T.C. Wright

Application of iTRAQ to catalogue the skeletal muscle proteome in pigs and assessment of effects of gender and diet dephytinization.

In this study iTRAQ was used to produce a highly confident catalogue of 542 proteins identified in porcine muscle (false positive<5%). To our knowledge this is the largest reported set of skeletal muscle proteins in livestock. Comparison with human muscle proteome demonstrated a low level of false positives with 83% of the proteins common to both proteomes. In addition, for the first time we assess variations in the muscle proteome caused by sexually dimorphic gene expression and diet dephytinization. Preliminary analysis identified 19 skeletal muscle proteins differentially expressed between male and female pigs (≥1.2‐fold, p<0.05), but only one of them, GDP‐dissociation inhibitor 1, was significant (p<0.05) after false discovery rate correction. Diet dephytinization affected expression of 20 proteins (p<0.05). This study would contribute to an evaluation of the suitability of the pig as a model to study human gender‐related differences in gene expression. Transgenic pigs used in this study might also serve as a useful model to understand changes in human physiology resulting from diet dephytinization.

Microbial fatty acid conversion within the rumen and the subsequent utilization of these fatty acids to improve the healthfulness of ruminant food products

Consumers are aware of foods containing microcomponents that may have positive effects on health maintenance and disease prevention. In ruminant milk, meat, and milk products; these functional food components include eicosapentaenoic acid (20:5n3), docosahexaenoic acid (22:6n3), 9c11t-conjugated linoleic acid, and vaccenic acid (11t-18:1). Modifying ruminal microbial metabolism of fatty acid in rumen through animal diet formulation is an effective way to enhance these functional fatty acids in ruminant-derived food products. However, it requires an understanding of the interrelationship between supply of lipid through the diet and rumen fermentation. Lipids in ruminant diets undergo extensive hydrolysis and biohydrogenation in the rumen. Apparent transfer efficiency of eicosapentaenoic acid and docosahexaenoic acid from feed to milk is very low (1.9 to 3.3%), which is, to a large extent, related to their extensive biohydrogenation in the rumen. Therefore, feeding a rumen-protected supplement containing eicosapentaenoic acid and docosahexaenoic acid, can be used to bypass the rumen. Ruminant-derived foods also contain different types of conjugated linoleic acid isomers, which are intermediates of rumen biohydrogenation of linoleic acid (9c12c-18:2). The predominant isomer of conjugated linoleic acid is 9c11t, which has numerous health benefits in animal models. The concentration of conjugated linoleic acid in ruminant-derived food products can be significantly enhanced through animal diet modification. We conclude that most current functional food products from ruminants have potential for their health-supporting properties, and for this market to succeed, an evidence-based approach should be developed in humans.

Effect of enhanced whole-milk feeding in calves on subsequent first-lactation performance.

The objective of this study was to determine the effect of enhanced whole-milk (WM) feeding systems in calves from birth to 8wk of age on subsequent first-lactation performance. The experiment was conducted as a completely randomized design consisting of 2 treatment groups. At birth, 152 Holstein heifer calves were randomly assigned to 1 of 2 treatments: (i) 4L of WM/d or (ii) 8L of WM/d. The calves were bucket fed 2 or 4L of WM twice daily at 0700 and 1600h. Each calf was housed individually in temperature-controlled nurseries and had ad libitum access to water and textured calf starter daily. Calves consumed greater volumes of textured calf starter when fed 4 versus 8L of WM/d. Water intakes mirrored starter intakes, leading to greater water consumption at weaning. Calves reared on 8L of WM/d were heavier at d 56 than calves reared on 4L of WM/d. The average daily gain of the calves offered 8L of WM/d from d 0 to 56 was greater than that of calves offered 4L of WM/d. Structural measurements were significantly greater for calves that consumed 8L of WM/d. The differences observed in withers height and live BW due to WM feeding level were not apparent by 3 and 12mo of age, respectively. Rumen pH was higher in calves that consumed 8L of WM/d than in calves that consumed 4L of WM/d. Whole-milk feeding level did not affect age at first calving or milk-production parameters. These results suggest that enhanced WM feeding improved growth performance until 3mo of age. However, first-lactation results indicated no lactation-performance benefits of increased nutrition and growth performance during the milk-fed period in dairy calves.

Apparent transfer efficiency of docosahexaenoic acid from diet to milk in dairy cows

Apparent transfer efficiency of docosahexaenoic acid from diet to milk in dairy cattle was investigated. Three levels of a fish meal-containing supplement, 4.5, 14.9, and 29.1% of dry matter intake were added to a basal diet. A linear decline (from 34% to 11%) in apparent transfer efficiency was observed, as level of supplement increased. Key words: Docosahexaenoic acid, fish meal, milk composition

Fatty acid profile of bovine milk naturally enhanced with docosahexaenoic acid.

Recent studies have shown that the fatty acid profile of dietary lipid has the potential for improving the health of consumers. The present study was conducted to determine the fatty acid composition of commercial milks, namely, Dairy-Oh! Homo-Milk (DOHM), which is naturally enhanced with docosahexaenoic acid (DHA), or regular Homo-Milk (HM). The milk was collected from local supermarkets. The most abundant saturated fatty acids in the milk were butyric (C4:0), lauric (C12:0), myristic (C14:0), palmitic (C16:0), and stearic (C18:0) acids. Among unsaturated fatty acids, oleic acid (cis-9-C18:1) was also considerably high (502.7 mg/100 mL of milk). The concentration of total trans-18:1 was higher (P < 0.05) in DOHM than in HM (134.7 vs 107.0 mg/100 mL of milk, respectively), whereas total cis-18:1 was higher (P < 0.05) in HM than in DOHM (566.4 vs 508.4 mg/100 mL of milk, respectively). The concentration of DHA was 24.0 times higher (P < 0.05) in DOHM than in HM. DOHM contained 2.8 times higher (P < 0.05) eicosapentaenoic acid (EPA) compared to HM. Milk fat from DOHM contained a greater concentration of cis-9,trans-11 conjugated linoleic acid (CLA, 16.4 vs 11.6 mg/100 mL of milk, DOHM vs HM, respectively). The total omega-3 polyunsaturated fatty acids content was 2.23 times greater (P < 0.05) in DOHM compared with HM, due to an increase in C18:3n-3, EPA, and DHA. The result of the milk fatty acid analyses indicates that milk fat from DOHM had increased contents of EPA, DHA, and cis-9,trans-11 CLA, which could have a more favorable impact on diet composition and healthfulness.

Modeling intermittent digesta flow to calculate glucose uptake capacity of the bovine small intestine

To test the hypothesis that the uptake capacity of the bovine small intestine for glucose is upregulated to match or slightly exceed glucose delivery, glucose was continuously infused into the proximal duodenum of four cannulated holstein heifers. Every 3 days, infusion rates were increased by an average of 34 mmol/h. A model of glucose disappearance from multiple boluses of intestinal digesta was used to estimate the transporter maximum velocity and functional maximum uptake capacity for the entire small intestine from average ileal glucose flows during the third day of each period. Because of its intermittency, digesta flow remained independent of simulated transit time. For each unit increase in glucose infusion rate, uptake capacity increased by only 0.55 units. Excess capacity for glucose uptake was approximately twofold in forage-fed cattle and declined to below delivery at infusions of >208 mmol/h added glucose, approximately three times the normal load. Calculations for cattle, sheep, and rats indicate that the glucose transport capacity of the small intestine is typically underutilized because of a fraction of time that transporters are not in contact with digesta.To test the hypothesis that the uptake capacity of the bovine small intestine for glucose is upregulated to match or slightly exceed glucose delivery, glucose was continuously infused into the proximal duodenum of four cannulated holstein heifers. Every 3 days, infusion rates were increased by an average of 34 mmol/h. A model of glucose disappearance from multiple boluses of intestinal digesta was used to estimate the transporter maximum velocity and functional maximum uptake capacity for the entire small intestine from average ileal glucose flows during the third day of each period. Because of its intermittency, digesta flow remained independent of simulated transit time. For each unit increase in glucose infusion rate, uptake capacity increased by only 0.55 units. Excess capacity for glucose uptake was approximately twofold in forage-fed cattle and declined to below delivery at infusions of >208 mmol/h added glucose, approximately three times the normal load. Calculations for cattle, sheep, and rats indicate that the glucose transport capacity of the small intestine is typically underutilized because of a fraction of time that transporters are not in contact with digesta.

Lactation induces upregulation of the ubiquitin-mediated proteolytic pathway in skeletal muscle of dairy cows but does not alter hepatic expression

The current study investigates regulation of mRNA expression of components of ubiquitin-mediated proteolysis in transition dairy cows. Longissimus dorsi muscle (exp. 1) and liver (exp. 2) biopsies were collected from Holstein dairy cows at 27 and 16 d pre-partum, respectively, and 3 and 10 d post-partum, respectively. Regulation of C8, E2, and ubiquitin mRNA expression was determined. Upregulation of skeletal muscle C8 (P = 0.09) and ubiquitin (P = 0.004) mRNA expression occurred post-partum compared with pre-partum. No regulation of hepatic mRNA expression was observed. In conclusion, ubiquitin-mediated proteolysis may contribute to skeletal muscle protein degradation during the periparturient period, and could provide a potential mechanism for attenuation of body protein loss at the onset of lactation.Key words: Lactation, ubiquitin-mediated proteolytic pathway, muscle (skeletal), liver

Use of metabolic control analysis in lactation biology

Sensitivity analysis is routinely carried out in the evaluation of simulation models to identify the degree to which parameters influence model outputs. This type of sensitivity analysis is much less frequently applied to real systems, but a technique called metabolic control analysis (MCA) was developed in the 1970s for the purpose of experimentally identifying the degree to which individual enzymes in a metabolic pathway influence flux through the pathway. MCA is applied to the results of inhibition, activation or genetic manipulation of enzymatic steps in a biochemical pathway. Flux control coefficients for each enzyme are defined as the fractional change in steady-state flux through the entire pathway for an infinitesimal change in the activity of that one enzyme. The sum of control coefficients in a linear, non-branching pathway is equal to one. It is a common finding in MCA that the control, or sensitivity, is distributed over multiple enzymes and not in a single rate-limiting enzyme. The fundamental principles of MCA are reviewed and an overview of experimental methods to measure control coefficients is provided, with the objective of introducing this approach to the fields of agricultural biochemistry and modelling, where it is little known. The application of MCA to the study of glucose metabolism and fatty acid synthesis in bovine mammary tissue are reviewed. The analyses indicated that mammary hexokinase activity exerts more control than transmembrane transport of glucose over lactose synthesis, and that control of cytosolic fatty acid synthesis is shared between acetyl-CoA carboxylase and fatty acid synthase, contrary to the widely held view that acetyl CoA carboxylase is the rate-limiting enzyme. It is suggested that MCA could be a valuable aid in the integration of proteomic and metabolomic data with metabolic flux measurements to engineer desired changes in the composition of milk from dairy animals.

Addition of glycerol to lactating cow diets stimulates dry matter intake and milk protein yield to a greater extent than addition of corn grain

The objective of this study was to determine if the addition of glycerol to the diet of dairy cows would stimulate milk protein yield in the same manner as the addition of corn grain. Twelve multiparous lactating dairy cows at 81 ± 5 d in milk were subjected to 3 dietary treatments in a replicated 3 × 3 Latin square design for 28-d periods. The diets were a 70% forage diet considered the basal diet, the basal diet with 19% ground and high-moisture corn replacing forages, and the basal diet with 15% refined glycerol and 4% added protein supplements to be isocaloric and isonitrogenous with the corn diet. Cows were milked twice a day and samples were collected on the last 7 d of each period for compositional analysis. Within each period, blood samples were collected on d 26 and 27, and mammary tissue was collected by biopsy on d 28 for Western blot analysis. Dry matter intake increased from 23.7 kg/d on the basal diet to 25.8 kg/d on the corn diet and 27.2 kg/d on the glycerol diet. Dry matter intake tended to be higher with glycerol than corn. Milk production increased from 39.2 kg/d on the basal diet to 43.8 kg/d on the corn diet and 44.2 kg/d on the glycerol diet. However, milk yield did not differ between corn and glycerol diets. Milk lactose yields were higher on the corn and glycerol diets than the basal diet. Milk fat yield significantly decreased on the glycerol diet compared with the basal diet and tended to decrease in comparison with the corn diet. Mean milk fat globule size was reduced by glycerol feeding. Milk protein yield increased 197 g/d with addition of corn to the basal diet and 263 g/d with addition of glycerol, and the glycerol effect was larger than the corn effect. The dietary treatments had no effects on plasma glucose concentration, but plasma acetate levels decreased 27% on the glycerol diet. Amino acid concentrations were not affected by dietary treatments, except for branched-chain amino acids, which decreased 22% on the glycerol diet compared with the corn diet. The decreases in plasma acetate and branched-chain amino acid concentrations with glycerol and the larger effects of glycerol than corn on milk protein and fat yields suggest that glycerol is more glucogenic for cows than corn grain.