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The periparturient period is the most critical phase in the productive cycle of dairy cows and is characterized by impairment of the immune system. Our objective was to evaluate the effect of feeding ethyl-cellulose rumen-protected methionine (RPM) starting at d −28 from expected parturition through 60 d in milk on biomarkers of inflammation, oxidative stress, and liver function as well as leukocyte function. Sixty multiparous Holstein cows were used in a block design and assigned to either a control or the control plus ethyl-cellulose RPM (Mepron, Evonik Nutrition & Care GmbH). Mepron was supplied from −28 to 60 d in milk at a rate of 0.09% and 0.10% dry matter during the prepartum and postpartum period. That rate ensured that the ratio of Lys to Met in the metabolizable protein was close to 2.8:1. Blood samples from 15 clinically healthy cows per treatment were collected at d −30, −14, 1, 7, 21, 30, and 60 and analyzed for biomarkers of liver function, inflammation, and oxidative stress. Neutrophil and monocyte function in whole blood was measured in vitro at −14, 1, 7, 21, and 30 d in milk. The statistical model included the random effect of block and fixed effect of treatment, time, and its interaction. Compared with control, ethyl-cellulose RPM increased plasma cholesterol and paraoxonase after parturition. Among the inflammation biomarkers measured, ethyl-cellulose RPM led to greater albumin (negative acute-phase protein) and lower haptoglobin than control cows. Although concentration of IL-1Î2 was not affected by treatments, greater IL-6 concentration was detected in response to ethyl-cellulose RPM. Cows supplemented with ethyl-cellulose RPM had greater plasma concentration of ferric-reducing antioxidant power, Î2-carotene, tocopherol, and total and reduced glutathione, whereas reactive oxygen metabolites were lower compared with control cows. Compared with control, ethyl-cellulose RPM enhanced neutrophil phagocytosis and oxidative burst.

Feeding a higher-energy diet by increasing cereal grains at the expense of forage during the last 3 to 4 wk prepartum is a traditional approach to help the rumen “adapt” to the traditional diets fed at the onset of lactation. Increasing grain/concentrate in the diet changes ruminal fermentation and in sheep and goats elicits marked changes in mRNA expression of immune-related genes in ruminal epithelium. Whether such changes at the epithelial and systemic levels occur in dairy cows when the dietary energy content increases at a fixed level of concentrate is unknown. Fourteen nonpregnant, nonlactating Holstein cows were fed a control lower-energy (CON, 1.30 Mcal/kg of dry matter) diet to meet 100% of estimated nutrient requirements for 3 wk, after which half of the cows were assigned to a higher-energy diet (OVE, 1.60 Mcal/kg of dry matter) and half of the cows continued on CON for 6 wk. Levels of forage and concentrate for CON and OVE were 80 and 79% and 20 and 21%, respectively. Plasma samples were collected 1 d before slaughter to examine biomarkers of metabolism, liver function, inflammation, and oxidative stress. The reticulo-rumen mass was recorded at slaughter, and samples of epithelium were harvested from all cows. The expression of 29 genes associated with tight junctions, immune function, and nutrient transport (volatile fatty acids, urea, and trace minerals) was examined. Overfeeding energy led to consistently greater dry matter intake over time, and lowered plasma concentrations of haptoglobin, paraoxonase, bilirubin, fatty acids, and myeloperoxidase (secreted by neutrophils). In contrast, OVE resulted in greater hydroxybutyrate and cholesterol concentrations. A greater reticulo-rumen mass in cows fed OVE did not alter genes associated with tight junctions (CDLN1, CDNL4, OCLN, TJP1), immune function (IL1B, IL10, NFKB1, TLR2, TLR4, TNF), oxidative stress (SOD1, SOD2), or most nutrient transporters. However, feeding OVE upregulated the acute-phase pro

Assessing dispersal events in plants faces important challenges and limitations. A methodological issue that limits advances in our understanding of seed dissemination by frugivorous animals is identifying 'which species dispersed the seeds'. This is essential for assessing how multiple frugivore species contribute distinctly to critical dispersal events such as seed delivery to safe sites, long-distance dispersal and the colonization of non-occupied habitats. Here, we describe DNA-barcoding protocols successfully applied to bird-dispersed seeds sampled in the field. Avian DNA was extracted from the surface of defecated or regurgitated seeds, allowing the identification of the frugivore species that contributed each dispersal event. Disperser species identification was based on a 464-bp mitochondrial DNA region (COI: cytochrome c oxidase subunit I). We illustrate the possible applications of this method with bird-dispersed seeds sampled in the field. DNA-barcoding provides a non-invasive technique that allows quantifying frugivory and seed dispersal interaction networks, assessing the contribution of each frugivore species to seed rain in different microhabitats, and testing whether different frugivore species select different fruit/seed sizes. DNA barcoding of animal-dispersed seeds can resolve the distribution of dispersal services provided by diverse frugivore assemblages, allowing a robust and precise estimation of the different components of seed dispersal effectiveness, previously unattainable to traditional field studies at individual seed level. Given that seeds are sampled at the end of the dispersal process, this technique enables us to link the identity of the disperser species responsible for each dispersal event to plant traits and environmental features, thereby building a bridge between frugivory and seed deposition patterns. © 2014 British Ecological Society.

The protection of sunflower meal (SFM) proteins by treatments with solutions of malic acid (1 M) or orthophosphoric acid (0.67 M) and heat was studied in a 3 × 3 Latin-square design using three diets and three rumen and duodenum cannulated wethers. Acid solutions were applied to SFM at a rate of 400 ml/kg under continuous mixing. Subsequently, treated meals were dried in an oven at 150C for 6 h. Diets (ingested at 75 g/kg BW0.75) were isoproteic and included 40% Italian ryegrass hay and 60% concentrate. The ratio of untreated to treated SFM in the concentrate was 100 : 0 in the control diet and around 40 : 60 in diets including acid-Treated meals. The use of acid-Treated meals did not alter either ruminal fermentation or composition of rumen contents and led to moderate reductions of the rumen outflow rates of untreated SFM particles, whereas it did not affect their comminution and mixing rate. In situ effective estimates of by-pass (BP) and its intestinal effective digestibility (IED) of dry matter (DM), CP and amino acids (AAs) were obtained considering both rates and correcting the particle microbial contamination in the rumen using 15N infusion techniques. Estimates of BP and IED decreased applying microbial correction, but these variations were low in agreement with the small contamination level. Protective treatments increased on average the BP of DM (48.5%) and CP (267%), mainly decreasing both the soluble fraction and the degradation rate but also increasing the undegradable fraction, which was higher using orthophosphoric acid. Protective treatments increased the IED of DM (108%) and CP, but this increase was lower using orthophosphoric acid (11.8%) than malic acid (20.7%). Concentrations of AA were similar among all meals, except for a reduction in lysine concentrations using malic acid (16.3%) or orthophosphoric acid (20.5%). Protective treatments also increased on average the BP of all AA, as well as the IED of most of them. Evidence of higher increases

Effects of considering the particle comminution rate (k(c)) in addition to particle rumen outflow (k(p)) and the ruminal microbial contamination on estimates of by-pass and intestinal digestibility of DM, organic matter and crude protein were examined in perennial ryegrass and oat hays. By-pass k(c)-k(p)-based values of amino acids were also determined. This study was performed using particle transit, in situ and N-15 techniques on three rumen and duodenum-cannulated wethers. The above estimates were determined using composite samples from rumen-incubated residues representative of feed by-pass. Considering the comminution rate, k(c), modified the contribution of the incubated residues to these samples in both hays and revealed a higher microbial contamination, consistently in oat hay and only as a tendency for crude protein in ryegrass hay. Not considering k(c) or rumen microbial contamination overvalued by-pass and intestinal digestibility in both hays. Therefore, non-microbial-corrected k(p)-based values of intestinal digested crude protein were overestimated as compared with corrected and k(c)-k(p)-based values in ryegrass hay (17.4 vs 4.40%) and in oat hay (5.73 vs 0.19%). Both factors should be considered to obtain accurate in situ estimates in grasses, as the protein value of grasses is very conditioned by the microbial synthesis derived from their ruminal fermentation. Consistent overvaluations of amino acid by-pass due to not correcting microbial contamination were detected in both hays, with large variable errors among amino acids. A similar degradation pattern of amino acids was recorded in both hays. Cysteine, methionine, leucine and valine were the most degradation-resistant amino acids.

Efficacy of combined acid-heat treatments to protect crude protein (CP) against ruminal degradation has not been extensively researched. Four in vitro trials (Daisy technology) with orthophosphoric and malic acids were performed to examine effects on protection of sunflower meal protein. In Trial 1, effects of the solution volume for adding two doses of orthophosphoric acid (0.4 and 1.2 eq/kg sunflower meal) were tested using five dilution volumes (80, 160, 240, 320 and 400 ml/kg of feed) for each acid dose. Samples were heated at 60°C. The quantity of CP that remained undegraded after 20 h in vitro (IVUCP) increased with the amount of acid added (P = 0.01). Increasing the dilution volume also tended (P = 0.065) to increase IVUCP. Therefore, a dilution volume of 400 ml/kg was employed in all further trials. In Trial 2, treatments with solutions of orthophosphoric and malic acids (1.2, 2.4, 3.6 and 4.8 eq/kg) and 60°C of drying temperature were used. Increased CP protection with increased acid doses was described. In this and further trials, higher protective effects of malic acid than orthophosphoric acid were also shown. In Trial 3, the effects of both these acids, four acid concentrations (0.6, 1.2, 1.8 and 2.4 eq/kg) and three levels of heat treatment required for drying the samples (100, 150 and 200°C for 60, 30 and 20 min, respectively) were evaluated. An interaction acid type × concentration × temperature was shown. In addition, interactions concentration × temperature was shown in each acid. With heat treatments of 100°C to 150°C, benefits were not obtained after increasing the acid dose over 0.8 eq/kg. The increase of the heat treatments to 200°C and the acid dose up to 1.2 eq/kg increased protection, but to exceed this dose did not improve protection. In Trial 4, available lysine, CP solubility in McDougall buffer and IVUCP were compared after treatment with water or solutions (0.8 eq/kg) of orthophosphoric or malic acids using 100°C and 150°C heat treatmen