Fish oil supplemented for 9 months does not improve glycaemic control or insulin sensitivity in subjects with impaired glucose regulation: a parallel randomised controlled trial.

The effects of fish oil (FO) supplementation on glycaemic control are unclear, and positive effects may occur only when the phospholipid content of tissue membranes exceeds 14% as n-3 PUFA. Subjects (n 36, thirty-three completed) were paired based on metabolic parameters and allocated into a parallel double-blind randomised trial with one of each pair offered daily either 6 g of FO (3·9 g n-3 PUFA) or 6 g of maize oil (MO) for 9 months. Hyperinsulinaemic-euglycaemic-euaminoacidaemic (HIEGEAA) clamps (with [6,6 2H2 glucose]) were performed at the start and end of the intervention. Endogenous glucose production (EGP) and whole-body protein turnover (WBPT) were each measured after an overnight fast. The primary outcome involved the effect of oil type on insulin sensitivity related to glycaemic control. The secondary outcome involved the effect of oil type on WBPT. Subjects on FO (n 16) had increased erythrocyte n-3 PUFA concentrations >14%, whereas subjects on MO (n 17) had unaltered n-3 PUFA concentrations at 9%. Type of oil had no effect on fasting EGP, insulin sensitivity or total glucose disposal during the HIEGEAA clamp. In contrast, under insulin-stimulated conditions, total protein disposal (P=0·007) and endogenous WBPT (P=0·001) were both increased with FO. In an associated pilot study (n 4, three completed), although n-3 PUFA in erythrocyte membranes increased to >14% with the FO supplement, the enrichment in muscle membranes remained lower (8%; P<0·001). In conclusion, long-term supplementation with FO, at amounts near the safety limits set by regulatory authorities in Europe and the USA, did not alter glycaemic control but did have an impact on WBPT.

Effects of supplementary folic acid and vitamin B(12) on hepatic metabolism of dairy cows according to methionine supply.

The present experiment was undertaken to study the interactions between dietary supplements of rumen-protected methionine (RPM) and intramuscular injections of folic acid and vitamin B(12), given from 3 wk before calving to 16 wk of lactation, on hepatic metabolism of lactating dairy cows. Sixty multiparous Holstein cows were assigned to 10 blocks of 6 cows each according to their previous milk production. Within each block, 3 cows were fed a diet calculated to supply Met as 1.83% of metabolizable protein, whereas the 3 other cows were fed the same diet supplemented with 18g of RPM calculated to provide Met as 2.23% of metabolizable protein. Within each level of Met, the cows received no vitamin supplement or weekly intramuscular injections of 160mg of folic acid alone or combined with 10mg of vitamin B(12). Liver biopsies were taken at 2, 4, 8, and 16 wk of lactation. Liver concentrations of folates and vitamin B(12) were increased by their respective supplements but this response to vitamin supplements was altered by methionine supply. Concentrations of total lipids and triglycerides increased in livers of cows fed RPM, whereas concentrations of cholesterol ester, cholesterol, diglycerides, phosphatidylethanolamine, and phosphatidylcholine were not affected. Folic acid, alone or combined with vitamin B(12), tended to increase the ratio of phosphatidylcholine to phosphatidylethanolamine. Gene expression of 5,10-methylene-tetrahydrofolate reductase, microsomal transfer protein, and phosphatidylethanolamine methyltransferase were higher in liver of cows fed RPM supplements. The relative mRNA abundance of 5,10-methylene-tetrahydrofolate reductase and methylmalonyl-CoA mutase were increased by the combined injections of folic acid and vitamin B(12), whereas those of methionine synthase and methionine synthase reductase were not affected by treatments. These results suggest that increasing supply of methyl groups, as preformed labile methyl groups or through methylneogenesis, affected the methylation cycle but had a limited effect on dairy cow performance. The observed effects of the combined supplement of folic acid and vitamin B(12) on lactational performance of dairy cows probably result from an improvement of energy metabolism during early lactation.

Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole body metabolism of methionine and glucose in lactating dairy cows.

The present experiment was undertaken to determine the effects of dietary supplements of rumen-protected methionine and intramuscular injections of folic acid and vitamin B(12), given 3 wk before to 16 wk after calving, on glucose and methionine metabolism of lactating dairy cows. Twenty-four multiparous Holstein cows were assigned to 6 blocks of 4 cows each according to their previous milk production. Within each block, 2 cows were fed a diet estimated to supply methionine as 1.83% metabolizable protein, equivalent to 76% of methionine requirement, whereas the 2 other cows were fed the same diet supplemented daily with 18 g of rumen-protected methionine. Within each diet, the cows were administrated either no vitamin supplement or weekly intramuscular injections of 160 mg of folic acid plus 10 mg of vitamin B(12.) To investigate metabolic changes at 12 wk of lactation, glucose and methionine kinetics were measured by isotope dilution using infusions of 3[U-(13)C]glucose, [(13)C]NaHCO(3) and 3[1-(13)C,(2)H(3)] methionine. Milk and plasma concentrations of folic acid and vitamin B(12) increased with vitamin injections. Supplementary B-vitamins increased milk production from 34.7 to 38.9 +/- 1.0 kg/d and increased milk lactose, protein, and total solids yields. Whole-body glucose flux tended to increase with vitamin supplementation with a similar quantitative magnitude as the milk lactose yield increase. Vitamin supplementation increased methionine utilization for protein synthesis through increased protein turnover when methionine was deficient and through decreased methionine oxidation when rumen-protected methionine was fed. Vitamin supplementation decreased plasma concentrations of homocysteine independently of rumen-protected methionine feeding, although no effect of vitamin supplementation was measured on methionine remethylation, but this could be due to the limitation of the technique used. Therefore, the effects of these B-vitamins on lactation performance were not mainly explained by methionine economy because of a more efficient methylneogenesis but were rather related to increased glucose availability and changes in methionine metabolism.

Influence of methionine supply on the response of lactational performance of dairy cows to supplementary folic acid and vitamin B12.

The present experiment was undertaken to determine if the effects of supplementary folic acid on lactational performance were caused by improved methylneogenesis and if the supply in vitamin B(12) could affect this metabolic pathway. In this eventuality, supplementary Met, a major source of preformed methyl groups, should reduce the requirements for these vitamins. Sixty multiparous Holstein cows were assigned to 10 blocks of 6 cows each according to their previous milk production. Within each block, 3 cows were fed a diet estimated to supply Met as 1.83% metabolizable protein and 3 cows were fed the same diet supplemented with 18 g of rumen-protected methionine (RPM) to supply Met as 2.23% of metabolizable protein. Within each level of Met, cows received no vitamin supplement or weekly intramuscular injections of 160 mg of folic acid alone or combined with 10 mg of vitamin B(12) from 3 wk before to 16 wk after calving. There was no treatment effect on dry matter intake during pre- and postcalving periods: 13.4 +/- 0.4 and 21.8 +/- 0.4 kg/d, respectively. Milk production was not affected by RPM supplementation. Folic acid and vitamin B(12) given together tended to increase milk production during the 16 wk of lactation. This effect was more pronounced during the first 4 wk of lactation: 37.5, 37.7, and 40.3 +/- 0.9 kg/d for cows receiving no vitamin supplement, folic acid alone, or folic acid combined with vitamin B(12), respectively. Milk fat yield was not affected by treatments. Lactose, crude protein, and total solid yields were greater, in early lactation, in cows injected with folic acid and vitamin B(12) together but this effect diminished as lactation progressed. Intramuscular injections of folic acid alone or combined with vitamin B(12) tended to decrease plasma concentrations of homocysteine from 5.51 microM with no vitamin supplement to 4.54 and 4.77 +/- 0.37 microM, respectively. Results of the present experiment suggest that the effects of the combined supplement of folic acid and vitamin B(12) on lactational performance of dairy cows were not due to an improvement in methyl groups supply, because RPM supplement, a source of preformed methyl groups, did not alter the cow responsiveness to vitamin supplements.

Effect of ruminally protected methionine on splanchnic metabolism of amino acids in lactating dairy cows.

The effect of ruminally protected Met (RPM) on splanchnic metabolism was measured in 3 primiparous and 3 multiparous Holstein cows. Doses of RPM (0, 36, and 72 g/d) were tested in a replicated 3 x 3 Latin square design, over 3 consecutive 14-d experimental periods. A mixed ration was fed in 12 equal meals per d (average dry matter intake: 17.5 +/- 0.08 kg/d). Indwelling catheters were surgically implanted in the mesenteric artery and the portal and hepatic veins for blood collection, as well as in 2 distal branches of the mesenteric vein for infusion of p-aminohippurate to determine blood flow. On d 14 of each period, a temporary catheter was inserted into a mammary vein and 6 hourly blood samples were collected to determine plasma concentrations of metabolites, hormones, and their respective fluxes across the splanchnic bed and mammary glands. Yields of milk (32.8, 32.0, and 32.9 +/- 0.92 kg/d) and protein (1,028, 1,053, and 1,075 +/- 28.7 g/d) were unaffected by level of RPM. However, the true protein content in milk from primiparous cows increased linearly (2.92, 3.09, and 3.34 +/- 0.077%). The addition of RPM linearly increased the net flux of Met across the portal-drained viscera, which resulted in increased arterial Met concentrations (25, 29, and 40 +/- 1.1 microM). Although it had no significant effect on net portal and hepatic fluxes of other essential amino acids, RPM resulted in a linear increase in the total splanchnic output of Ile, Leu, Phe, and Thr. These results suggest that feeding RPM triggered a homeostatic response resulting in less utilization of certain essential amino acids through the gastrointestinal tract and liver. Net mammary uptake of Met did not change with the addition of RPM. However, mammary extraction of Met decreased in a linear fashion in response to increased arterial inflow.

Regulation of glucose and protein metabolism in growing steers by long-chain n-3 fatty acids in muscle membrane phospholipids is dose-dependent.

A previous study showed that long-chain n-3 polyunsaturated fatty acids (LCn-3PUFA; >18 carbons n-3) exert an anabolic effect on protein metabolism through the upregulation of insulin sensitivity and activation of the insulin signaling pathway. This study further delineates for the first time whether the anabolic effect of LCn-3PUFA on metabolism is dose responsive. Six steers were used to test three graded amounts of menhaden oil rich in LCn-3PUFA (0%, 2% and 4%; enteral infusions) according to a double 3 × 3 Latin square design. Treatment comparisons were made using iso-energetic substitutions of control oil for menhaden oil and using 6-week experimental periods. The LCn-3PUFA in muscle total membrane phospholipids increased from 8%, 14% to 20% as dietary menhaden oil increased. Feeding graded amounts of menhaden oil linearly decreased plasma insulin concentration (49, 35 and 25 µU/ml, P = 0.01). The insulin-stimulated amino acid disposal rates as assessed using hyperinsulinemic-euglycemic-euaminoacidemic clamps (20, 40 and 80 mU/kg per h) were linearly increased by the incremental administrations of menhaden oil from 169, 238 to 375 µmol/kg per h (P = 0.005) during the 40 mU/kg per h clamp, and from 295, 360 and 590 µmol/kg per h (P = 0.02) during the 80 mU/kg per h clamp. Glucose disposal rate responded according to a quadratic relationship with the incremental menhaden oil amounts (P < 0.05). A regression analysis showed that 47% of the amino acid disposal rates elicited during the hyperinsulinemic clamp was related to muscle membrane LCn-3PUFA content (P = 0.003). These results show for the first time that both protein and glucose metabolism respond in a dose-dependent manner to menhaden oil and to muscle membrane LCn-3PUFA.

External pudic venous reflux into the mammary vein in lactating dairy cows.

A representative blood sample from the mammary vein depends on the functional integrity of the valves in the external pudic vein (EPV). To determine if the EPV valves maintain blood flow into the inguinal direction during the second and subsequent lactations, we used eight lactating cows catheterized in the EPV, the lateral branch of the cranial mammary vein (MV), and the external pudic artery (EPA). The averaged daily milk yields were 25.0 +/- 1.8 kg in cows in second lactation and 31.5 +/- 2.9 kg in older cows. The relative time taken by a pulse dose of p-amino hippuric acid (PAH) injected into the EPV, to reach the EPA and the MV, was measured in a first trial. In a second trial, we assessed the extent of alteration of the mammary PAH blood concentration with blood originating from other tissues using a continuous infusion of PAH into the EPA simultaneously with blocking or not any EPV backflux. From the first experiment, the PAH injected into the EPV appeared first in the EPA and then in the MV in cows in second lactation, suggesting that blood flow was towards the inguinal region. But in a third-lactation cow, the order of appearance was reversed. In parallel, the occlusion trial demonstrated that the concentration of PAH in the MV was diluted by 14 to 39% with blood draining nonmammary tissues only in cows in third or fourth lactation. This resulting reversed flow from the EPV towards the MV would have a detrimental impact on conclusions of mammary gland metabolism studies conducted with cows in their third lactation or higher.

Variations in mammary metabolism during the natural filling of the udder with milk over a 12-h period between two milkings.

Two groups of four Holstein cows, one in their second and the other in their third or fourth lactation, were used to study temporal variations of mammary metabolism over a 12-h period between two milkings. Blood samples were collected every 30 min from an artery and a mammary vein during a 12-h interval between two milkings. Isoleucine, leucine, lysine, methionine, and phenylalanine mammary net fluxes varied or tended to change over time after milking with a similar pattern between whole blood and plasma. For these amino acids, whole blood and plasma net fluxes reached their maximum over the first 8 h after milking. Simultaneously, respiratory quotients decreased linearly and varied from 2.31 to 2.01 during the first 8 h of the period, suggesting active mammary lipogenesis. From 8 to 12 h after milking, mammary amino acid net fluxes decreased, while mammary oxygen uptake tended to increase with a concomitant decrease in the respiratory quotient reaching 1.84 to 1.40. These findings suggest that, beginning 8 h after milking, mammary uptake of amino acids starts to decrease and catabolic processes appear promoted; this phenomenon could help to explain the increase in milk production reported in the literature with increased milking frequency.

Variations in mammary protein metabolism during the natural filling of the udder with milk over a 12-h period between two milkings: leucine kinetics.

To define the temporal variations of whole body and mammary leucine kinetics over a 12-h period between two milkings, we used two groups of four Holstein cows, one in their second and the other in their third or fourth lactation. Cows were infused with L-[1-13C]leucine during the 12-h interval between two milkings. Blood was sampled every 30 min during that period from arterial and mammary sources. Timeafter milking did not affect whole body irreversible loss rate of leucine but affected whole body leucine oxidation, which broadly followed variations in arterial plasma leucine concentration. Similarly, mammary leucine irreversible loss rate and leucine used for protein synthesis were not affected by time after milking. Leucine oxidation by the mammary gland was, however, affected by time after milking. It increased by 15% from the first 2-h period to the following 4-h period and then decreased by 13% over the following 2-h period. A 21% increase in leucine oxidation was observed from 8 to 10 h after milking, and then it decreased by 26% over the last 2-h period. Protein degradation expressed as percentage of mammary leucine flux followed a similar temporal pattern. Leucine used for protein synthesis by the mammary gland was unaltered over time after milking, suggesting that the increased availability of leucine resulting from mammary protein breakdown would increase intracellular concentrations of leucine, which would have favored its catabolism. Overall, these results confirm the high metabolic activity of the mammary gland, as protein synthesis by the mammary gland averaged 43% of whole body protein synthesis.

Peripheral leptin administration alters hormone and metabolite levels in the young pig.

The present study was conducted to determine if peripheral leptin administration can alter GH secretion or feed intake in young pigs. Six, 6 kg female pigs were fasted overnight and randomly chosen to receive porcine recombinant leptin or saline injections in a crossover design. Three leptin dosages were tested over a 10 day period, 100, 200 or 500 microg/kg body mass (L100, L200 or L500). Leptin was administered in 0.2% bovine serum albumin as a bolus injection into the carotid artery. Blood samples were obtained from the jugular vein over a 24 h period. Leptin delayed feeding in pigs treated with L200 and L500 (P<0.05), while reducing overall intake in pigs treated with L100 (P<0.05). L200 or L500 depressed blood glucose (P<0.05). Plasma insulin levels were elevated by feeding in control animals, while insulin levels were depressed in pigs treated with L200 or L500 (P<0.05). L200 elevated plasma growth hormone (P<0.05) with three peaks apparent at 5, 8, and 13 h post injection. The ability for a single injection of leptin to produce significant changes in hormone and metabolite levels suggests that this peptide has a role in regulation of peripheral metabolism.

Effects of buffers on milk fatty acids and mammary arteriovenous differences in dairy cows fed Ca salts of fatty acids.

Ten Holstein cows in early lactation were used in a replicated 5 x 5 Latin square design to study the effects of MgO and three buffers added to diets containing Ca salts of canola oil fatty acids. Treatments were 1) control (basal diet; no buffer). 2) 1.1% NaHCO3 plus 1.1% KHCO3, 3) 1.9% NaHCO3, 4) 0.5% MgO, and 5) 2.0% Na sesquicarbonate (percentage of dry matter). The control diet contained 53% grass silage, 43% concentrate, and 4% Ca salts. Body weight, intake, milk yield, and percentages of milk fat, protein, and lactose were unaffected by treatments. Buffers and MgO tended to increase triacylglycerol extraction by the mammary gland and changed the proportions of some fatty acids in milk. Arterial concentrations of acetate and triacylglycerol were correlated with their respective arteriovenous differences. Extraction by the mammary gland was high for acetate (approximately equal to 58.2%), triacylglycerol (approximately equal to 47.3%) propionate (approximately equal to 34.6%), and glucose (approximately equal to 24.3%). Extraction of free fatty acids, phospholipids, or cholesterol was negligible. Mammary triacylglycerol arteriovenous difference tended to be higher than when MgO was fed than when NaHCO3 was fed. Sodium sesquicarbonate, NaHCO3, and the blend of bicarbonate buffers increased C18:2 in milk fat when compared with the control treatment. The concentration of C18:2 in milk fat decreased when MgO was fed, but the ratio of cis-C18:1 to trans-C18:1 increased compared with effects of dietary NaHCO3. Medium-chain fatty acids in milk fat tended to be higher than Na sesquicarbonate than with NaHCO3. Buffers and MgO modified the profiles of fatty acids in milk.