996; Rogers et al., 1987) and abomasally (Varvikko et al., 1999) or duodenally (Pisulewski et al., 1996) infused Met have also been reported by others. Assuming that there was no change in the rate of Met utilization by the tissues, induced by the amount of the dose administered, the increase in plasma Met concentration delivered by RPMet relative to the increase in a known quantity of duodenally delivered Met provided a sensitive, quantitative indicator of Met bioavailability. CONCLUSIONS The RPMet product has the characteristics to make it a highly effective source of rumen-protected Met. These characteristics are primarily due to the high load of Met (minimum 85% D,L-Met) and excellent ruminal resistance to degradation. However, the protection from degradation that is afforded in rumen is also apparent in the small intestine. Bioavailability, defined as the amount of available Met delivered to the intestine, averaged 28% when based on the response of plasma Met.
BIOAVAILABILITY OF PROTECTED METHIONINE
High producing dairy cows require 50 to 60 g of Met per day on typical diets, and may be deficient by up to 5 to 10 g of Met per day. Based on the mean estimate of bioavailability, 20 g of RPMet would provide 5.6 g of absorbable D,L-Met. ACKNOWLEDGMENTS The experiment was financially supported in part by Degussa Corporation (Allendale, NJ), the Matching Investment Initiative Fund (Agriculture and Agri-Food Canada, Lethbridge, AB, Canada), and the Canada/ Alberta Livestock Research Trust (Lethbridge, AB). The authors thank K. A. Andrews for the assistance in conducting the experiments, A. F. Furtado for performing the Met analysis, and the staff of the Lethbridge Research Centre dairy unit (Lethbridge, AB, Canada) for the care and management of the cows. REFERENCES Bergen, W. G. 1979. Free amino acids in blood of ruminants—physiological and nutritional regulation. J. Anim. Sci. 49:1577–1589. Berthiaume, R., H. Lapierre, M. Stevenson, N. Cote´, and B. W. McBride. 2000. Comparison of the in situ and in vivo intestinal disappearance of ruminally protected methionine. J. Dairy Sci. 83:2049–2056. Blum, J. W., R. M. Bruckmaier, and F. Jans. 1999. Rumen-protected methionine fed to dairy cows: Bioavailability and effects on plasma amino acid pattern and plasma metabolite and insulin concentrations. J. Dairy Sci. 82:1991–1998. Broderick, G. A., L. D. Satter, and A. E. Harper. 1974. Use of plasma amino acid concentration to identify limiting amino acids for milk production. J. Dairy Sci. 57:1015–1023. Canadian Council on Animal Care. 1993. Guide to the Care and Use of Experimental Animals. Vol. 1. 2nd ed. CCAC, Ottawa, ON, Canada. Cohen, S. A., M. Meys, and T. L. Tarvin. 1989. The Pico-Tag method. A manual of advanced techniques for amino acid analysis. WM02 Revision 1. Waters Corporation, Milford, MA. de Boer, G., J. J. Murphy, and J. J. Kennelly. 1987. Mobile nylon bag for estimating intestinal availability of rumen undegraded protein. J. Dairy Sci. 70:977–982.
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Goering, H. K., and P. J. Van Soest. 1970. Forage fiber analysis. Apparatus, reagents, procedures and some applications. USDA, ARS Agriculture Handbook, Number 379. Greissinger, D., H. Kniesel, W. Heimbeck, and H. Tanner, inventors. 1994. Active-substance preparation for oral administration, especially to ruminants. Degussa AG, assignee. US Pat. No. 5,279,832. Nocek, J. E. 1988. In situ and other methods to estimate ruminal protein and energy digestibility: A review. J. Dairy Sci. 71:2051–2069. Orskov, E. R., and I. McDonald. 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. (Cambr.) 92:499–503. Overton, T. R., D. W. LaCount, T. M. Cicela, and J. H. Clark. 1996. Evaluation of a ruminally protected methionine product for lactating dairy cows. J. Dairy Sci. 79:631–638. Papas, A. M., C. J. Sniffen, and T. V. Muscato. 1984a. Effectiveness of rumen-protected methionine for delivering methionine postruminally in dairy cows. J. Dairy Sci. 67:545–552. Papas, A. M., J. L. Vicini, J. H. Clark, and S. Peirce-Sandner. 1984b. Effect of rumen-protected methionine on plasma free amino acids and production by dairy cows. J. Nutr. 114:2221–2227. Piepenbrink, M. S., T. R. Overton, and J. H. Clark. 1996. Response of cows fed a low crude protein diet to ruminally protected methionine and lysine. J. Dairy Sci. 79:1638–1646. Pisulewski, P. M., H. Rulquin, J. L. Peyraud, and R. Verite. 1996. Lactational and systemic responses of dairy cows to postruminal infusions of increasing amounts of methionine. J. Dairy Sci. 79:1781–1791. Rogers, J. A., U. Krishnamoorthy, and C. J. Sniffen. 1987. Plasma amino acids and milk protein production by cows fed rumenprotected methionine and lysine. J. Dairy Sci. 70:789–798. Rulquin, H., P. M. Pisulewski, R. Ve´rite´, and J. Guinard. 1993. Milk production and composition as a function of postruminal lysine and methionine supply: A nutrient-response approach. Livest. Prod. Sci. 37:69–90. SAS/STAT User’s Guide, Release 6.03 Edition. 1988. SAS Inst., Inc., Cary, NC. Sauer, W. C., H. Jørgensen, and R. Berzins. 1983. A modified nylon bag technique for determining apparent digestibilities of protein in feedstuffs for pigs. Can. J. Anim. Sci. 63:233–237. Schwab, C. G., C. K. Bozak, N. L. Whitehouse, and M.M.A. Mesbah. 1992. Amino acid limitation and flow to duodenum at four stages of lactation. 1. Sequence of lysine and methionine limitation. J. Dairy Sci. 75:3486–3502. Varvikko, T., and A. Vanhatalo. 1990. The effect of differing types of cloth and of contamination by non-feed nitrogen on intestinal digestion estimates using porous synthetic-fibre bags in cows. Br. J. Nutr. 63:221–229. Varvikko, T., A. Vanhatalo, T. Jalava, and P. Huhtanen. 1999. Lactation and metabolic responses to graded abomasal doses of methionine and lysine in cows fed grass silage diets. J. Dairy Sci. 82:2659–2673.
Journal of Dairy Science Vol. 84, No. 6, 2001
Ruminal Degradability, Intestinal Disappearance, and Plasma Methionine Response of Rumen-Protected Methionine in Dairy Cows1 K. M. Koenig and L. M. Rode2 Agriculture and Agri-Food Canada, Research Branch Lethbridge, AB, Canada T1J 4B1
ABSTRACT Bioavailability of Met from a rumen-protected Met product was evaluated in two experiments using three ruminally and duodenally cannulated lactating (experiment 1) and nonlactating (experiment 2) dairy cows. In the first experiment, the ruminal in situ and mobile bag technique was used to assess ruminal degradability and intestinal disappearance of Met from the protected Met product. Effective ruminal degradability of Met at a ruminal outflow rate of 0.11/h was 21.7%. Combining effective ruminal degradability with intestinal digestibility yielded an estimate of Met availability of 25%. In the second experiment, designed as a 3 × 3 Latin square, Met availability was assessed by determining the response of plasma Met to supplementation of the protected Met product relative to that of duodenally administered Met. The periods were 1 wk with cows fed a meal containing 0, 20, or 63 g of protected Met on d 1 and infused intraduodenally with 10.7 g of Met on d 4. Blood was collected at various times relative to the time of oral dosing and the commencement of the duodenal infusion. Plasma Met response measured as area under the curve increased linearly with increasing protected Met. The response of plasma Met increased by 33 and 65.5% of the control values for 20 and 63 g of protected Met, respectively. Intestinal bioavailability of Met in the protected Met product ranged from 22 to 34%. (Key words: rumen-protected methionine, intestinal availability, dairy cow) Abbreviation key: AUC = area under the curve, ERD = effective ruminal degradability, RPAA = rumen-protected AA, RPMet = rumen-protected Met. INTRODUCTION Protein is one of the major limiting nutrients in the diets of lactating dairy cows. This is particularly the Received August 25, 2000. Accepted January 4, 2001. Corresponding author: K. M. Koenig; e-mail: [email protected]. 1 Contribution Number 3870043 of the Lethbridge Research Centre. 2 Current address: Biovance Technologies, Inc., 3303 Beauvais Place S., Lethbridge, Alberta, T1K 3J5.
case for cows in early lactation when DMI is relatively low and protein requirement is high. Feeding a diet containing more protein is not a satisfactory solution because the breakdown of dietary protein in the rumen is one of the most inefficient processes in ruminant nutrition and will lead to the excretion of excess nitrogen. In typical North American dairy rations, only 25 to 35% of the feed protein reaches the small intestine for absorption. In an attempt to overcome this inefficiency, dietary protein sources that are considered to be good sources of bypass or rumen undegradable intake protein have been used. An alternate strategy is to feed rumen-protected AA (RPAA) so that any AA imbalances are corrected and overall utilization of dietary protein is improved. Dairy diets formulated for high milk production are often deficient in Met or Lys (Rulquin et al., 1993; Schwab et al., 1992) and possibly several additional AA. Protein supplements such as fish meal, which is relatively high in rumen undegradable Met and Lys, and blood meal, which is moderate in rumen undegradable Met but high in Lys, also contribute to additional alpha-amino N beyond the animal’s requirement. This extra N must be excreted as urea at an additional energy cost to the animal. If AA can be protected from ruminal breakdown and be absorbed in the intestine, then the total amount of protein absorbed by the dairy cow will be utilized more efficiently. This will result in a potential reduction in the protein levels required in dairy cow diets. Rumen-protected AA products must be resistant to degradation until they escape the rumen to the small intestine. In the small intestine, they must then be available for absorption to be of value to the animal. While several RPAA products are commercially available, the efficacy of many of these products is not well established. The objective of this study was to quantitate the bioavailability of a rumen-protected methionine (RPMet) product based on 1) in situ ruminal degradability and intestinal disappearance, and 2) the response of blood plasma Met concentrations to RPMet and duodenally administered Met.
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%, DM basis
Barley silage Alfalfa cubes Barley grain (medium roll) Beet pulp Blood meal Soybean meal Canola meal Dried molasses NaHCO3 CaHPO4 Mineral premix1 Vitamin premix2 Flavor3 Liquid molasses Canola oil
29.80 20.11 29.07 7.26 6.25 2.23 2.17 0.43 0.89 0.57 1.18 0.015 0.009 0.0009 0.0020
1 Supplies per kilogram of TMR: 1.0% NaCl, 92 mg/kg of Zn, 87 mg/ kg of Mn, 24 mg/kg of Cu, 1.2 mg/kg of I, 0.48 mg/kg Se, and 0.37 mg/kg Co. 2 Supplies per kilogram of TMR: 1500 IU of vitamin A, 150 IU of vitamin D, and 1.5 IU of vitamin E. 3 Cattle feeding flavor (Alltech, Inc., Guelph ON, Canada).
MATERIALS AND METHODS Experiment 1 Animals and diet. Three lactating Holstein cows (97 ± 30 DIM and 629 ± 16 kg BW; X ± SD) with ruminal (Bar Diamond, Parma, ID) and duodenal T-type cannulas (placed proximal to the common bile and pancreatic duct approximately 10 cm distal to the pylorus) were used to evaluate ruminal degradation and intestinal disappearance of Met from an RPMet product. The cows were housed in individual tie stalls on mattresses bedded with wood shavings and were milked twice daily in their stalls at 0700 and 1700 h. The cows were offered a TMR (Table 1) twice daily at 0630 and 1530 h, at a level that permitted approximately 10% orts. Feed offered and orts were recorded daily. The TMR and barley silage were sampled weekly, and orts were collected daily and composited weekly to calculate DMI. The DM content of the feed and orts was determined by drying in a forced-air oven at 55°C for 48 h. The cows were adapted to the diet for 28 d before commencing three consecutive 14-d periods. Measurements and samples were obtained from d 4 to 14 of each period. During the experiment, the cows averaged 26 ± 3.1 kg/d of DMI and 24.6 ± 5.53 kg/d of milk. Between h 0900 and 1100 the cows were released outside to an exercise pen, except when measurements were required during this time. The cows were cared for according to the guidelines of the Canadian Council on Animal Care (1993). In situ rumen and mobile bag procedure. Bags measuring 3.0 × 3.5 cm were constructed from polyester
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monofilament fabric (PeCAP, 51 micron, Tetko, Inc., Elmford, NY). Forty polyester bags containing 1.0 g of Mepron M85 (85% minimum D,L-Met content, Degussa Corporation, Allendale, NJ) were heat sealed and distributed among five mesh retaining bags (eight polyester bags per mesh bag) and incubated in the rumen to determine DM and Met degradation. The mesh bags with 3- × 5-mm pores permitted ruminal fluid to percolate freely and contained a weight to control the position of the bags within the ruminal contents. One mesh bag, containing eight polyester bags, was removed from the rumen after 3, 6, 12, 24 and 96 h of incubation in the rumen. Upon removal, the polyester bags were washed under cold running tap water until there was no color visible in the rinse water. Four of the bags (mobile bags) were placed one at a time at 15-min intervals into 200 ml of pepsin-HCl solution (2 g of pepsin A (Sigma Chemical Co., St. Louis, MO) per liter of 0.01 N HCl) for 1 h at 39°C to simulate digestion within the abomasum. Bags that were not placed in the pepsin-HCl solution immediately after rumen withdrawal were stored at 4°C. Following the 1-h pepsin-HCl incubation, the bags were introduced at 15-min intervals, through the neck of the duodenal cannula and into the duodenum. Within 12 to 24 h after placement into the duodenum, the mobile bags were recovered from the feces and washed under cold running tap water as described previously for the bags removed from the rumen. Zero-hour bags were incubated in 600 ml of artificial rumen fluid prepared according to the method of Goering and Van Soest (1970), but excluding the microminerals, for 30 min at 39°C. After incubation, four 0-h bags were washed and incubated in pepsin-HCl and then placed in the duodenum and recovered from the feces as previously described for the other time points. The polyester bags containing the residue from rumen and mobile bags were oven-dried at 55°C for 48 h and then weighed to determine DM disappearance. The rumen and mobile bag procedure was repeated in each cow for a second and third period. Rumen and mobile bag residues were ground to a fine powder using a mortar and pestle, mixed with 0.1 N HCl to solubilize the Met, diluted with 0.1 N HCl, and filtered through a 0.45-micron syringe-tip filter (ColeParmer, Chicago, IL). Methionine was quantified by HPLC based on precolumn derivatization with phenylisothiocyanate (Cohen et al., 1989). The methionine content of the RPMet as assessed from the extraction and analysis of triplicate samples of 1.0 g was 87.1% and was marginally higher than the product specification of 85% minimum. The amount of RPMet (DM basis) that was initially placed in the polyester bags before incubation was multiplied by the measured Met concentration (87.1%) to calculate the Met content. Journal of Dairy Science Vol. 84, No. 6, 2001
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Ruminal DM and Met disappearance at each individual incubation time were calculated as the difference between the DM and Met content of the initial samples and the residues remaining after incubation in the rumen and expressed as a percentage of the DM and Met content of the initial sample, respectively. Kinetic parameters of DM and Met disappearance in the rumen were estimated using an iterative least squares method of the nonlinear regression procedure of SAS (1988) by fitting the following model (Orskov and McDonald, 1979) to the means of quadruplicate values for each cow in each period: y = a + b(1 − e−ct ), where y is the percentage of DM or Met that disappears at time t (%), a is the fraction of DM or Met rapidly solubilized (%), b is the fraction of DM or Met potentially degradable (%), c is the fractional rate constant for the disappearance of fraction b (/h), and t is the time of incubation (h). The potentially degradable fraction (%) was calculated as (a + b). Effective ruminal degradability (ERD) was calculated from the equation: ERD = a + (b × c)/(c + k), where k is the fractional passage rate from the rumen and constants a, b, and c are as defined previously. Effective ruminal degradabilities of DM and Met were estimated for hypothetical ruminal passage rates of 0.05, 0.08, and 0.11/h. Total tract DM and Met disappearance of RPMet at each individual incubation time were calculated as the difference between the DM and Met content of the initial sample and the residue remaining after recovery of the mobile bag in the feces and expressed as a percentage of DM and Met content of the initial sample, respectively. Dry matter and Met disappearance of RPMet in the intestinal tract was calculated as the difference between the average DM and Met disappearance at each time point in the rumen-incubated bags and the mobile bags for each cow, in each period, at each incubation time. Intestinal DM and Met disappearance of RPMet was also expressed as a percentage of the RPMet entering the intestine. An estimate of bioavailability (%) was calculated by combining effective ruminal degradability and intestinal digestibility as follows: (100 − ERD) × fractional intestinal digestibility. Experiment 2 Animals and diet. Three nonlactating Holstein dairy cows (750 ± 89.0 kg of BW, 11.9 ± 0.7 kg of DMI; Journal of Dairy Science Vol. 84, No. 6, 2001
x¯ ± SD) with ruminal and duodenal cannula were used to assess the gastrointestinal availability of RPMet by comparing the response of plasma Met to an oral dose of RPMet and duodenally administered D,L-Met. The experiment consisted of three periods of 6 d (d 1 to 3 for oral dosing and sampling and d 4 to 6 for duodenal dosing and sampling). One day of rest separated the periods. Cows were weighed 1 d before the beginning of the first period and at the end of each period at the same time each day to determine the mean BW for each cow. Cows were housed and cared for as described for the previous experiment. Two weeks before the experiment, the cows began receiving the experimental diet which consisted (DM basis) of 3.3 kg of barley silage, 2.9 kg of grass hay, and 3.6 kg of barley grain concentrate daily (Table 2). One half of the daily allotment of barley silage was offered at 0600 h and the other half along with the grass hay was offered at 1530 h. The concentrate was fed at 1430 h. Samples of the barley silage, hay, and concentrate were collected weekly and composited. Feed refused was weighed daily and sampled, and the samples were composited for each period. Feed and orts samples were dried at 55°C in an oven for 48 h. Feed intake was determined as the difference in the DM of feed offered and refused. Dosing of RPMet and D,L-Met. In each of the three periods, the cows received one of three oral doses: 0 (control), 20, and 63 g of RPMet (Mepron M85); equivalent to 0, 17.4 and 54.9 g of D,L-Met (based on 87.1% Met). Coarsely ground barley grain served as the carrier for the dose. Cows began receiving 0.5 kg of the grain carrier at 0600 h, 4 d before the beginning of the first period and continued to receive it throughout the experiment. On the first day of each period, the oral dose of RPMet was mixed with the barley grain. On this day, the 0600 h feed allotment was withheld from cows until the dose mixed with the barley grain was administered. The cows were expected to consume the dose within 10 Table 2. Ingredient composition of the diet of experiment 2. Item
%, DM basis
Barley silage Grass hay Barley grain (medium roll) Canola meal Canola oil Liquid molasses Mineral premix1 Vitamin premix2
33.55 29.65 30.51 3.98 0.17 0.42 1.7 0.02
1 Supplies per kilogram of concentrate: 4.1% NaCl, 360 mg/kg of Zn, 340 mg/kg of Mn, 93 mg/kg of Cu, 4.7 mg/kg of I, 1.9 mg/kg of Se, and 1.4 mg/kg of Co. 2 Supplies per kilogram of concentrate: 54,500 IU of vitamin A, 5450 IU of vitamin D, and 54 IU of vitamin E.
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min. If, however, the dose was not consumed within 30 min, the dose that remained was placed into the rumen under the mat layer. Blood was collected via jugular venipuncture into sterile heparinized Vacutainer tubes (Vacutainer Systems, Becton Dickinson, Rutherford, NJ) at 0, 1, 3, 6, 9, 12, 24, and 48 h relative to the time of oral dosing. Seventy-two hours after oral dosing, each cow received Met by infusion into the duodenum. An indwelling catheter was placed in the jugular vein of the cows 10 to 15 h before infusion. To simulate the rate of Met delivery to the intestine from orally administered RPMet, D,L-Met was continuously infused at an exponential rate of decline in concentration into the lumen of the duodenum over a 12-h period beginning at 0600 h on d 4 of each period. The D,L-Met (BDH Chemicals Ltd., Poole, London), 13.7 g, was dissolved in 400 ml of 0.1 N HCl. Once dissolved, the volume of the Met infusate was adjusted to 450 ml with 0.2 M NaOH to yield a pH of 2.8. A volume of approximately 450 ml was maintained in the Met infusate reservoir by a constant inflow of 0.0018 N HCl, pH 2.8, at a rate equivalent to the outflow that entered the intestine. After 12 h, the volume of fluid remaining in the infusate reservoir was measured and analyzed for Met to determine the exact quantity of Met administered to the duodenum. Blood was collected via the catheter into heparinized tubes for 0, 1, 2, 3, 4.5, 6, 7.5, 9, 10.5, 12, and 24 h relative to the commencement of the duodenal Met infusion. At 48 h, blood was collected by jugular venipuncture. Plasma was separated from cells by centrifuging at 3000 × g for 15 min and collecting the supernatant. The plasma supernatant was deproteinized by adding an equal volume of acetonitrile and centrifuging at 3000 × g. The protein-free supernatant was collected and stored at −70°C until analyzed for Met. Samples of deproteinized plasma and the remaining infusate were analyzed for Met by HPLC as described previously. Calculations and statistical analysis. The response to the various oral doses of RPMet and the duodenal administration of D,L-Met was determined as the area under the curve (AUC) for plasma Met concentration over time by the trapezoidal method using SAS (1988). Data for AUC was analyzed using the GLM procedure of SAS (1988) to account for the effects of cow, period, and dose. Linear and quadratic effects were tested using orthogonal contrasts. Differences were considered significant at P < 0.05, and trends were discussed at 0.05 < P < 0.15. The response of plasma Met to 20 and 63 g of RPMet and the duodenally infused Met was expressed as a percentage of the AUC above the baseline response at 0 g of RPMet:
Figure 1. In situ ruminal DM (䊐) and Met (•) disappearance from rumen-protected methionine. Each time point represents the mean of three cows, where the value for each cow was calculated as the mean of three periods, each of which were estimated from the incubation of four bags. The vertical bars represent the SEM. Where the vertical bars are not visible, the SEM is smaller than the size of the symbol representing the mean.
100 × [(AUC20 or 63 RPMet or Met − AUC0 g RPMet)/AUC0 g RPMet]. The amount of absorbable Met delivered to the intestine was then calculated from the ratio of the plasma response of Met (percentage above the baseline response) for the RPMet and the duodenally infused Met multiplied by the amount of Met infused into the lumen of the duodenum: (AUC20 or 63 RPMet % above baseline/AUCMet % above baseline) × g of Met infused into the duodenum. RESULTS AND DISCUSSION In Situ Ruminal Digestion Kinetics One cow was removed from the first experiment before completion of the third period and, therefore, her data for this period were not included in the study. In situ ruminal DM and Met disappearance curves are presented in Figure 1 and the ruminal digestion kinetics in Table 3. The ruminal fractional degradation rate (parameter ‘c’) was 0.0224 and 0.0225/h for DM and Met, respectively. The low fractional degradation rate is indicative of a stable RPMet product. Potential degradability was 93.6% for DM and 95.7% for Met. These values are consistent with a product that is potentially high in digestibility and bioavailability. Effective ruminal degradability, and ruminal escape or bypass, are a function of the ruminal outflow rate. At a ruminal outflow rate of 0.05/h, ERD was calculated as 29.5% for DM and 34.0% for Met. At a ruminal outflow rate of Journal of Dairy Science Vol. 84, No. 6, 2001
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0.08/h, ERD was 21.3 and 26.1% for DM and Met, respectively. The ruminal retention time of the RPMet product is reported to be about 6 h (Greissinger et al., 1994), corresponding to a fractional turnover rate of approximately 0.11. At a ruminal outflow rate of 0.11/ h, ERD was calculated as 16.8% for DM and 21.7% for Met. For a RPMet product, particle size and specific gravity (density) are critical factors affecting ruminal outflow rate. If particles are too dense, they will sink in the rumen, and if density is too low, then particles will float. In either case, ruminal outflow rate will decrease to the range of 0.08 to 0.05/h or lower, significantly increasing the effective degradability of the product. Ruminal, Intestinal, and Total Tract Disappearance Ruminal and total tract disappearance of DM and Met (Table 4) were similar, which is consistent with the high Met content of RPMet (85% minimum D,LMet). Ruminal disappearance of Met was marginally higher than ruminal DM disappearance. Because small flaws or cracks in the RPMet coating would allow for solubilization of the Met and since the coating material appears to be of low digestibility, these results are to be expected. The extent of ruminal RPMet disappearance observed in the present study is in good agreement with the results of Overton et al. (1996), but slightly lower than the results of Berthiaume et al. (2000). Total tract DM and Met disappearance of RPMet ranged from 39 to 50% for all ruminal incubation times, except for 24 and 96 h, where total tract disappearance was higher and ranged from 55 to 90%. Higher total tract disappearance values for these time points reflected the higher ruminal disappearance observed for these times. Intestinal tract disappearance, measured as total tract minus ruminal disappearance, declined Table 3. In situ ruminal digestion kinetics and effective degradability of rumen-protected methionine. Parameter1
DM
SEM
Met
SEM
a, % b, % c, /h a+b ERD, k = 0.05/h k = 0.08/h k = 0.11/h
1.45 92.17 0.0224 93.63 29.45 21.28 16.81
0.43 1.34 0.0031 1.12 2.13 1.65 1.30
6.77 88.97 0.0225 95.73 33.98 26.06 21.71
0.70 1.61 0.0034 2.01 2.57 2.11 1.77
1 Parameters were calculated from the fitted equation y = a + b(1−e− ), where y = percentage of DM or Met disappearance from the polyester bag at time t, a = soluble fraction (%), b = slowly degradable fraction (%), c = fractional rate constant at which b is degraded (/h), and t = time of incubation (h). The potentially degradable fraction (%) was calculated as (a + b). Effective ruminal degradability (ERD) was calculated using the equation a + bc/(c + k), where k = 0.05, 0.08, and 0.11/h. ct
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Table 4. Dry matter and methionine disappearance from rumenprotected methionine in the rumen, intestine and total tract of dairy cows.1 Hours of ruminal incubation
DM Rumen2, % SEM Intestine,3 % SEM Intestine,4 % SEM Total tract,5 % SEM Methionine Rumen, % SEM Intestine,3 % SEM Intestine,4 % SEM Total tract, % SEM
0
3
6
12
24
96
2.7 0.1 37.9 3.9 39.2 4.2 40.5 4.0
6.5 0.2 33.6 1.9 36.1 2.2 40.1 1.8
12.5 0.9 26.6 1.4 30.7 2.0 39.1 2.0
21.4 0.9 24.4 1.5 31.1 1.9 45.7 0.7
39.1 1.5 16.7 2.4 27.7 4.2 55.8 2.5
82.1 0.2 3.8 0.3 20.2 2.6 85.9 0.2
8.6 0.4 35.9 3.8 39.5 4.4 44.5 4.0
14.7 1.9 28.7 1.3 34.7 2.3 43.4 2.3
17.8 0.8 24.4 1.5 29.6 1.8 42.2 0.8
25.0 1.3 25.0 1.3 33.4 1.4 50.1 1.1
42.9 1.3 15.8 2.0 28.1 3.7 58.8 1.7
85.5 1.3 4.0 1.3 26.1 6.6 89.6 0.3
1 n = 3. Observations were obtained from three cows, replicated for three periods with four bags per incubation time. 2 Ruminal disappearance (%) = [(initial − residue remaining after ruminal incubation)/initial] × 100%. 3 Intestinal tract disappearance (%) = total tract disappearance (%) − ruminal disappearance (%). 4 Intestinal tract disappearance as a proportion of the ruminally protected methionine leaving the rumen (%) = [(total tract disappearance (%) − ruminal disappearance (%)/(100 − ruminal disappearance (%))] × 100%. 5 Total tract disappearance (%) = [(initial − residue remaining after recovery of mobile bag from feces)/initial] × 100%.
steadily as ruminal incubation time increased. When intestinal disappearance was expressed as a percentage of the RPMet entering the intestine, Met disappearance ranged from 26.1 to 39.5% with an average value of 31.9%. The length of ruminal incubation did not appear to affect digestibility of Met entering the intestine. Combining ERD and intestinal digestibility will yield an estimate of bioavailability. At a ruminal outflow rate of 0.08/h, Met bioavailability was estimated to be 23.6% ([100 − ERD] × intestinal digestibility). At 0.11/h, Met bioavailability was 25%. Generally, the better the ruminal protection, the lower the intestinal availability for encapsulated RPAA. Therefore, there is often a tradeoff between ruminal resistance and intestinal availability. The exception to this would be with products with pHsensitive polymer coatings (Blum et al., 1999). The samples size to surface area used in the mobile bag technique is generally larger (de Boer et al., 1987; Sauer et al., 1983; Varvikko and Vanhatalo, 1990) than that used for the ruminal in situ technique. A smaller bag is favored for the mobile bag technique to avoid disruption of the flow of digesta through the intestine. As the sample size to surface area increases, however,
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feed particles within the bags can become compacted, restricting solubilization and access of digestive enzymes to the feed particles, which will thereby underestimate in situ digestibility. Underestimation of in situ ruminal digestibility is apparent particularly at the early time points of the ruminal incubation (Nocek, 1988). The larger sample size to surface area may, however, have less of an effect on intestinal digestibility, due to the length of time the bags remain in the intestine before the measurement of substrate disappearance. The mobile bag technique has compared favorably with true protein digestibility measured conventionally in swine (Sauer et al., 1983) and cattle (de Boer et al., 1987). Although, in a recent study, the mobile nylon bag technique was reported to underestimate the bioavailability of Met from RPMet (Berthiaume et al., 2000). The mobile nylon bag technique used in this evaluation does not include any preintestinal loss of Met due to mastication. Mastication losses are difficult to measure, but they are a function of particle size and density. Estimates for RPAA with physical characteristics similar to the product used herein suggest that mastication and the preintestinal losses associated with mastication may be 10 to 15% of the oral dose (unpublished). However, without direct measurements for these mastication losses, it would be inappropriate to include the values in calculation of bioavailability. Intestinal disappearance was 25 to 36% for ruminal incubation times of 0 to 12 h in the present study, compared with values of 52 to 63% in the study of Overton et al. (1996) and 48% (for 4.5 h of incubation) in the study of Berthiaume et al. (2000). There were some methodological differences between the studies with regards to the sample size to surface area of the polyester bags, and the length of the time mobile bags were incubated in pepsin-HCl before insertion into the duodenum. The similarity between the present study and that of Overton et al. (1996) for ruminal degradability of RPMet suggests that access of digestive fluids was probably not a contributing factor to the differences observed for intestinal digestibility. Incubation of mobile bags in pepsin HCl for 1 or 2.5 h would have had little effect on the extent of intestinal degradation of RPMet and release of Met due to the acid-insensitive coating. Residence time within the intestinal tract could, however, be expected to affect the extent of Met release from the RPMet product. The incubation of the material within the rumen indicated that the extent of Met degradation increased with increasing time of exposure to digestive fluids. Higher DMI by cows in the present study, if coupled with a faster rate of passage through the lower tract, may have reduced intestinal disappearance and hence bioavailability of RPMet rela-
Figure 2. Response of plasma methionine to a 0 (•), 20 (䊏), and 63 (▲) g oral dose of rumen-protected methionine and the intraduodenal infusion of 10.7 g of D,L-methionine (䊐) in nonlactating dairy cows. The vertical bars represent the SEM (n = 3).
tive to the other two studies (Berthiaume et al., 2000; Overton et al., 1996). Response of Plasma Methionine to RPMet The response of plasma Met to a 20 and 63 g oral dose of RPMet was evaluated in the second study, relative to the direct administration of 10.7 ± 0.2 g of Met to the duodenum over a 12-h infusion. Twenty grams of RPMet was investigated, as 10 to 20 g is the dose recommended by the manufacturer of the RPMet product to meet the requirements of high producing dairy cattle. The cows readily consumed the smaller, 20-g dose of RPMet and the carrier, but two of the three cows refused to consume the higher dose of RPMet (63 g) within the 30 min permitted, and, therefore, the dose and carrier remaining was placed directly into the rumen through the rumen cannula. In other studies in which cows were fed similar amounts of RPMet mixed or top-dressed on rations, no negative effects on DMI have been reported (Blum et al., 1999; Papas et al., 1984b; Rogers et al., 1987). Plasma Met concentration peaked between 9 to 12 h after oral dosing of RPMet, and 3 h after initiation of the duodenal infusion of Met (Figure 2). The delay in reaching peak concentration following oral dosing of RPMet is due in part to the residence time in the rumen (6 to 9 h), and the time required for absorption and accumulation of Met in the plasma pool. Plasma Met concentrations had returned to baseline levels at 24 h after receiving the oral dose of RPMet and the duodenal infusion of Met, and, therefore, the response measured as AUC for plasma Met was determined from 0 to 24 h (Figure 2). Plasma Met response Journal of Dairy Science Vol. 84, No. 6, 2001
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(AUC) tended (P = 0.125) to increase linearly with increasing levels of RPMet (data not presented). The increase in plasma Met above the control level (0 g of RPMet) reached 32.5 and 65.5% for cows receiving 20 and 63 g of RPMet, respectively. As expected, plasma Met responded similarly (P > 0.05) to the duodenal infusion of Met when administered 24 h after the return to baseline of the plasma Met from the three oral treatment levels of RPMet. The increase in plasma Met response following duodenal administration of Met, ranged from 51 to 71% above the baseline response (plasma Met response to oral supplementation of 0 g RPMet), with an average response of 59.2%. To estimate the amount of Met delivered to the intestine by the RPMet product, the response (as a percentage above the baseline response at 0 g of RPMet) of plasma Met to oral RPMet supplementation was expressed as a proportion of the average response due to the duodenal administration of Met, and then multiplied by the amount of Met administered to the duodenum. Thus, 20 g of RPMet provided approximately 5.9 g of absorbable Met (32.5%/59.2% × 10.7 g of Met), and 63 g of RPMet delivered 11.8 g of absorbable Met (65.5%/59.2% × 10.7 g Met) to the intestine. An estimate of Met bioavailability was then calculated by expressing the quantity of absorbable Met delivered to the intestine as a percentage of the Met content of the oral dose of RPMet. Thus, the bioavailability of Met in RPMet, based on the response of plasma Met ranged from 22 to 34% [100% × 11.8 g of Met absorbed/(63 g of RPMet × 87.1% Met) and 100% × 5.9 g of Met absorbed/(20 g of RPMet × 87.1% Met)], and averaged 28%. The above estimate of RPMet bioavailability was based on a single-point calibration for the response of plasma Met to duodenally delivered Met. The variation of the response of plasma Met to a single level of duodenal Met was reduced for each animal at the expense of measuring the response to several levels with greater variation. The assumption was made that the plasma Met response to increasing duodenal Met is linear up to the amount of Met that was investigated. As previously discussed, a linear relationship was obtained for the response of plasma Met to increasing RPMet. The amount of Met delivered to the intestine (determined by the amount of Met remaining in the infusate reservoir after 12 h of administration) was less than the amount that was originally predicted to be delivered. As a result, the plasma Met response to the higher dose of RPMet (11.8 g of absorbable Met) was beyond the range of response to the Met administered duodenally (10.7 g of absorbable Met), but only marginally. The estimate of Met bioavailability based on plasma Met response may also be prone to sampling error if the Journal of Dairy Science Vol. 84, No. 6, 2001
peak plasma Met response after oral administration occurred between the times of plasma sampling. Missing the peak plasma Met response could result in the underestimation of the calculated AUC and hence the estimate of Met bioavailability. Amino acid concentrations in plasma or serum provide a qualitative measure of the postruminal delivery of essential AA from RPMet products (Blum et al., 1999; Papas et al., 1984a, 1984b). A quantitative measure was obtained in the present experiment by relating the plasma Met response of RPMet to the response of a known quantity of Met delivered directly to the intestine, the major site of AA absorption. The quantitative estimation of Met bioavailability required that a linear or other definable relationship exist between the plasma Met concentration and the amount of Met delivered postruminally. Identification of an inflection point or break point in a curve for the response of plasma AA concentrations to postruminally delivered AA has been used to identify the requirements of essential AA (Bergen, 1979). When the provision of the essential AA is below the requirement, the AA concentration in plasma may either increase marginally or not at all, but once the requirement for the AA has been met, plasma concentration increases more rapidly (Bergen, 1979; Broderick et al., 1974). The Met requirements of the nonlactating cows used in the present study were considered adequately met by the experimental diet (Cornell-PennMiner Dairy, version 1), and thus, the response of plasma Met tended to increase linearly (P = 0.125) with the postruminal supply of Met. Linear responses in plasma Met to incremental amounts of RPMet (Papas et al., 1984a; Piepenbrink et al., 1996; Rogers et al., 1987) and abomasally (Varvikko et al., 1999) or duodenally (Pisulewski et al., 1996) infused Met have also been reported by others. Assuming that there was no change in the rate of Met utilization by the tissues, induced by the amount of the dose administered, the increase in plasma Met concentration delivered by RPMet relative to the increase in a known quantity of duodenally delivered Met provided a sensitive, quantitative indicator of Met bioavailability. CONCLUSIONS The RPMet product has the characteristics to make it a highly effective source of rumen-protected Met. These characteristics are primarily due to the high load of Met (minimum 85% D,L-Met) and excellent ruminal resistance to degradation. However, the protection from degradation that is afforded in rumen is also apparent in the small intestine. Bioavailability, defined as the amount of available Met delivered to the intestine, averaged 28% when based on the response of plasma Met.
BIOAVAILABILITY OF PROTECTED METHIONINE
High producing dairy cows require 50 to 60 g of Met per day on typical diets, and may be deficient by up to 5 to 10 g of Met per day. Based on the mean estimate of bioavailability, 20 g of RPMet would provide 5.6 g of absorbable D,L-Met. ACKNOWLEDGMENTS The experiment was financially supported in part by Degussa Corporation (Allendale, NJ), the Matching Investment Initiative Fund (Agriculture and Agri-Food Canada, Lethbridge, AB, Canada), and the Canada/ Alberta Livestock Research Trust (Lethbridge, AB). The authors thank K. A. Andrews for the assistance in conducting the experiments, A. F. Furtado for performing the Met analysis, and the staff of the Lethbridge Research Centre dairy unit (Lethbridge, AB, Canada) for the care and management of the cows. REFERENCES Bergen, W. G. 1979. Free amino acids in blood of ruminants—physiological and nutritional regulation. J. Anim. Sci. 49:1577–1589. Berthiaume, R., H. Lapierre, M. Stevenson, N. Cote´, and B. W. McBride. 2000. Comparison of the in situ and in vivo intestinal disappearance of ruminally protected methionine. J. Dairy Sci. 83:2049–2056. Blum, J. W., R. M. Bruckmaier, and F. Jans. 1999. Rumen-protected methionine fed to dairy cows: Bioavailability and effects on plasma amino acid pattern and plasma metabolite and insulin concentrations. J. Dairy Sci. 82:1991–1998. Broderick, G. A., L. D. Satter, and A. E. Harper. 1974. Use of plasma amino acid concentration to identify limiting amino acids for milk production. J. Dairy Sci. 57:1015–1023. Canadian Council on Animal Care. 1993. Guide to the Care and Use of Experimental Animals. Vol. 1. 2nd ed. CCAC, Ottawa, ON, Canada. Cohen, S. A., M. Meys, and T. L. Tarvin. 1989. The Pico-Tag method. A manual of advanced techniques for amino acid analysis. WM02 Revision 1. Waters Corporation, Milford, MA. de Boer, G., J. J. Murphy, and J. J. Kennelly. 1987. Mobile nylon bag for estimating intestinal availability of rumen undegraded protein. J. Dairy Sci. 70:977–982.
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