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Our objective was to evaluate the lactational responses of dairy cows to methionine provided from 2 ruminally protected sources of methionine activity. Twenty-one Holstein dairy cows [11 primiparous (634 kg of body weight, 140 d in milk) and 10 second-parity (670 kg of body weight, 142 d in milk)] were assigned to a treatment sequence in 4 replicated 5 × 5 Latin squares plus 1 cow, with 14-d periods. Treatments were as follows: control; 7.5 or 15 g/d of a ruminally protected product of 2-hydoxy-4-methylthio-butyric acid (NTP-1401; Novus International Inc., St. Charles, MO); or 7.5 or 15 g/d of a ruminally protected dl-methionine product (Smartamine M; Adisseo, Alpharetta, GA). The diet was predicted to meet metabolizable protein and energy requirements. Diets contained 16.1% crude protein, and the control diet was predicted to be deficient in metabolizable methionine (1.85% of metabolizable protein) but sufficient in lysine (6.8% of metabolizable protein). Feed intake and milk yield were measured on d 11 to 14. Blood was collected on d 14. Dry matter intake, milk yield, energy-corrected milk, milk fat yield and percentage, and efficiencies of milk and energy-corrected milk yield were not affected by treatment. Milk protein percentage and milk protein yield increased linearly with supplementation, without differences between methionine sources or interactions between source and level. Linear regressions of milk protein percentage and milk protein yield against supplement amount within source led to slope ratios (NTP-1401:Smartamine M) of 95% for protein percentage and 84% for protein yield, with no differences between sources for increasing milk protein. Plasma methionine concentrations were increased linearly by methionine supplementation; the increase was greater for Smartamine M than for NTP-1401. Plasma d-methionine was increased only by Smartamine M. Plasma 2-hydoxy-4-methylthio-butyric acid was increased only by NTP-1401. Our data demonstrated that supplementation with these methionine sources can improve milk protein percentage and yield, and the 2 methionine sources did not differ in their effect on lactation performance or milk composition.

The objective of this experiment was to test the effect of a Met analog, 2-hydroxy-4-methylthio-butanoic acid (HMTBa), on ruminal fermentation and microbial protein synthesis, nutrient digestibility, urinary N losses, and performance of dairy cows. Eight multiparous lactating Holstein dairy cows were assigned to 4 levels of HMTBa [0 (control), 0.05, 0.10, and 0.15% (dry matter basis)] in a replicated 4×4 Latin square trial. Experimental periods were 28 d, including 21 d for adaptation. Ruminal ammonia and microbial N were labeled through a 6-d intraruminal infusion of (15)NH4Cl, and microbial protein synthesis in the rumen was estimated using the reticular sampling technique. Treatment had no effect on dry matter intake (28.4 to 29.8kg/d), milk yield (44.1 to 45.3kg/d), feed efficiency, and milk composition. Total-tract apparent digestibility of nutrients was generally not affected by treatment, except digestibility of crude protein and starch decreased quadratically with HMTBa supplementation. Fecal, but not urinary, and total excreta N losses were increased quadratically by HMTBa. Ruminal pH, ammonia concentration, protozoal counts, and the major volatile fatty acids were not affected by treatment. Microbial N outflow from the rumen was linearly increased by HMTBa. 2-Hydroxy-4-methylthio-butanoic acid linearly increased the proportion of Fecalibacterium and quadratically decreased the proportion of Eubacterium in ruminal contents. Of the individual bacterial species, HMTBa increased or tended to increase Prevotella loescheii and Prevotella oralis. 2-Hydroxy-4-methylthio-butanoic acid linearly increased the concentration (and yield) of 15:0 in milk fat. In the conditions of this crossover experiment, HMTBa had no effect on feed intake and performance of dairy cows, decreased dietary crude protein digestibility, and increased microbial N outflow from the rumen.

The objectives of our study were to evaluate the productive response to methionine supplementation in lactating dairy cows and to define a relationship between metabolizable Met (MP Met) intake and production. A database of 64 papers meeting the selection criteria was developed evaluating postruminally infused dl-methionine (9 papers with 18 control diets and 35 treatment comparisons), 2-hydroxy-4-methylthio butanoic acid (HMTBa) provided as either a liquid or Ca salt form (17 papers with 34 control diets and 46 treatment comparisons), Mepron (Evonik Industries, Essen, Germany; 18 papers with 35 control diets and 42 treatment comparisons), and Smartamine (Adisseo Inc., Antony, France; 20 papers with 30 control diets and 39 treatment comparisons). Dietary ingredients and their accompanying nutritional compositions as described in the reports were entered into the Cornell-Penn-Miner software to model the diets and to predict nutrients that were not reported in the original publication. Data were analyzed using a weighted analysis of response to supplementation compared with the intraexperiment control, as well as through a regression analysis to changing dietary MP Met. Data included in the analysis were from experiments published between 1970 and 2011 with cows supplemented with between 3.5 and 67.9 g of Met or its equivalent from HMTBa. Cows supplemented with Smartamine consumed more, whereas cows supplemented with Mepron consumed less DM compared with controls. Milk yield did not significantly respond to Met supplementation, although it tended to increase for cows supplemented with HMTBa and Mepron. Milk protein yield was increased due to supplementation from all sources or from infusion, and protein concentration was greater for all supplements or infusion of dl-Met, except for cows supplemented with HMTBa. Irrespective of Met source, milk protein yield increased 2.23 g of protein/g of MP Met until reaching the breakpoint. Milk fat yield was increased for Mepron and HMTBa, whereas milk fat concentration was increased for infused dl-Met and for cows supplemented with HMTBa. Based on regression analysis, response of milk fat yield to Met supplementation was not different for infused dl-Met, Mepron, and Smartamine (1.87 g of fat/g of MP Met), whereas the response to HMTBa was significantly greater at 5.38 g of fat/g of MP Met.

The objectives of the current study were to determine the fate and contribution to Met kinetics of 2-hydroxy-4-(methylthio)butanoate (HMTBA) at the whole-body, splanchnic, and mammary levels. Four multicatheterized cows (31.3 kg of milk/d; 17.7 kg of DMI/d) were used in a crossover design, with two 1-wk periods, to determine the metabolic fate of HMTBA and its effect on Met metabolism. Over the last 2 d of each period, cows were infused, via a jugular vein, with saline or HMTBA (Alimet, Novus International Inc., St. Louis, Mo) at the rate of 36 g/d. During the last 8h, the HMTBA infusion was substituted by equimolar [1-(13)C]HMTBA (8.79 mmol/h) and l[methyl-(2)H(3)]Met (1.31 mmol/h) was infused in all cows. During the last 5h, hourly samples (n=6) were collected to determine plasma flows plus the isotopic enrichments (IE) and concentrations of HMTBA ((13)C) and Met (both (13)C and (2)H(3)) in plasma from an artery plus portal, hepatic, and mammary veins. The IE of [(13)C] and [(2)H(3)]Met were also determined in milk protein taken over the last 1h of infusion in HMTBA-infused cows. The infused HMTBA increased whole-body plasma flux of Met by 6.5 mmol/h (from 17.9 to 24.4 mmol/h). Based on enrichments of (13)C-labeled Met, 3.8 mmol/h of Met flow through plasma was derived directly from HMTBA. These 2 estimates accounted for between 43 to 74% of the HMTBA dose infused, contributing to increased whole-body Met availability. Although the portal-drained viscera, liver, and mammary gland (MG) extracted 11, 37, and 3.4%, respectively, of the infused HMTBA, tissue net Met fluxes were either unchanged (portal-drained viscera, MG) or even reduced (liver: -7.9 vs. -2.4±0.6 mmol/h). Therefore, net postsplanchnic supply of Met decreased from 7.0 to 2.9 mmol/h between control and HMTBA-infused cows, compared with needs for milk protein secretion of 7.6 and 8.1 mmol/h, respectively. The HMTBA provided directly 15% of the Met required for milk protein secretion, with 0.2 mmol/h synthesized within the MG, whereas 1.1 mmol/h originated from Met produced in other tissues and transported to the MG through blood circulation. Most of the remainder needed by the MG arose from unlabeled Met released from protein breakdown in extra-splanchnic tissues and that was not reused to support intracellular protein synthesis, as this function was performed by Met synthesized from HMTBA in situ. Absorbed HMTBA, therefore, both produces and spares Met for use by the MG.

The objective of the study was to evaluate the effect of diets supplemented with fatty acids of different degrees of saturation, in the absence or presence of an antioxidant (AOX; Agrado Plus, Novus International Inc., St. Charles, MO), on dairy cow lactation performance. Calcium salts of long-chain fatty acids were supplemented as a source of lower saturation fatty acid, and a palm acid product was supplemented as the higher saturation fatty acid source. Sixty early-lactation Chinese Holstein cows (100+/-23 d in milk) were randomly allocated to 4 dietary treatments in a 2 x 2 factorial design: (1) lower saturation fatty acid (LS), (2) LS and AOX, (3) higher saturation fatty acid (HS), and (4) HS and AOX. The Ca salts of long-chain fatty acids and palm acid product were supplied at 1.8 and 1.5% on a dry matter basis, respectively, to form isoenergetic diets. The AOX was added at 0.025% in the ration. The experiment lasted 9 wk, including 1 wk for adaptation. Lactation performance was recorded and milk was sampled and analyzed weekly. Blood samples were taken from the coccygeal vein to determine metabolism parameters on d 16, 36, and 56 during the experiment. Neither fatty acid type nor AOX supplementation showed a significant effect on dry matter intake during the study. Milk yield was lower in the LS-fed cows compared with the cows fed HS. Milk fat and milk protein concentrations were not affected by fatty acid type or AOX supplementation. Adding AOX increased the yield of milk in the LS-fed cows, but did not affect those fed HS. Activity of plasma superoxide dismutase was significantly lower, plasma glucose tended to be lower, and plasma malondialdehyde was higher in the LS-fed animals compared with those fed HS. Addition of AOX decreased both plasma nonesterified fatty acids and hydrogen peroxide contents and increased total antioxidant capacity across the fatty acid types. Plasma beta-hydroxybutyrate was not affected by fatty acid type or AOX treatment. Cows fed LS had higher cis-9C(18:1) and trans-10, cis-12C(18:2) in milk at the expense of C(18:0), whereas AOX addition increased milk cis-9C(18:1) at the expense of milk C(12:0), C(16:0), and trans-10, cis-12C(18:2). It is inferred that feeding LS resulted in inferior lactation performance, whereas addition of antioxidant partially alleviated these negative effects.

The objective of the study was to evaluate the effect of feeding the dietary antioxidant Agrado Plus (AOX; Novus International, St. Louis, MO) in diets that contained 2% fresh fat (FF) or oxidized fat (OF) on milk production and composition and antioxidant status of cows during mid to late lactation. Forty-four mid to late lactating primiparous cows housed in a tie-stall barn were fed a diet that contained 2% FF for 15 d as adaptation period and then randomly allocated to 1 of the 4 dietary treatments (FF, FF+AOX, OF, OF +AOX) for 6 wk. Feeding AOX increased dry matter intake, 3.5% fat-corrected milk, and milk fat yield, and decreased milk protein content but not yield. Feeding OF increased milk fat yield, but decreased dry matter intake and body weight gain. Milk fat composition changed with treatments: AOX increased cis 18:1 and decreased trans-11 18:1, whereas OF decreased trans-9 and trans-11 18:1 and cis-9, trans-11 18:2 in milk. Plasma antioxidant enzymes and status were affected by treatments. Feeding OF increased superoxidase dismutase activity but decreased plasma antioxidant status, whereas AOX supplementation increased glutathione peroxidase activity across fat types and increased the antioxidant status and superoxidase dismutase activity when feeding OF diets. It can be concluded that feeding AOX improved lactation performance and the antioxidant status of the cow across fat types, and feeding OF increased milk fat yield but decreased dry matter intake, body weight gain, and antioxidant status. The negative effects of feeding OF were partially alleviated by AOX.

Three soybean meal, 3 SoyPlus (West Central Cooperative, Ralston, IA), 5 distillers dried grains with solubles, and 5 fish meal samples were used to evaluate the modified 3-step in vitro procedure (TSP) and the in vitro immobilized digestive enzyme assay (IDEA; Novus International Inc., St. Louis, MO) for estimating digestibility of AA in rumen-undegraded protein (RUP-AA). In a previous experiment, each sample was ruminally incubated in situ for 16 h, and in vivo digestibility of AA in the intact samples and in the rumen-undegraded residues (RUR) was obtained for all samples using the precision-fed cecectomized rooster assay. For the modified TSP, 5 g of RUR was weighed into polyester bags, which were then heat-sealed and placed into Daisy(II) incubator bottles. Samples were incubated in a pepsin/HCl solution followed by incubation in a pancreatin solution. After this incubation, residues remaining in the bags were analyzed for AA, and digestibility of RUP-AA was calculated based on disappearance from the bags. In vitro RUP-AA digestibility estimates obtained with this procedure were highly correlated to in vivo estimates. Corresponding intact feeds were also analyzed via the pepsin/pancreatin steps of the modified TSP. In vitro estimates of AA digestibility of the feeds were highly correlated to in vivo RUP-AA digestibility, which suggests that the feeds may not need to be ruminally incubated before determining RUP-AA digestibility in vitro. The RUR were also analyzed via the IDEA kits. The IDEA values of the RUR were good predictors of RUP-AA digestibility in soybean meal, SoyPlus, and distillers dried grains with solubles, but the IDEA values were not as good predictors of RUP-AA digestibility in fish meal. However, the IDEA values of intact feed samples were also determined and were highly correlated to in vivo RUP-AA digestibility for all feed types, suggesting that the IDEA value of intact feeds may be a better predictor of RUP-AA digestibility than the IDEA value of the RUR. In conclusion, the modified TSP and IDEA kits are good approaches for estimating RUP-AA digestibility in soybean meal products, distillers dried grains with solubles, and fish meal samples.

A dual-effluent continuous culture system was used to investigate, in a 2 x 2 factorial design, the effect of feeding a fresh (FF) or oxidized (OF) blend of unsaturated fats (33% fish oil, 33% corn oil, 26% soybean oil, and 7% inedible tallow) when supplemented with a blend of antioxidants (AO; Agrado Plus, Novus International Inc.; Agrado Plus is a trademark of Novus International Inc. and is registered in the United States and other countries) on nutrient digestibility, bacterial protein synthesis, and fatty acid metabolism. Twice a day for 10 d, 12 fermenters were fed a diet that consisted of 52% forage and 48% grain mixture that contained 3% (dry matter basis) FF or OF, with or without AO. The OF contained a higher concentration of peroxides (215 vs. 3.5 mEq/kg), and a lower concentration of unsaturated fatty acids than the FF. Feeding OF reduced nitrogen digestibility, microbial nitrogen yield, and efficiency (expressed as kilograms of dry matter digested) and increased the outflow of saturated fatty acids in the effluent when compared with feeding FF. Adding AO improved total carbohydrate, neutral, and acid detergent fiber digestibilities and the amount of digested feed nitrogen converted to microbial nitrogen across the types of fats. From this study, we concluded that feeding OF reduced microbial nitrogen and increased the outflow of saturated fatty acids. Feeding AO improved fiber digestibility by rumen microorganisms, regardless of the type of fat.

The objective of this study was to measure net portal absorption of 2-hydroxy-4-methylthiobutanoate (HMTBA) in dairy cows. Four multicatheterized lactating cows were used in a cross-over design with 7-d experimental periods. They were fed every other hour a total mixed ration and received, in addition, twice a day, 12.5 or 25 g/meal of HMTBA. On the last day of treatment, net portal absorption of HMTBA was numerically greater after the 25-g compared with the 12.5-g bolus meal of HMTBA, and the amount absorbed relative to the dose ingested was unchanged between treatments averaging 11.2 +/- 4.7% of the dose. This represents a minimum value of HMTBA availability because it does not take into account any HMTBA metabolized to Met by gut tissues (in sheep this amounted to another 5% of the dose). A rapid method to estimate net portal absorption based on temporal variations of the peripheral plasma HMTBA concentrations following the HMTBA meal is also presented. Based on the good relationship (concordance correlation coefficient = 0.97) observed between direct measurements and estimations, this simplified approach offers a reasonable approach to assess HMTBA absorption under different feeding situations.

Anabolic availability of the hydroxyl methionine analog, 2-hydroxy-4-methylthiobutanoic acid (HMTBA), given as oral doses to lambs, was quantified both directly as appearance in the portal vein and as synthesis to Met by digestive tract tissues. Eight lambs, prepared with vascular catheters in the mesenteric and portal veins plus the aorta, received twice daily for 7 d either 0.46 g or 2 g of HMTBA. On d 7, [1-13C]HMTBA was supplied as 1 oral dose while [methyl-2H3]Met was infused into the jugular vein. Peak absorption as HMTBA occurred 70 to 90 min after the oral dose. All digestive tract tissues converted HMTBA to Met, equivalent to 24% of the Met provided by the diet for the larger HMTBA dose. Overall, total availability of HMBTA averaged 17.9% of the dose (range 10.6 to 27.9%), with 12.5% (range 7 to 22%) as absorbed HMBTA and the remainder as Met synthesized by digestive tract tissues. Release of 13CO2 into the portal vein accounted for another 23% of the dose. In all digestive tract tissues, the d-isomer was present in a smaller proportion than in the dose. In terms of whole-body kinetics, HMTBA loss from the plasma followed first-order kinetics, with a mean biological half-life of 76 min. Using this value, a simple model was devised to estimate HMTBA absorption based on peripheral plasma samples. When compared with direct measures of absorption, the model gave a slope of 0.81 (R2 = 0.68) and offers a practical means to test HMTBA availability to animals.

Absorption and metabolism of the Met hydroxy analog 2-hydroxy-4-methylthiobutyrate (HMTBA) was examined using stable isotopes. In the first trial, dl[1- 13 C]HMTBA was infused for 6h (7.4μmol/min) into the abomasum, and [ 2 H 3 ]Met was infused into the mesenteric vein, of 4 lambs prepared with vascular catheters across the splanchnic bed. Daily, lambs were offered 35g of a mixed forage-concentrate feed/kg. Recovery of HMTBA at the portal vein was 87%, and of this, 63% bypassed the liver. In contrast, hepatic extraction of Met equaled or exceeded net absorption. Only small quantities of Met synthesized from HMTBA were exported from either the digestive tract or liver, but there was substantial and significant input from posthepatic tissues. In a second experiment, 3 of the lambs were killed following 4-h infusions of dl[1- 13 C]HMTBA and [ 2 H 3 ]Met with enrichments monitored in 15 tissues. Only kidney showed [1- 13 C]Met enrichment higher than plasma, which suggests that it must be a primary source of plasma Met derived from HMTBA. Based on comparison of plasma and intracellular [1- 13 C]:[ 2 H 3 ]Met enrichments, all tissues synthesized Met from HMTBA but to significantly different extents. The lowest values were for muscle, skin, brain, and lung; intermediate conversions occurred in rumen, omasum, abomasum, duodenum, jejunum, ileum, and cecum; and the greatest synthesis, equivalent to 22 to 24% of Met entry into cells, was observed for liver and kidney. Therefore, although liver and kidney both converted HMTBA to Met, it was retained by the former and exported by the latter. Under these experimental conditions, synthesis of Met from HMTBA completely eliminated use of dietary Met.

This study was undertaken to determine how, and where, 2-hydroxy-4-methylthiobutyrate (HMTBA) can augment Met metabolism in lambs. Four lambs (initial body weight of 50 kg, SE = 2, and 6 mo of age) prepared with catheters in the mesenteric, portal, hepatic, and jugular veins plus the aorta, were fed at 1.5x maintenance on a grass hay, barley, fish meal, molasses/pre-mix (5:3:1:1, as fed) diet, supplied as hourly meals. Lambs were infused for 10 h with [methyl-2H3]Met (0.11 mmol/h) in a jugular vein and p-aminohippurate into the mesenteric vein. From 1 h onwards, successive 3-h infusions of saline (control), 0.55 mg/min (3.67 micromol/min), and 4.44 mg/min (29.6 micromol/min) of HMTBA were also infused into the mesenteric vein. Plasma, sampled continuously, was collected every 20 min during the last 60 min of each infusion. All infused HMTBA was recovered at the portal vein with 25% extracted subsequently by the liver. Portal appearance of total Cys and Met was unaltered by HMTBA infusion, but net splanchnic appearance of Cys increased (0.04, 0.08, 0.23 mmol/h, SEM = 0.05), whereas Met decreased (0.14, -0.01, -0.21 mmol/h, SED = 0.05). Despite this, arterial Met increased (27.0, 30.7, 51.5 microM, SEM = 2.1) as did Met irreversible loss rate (27.6, 28.7, 40.1 micromol/h, SEM = 0.51), equivalent to 40% of the HMTBA reentering the plasma after conversion to Met. These data indicate that, in ruminants, HMTBA is probably converted to Met within peripheral tissues; that is, where the metabolic need for Met exists.

Ruminal escape of various amounts of methionine hydroxy analog [D,L-2-hydroxy-4-(methylthio)-butanoic acid (HMB)] was measured in an experiment designed as a 4 x 4 Latin square using four lactating dairy cows with cannula in the rumen and duodenum. The cows were fed a diet composed of corn silage, alfalfa haylage, rolled barley grain, canola meal, and blood meal, three times per day. The cows were fed the liquid analog each day for 1 wk before the experiment was started. On the day of the experiment, each cow received an intraruminal bolus dose of 0, 25, or 50 g of the liquid analog (Alimet feed supplement, 88% HMB) or 51.2 g of a dry calcium salt of the analog (86% HMB; MHA) mixed with 0.5 kg of ground barley grain. A liquid phase marker (Co-EDTA) was administered as a bolus dose into the rumen at the time of administration of the methionine hydroxy analogs. Rumen and duodenal contents, and blood serum were collected at 0, 1, 3, 6, 9, 12, and 24 h relative to the time of dosing. Rumen and duodenal samples were analyzed for Co and HMB, and serum was analyzed for free methionine. Fractional rate constants for the passage of the liquid marker (k(p)) and the decline of HMB concentration in the rumen (k(rHMB)) were determined by nonlinear regression. Liquid passage from the rumen was similar among the four analog treatments (0.136 +/- 0.012/h; mean +/- SEM). Ruminal escape of HMB as a percentage of the dose (100% x k(p)/k(rHMB)) did not differ between cows receiving 25, 50, and 51.2 g of the methionine analogs (42.5, 41.0, and 34.9 +/- 9.0%, respectively) and averaged 39.5%. Duodenal appearance of HMB as a percentage also did not differ between cows receiving 25, 50, and 51.2 g of the methionine analogs (16.2, 26.8, and 22.7%, respectively) and averaged 22%. Omasal absorption of HMB was variable ranging from 12.3 to 26.3% and averaged 17.6%. Serum methionine concentration peaked at 3 and 6 h after dosing and increased in proportion to the amount of the analog administered. It was concluded that 39.5% of the methionine hydroxy analog escaped rumen degradation, the percentage of the dose that escaped the rumen was not affected by the amount or form of the methionine analog fed, and the analog that escaped ruminal degradation was likely absorbed and metabolized to methionine.