Measuring resistance to ruminal degradation and
bioavailability of ruminally protected methionine
Alex Bach
1, Marshall D. Stern
*Department of Animal Science, University of Minnesota, 180A Haecker Hall, 1364 Eckles Ave., St. Paul, MN 55108, USA
Received 9 February 1999; received in revised form 11 May 1999; accepted 26 January 2000
Abstract
The objectives of this study were to evaluate ruminal degradation and intestinal digestion of two ruminally protected methionine (RPM) products and to assess the potential use of changes in plasma methionine concentrations as an indication of methionine availability to the animal. Ruminal degradation of the protected methionine was assessed using the in situ technique. The intestinal availability of methionine after ruminal incubation was determined in vitro using an enzymatic procedure. Four Holstein cows receiving a typical mid-lactation ration (16.5% CP, 1.6 Mcal NEL/kg) were supplemented with 0, 30, and 60 g per day of a slowly degraded ruminally
protected methionine (SDM), or 60 g per day of a moderately slowly degradable ruminally protected methionine (MSDM) in a 44 Latin square design. Blood samples were collected from the jugular vein at 0, 6, 12, 18, 24, and 36 h after feeding the RPM sources. Ruminal degradation rates of SDM and MSDM were 0.03 hÿ1 and 0.07 hÿ1, respectively. The calculated amount of methionine available for absorption, based on the in situ and in vitro results, was 17.9, 11.9 and 23.8 g per day when dosing 60 g of MSDM, 30 and 60 g of SDM, respectively. The highest (p<0.05) methionine plasma concentration (133.9mM) was measured with 60 g of SDM, followed by 30 g of SDM, and 60 g of MSDM. Plasma methionine concentrations were affected by an interaction (p<0.05) between time after dosing methionine and rate of ruminal degradation of the methionine dosed. Methionine plasma concentration peaked 12 h after dosing SDM, whereas methionine plasma concentration appeared to peak between 6 and 12 h after feeding MSDM. There was a good relationship (r20.86) between the grams of methionine escaping from the RPM products and the greatest area under the curve describing plasma methionine concentration. Data from this study show that the lower the ruminal degradation rate, the later the maximum plasma concentration of methionine will occur, and that the plasma methionine concentrations or their area
84 (2000) 23±32
*Corresponding author. Tel.:1-612-624-9296; fax:1-612-625-1283.
E-mail address: stern002@tc.umn.edu (M.D. Stern)
1Current address: Agribrands Europe. 197-199 Aribau, 08021 Barcelona, Spain.
under the curve can be successfully used to predict bioavailability of ruminally protected methionine.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Methionine; Rumen; Digestion; Bioavailability; Dairy
1. Introduction
Amino acids protected from degradation in the rumen have the potential to improve the balance of amino acids available for absorption from the small intestine. Methionine has been shown to be a common limiting amino acid for milk production with different types of rations (Schwab et al., 1976, 1992; Rulquin, 1992). Dietary supplementation with ruminally protected methionine (RPM) has the potential to increase milk production, but would only be expected to have such an effect when its resistance to ruminal degradation and its intestinal digestibility are high. Therefore, these two variables need to be determined to assess the quality of any given RPM. There are various techniques for measuring the ruminal rate of degradation of feedstuffs. One of the most common techniques is in situ incubation of feedstuffs in the rumen (Stern et al., 1997). This technique is not suitable to measure availability of soluble products, because they escape from the bag independently of their ruminal degradation, and therefore cannot be used to assess the ruminal degradation of soluble forms of RPM, such as methionine hydroxy analog (Patterson and Kung, 1988). The other variable that needs to be evaluated is the intestinal digestion of the RPM. This can be determined in vitro using the three-step procedure of Calsamiglia and Stern (1995), but this technique also cannot be used with soluble forms of RPM. As an alternative for measuring the bioavailability of soluble amino acids, several authors (Brookes et al., 1973; Reis et al., 1978; Strath and Shelford, 1978; Doyle and Adams, 1980; Cottle and Velle, 1989) have used amino acid concentrations in plasma as an indication of ruminal degradation and intestinal digestion. However, these studies have not directly compared plasma concentrations with other methods of assessing the bioavailability of amino acids.
The objectives of this study were:
1. to assess the ruminal degradation and intestinal digestion of two different RPM products in situ and in vitro;
2. to evaluate the potential of changes in plasma methionine concentration as an indication of the availability of RPM; and
3. to compare the results obtained with the in situ and in vitro methods and those derived from plasma concentrations.
2. Materials and methods
2.1. Ruminal degradation and intestinal digestion
were studied using the in situ Dacron polyester bag technique and the in vitro three-step technique (Calsamiglia and Stern, 1995), respectively. The SDM consisted of a matrix of
DL-methionine (65%), calcium salts of long chain fatty acids, a fatty acid and talc,
whereas the MSDM consisted of a matrix ofDL-methionine (66%), calcium salts of long
chain fatty acids and a fatty acid. The advantage of the matrix form is that the resistance to ruminal degradation persists even if the surface is physically damaged. Both the products had a mean particle size of 0.8±1.4 mm, and a density of 1.2 g/cm3. A ruminally cannulated Holstein cow in mid-lactation, fed a 60:40 concentrate:forage diet, was used for the in situ incubations. An average of 0.50.04 g of each RPM was weighed into Dacron polyester (Erlanger, Blumgart, New York) bags measuring 6 cm10 cm, with a 5216mm average pore size. Samples were incubated in the rumen for 2, 8, 16, 24, and 48 h after being soaked in distilled water (388C) for 10 min. Each sample was incubated in duplicate bags for each incubation time on four consecutive days. A pair of bags was also incubated in distilled water at 388C for 10 min to estimate washout at time zero for each sample. After removal from the rumen, bags were hand-washed with cold tap water for at least 20 min and dried for 24 h in a 608C forced-air oven. Bags used to determine the washout at time zero were rinsed following the same procedure. Nitrogen determination was performed according to AOAC (1984) using a Tecator 1035 autoanalyzer (Tecator, Herndon, VA).
The linear model described by Mathers and Miller (1981) was used to estimate the rate of nitrogen disappearance from the Dacron bags. This model assumes there is no fraction completely resistant to ruminal degradation, or that it is insigni®cant. Therefore, the nitrogen remaining after 48 h of ruminal incubation, which was considered completely resistant to ruminal degradation, was subtracted from each data point before natural logarithm transformation and regression. The extent of ruminal N disappearance (% of initial N) was calculated as follows:
Extent of ruminal degradationa bÿa kd kdkp
where:ais the soluble fraction (% of total),b the potentially degradable fraction (100-fraction completely resistant to ruminal degradation, % of the total).kdthe rate of ruminal
degradation (hÿ1), and kpthe rate of passage (assumed to be 0.06 hÿ1).
2.2. Plasma methionine concentration
Four Holstein cows in mid-lactation were allocated in a 44 Latin square design. Each period had a duration of 9 days. Sampling was completed during the last 2 days of each period, and the initial 7 days were used to assure that all dosed methionine from the previous treatment was metabolized. Treatments were a basal diet supplemented with 30 g of SDM, 60 g of SDM, 60 g of MSDM, and a Control treatment, where no methionine was supplemented. The basal diet offered to the cows is in Table 1. The methionine supplementation was performed by esophageal administration of the appropriate dose using a bolus gun. A catheter (14 g and 13.3 cm long; Angiocath, Becton&Dickinson, Sandy, UT) was placed in the jugular vein of the experimental cows the day before sampling. Jugular blood samples were collected into heparinized tubes at
0, 6, 12, 18, 24, and 36 h after administering RPM. Blood samples (10 ml) were immediately placed on ice and centrifuged (3000g) to obtain plasma. Plasma was deproteinized with sulfosalicylic acid (15% wt./vol.) and plasma methionine concentra-tions were determined on a Beckman 6300 amino acid analyzer (Beckman, Fullerton, CA) with a 10 cm cation±ion exchange column. Allo-isoleucine was included as an internal standard to correct for methionine recovery.
The area under the curve describing plasma methionine concentrations was determined by integration with Mathematica (Wolfram Research, Champaign, IL; Wolfram, 1991). The mathematical description of the curve to integrate was obtained with polynomial regression of degree ®ve (number of observations minus one) which resulted in an equation describing a line between each of the observed points. This technique was chosen instead of curve smoothing, because of the relatively low number of observations.
2.3. Statistical analyses
Solubility, rate, and extent of ruminal degradation as well as intestinal digestion and intestinally absorbable methionine values for both types of RPM were compared using a t-test. Methionine plasma concentrations were analyzed as a split-plot design using MacAnova (version 4.04, see MacAnova User's Guide, 1997), with a whole-plot Table 1
Ingredient and chemical composition of the basal diet fed to the cows
Item
consisting of cow, period and treatment, and a sub-plot consisting of time after feeding RPM and the interaction between treatment and time.
The model was as follows:
plasma methionine concentrationmCPTE PT H HT e
wheremis the grand mean, C the cow effect, P the period effect, T the treatment effect, H the effect of the hour after feeding, E(PT) the error term used for the whole-plot, ande the random error. The least square means of methionine plasma concentrations obtained with each treatment at each time point were separated using the ScheffeÂ's method (ScheffeÂ, 1953) for computing all possible contrasts.
3. Results and discussion
3.1. Ruminal degradation and intestinal digestion
Both the RPM products used in this study had very low solubilities in rumen ¯uid, which allowed the use of the in situ technique to estimate their rate of ruminal degradation. Ruminal degradation patterns of both RPMs are summarized in Table 2 and the nitrogen residues at different times of ruminal incubation are shown in Fig. 1. The SDM had a similar (p>0.05) N-solubility value, but a slower (p<0.05) N-degradation rate and a greater (p<0.05) ruminal undegradable N fraction than MSDM. After a 16-h incubation in the rumen, MSDM retained 47.8% of initial N, and SDM had 71.3% of initial N. The estimated digestion in the small intestine (% of RUM) was above 90% for both the samples (Table 2). The intestinally absorbable dietary methionine (as a percentage of methionine in the original sample) available from MSDM was lower (p<0.05) than the amount available from SDM, which was primarily due to the greater resistance to ruminal microbial degradation of SDM compared with the MSDM.
3.2. Methionine plasma concentrations
The basal plasma methionine concentration (at time 0) averaged 19.1mM, which was similar to the concentration found at 36 h after dosing RPM (19.9mM). However, the
Table 2
Nitrogen solubility, rate, and extent of N disappearance in the rumen and intestinal digestion of slowly degradable methionine (SDM) and moderately slowly degradable methionine (MSDM)
Sample N solubility
aID, Intestinal digestion; RUM, ruminal undegradable methionine. bIntestinally absorbable dietary methionine (% of initial methionine). cDifferent letters within columns indicate differences (p<0.05).
dose of any RPM affected plasma methionine concentrations (p<0.05). The greatest measured plasma methionine concentration occurred with 60 g of SDM, followed by 30 g of SDM, and 60 g of MSDM (Table 3). Based on in situ and in vitro intestinal digestion (Table 2), the estimated amount of methionine available for absorption when dosing 60 g of MSDM was 17.9 g (60 g of MSDM0.66 (methionine content)0.48 (undegradable fraction)0.94 (digestible fraction)17.9), which is greater than the estimated 11.9 g available for absorption when dosing 30 g of SDM, and lower than the estimated 23.8 g/ day available for absorption when dosing 60 g of SDM.
Fig. 1. Nitrogen remaining after ruminal incubation of a slowly degradable methionine (&) and a moderately slowly degradable methionine (*). Error lines indicate standard deviations.
Table 3
Methionine plasma concentrations (mM) after the administration of various amounts of ruminally protected methionine differing in rate of ruminal degradation
Time (h) Treatmenta S.E.
Control 60 SDM 60 MSDM 30 SDM
0 17.0 21.30 15.45 22.45 2.50
6 22.47 db 77.00 b 60.65 b 41.15 c 8.13
12 24.15 e 133.89 b 59.75 d 70.33 c 9.96
18 24.53 c 69.30 b 31.59 c 44.21 c 8.88
24 19.30 c 38.75 b 23.81 c 26.13 c 4.27
36 18.20 23.15 21.82 20.67 4.17
aControl, No methionine was given; 60 SDM, 60 g of slowly degradable methionine, 60 MSDM, 60 g of
moderately slowly degradable methionine; 30 SDM, 30 g of slowly degradable methionine.
Plasma methionine concentrations were affected by an interaction (p<0.05) between treatment and time after dosing RPM. Plasma methionine concentrations peaked 12 h after dosing SDM, whereas plasma methionine concentrations appeared to peak between 6 and 12 h after dosing MSDM, although the actual peak may have been missed. However, the maximum plasma methionine concentrations achieved with MSDM were much smaller than the maximum plasma methionine concentrations recorded when an equal quantity of SDM was dosed. Because the maximum plasma methionine concentrations with MSDM probably occurred between 6 and 12 h, and with SDM around 12 h after dosing RPM (Fig. 2), the most critical blood samples needed to study the availability of these types of RPM using plasma methionine concentrations were between 6 and 12 h after administration. However, these time points will vary depending on the rate of ruminal degradation of the RPM that is being studied. From the data presented in this study, it can be concluded that the faster the rate of ruminal degradation of methionine, the faster the maximum plasma methionine concentrations will be achieved. This conclusion is supported by previous studies, such as that of Robert et al. (1997) who found a maximum plasma methionine concentration 14 h after offering a protected form of methionine, and Cottle and Velle (1989) as well as Koenig et al. (1999) who reported that the greatest plasma methionine concentrations were obtained about 6 h after intraruminal infusion of methionine. The methionine products used in these two last studies were not protected against ruminal degradation, and according to the observations Fig. 2. Plasma methionine concentrations after the administration of various amounts of ruminally protected methionine differing in rate of ruminal degradation. Control (&); 60 g of slowly degradable methionine (^); 30 g of slowly degradable methionine (~); 60 g of moderately slowly degradable methionine (*). (Depicts data summarized in Table 3).
from the current study, a much shorter lag time before maximum plasma concentrations are achieved, was expected. Therefore, it seems clear that to precisely utilize plasma methionine concentrations as a means of ranking the bioavailability of RPM, collection of blood samples needs to be performed with greater frequency, especially during the ®rst 12 h after offering the methionine to the animal, in order to avoid missing the peak of methionine plasma concentration.
An alternative approach to utilizing individual methionine plasma concentrations to predict the relative intestinal absorption of Met from PRM products is to investigate the area under the curve that describes the evolution of the plasma methionine concentrations. Using this approach, the administration of 60 g of SDM resulted in the greatest (p<0.05) area under the curve (2744mmol h/l), followed by 30 g of SDM (1906mmol h/l) and 60 g of MSDM (1658mmol h/l). All of these areas were different (p<0.05) than the area obtained with the control treatment (688mmol h/l). Similar to the results obtained with peak plasma methionine concentrations, there was an apparent disagreement between the estimates of methionine availability obtained in situ and in vitro with those obtained from integrating plasma methionine concentrations for the doses of 60 g of SDM and 30 g of MSDS. This was probably due, in part, to the low integrated area under the curve, that resulted from missing the presumed maximum plasma methionine concentration with the dose of 60 g of MSDM. Despite this discrepancy, there was a good relationship (r20.86) between the grams of methionine escaping from the RPM products and the greatest area under the curve describing plasma methionine concentration (Fig. 3). Therefore, the use of the integrated area under the curve to assess the bioavailability of methionine, although requiring more blood collection times in order to capture the greatest concentrations of methionine to allow accurate calculation of the
area under the curve, may be a very valuable method of comparing different types of RPM within the same experiment.
4. Conclusions
Results obtained in this study showed that measuring plasma methionine concentra-tions or their area under the curve can be a practical tool to qualitatively determine the bioavailability of ruminally protected methionine, because there is a good relationship between in situ and in vitro estimates of bioavailability and the estimates derived from blood concentrations. It is important to use suf®cient blood collection times to capture the plasma peak concentration of methionine. The most important blood collection times with the products used in the current study were between 6 and 18 h after feeding the ruminally protected methionine, because they coincided with the maximum plasma methionine concentrations. However, these time points may change depending on the rate of degradation of the methionine under study, being shorter for rapidly degradable methionine products and longer for slowly degradable methionine products.
Acknowledgements
Published as paper number 991164805 of the scienti®c series of the Minnesota Agric. Exp. Sta. on research conducted under Minnesota Agric. Exp. Sta. project number 16-050 supported by the College of Agricultural, Food and Environmental Sciences. The authors are grateful to Nisso America Inc. (New York, NY 10017) for providing the ruminally protected methionine products and for the partial ®nancial support of this research.
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