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,1
* Unité Mixte de Recherches Production du Lait, Institut National de la Recherche Agronomique, 35590, Saint-Gilles, France
Center of Evaluation and Research in Nutrition, Adisseo France SAS, 03600, Commentry, France
1 Corresponding author: benoit.graulet{at}clermont.inra.fr
| ABSTRACT |
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Key Words: rumen-protected methionine bioavailability milk protein dairy cow
| INTRODUCTION |
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Several technologies have been developed to achieve efficient Met protection against ruminal degradation by microorganisms. Determination of the efficacy of these protection technologies has varied according to the mode of protection, but also according to the experimental approach used by the authors. Among physically protected products, the best results have been obtained by coating a core of Met with a matrix of pH-sensitive polymer, such as in Smartamine M (SmM; Adisseo SAS, Antony, France; 78% Met content). Indeed, the Met bioavailability in SmM was first estimated at 80% from the determination of its rumen resistance and its intestinal release by nylon bag studies, as reported by Schwab (1995). Methionine bioavailability in SmM was also estimated in lactating dairy cows at 75 to 97% by digestibility tests (Robert and Williams, 1997) and at approximately 75% by blood tests (Rulquin and Kowalczyk, 2003). Another proposed strategy for Met protection from rumen degradation was to supply it in the form of chemically modified molecules of Met. Numerous derivatives and analogs of Met (for a detailed list, see Schwab, 1995) have been tested for their resistance to degradation, with the Met hydroxy analog, or D,L-2-hydroxy-4-(methylthio)-butanoic acid (HMB), being the most studied because it is currently used successfully for Met supply to monogastrics.
As a liquid source, HMB is easy to handle and it can be integrated into pellets. Moreover, HMB is more resistant to rumen degradation than Met, according to in vitro (Vazquez-Añón et al., 2001) and in vivo studies (Langar et al., 1978; Koenig et al., 1999, 2002). However, a controversy exists about the value of the HMB rumen bypass rate. Indeed, relatively low values (9 to 16%) were initially observed by Langar et al. (1978) in sheep, and in one study using dairy cows in late lactation, less than 1% of the dose of HMB supplied reached the small intestine (Jones et al., 1988). By contrast, Koenig et al. (1999, 2002) reported HMB bypass values closer to 40 to 50% in the rumen of dairy cows in early lactation. This discrepancy could be explained by the difference in the dose level used in the studies, the mode of supplementation, or both by species particularities or by the physiological status of the cows. Moreover, HMB supplementation has a very moderate effect on plasma Met concentrations, which is in contradiction with a rumen bypass value of 50% (Koenig et al., 1999, 2002). Furthermore, its main effects on milk production and composition were an increase in fat yield, especially when HMB was given repeatedly at doses of 25 to 35 g/d to multiparous dairy cows in early lactation receiving an adequate protein supply in the diet, whereas protein yield was not modified and milk production was rarely increased by HMB supplementation (St-Pierre and Sylvester, 2005). Thus, HMB does not seem to be able to effectively meet the Met requirements of lactating cows, at least for milk protein synthesis. Recently, it has been demonstrated that the isopropyl ester of HMB (HMBi) allows a significant Met supply to cows (Graulet et al., 2005) and an increase in their milk protein yield (Noftsger et al., 2005; St-Pierre and Sylvester, 2005).
The objective of this trial was to compare the effects of HMBi, HMB, and SmM on milk production and composition (especially protein content) in conditions in which the 2 Met hydroxyl analogs were integrated into pellets and SmM, used as a positive control, was supplied as a top dressing with the ration.
| MATERIALS AND METHODS |
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Sample Collection and Measurements
The amount of feed offered and orts were measured daily. Feed samples were collected daily for corn silage and weekly for other feeds. Body weight was measured once a week. Milk yield was measured every day at 0700 and 1630 h with Metatron 12 automatic samplers (Westfalia Separator, Château Thierry, France). A milk sample was collected mechanically at each milking over 5 d in each experimental week for analysis of the fat and protein contents of the milk. Milk (150 mL) was sampled at the morning milking on the fifth day of the last week of each period for analysis of fatty acids (FA) and CN. Blood samples (10 mL) were drawn at 30 min before and at 2 and 6 h after the morning feeding by caudal venipuncture on the fifth day of the last week of each period using heparinized Vacutainers (Beckton Dickison, Meylan, France). After centrifugation for 10 min at 1,450 x g, plasma was collected and a mean representative sample for each cow in each period was made by mixing equal amounts of each of the 3 collected samples per cow in the same day. The pooled samples were stored at 20°C until subsequent metabolite analysis.
Chemical Analysis
Determinations of DM, ash, total N, ADF, NDF, and acid-detergent lignin in feedstuffs as well as the rumen degradability of protein were performed as previously described by Pisulewski et al. (1996). This degradability was used to calculate the PDI values of the feeds (Table 1
) according to equations of the Institut National de la Recherche Agronomique (1989). The HMBi content in the soybean meal concentrate was assayed from a 5.0-g sample of the feed ground to 0.5 mm. The HMBi was extracted from the meal by a 30-min incubation at room temperature under agitation in 250 mL of a solution of methyl alcohol:water:phosphoric acid (500:500:1). The preparation was filtered on a 0.45-µm filter (Millipore, Molsheim, France), and HMBi was quantified in the samples by UV detection at 210 nm after separation by HPLC on a Hypersil Elite HyPURITY C18 column (150 x 4.6 mm, 5 µm; Thermo Electron Corp., Madison, WI) under isocratic conditions. The mobile phase was a mix (80:20) of acetonitrile and water acidified to pH 2.0 with phosphoric acid distributed at a flow rate of 0.9 mL/min. The injection volume of the samples was 10 µL. The quantification limits were 0.5 and 60 g/kg, with a mean recovery yield of 96%. Intra- and interassay variations were lower than 0.4% and 7%, respectively. The HMB content in the soybean meal concentrate was assayed from a 5.0-g sample of the feed ground to 0.5 mm. The HMB was extracted from the resulting flour by incubation for 60 min at 45°C in a shaking water bath with 50 mL of a solution of acetonitrile:water (100:900). The preparation was cooled by incubation in cold water for several min and then centrifuged at 3,000 x g. The supernatant was collected and the pellet was treated one more time with an additional 50 mL of solution (incubation and centrifugation). The supernatants were pooled and then treated with 1 mL of 50% potassium hydroxide, with shaking for 1 min. The solution was mixed for 30 s with 1 mL of 85% orthophosphoric acid and centrifugated for 10 min at 8,100 x g. Finally, the supernatant was collected and filtered on a 0.22-µm filter (Millipore). The HMB was quantified in the samples by UV detection at 214 nm after separation on a Hibar prepacked RT-2504 LichroSorb RP-18 column (5 µm; Merck KgA, Darmstadt, Germany) under isocratic conditions with an HP 1100 HPLC apparatus (Agilent Technologies, Palo Alto, CA). The mobile phase was a solution of acetonitrile:water acidified to pH 2.0 with 85% orthophosphoric acid (8:92) distributed at a flow rate of 0.8 mL/min. The injection volume of the samples was 20 µL. The linearity of HMB quantification was demonstrated between 0.2 and 5.1 g/kg. The intra-and interassay variations were less than 5% and less than 7%, respectively.
Plasma concentrations of glucose, NEFA, BHBA, and urea were determined as described by Pisulewski et al. (1996). The total glycerol (assimilated to triacylglycerols) was assayed with an enzymatic kit (Biotrol Diagnostic, Chennevièreslès-Louvres, France). The Met assay was performed as previously reported (Südekum et al., 2004; Graulet et al., 2005). Briefly, the macromolecular nitrogen in 1,000 ± 0.1 mg of plasma sample was precipitated by treatment with 10% (vol/wt) of 3.15 M 5-sulfosalicylic acid. After centrifugation for 10 min at room temperature and 11,670 x g, the supernatant was collected. The pellet was washed several times with a lithium salt buffer, pH 2.2, containing 33.3 mM trilithium citrate (Merck KGaA), 35.2 mM citric acid, 0.5% thiodiethylene glycol (Sigma Chemical Co., St. Louis, MO), 0.28% hydrochloric acid, and finally octanoic acid (2 drops for 2 L of buffer), and the supernatants were pooled. The final volume was adjusted to 5.0 mL with the lithium salt buffer and the preparation was filtered on a 0.22-µm filter before storage at 4°C until assay. The Met concentration was determined in 50 µL of sample by ion-exchange chromatography using a Beck-man 6300 AA analyzer (Beckman Instruments, Palo Alto, CA) at 570 nm after postcolumn ninhydrin derivatization at 125°C. Methionine concentrations in the plasma samples were expressed as µM after correction for calibration adjustment, internal standard variations, and dilution values.
Fat and protein contents of milk were determined by infrared analysis (MilkoScan; Foss Electric, Hillerød, Denmark). Milk CN content and FA composition were analyzed as described by Pisulewski et al. (1996).
Statistical Analysis
Statistical analysis of data was performed according to ANOVA using the GLM procedure of SAS (SAS Institute, 1987) and the following model Yijk =
i + ßj +
k +
ijk, where the main sources of variation were
i, the cow effect; ßj, the period effect;
k, the treatment effect; and
ijk, the residual effects. When the treatment effect was significant, differences were assessed by use of Fishers protected least significant differences test.
| RESULTS |
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The nature of rumen-protected Met supplementation did not affect total DM or nutrient intake (Table 2
). The cows were not fed 100 and 115% of their energy and protein requirements as expected, but 95 and 106%, respectively. However, the NEL balance remained similar between treatments and was not significantly different from zero (Table 3
).
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Milk FA composition was affected by the Met source (Table 5
). Compared with the control, HMB increased the proportion in 15:0 by 0.23 percentage units (P < 0.05), and SmM increased the level of saturation of total C18 FA, because the 18:0 content increased by 0.40 percentage units and 18:2 decreased by 0.08 percentage units (P < 0.05), whereas 18:1 was not affected. The HMBi treatment induced several modifications in the milk FA composition. Like SmM, HMBi induced an increase in the level of saturation of C18 FA: The 18:0 content increased by 0.66 percentage units and 18:2 decreased by 0.12 percentage units (P < 0.05) without affecting the 18:1 content. Moreover, like HMB, HMBi increased the proportion in 15:0 (+0.32 percentage units, P < 0.05). Furthermore, it increased the 17:0 content (+0.11%), although not significantly. Finally, HMBi treatment also induced a general reduction (1.51%) in short- and medium-chain FA in the milk (C6 to C14).
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| DISCUSSION |
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In the current study, MetDI and Lys contents in the basal ration where 1.78 and 7.13% of the PDI (Table 1
), whereas the requirements proposed for lactating cows are 2.5 and 7.3% (Rulquin et al., 1993), indicating that the diet was deficient in Met and not in Lys.
Lactation Performance
In the present work, supplementation of the Met-deficient diet with SmM or HMBi improved milk protein yield and true protein content. The beneficial effect of SmM on milk protein synthesis has been reported previously, both in energy-deficient and in energy-sufficient diets (Schwab, 1995; Rulquin and Delaby, 1997). The increase in milk protein yield (+41 g/d) obtained with the SmM treatment (13.3 g of Met equivalent/d) was similar to the response (+43 g/d) when 12 g/d of Met was infused into the duodenum (Pisulewski et al., 1996) and was higher than responses (average of 20 g/d) obtained by Rulquin and Delaby (1994) with 24 g/d of SmM. Moreover, SmM supplementation induced an increase in plasma Met concentrations, as previously observed by several authors (Rulquin and Delaby, 1997; Südekum et al., 2004). These observations confirm the efficiency of SmM in providing Met to the dairy cow, especially for milk protein synthesis by the mammary gland (Schwab, 1995; Armentano et al., 1997). Supplementation with HMBi (16.9 g Met equivalent/d) also increased plasma Met concentrations (x2) and milk protein yield (+32 g/d) and content (+0.10%), indicating that HMBi also provided significant amounts of Met to the cow. In 2 recent studies, it was demonstrated that supplementing the diets of lactating dairy cows with liquid HMBi mixed with a clay (powder presentation) improved the milk protein performance (Noftsger et al., 2005; St-Pierre and Sylvester, 2005). Moreover, in the present paper, HMBi supplementation supplied Met available for milk protein synthesis by lactating dairy cows when it was provided in a liquid form integrated into the diet in pelleted soybean meal. From the Met equivalent supplied and respective responses in milk protein yield obtained with SmM and HMBi in this trial, the Met bioavailability in HMBi was calculated to be 61% that of SmM. Moreover, by assuming a mean Met bioavailability of SmM of 80%, the Met bioavailability in HMBi could be estimated at 49%. This estimation is in agreement with the Met bioavailablity value recently determined by a blood test approach (Graulet et al., 2005). In the present study, the dietary supplementation with HMBi induced an increase in the plasma Met concentration that confirmed its efficiency in supplying Met to the lactating cow. Surprisingly, this increase was largely higher than the one observed after SmM supplementation. However, the present experiment was not designed to quantify the rumen-protected Met supply through the plasma Met concentration. Thus, this unexpected result could be explained by the schedule of blood sampling, because plasma Met concentrations after HMBi or SmM supplementation followed distinct evolutions, as previously reported (Graulet et al., 2005).
The HMBi significantly increased the CN percentage in milk protein. This latter result was in agreement with previous observations realized in our laboratory demonstrating that the increase in protein content after Met postruminal infusions resulted from an increase in the CN (essentially the ß-CN) but not the whey protein content (Pisulewski et al., 1996). In the present paper, SmM supplementation did not increase the CN content in milk or the ratio of CN to milk protein, whereas the opposite results have previously been observed (Rulquin and Delaby, 1994, 1997). This discrepancy is not fully understood because experimental conditions (lactation stage, energy and protein balance, supplement doses) were very similar to those of Rulquin and Delaby (1994, 1997).
In our experiment, HMB supplementation did not increase the milk yield or affect the milk composition, including the percentage of fat. The most consistent response to HMB supplementation of lactating dairy cow rations, based in the literature, is an increase in milk fat percentage (Hansen et al., 1991) and sometimes milk yield (Piepenbrink et al., 2004). We could suppose that the Latin square design (short-term period) of our study did not allow HMB to produce significant effects on fat metabolism because these latter ones would be mediated by long-term modifications of the rumen fermentation (Schwab, 1995). By contrast, the ineffectiveness of HMB in increasing the milk protein content was similar to previous findings (Piepenbrink et al., 2004) and consistent with the absence of an effect of HMB on the plasma Met concentration. Indeed, effects of HMB on milk protein and plasma Met concentrations have been rare and minimal (NRC, 2001), indicating that HMB is not a good substitute for Met in ruminants.
Milk FA
Supplementation of the dairy cow ration with SmM or HMBi increased the 18:0 content and reduced the 18:2 content in milk lipids. Rulquin and Delaby (1997) also reported a reduction in 18:2 in milk with SmM supplementation, whereas Pisulewski et al. (1996) demonstrated a quadratic effect of the dose of Met supplementation (with the optimal value at 12 g/d) on the 18:0 content in milk. Milk FA with carbon numbers greater than 16 are not produced in the mammary gland but originate from the plasma lipids as NEFA because of mobilization of the body fat stores (adipose tissues) or as part of triacylglycerols circulating in lipoproteins (chylomicrons from the digestive tract of very low density lipoproteins from the liver). It is unlikely that the biohydrogenation of FA by the ruminal microorganisms would be affected by Met postruminal infusions (Pisulewski et al., 1996) or SmM supplementation (Rulquin and Delaby, 1997). The stearoyl-coenzyme A desaturase activity is not very efficient in the bovine liver (St-John et al., 1991); consequently, it is the most probable that Met supplementation by SmM or HMBi would decrease its activity in the mammary gland or in the adipose tissues.
Supplementation of the ration by pellets containing Met derivatives (HMB or HMBi) induced an increase in saturated odd-numbered FA (mainly 15:0 but also 17:0 for HMBi, although not significantly). These FA are considered as markers of rumen microbial activity (Vlaeminck et al., 2005). Consequently, the increase in these FA in milk would indicate a stimulation of rumen microbial growth in cows supplemented with HMBi and HMB. This conclusion is supported by the increases in digestibility of NDF and OM substrates previously observed in vitro with HMBi or HMB (Noftsger et al., 2005).
Finally, a decrease in the percentage of even-numbered short- and medium-chain FA (C6 to C14) in milk fat was also observed after HMBi supplementation. Taken together, these results suggest that the mammary gland of lactating dairy cows supplemented with HMBi would reduce de novo FA synthesis inversely proportional to the increase in the uptake of plasma NEFA, including odd-chain FA of rumen microbial origin.
| CONCLUSIONS |
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Received for publication November 22, 2005. Accepted for publication May 31, 2006.
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