Effects of trace mineral amount and source on aspects of oxidative metabolism and responses to intramammary lipopolysaccharide challenge in midlactation dairy cows.

Trace minerals have important roles in immune function and oxidative metabolism; however, little is known about the relationships between supplementation level and source with outcomes in dairy cattle. Multiparous Holstein cows (n=48) beginning at 60 to 140 days in milk were utilized to determine the effects of trace mineral amount and source on aspects of oxidative metabolism and responses to intramammary lipopolysaccharide (LPS) challenge. Cows were fed a basal diet meeting National Research Council (NRC) requirements except for no added zinc (Zn), copper (Cu) or manganese (Mn). After a 4-week preliminary period, cows were assigned to one of four topdress treatments in a randomized complete block design with a 2×2 factorial arrangement of treatments: (1) NRC inorganic (NRC levels using inorganic (sulfate-based) trace mineral supplements only); (2) NRC organic (NRC levels using organic trace mineral supplements (metals chelated to 2-hydroxy-4-(methythio)-butanoic acid); (3) commercial inorganic (approximately 2×NRC levels using inorganic trace mineral supplements only; and (4) commercial organic (commercial levels using organic trace mineral supplements only). Cows were fed the respective mineral treatments for 6 weeks. Treatment effects were level, source and their interaction. Activities of super oxide dismutase and glutathione peroxidase in erythrocyte lysate and concentrations of thiobarbituric acid reactive substances (TBARS) and total antioxidant capacity (TAC) in plasma were measured as indices of oxidative metabolism. Effects of treatment on those indices were not significant when evaluated across the entire experimental period. Plasma immunoglobulin G level was higher in cows supplemented with organic trace minerals over the entire treatment period; responses assessed as differences of before and after Escherichia coli J5 bacterin vaccination at the end of week 2 of treatment period were not significant. Cows were administered an intramammary LPS challenge during week 5; during week 6 cows fed commercial levels of Zn, Cu and Mn tended to have higher plasma TAC and cows fed organic sources had decreased plasma TBARS. After the LPS challenge, the extent and pattern of response of plasma cortisol concentrations and clinical indices (rectal temperature and heart rate) were not affected by trace mineral level and source. Productive performance including dry matter intake and milk yield and composition were not affected by treatment. Overall, results suggest that the varying level and source of dietary trace minerals do not have significant short-term effects on oxidative metabolism indices and clinical responses to intramammary LPS challenge in midlactation cows.

Immune responses in lactating Holstein cows supplemented with Cu, Mn, and Zn as sulfates or methionine hydroxy analogue chelates.

The aim of this study was to compare effects of inorganic sulfate versus chelated forms of supplemental Cu, Mn, and Zn on milk production, plasma and milk mineral concentrations, neutrophil activity, and antibody titer response to a model vaccination. Holstein cows (n=25) were assigned in 2 cohorts based on calving date to a 12-wk randomized complete block design study. The first cohort consisted of 17 cows that had greater days in milk (DIM; mean of 77 DIM at the start of the trial) than the second cohort of 8 cows (32 DIM at the start of the trial). Diets were formulated to supplement 100% of National Research Council requirements of Cu, Mn, and Zn by either inorganic trace minerals (ITM) in sulfate forms or chelated trace minerals (CTM) supplied as metal methionine hydroxy analog chelates, without accounting for trace mineral contribution from other dietary ingredients. Intake and milk production were recorded daily. Milk composition was measured weekly, and milk Cu, Mn, and Zn were determined at wk 0 and 8. Plasma Cu and Zn concentrations and neutrophil activity were measured at wk 0, 4, 8, and 12. Neutrophil activity was measured by in vitro assays of chemotaxis, phagocytosis, and reactive oxygen species production. A rabies vaccination was administered at wk 8, and vaccine titer response at wk 12 was measured by both rapid fluorescent focus inhibition test and ELISA. Analyzed dietary Cu was 21 and 23mg/kg, Mn was 42 and 46mg/kg, and Zn was 73 and 94mg/kg for the ITM and CTM diets, respectively. No effect of treatment was observed on milk production, milk composition, or plasma minerals. Dry matter intake was reduced for CTM compared with ITM cows, but this was largely explained by differences in body weight between treatments. Milk Cu concentration was greater for CTM than ITM cows, but this effect was limited to the earlier DIM cohort of cows and was most pronounced for multiparous compared with primiparous cows. Measures of neutrophil function were unaffected by treatment except for an enhancement in neutrophil phagocytosis with the CTM treatment found for the later DIM cohort of cows only. Rabies antibody titer in CTM cows was 2.8 fold that of ITM cows as measured by ELISA, with a trend for the rapid fluorescent focus inhibition test. Supplementation of Cu, Mn, and Zn as chelated sources may enhance immune response of early lactation dairy cows compared with cows supplemented with inorganic sources.

Determining the methionine activity of Mintrex organic trace minerals in broiler chicks by using radiolabel tracing or growth assay.

Mintrex Zn, Mintrex Cu, and Mintrex Mn organic trace minerals contain 16% Zn, 15% Cu, and 13% Mn with 80, 78, and 76% 2-hydroxy-4-(methylthio)butanoic acid (HMTBA) by weight as the organic ligand, respectively. Our objective was to determine if HMTBA from Mintrex was fully available as a Met source. In experiment 1, thirty-six broilers (7 to 10 d old) were orally gavaged with methyl-(14)C-labeled HMTBA, either as free HMTBA (Alimet feed supplement) or Zn bis(-2-hydroxy-4-methylthiobutyrate) (Mintrex Zn). Radiolabel incorporation from either source into protein was measured as a marker of bioavailable Met activity. Results demonstrated that the HMTBA from Mintrex Zn was equally available as free HMTBA to support protein synthesis. In experiment 2, five hundred seventy-six 1-d-old broilers were allotted to 12 dietary treatments (TRT) for a 21-d growth assay. A TSAA-deficient diet containing 0.70% total TSAA (TRT 1) was supplemented with 0.05, 0.10, 0.15, and 0.20% free HMTBA (TRT 2 to 5) to establish the standard Met response curve. Treatment 6 was analogous to TRT 2 but had an additional 160 ppm Zn, 80 ppm Cu, and 160 ppm Mn as sulfates. Treatments 7 to 12 were identical to TRT 2 but supplemented with 40 or 160 ppm Zn from Mintrex Zn, 20 or 80 ppm Cu from Mintrex Cu, or 40 or 160 ppm Mn from Mintrex Mn, respectively. For TRT 1 through 6, growth performance increased due to increasing Met addition (P < 0.01) but not to increasing inorganic trace minerals. For Mintrex Zn, Cu, and Mn (TRT 7 to 12), there was a linear increase in cumulative gain:feed ratio (P < 0.04), and for Mintrex Zn and Mn, there was a linear increase in cumulative gain (P < 0.03) to increasing Mintrex addition. A 1-slope broken-line model was used to calculate bioavailable Met activity from Mintrex for comparison with actual intake values. Results indicated that HMTBA from Mintrex was fully available as a Met source.

Comparative in vitro and in vivo absorption of 2-hydroxy-4(methylthio) butanoic acid and methionine in the broiler chicken.

Poultry diets are typically supplemented with DL-2-hydroxy-4(methylthio) butanoic acid (HMTBA, or the hydroxy analog of methionine) or DL-methionine (DLM). Although HMTBA and DLM provide methionine activity, they are structurally distinct molecules with different physiological characteristics until they are converted to L-methionine. The relative rates of intestinal HMTBA vs. DLM absorption have been controversial, and it has been claimed that HMTBA is not fully absorbed. We measured the uptake of HMTBA and DLM in an in vitro everted intestinal slice model. Sections of intestinal slices (jejunum and ileum) were incubated with 0.1 to 50 mM HMTBA that was radiolabeled or DLM that was radiolabeled, and absorption was measured by scintillation counting. The HMTBA uptake was equal to or greater than DLM absorption in each tissue and at every time point with one exception. Furthermore, the rates of HMTBA absorption were always equal to or significantly greater than DLM uptake. In a separate in vivo experiment, absorption of HMTBA and L-methionine was monitored along the entire gastrointestinal (GI) tract. Broilers were fed commercial-type corn-soy diets supplemented with 0.21% HMTBA. Digesta was collected from crop, proventriculus, gizzard, duodenum, jejunum, ileum, large intestine, and cloaca and analyzed for the concentration of free HMTBA and free methionine in each compartment. These studies demonstrated that HMTBA is absorbed completely and along the entire GI tract, especially the upper GI tract. Furthermore, there was a higher concentration of free L-methionine than HMTBA in the digesta from every segment distal to the gizzard.

The relative effectiveness of 2-hydroxy-4-(methylthio) butanoic acid and DL-methionine in young swine.

We compared the effectiveness of 2-hydroxy-4-(methylthio) butanoic acid (HMB) and DL-methionine (DLM) as sources of L-methionine activity in methionine-deficient primary cultures of pig liver cells and methionine-deficient early-weaned pigs. Viable hepatocytes were obtained from minced pig liver and maintained in a high density, differentiated, nonproliferation cell culture system. Culture medium was supplemented with HMB, DLM, or L-methionine, and cells were pulse-dosed with L-[14C(U)]leucine for 24 h to determine the level of protein synthesis. Leucine incorporation per milligram of protein indicated a six-to eightfold increase in protein synthesis (P < .01) with methionine levels between 5 and 10 microM, regardless of source of methionine activity. Two 24-pen replicate methionine dose titrations were conducted with 95 early-weaned commercial crossbred pigs. The pelleted corn, dried whey, and porcine plasma basal diet contained 1.5% lysine, .23% methionine, and .48% cystine and was supplemented with 0, .05, or .10% methionine activity as DLM or HMB for 21 d. There was a 134, 104, and 61% increase (P < .01) in cumulative ADG for each successive week on study with a 30 and 19% improvement in feed/gain (P < .01) after 7 and 14 d. Performance responses due to source of methionine activity did not differ and slope ratio potency determinations (gain vs intake of methionine source) of HMB vs DLM indicated a 119, 111, and 95% relative activity for cumulative weekly performance. These results support the hypothesis that HMB and DLM provide equimolar levels of methionine activity in swine.

Effect of intermittent periods of high environmental temperature on broiler performance responses to sources of methionine activity.

The objective of these experiments was to study the effect of intermittent high temperature (IHT) on performance of broilers fed different sources of methionine activity. Two 20-d experiments were conducted in which individually caged 28-d-old cockerels were exposed to a 5-d period of constant high temperature (HT, 30 to 32 C) followed by an equal period of thermoneutral (TN) temperatures (22 C) with the 10-d temperature cycle repeated twice. Birds held at 22 C were TN controls. Grower diets contained 3,275 kcal/kg ME and 20% CP and were supplemented with either 2-hydroxy-4-(methylthio)butanoic acid (HMB, Alimet Feed Supplement) or DL-Met up to a maximum of .88 to .90% total sulfur amino acids (TSAA). In Experiment 1, gain:feed but not average daily gain was greater (P < .05) for HMB than DL-Met birds subjected to IHT (.451 vs .413, respectively), but no treatment differences were observed for TN birds. Results of Experiment 2 demonstrated a linear response to HMB and DL-Met dose in TN (P < .01); however, only HMB-supplemented birds responded similarly in IHT (P < .01). These results are consistent with lower availability of DL-Met as a result of IHT. In vitro experiments indicated that capacity for uptake of DL-Met into intestinal epithelial cells was reduced in heat-stressed birds. Uptake of D-Met was more severely affected than was L-Met. Consequently, a third performance experiment compared the ability of D-Met and L-Met to support growth under conditions of HT. The results indicated that the effect of HT on broiler performance was mediated through reduced utilization of D-Met.

Effect of heat stress on 2-hydroxy-4-(methylthio)butanoic acid and DL-methionine absorption measured in vitro.

The objective of the present experiments was to determine the biochemical basis for preliminary chick performance data, which indicate an ameliorative effect of 2-hydroxy-4-(methylthio)butanoic acid (HMB) when compared with DL-methionine (DLM) fed during hot conditions. In vitro passage of HMB or DLM across intact segments of small intestine from either control (thermoneutral, TN) or heat-stressed (HS) birds was used as a model for intestinal absorption. For DLM placed in the lumen, appearance in the outside buffer was reduced when using intestine from HS birds compared with tissue from TN birds. In contrast, the appearance of HMB in the outside buffer was greater using HS intestine, resulting in a substrate by environment interaction (P < .01). Slices of everted small intestine from TN and HS birds were used to study epithelial uptake of methyl labeled 14C-DLM by three transport pathways: diffusion, carrier-specific energy- and sodium-independent uptake (ESI), and carrier-specific energy- and sodium-dependent uptake (ESD). Correcting for extracellular volume, total epithelial uptake of 14C-DLM (diffusion plus ESI plus ESD) was reduced by 34% in HS intestine (P < .05). Energy-dependent uptake was observed to decrease by 87% in HS (P < .05). Energy-independent uptake was increased (136%, HS versus TN, P < .05), but not enough to compensate for the decrease in ESD uptake. Intestinal transport systems for glucose and leucine were also observed to change during HS, suggesting a role for cellular transport changes in the performance reduction associated with HS.