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The dairy industry demand for rumen-protected methionine (RP-Met) supplements has been competitive because of the constant emergence of new products. To evaluate performances, our study was designed to characterize the production response of 3 RP-Met supplements in mid-lactation dairy cows. Twelve multiparous Holstein cows were used in a replicated 4 × 4 Latin square design with 21-d treatment period. Treatments included control [(basal) diet based on corn silage and alfalfa haylage, supplemented with 0.025% of ration DM of lysine (Lys; Ajipro)] or 1 of 3 RP-Met supplements [Smartamine M (SMM), Mepron M85 (MM85), or Novimet (NVM)]. For RP-Met groups, Met and Lys were supplemented to the basal diet at 0.03 and 0.20% of ration DM, respectively. Treatments had no effect on DMI or milk yield. Treatment did not modify milk fat or lactose concentration; however, milk protein content was elevated with SMM, relative to control or NVM (3.30% vs. 3.24 or 3.24%, respectively; P< 0.05). Milk fat, protein, and lactose yield were not modified by treatments. Treatments tended (P= 0.12) to affect milk urea nitrogen. Serum Met concentration increased for SMM compared with control, MM85, or NVM (27.3 μMvs. 21.2, 23.3, or 22.7 μM, respectively; P< 0.001). Similarly, supplementation of SMM reduced the serum Lys:Met ratio (4.5:1) compared with control (5.2:1), MM85 (5.1:1), or NVM (5.2:1) (P< 0.05). Treatment did not modify the serum levels of all other EAA. We conclude that SMM increased circulating Met and milk protein content more effectively than NVM or MM85.

Previous in vitro data showed that Fusobacterium necrophorumwas inhibited by limonene. We further evaluated effects of limonene on growth of F. necrophorumin vitro as well as on ruminal concentrations of F. necrophorumin vivo. With in vitro cultivation in anaerobic brain-heart infusion broth, limonene decreased growth of F. necrophorum. Thymol also reduced growth of F. necrophorum, but it was less effective than limonene. Tylosin effectively reduced growth of F. necrophorumin vitro. Although the response over fermentation times and concentrations of antimicrobials differed somewhat between tylosin and limonene, the 2 antimicrobial agents yielded similar inhibitory effects on growth of F. necrophorumat concentrations ranging from 6 to 24 mg/L. The effects of limonene on ruminal F. necrophorumconcentration in vivo were tested in 7 ruminally cannulated heifers (225 kg initial BW) used in a 7 · 4 Youden square design. Treatments included: 1) control, 2) limonene at 10 mg/kg diet DM, 3) limonene at 20 mg/kg diet DM, 4) limonene at 40 mg/kg diet DM, 5) limonene at 80 mg/kg diet DM, 6) CRINA-L (a blend of essential oil components) at 180 mg/kg diet DM, and 7) tylosin at 12 mg/kg diet DM. Each period included 11 d with 10 d washouts between periods. Samples of ruminal contents were collected before treatment initiation and after 4, 7, and 10 d of treatment for measuring F. necrophorumby the most probable number method using selective culture medium. Limonene linearly decreased (P= 0.03) ruminal F. necrophorumconcentration, with the lowest concentration achieved with 40 mg of limonene/kg dietary DM. Limonene tended (P≤ 0.07) to linearly reduce ruminal molar proportions of propionate and valerate while tending to linearly increase (P≤ 0.10) those of butyrate and 2-methyl butyrate. Limonene did not affect ruminal NH3concentrations or degradation rates of lysine. Neither CRINA-L (P= 0.52) nor tylosin (P= 0.19) affected ruminal F. necrophorumconcentrations. CRINA-L significantly decreased ruminal concentrations of NH3and molar proportions of 3-methyl butyrate, whereas tylosin significantly decreased molar proportions of propionate while increasing those of butyrate and tending to increase those of acetate. Limonene supplementation reduced ruminal concentrations of F. necrophorumsuggesting that it may have the potential to reduce the prevalence of liver abscesses, although further research is needed to assess the effect of limonene in feedlot cattle.

In contrast to bifunctional reagents such as glutaraldehyde or polyfunctional reagents such as polyepoxides, carbodiimides belong to the class of zero-length crosslinkers which modify amino acid side-groups to permit crosslink formation, but do not remain as part of that linkage. The authors have compared the effects of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and glutaraldehyde (the de facto industrial standard crosslinker) on the hydrothermal, biochemical, and uniaxial mechanical properties of bovine pericardium. EDC crosslinking was optimized for maximum increase in collagen denaturation temperature using variables of pH, concentration, and ratio of EDC to N-hydroxysuccinimide (NHS): a reagent for formation of activated esters. EDC and glutaraldehyde crosslinked materials were subjected to hydrothermal denaturation tests, biochemical degradation by enzymes (collagenase, trypsin) and CNBr, amino acid analysis for unreacted lysine, and to high strain rate mechanical tests including: large deformation stress-strain studies (0.1 to 10 Hz), stress relaxation experiments (loading time 0.1 s) and small deformation forced vibration (1 and 10 Hz). A protocol for EDC crosslinking was developed which used 1.15% EDC (2:1 EDC:NHS) at pH 5.5 for 24 h. The increase in denaturation temperature for EDC (from 69.7±1.2°C to 86.0±0.3°C) was equivalent to that produced by glutaraldehyde (85.3±0.4°C). Both treatments equivalently increased resistance to collagenase and CNBr degradation; however, after denaturation, the EDC-treated tissue was slightly more resistant to collagenase, and markedly more resistant to trypsin. EDC-treated materials were more extensible and more elastic than glutaraldehyde-treated materials. Despite the differences in crosslinking mechanism, EDC and glutaraldehyde-treated materials are very similar. Subtle but intriguing differences in biochemical structure remain to be investigated.

Circulating sphingolipid ceramide is associated with the development of insulin resistance in overweight monogastrics. An increase in NEFA delivery to the liver can increase lipoprotein ceramide packaging and secretion. In turn, lipoprotein ceramide can antagonize insulin action in skeletal muscle. We recently discovered that ceramide concomitantly increases with hyperlipidemia in overweight cows transitioning from gestation to lactation. Considering evidence in monogastrics, our objective was to characterize the relationship between adiposity and liver and skeletal muscle ceramide concentrations. Multiparous Holstein cows were grouped by adiposity at d −28 prepartum as either lean (2.9 ± 0.1 BCS; n= 7; LEAN) or overweight (4.0 ± 0.2 BCS; n= 7; OVER). Diets were formulated to meet or exceed requirements. Blood samples were routinely collected from d −21 to 21, relative to calving, and liver and skeletal muscle biopsies were performed at d −21, −7, and 4. Liquid chromatography tandem mass spectrometry was used to quantify ceramide, monohexosylceramide (GlcCer), and lactosylceramide (LacCer). Data were analyzed as repeated measures using a mixed model with fixed effects of adiposity and time. Pearson's correlations were analyzed. Relative to LEAN, OVER experienced increased BCS loss, plasma NEFA, hepatic total lipid accumulation, and circulating ceramide (e.g., C24:0-ceramide) during the transition from gestation to lactation (P< 0.05). Comparable to plasma, C24:0-ceramide was the most abundant ceramide in the liver and muscle. To support a relationship between liver and plasma ceramides, plasma total ceramide and liver total ceramide were positively correlated during transition (r= 0.39, P< 0.05). Similarly, plasma C24:0-ceramide and liver C24:0-ceramide were positively correlated (r= 0.41, P< 0.05). Coinciding with an increase in circulating NEFA, hepatic total ceramide and C24:0-ceramide increased postpartum in OVER (P< 0.05). Liver total ceramide and C24:0-ceramide were positively correlated with plasma NEFA (r= 0.44 and 0.49, respectively; P< 0.05). Additionally, we observed an increase in postpartum hepatic C22:0-ceramide in OVER (P< 0.05). Hepatic total GlcCer tended to be increased postpartum for all cows (P= 0.10); however, hepatic total LacCer increased only in LEAN (P< 0.05). Muscle total ceramide and C24:0-ceramide levels were lower prepartum in OVER (P< 0.05). In contrast, postpartum muscle C16:0- and C24:0-ceramide were higher in OVER (P< 0.05). We conclude that enhanced lipolysis in overweight dairy cows increases hepatic ceramide synthesis to support ceramide accumulation in circulation. The ability of hepatic-derived ceramide to antagonize insulin action in extrahepatic tissues during early lactation requires further investigation.

Methionine (Met) and lysine (Lys) are limiting AA in dairy cow diets. Supplementation of rumen-protected (RP) Met and Lys can improve milk yield as well as yield and content of milk protein. Currently, multiple sources of RP-Met are available for supplementation; however, the comparative efficacy of these supplements to improve performance requires evaluation. Therefore, our objective was to characterize the production response of three RP-Met supplements in midlactation cows. Twelve multiparous Holstein cows (602 ± 46 kg BW and 174 ± 18 DIM) were used in a replicated 4 × 4 Latin square design with 21-d treatment periods. Dietary treatments included a corn silage and alfalfa haylage diet (control; no added Met) supplemented with one of three RP-Met sources (Novimet [Innovad], Smartamine M [Adisseo], and Mepron M85 [Evonik]). Treatments were designed to maintain a Lys:Met ratio of 2.9:1. For the control, Lys (RP-Lys; AjiPro) was added at 0.02% ration DM. For RP-Met supplementation, Met (RP-Met) was added at 0.03% ration DM. Cows fed RP-Met were provided Lys (RP-Lys) at 0.20% ration DM. Milk yields were recorded, and samples were collected during each period (d 19 to 21). Blood samples were collected on d 21 at 2, 4, and 6 h following feeding, and serum was pooled. Following solid-phase extraction, 21 serum AA were quantified using gas chromatography tandem mass spectrometry. Data were analyzed using a mixed model with repeated measures (fixed effects of treatment and period). Treatments had no effect on DMI or milk yield. Treatment did not modify milk fat, protein, or lactose yield; however, milk protein content was elevated with Smartamine M relative to control or Novimet (3.30 vs. 3.24 and 3.24% respectively; P< 0.05). Milk fat and lactose concentration were not modified with treatment. Treatment with RP-Met tended to increase milk urea nitrogen (P= 0.12). Smartamine M increased serum Met concentration (27.3 μM) compared with the control (21.2 μM), Novimet (22.7 μM), or Mepron M85 (23.3 μM) (P< 0.001). In a similar manner, Smartamine M lowered the serum Lys:Met ratio (4.5:1) compared with the control (5.2:1), Novimet (5.2:1), or Mepron M85 (5.1:1) (P< 0.05). Smartamine M was also able to enhance circulating glutamine relative to the control (320.8 vs. 289.5; P= 0.15). Treatment did not modify serum levels of all other AA, including Lys. We conclude that Smartamine M increased circulating Met and milk protein content more effectively than Novimet or Mepron M85.