Effects of parity and week after calving on the metabolic, redox and immune status of dairy cows.

At the onset of lactation in dairy cows, inflammation and oxidative stress may occur and result in a risk of pathologies and lower milk yield. To propose an innovative management strategy for cows during this period, it is essential to better understand these physiological variations. Our objective was to evaluate the metabolic, redox and immune status of 7 primiparous and 8 multiparous Holstein cows during late gestation and the first months of lactation. Blood samples were collected between 3 weeks before calving until 12 weeks postpartum. Milk samples were also collected, but only at the time points after calving. The metabolic (nonesterified fatty acids (NEFA), BHB, glucose, urea, calcium) and redox (reactive oxygen metabolites (ROM), oxidative stress index (OSI), glutathione peroxidase activity, vitamin E) statuses were analyzed in plasma or erythrocytes. The expression of genes related to antioxidant functions was determined in leukocytes collected from milk. For immune status, plasma cytokine levels and the production of reactive oxygen species (ROS) in classical and regulatory neutrophils were measured in 2 whole blood ex vivo challenges. The data were analyzed using a mixed model that included the fixed effects of parity and week and their interaction. Milk yield, plasma NEFA and BHB in wk 2 and 4 after calving were higher in multiparous cows than in primiparous cows, whereas glucose and calcium tended to be lower. Plasma ROM and OSI levels in wk 8 were higher in multiparous than in primiparous cows. Multiparous cows also displayed higher glutathione peroxidase activity in erythrocytes, and antioxidant transcription factor and superoxide dismutase-1 expression levels in milk leukocytes. Moreover, multiparous cows had higher plasma concentrations of vitamin E but lower plasma levels of cytokines CXCL10, CCL2, IL1Rα and IFNγ. Following ex vivo whole blood stimulation with Escherichia coli, lower IL1α and TNFα levels were measured in multiparous than in primiparous cows. Intracellular ROS production by neutrophils was lower in multiparous than in primiparous cows. These results thus indicated marked physiological changes in wk 8 compared with wk 2 and 4 of lactation. These differences in the physiological status of primiparous and multiparous cows offer interesting perspectives for potential dietary strategies to prevent pathologies which take account of parity and week relative to calving.

Methods and approaches to estimate B vitamin status in dairy cows: Knowledge, gaps and advances.

Clinical symptoms of B vitamin deficiency are rarely observed in ruminants because these vitamins are synthesized by the rumen microbiota. However, over the last decades, numerous reports of beneficial effects on production and metabolic efficiency of dairy cows have been published supporting that, under some conditions, B vitamin subclinical deficiency is present in these animals. Due to their roles as coenzymes or cofactors in major metabolic pathways, an adequate supply in B vitamins is critical to optimize metabolic efficiency. Nowadays, taking into account the growing interest for the Smart Farming concept, fulfilling ruminant requirements for B vitamins according to their physiological stage under different feeding management cannot be neglected. In dairy cows, B vitamin supply is greatly dependent of the activity of the ruminal microbiota. Indeed, the amount of vitamins reaching the small intestine is dependent of the utilization of the vitamins provided by the diet and their synthesis by the microorganisms present in the rumen. The two major challenges faced to determine B vitamin status of ruminants are the difficulty to estimate B vitamin supply due to the lack of knowledge on factors driving the fate of B vitamins in the digestive tract, especially in the rumen, and the choice and thresholds of biomarkers reflecting adequately the animal status. The present paper aims to present the actual state of knowledge on the methodological approaches used to estimate B vitamin supply and status of ruminant and to point out future research orientations.

Beta-carotene preferentially regulates chicken myoblast proliferation withdrawal and differentiation commitment via BCO1 activity and retinoic acid production.

The enzyme ß-carotene oxygenase 1 (BCO1) catalyzes the breakdown of provitamin A, including beta-carotene (BC), into retinal, prior to its oxidation into retinoic acid (RA). Allelic variation at the BCO1 locus results in differential expression of its mRNA and affects carotenoid metabolism specifically in chicken Pectoralis major muscle. In this context, the aim of this study was to evaluate the potential myogenic effect of BC and the underlying mechanisms in chicken myoblasts. BCO1 mRNA was detected in myoblasts derived from chicken satellite cells. Treating these myoblasts with BC led to a significant decrease in BrdU incorporation. This anti-proliferative effect was confirmed by a cell cycle study using flow cytometry. BC also significantly increased the differentiation index, suggesting a positive effect on the commitment of avian myoblasts to myogenic differentiation. Addition of DEAB, a specific inhibitor of RALDH activity, significantly reduced BC anti-proliferative and pro-differentiating effects, suggesting that BC exerted its biological effect on chicken myoblasts through activation of the RA pathway. We also observed that in myoblast showing decreased BCO1 expression consecutive to a natural mutation or to a siRNA treatment, the response to BC was inhibited. Nevertheless, BCO1 siRNA transfection increased expression of BCO2 which inhibited cell proliferation in control and BC treated cells.

Effects of dietary nitrogen levels and carbohydrate sources on apparent ruminal synthesis of some B vitamins in dairy cows.

Effects of nitrogen level and carbohydrate source on apparent ruminal synthesis (ARS) of thiamin, riboflavin, niacin, vitamin B6, folates, and vitamin B12 were evaluated using 4 lactating Holstein cows distributed in a 4 × 4 Latin square design with treatments following a 2 × 2 factorial arrangement. Cows were fitted with cannulas in the rumen and proximal duodenum. The treatments were 2 N levels and 2 carbohydrate sources. The diet with the high N level provided 14% crude protein, calculated to meet 110% of the protein requirements and an adequate supply in rumen-degradable protein, whereas the diet with the low N level contained 11% crude protein, calculated to meet 80% of the protein requirements with a shortage in rumen-degradable protein. Carbohydrate source treatments differed by their nature (i.e., high in starch from barley, corn, and wheat, or high in fiber from soybean hulls and dehydrated beet pulp). All 4 diets were isoenergetic, based on corn silage, and had the same forage-to-concentrate ratio (60:40, dry matter basis). Duodenal flow was determined using YbCl3 as a marker. Each B-vitamin ARS was calculated as duodenal flow minus daily intake. The intake of several B vitamins varied among treatments, but because the animals consumed a similar amount of feed every day (average of 20 kg of dry matter/d) the difference was mostly due to vitamin content of each ingredient and their relative proportion in the diets. Decreasing N concentration in the diet reduced vitamin B6 duodenal flow and increased its apparent ruminal degradation. It also decreased duodenal flow and ARS of folates. The high-starch diets increased duodenal flow and ruminal balance of riboflavin, vitamin B6, and folates, whereas the high-fiber diets increased vitamin B12 ARS and duodenal flow. These effects on apparent synthesis are possibly due to changes in ruminal fermentation.

Impacts of production conditions on goat milk vitamin, carotenoid contents and colour indices.

The content, composition and variation of vitamin compounds in goat milk have been little studied. An experimental design was based on 28 commercial farms, selected considering the main feeding system (based on main forage and especially pasture access), goat breed (Alpine vs Saanen) and reproductive management (seasonal reproduction), in the main French goat milk production area. Each farm received two visits (spring and autumn) that included a survey on milk production conditions and bulk milk sampling. Milk vitamins (A, E, B2, B6, B9, B12) and carotenoid concentrations plus colour indices were evaluated. A stepwise approach determined the variables of milk production conditions that significantly altered milk indicators. The main forage in the diet was the major factor altering goat milk vitamin and carotenoid concentrations and colour indices. Bulk milk from goats eating fresh grass as forage was richer in α-tocopherol (+64%), pyridoxal (+35%) and total vitamin B6 (+31%), and b* index (characterising milk yellowness in the CIELAB colour space) was also higher (+12%) than in milk from goats eating conserved forages. In milk from goats eating fresh grass, concentrations of pyridoxamine, lutein and total carotenoids were higher than in milk of goats fed corn silage (+24, +118 and +101%, respectively), and retinol and α-tocopherol concentrations were higher than in milk of goats fed partially dehydrated grass (+45 and +55%). Vitamin B2 concentration was higher in milk of goats eating fresh grass than in milk of goats fed hay or corn silage as forage (+10%). However, bulk milk when goats had access to fresh grass was significantly poorer in vitamin B12 than when fed corn silage (-46%) and in γ-tocopherol (-31%) than when fed conserved forage. Alpine goats produced milk with higher vitamin B2 and folate concentrations than Saanen goats (+18 and +14%, respectively). Additionally, the milk colour index that discriminates milks based on their yellow pigment contents was 7% higher in milk from Alpine than Saanen herds, but milk from Saanen goats was richer in lutein (+46%). Goat milks were richer in vitamins B2 and B12 and folates, but poorer in vitamin B6 in autumn than in spring (+12, +133, +15 and -13%, respectively). This work highlights that goat milk vitamin and carotenoid concentrations and colour indices vary mainly according to the main forage of the diet and secondly according to the breed and season.

Authentication of cow feeding and geographic origin on milk using visible and near-infrared spectroscopy.

The ability of near-infrared spectroscopy to trace cow feeding systems and farming altitude was tested on 486 bulk milk samples from France and northwestern Italy. Milks were grouped into feeding systems according to the main forage in the diet. Partial least square discriminant analysis correctly classified 95.5, 91.5, and 93.3% of pasture versus maize silage, hay, and fermented herbage feeding systems, respectively. Discrimination was slightly less successful when diets with large proportions of the nondominant forage were included in each group. Near-infrared spectroscopy correctly discriminated no-pasture from pasture milk, even with only 30% of pasture in the diet (5.4% cross-validation error), and the error stabilized when pasture exceeded 70% (2.5% error). Near-infrared spectroscopy did not reliably trace milk geographic origin when the feeding system effect was isolated from the altitude effect. These findings may be usefully exploited for the authentication of dairy products.

Simultaneous quantification of carotenoids, retinol, and tocopherols in forages, bovine plasma, and milk: validation of a novel UPLC method.

Simultaneous quantification of various liposoluble micronutrients is not a new area of interest since these compounds participate in the nutritional quality of feeds that is largely explored in human, and also in animal diet. However, the development of related methods is still under concern, especially when the carotenoid composition is complex such as in forages given to ruminants or in lipid-rich matrices like milk. In this paper, an original method for simultaneous extraction and quantification of all carotenoids, vitamins E, and A in milk was proposed. Moreover, a new UPLC method allowing simultaneous determination of carotenoids and vitamins A and E in forage, plasma and milk, and separation of 23 peaks of carotenoids in forages was described. This UPLC method using a HSS T3 column and a gradient solvent system was compared to a previously published reverse-phase HPLC using two C18 columns in series and an isocratic solvent system. The UPLC method gave similar concentrations of carotenoids and vitamins A and E than the HPLC method. Moreover, UPLC allowed a better resolution for xanthophylls, especially lutein and zeaxanthin, for the three isomers of beta-carotene (all-E-, 9Z- and 13Z-) and for vitamins A, an equal or better sensitivity according to gradient, and a better reproducibility of peak areas and retention times, but did not reduce the time required for analysis.

Effects of increasing amounts of extruded linseed in the diet on apparent ruminal synthesis of some B vitamins in dairy cows.

Many studies have shown that metabolic efficiency of ruminants can be significantly decreased when B-vitamin supply is insufficient. Under the present state of knowledge, the amounts of B vitamins available for intestinal absorption cannot be predicted based on diet composition. Therefore, in an attempt to increase our understanding of the effects of dietary factors, on B-vitamin supply for dairy cows, the effects of increasing amounts of extruded linseed in diets based on hay (permanent grassland hay, H; Experiment 1) or corn silage (CS; Experiment 2) on apparent ruminal synthesis (ARS) of thiamin, riboflavin, niacin, vitamin B6, folates and vitamin B12 were evaluated. In each experiment, four lactating Holstein cows fitted with cannulas in the rumen and the proximal duodenum were used in a 4 × 4 Latin square design. In both experiments, the dietary treatments consisted of an increasing supply of extruded linseed representing 0%, 5%, 10% or 15% of diet DM. The forage : concentrate ratios were 50 : 50 and 60 : 40 for Experiments 1 and 2, respectively. Duodenal flow was determined using YbCl3 as a marker. The ARS of each B vitamin was calculated as duodenal flow - daily intake. In both experiments, treatments did not affect thiamin, riboflavin, niacin and vitamin B12 duodenal flow or ARS. Increasing the dietary concentration of extruded linseed decreased folate intake in Experiment 1 and vitamin B6 intake in Experiment 2 but resulted in a greater duodenal flow of vitamin B6 and folates regardless of the forage used in basal diet. Greater dietary linseed concentrations decreased vitamin B6 apparent degradation in the rumen in CS-based diet only and increased folate ARS in both H- and CS-based diets. Increasing linseed concentration of isonitrogenous and isoenergetic diets increased vitamin B6 and folate supply to dairy cows, both with H- and CS-based diets.

Combinations of non-invasive indicators to detect dairy cows submitted to high-starch-diet challenge.

High-starch diets (HSDs) fed to high-producing ruminants are often responsible for rumen dysfunction and could impair animal health and production. Feeding HSDs are often characterized by transient rumen pH depression, accurate monitoring of which requires costly or invasive methods. Numerous clinical signs can be followed to monitor such diet changes but no specific indicator is able to make a statement at animal level on-farm. The aim of this pilot study was to assess a combination of non-invasive indicators in dairy cows able to monitor a HSD in experimental conditions. A longitudinal study was conducted in 11 primiparous dairy cows fed with two different diets during three successive periods: a 4-week control period (P1) with a low-starch diet (LSD; 13% starch), a 4-week period with an HSD (P2, 35% starch) and a 3-week recovery period (P3) again with the LSD. Animal behaviour was monitored throughout the experiment, and faeces, urine, saliva, milk and blood were sampled simultaneously in each animal at least once a week for analysis. A total of 136 variables were screened by successive statistical approaches including: partial least squares-discriminant analysis, multivariate analysis and mixed-effect models. Finally, 16 indicators were selected as the most representative of a HSD challenge. A generalized linear mixed model analysis was applied to highlight parsimonious combinations of indicators able to identify animals under our experimental conditions. Eighteen models were established and the combination of milk urea nitrogen, blood bicarbonate and feed intake was the best to detect the different periods of the challenge with both 100% of specificity and sensitivity. Other indicators such as the number of drinking acts, fat:protein ratio in milk, urine, and faecal pH, were the most frequently used in the proposed models. Finally, the established models highlight the necessity for animals to have more than 1 week of recovery diet to return to their initial control state after a HSD challenge. This pilot study demonstrates the interest of using combinations of non-invasive indicators to monitor feed changes from a LSD to a HSD to dairy cows in order to improve prevention of rumen dysfunction on-farm. However, the adjustment and robustness of the proposed combinations of indicators need to be challenged using a greater number of animals as well as different acidogenic conditions before being applied on-farm.

Plant polyphenols associated with vitamin E can reduce plasma lipoperoxidation in dairy cows given n-3 polyunsaturated fatty acids.

Diets rich in n-3 polyunsaturated fatty acids (PUFA) improve the nutritional value of ruminant products but also increase the risk of lipoperoxidation in plasma and tissues. The relative effectiveness of dietary antioxidants such as vitamin E (vit E) given alone or with plant extracts rich in polyphenols (PERP) containing rosemary, grape, citrus, and marigold was investigated in the plasma of mid-lactation dairy cows given diets enriched in 18:3 n-3. For a 30-d period, the animals were given a maize silage-based diet (control group C, n = 6) or the same basal diet supplemented with extruded linseed rich in 18:3 n-3 [50 g of oil/kg of diet dry matter (DM); group L, n = 6], extruded linseed + vit E (375 international units/kg of diet DM; 7,500 IU/cow per day; group LE, n = 6), or extruded linseed + vit E + PERP (10 g/kg of diet DM; group LEP, n = 5). Plasma susceptibility to lipoperoxidation was evaluated using in vitro parameters of conjugated diene formation (lag phase and maximum oxidation rate). Plasma indicators of lipoperoxidation and antioxidant status were analyzed in the 4 experimental groups as well as the fatty acid (FA) composition of total plasma lipids. At d 30, group L significantly increased plasma cholesterol esters (+57%) and phospholipids (+35%) compared with group C. It also increased plasma n-3 PUFA (4.7-fold increase) to the detriment of n-6 PUFA (-30%), leading to a higher peroxidizability index (+20%). Plasma in vitro lipoperoxidation was higher in group L (rich in 18:3 n-3) than in group C. Vitamin E alone had no effect on lipoperoxidation, whereas vit E in association with PERP lowered lipoperoxidation by increasing the resistance time against peroxidation (+47%) and by decreasing the oxidation rate (-48%) compared with group L at d 30. Surprisingly, in vivo plasma lipoperoxidation estimated by the plasma level of the major lipoperoxidation product (malondialdehyde) was not significantly increased in group L. This study shows, for the first time, that PERP supplied in association with vit E were able to reduce lipoperoxidation in lactating cows given a diet rich in 18:3 n-3, thereby helping to protect cows against the deleterious consequences of lipoperoxidation and potentially ensuring antioxidant potential for 18:3 n-3-enriched dairy products.

Variations in carotenoids, vitamins A and E, and color in cow's plasma and milk following a shift from hay diet to diets containing increasing levels of carotenoids and vitamin E.

This experiment was conducted to determine the variations in carotenoid, vitamins A and E concentrations, and color in the plasma and milk of dairy cows following a shift from a hay diet to diets containing increasing levels of carotenoids and vitamin E. This study was performed on 32 multiparous Montbéliarde dairy cows in midlactation. After a 6-wk preexperimental period on a diet based on hay and concentrates, the cows were allocated to 4 homogeneous groups, and thereafter fed for 6 wk on isoenergetic experimental diets where the hay was replaced by an experimental feed rich in carotenoids and vitamin E, consisting in 75% grass silage and 25% alfalfa protein concentrate (PX Agro Super Desialis, Châlons en Champagne, France). The hay-to-experimental feed ratios were 100/0 in group 1, 67/33 in group 2, 33/67 in group 3, and 0/100 in group 4, providing 1.6, 3.6, 5.4, and 7.4 g/d of total carotenoids, respectively. Variations in carotenoid, vitamins A and E concentrations as well as variations in color index (CI) were monitored from d -7 through to d 42 on the experimental diets. Zeaxanthin, lutein, 13-cis-beta-carotene, and all-trans-beta-carotene accounted for an average 3, 10, 9, and 78%, respectively, of total carotenoids in plasma and 0, 17, 12, and 71%, respectively, of total carotenoids in milk. The switch from preexperimental to experimental diets only slightly affected zeaxanthin, lutein, and vitamin A concentrations in plasma and milk. A rapid increase in vitamin E and beta-carotene (BC) was observed during the first week in both plasma and milk. For vitamin E, the time to reach a plateau was from 8 d (group 2) to 28 d (group 4) in plasma, and 5 d (groups 2-4) in milk. Plasma concentrations of BC had stabilized after 28 d in group 2 but were not stabilized after 42 d in groups 3 and 4, whereas milk concentrations of BC plateaued from d 21 in group 2 and d 28 in groups 3 and 4. At the end of the experimental period, BC and vitamin E concentrations in plasma and vitamin E concentrations in milk fat were linearly related to the proportion of experimental feed in the diet. In contrast, BC concentrations in milk fat did not differ between groups 2, 3, and 4, reflecting saturation at high levels of carotenoid intake (i.e., when plasma BC exceeded 5 mug/mL). These results suggested that under high-carotenoid diets, milk secretion of BC is not limited by the amount of plasma BC arriving to the mammary gland but by mechanisms involved in the BC transfer from plasma to milk. These mechanisms will need to be investigated. The BC concentrations were responsible for more than 80% of CI variations in plasma and 56% of CI variations in milk, where there was wide variability among individuals. Plasma CI appeared to be a more promising tool than milk CI as an indicator of the carotene content of the diets ingested by dairy cows.

Variations in carotenoids, vitamins A and E, and color in cow's plasma and milk during late pregnancy and the first three months of lactation.

The main aim of this work was to assess the effect of lactation period on the secretion of carotenoids in cow's milk. Our objective was to determine the variations in carotenoids in the plasma and milk of dairy cows from drying off to wk 12 of lactation, and to specify whether these variations depend on body stores of these micronutrients at calving. We also investigated the relationship between beta-carotene (BC) and color in plasma and milk to evaluate the methods based on direct or indirect characterization of these micronutrients for traceability of feeding management. The experiment was carried out on 18 dairy cows, which were dried off 8 wk before their expected date of parturition. They were then divided into 2 homogeneous groups and fed diets contrasting in carotenoid content, high (grass silage) vs. low (corn silage), from wk -7 until parturition. From parturition through wk 12 of lactation, both groups received a grass silage-based diet. Variations in concentrations of carotenoids and the color index (CI) in plasma and milk were monitored from drying off to wk 12 of lactation. Other components of nutritional interest (i.e., vitamins A and E) were also measured. Lutein, all-trans BC and cis-13 BC were the carotenoids found in plasma and milk. Plasma concentrations of carotenoids, vitamin A, and vitamin E decreased throughout the dry period and in the first week of lactation, then increased through the first 3 mo of lactation, parallel to grass silage intake. For both groups, carotenoid and vitamin concentrations in milk drastically decreased during the first week of lactation, then did not vary significantly throughout the remainder to the experiment (wk 12). Plasma concentrations of carotenoids and vitamins were higher in the high-carotenoid group than in the low-carotenoid group during the dry period. Those differences were also observed in colostrum and disappeared in both plasma and milk during the first 10 d of the lactation period. This work allowed us to conclude that, unlike in plasma, variations in carotenoids, vitamin A, and vitamin E in milk were only slight in early lactation. In both plasma and milk, the concentrations were only transitorily affected by the nature of forage fed during the dry period, showing that they depended mainly on the dietary supply, even during the lipid mobilization period. The relationship between concentrations of BC and the CI was linear in plasma (R2 = 0.51) and milk (R2 = 0.37) and reached a plateau in the milk + colostrum data set (R2 = 0.77). The changes in CI during the first 3 mo of lactation were not negligible compared with variations related to the nature of forage reported in previous studies. This implies that methods being developed for the traceability of feeding management of dairy cows based on direct or indirect characterization of these micronutrients in milk, plasma, or both will need to account for changes in relation to lactation stage, which requires further investigation.

Effects of dietary supplements of folic acid and vitamin B12 on metabolism of dairy cows in early lactation.

The present experiment was undertaken to determine the effects of dietary supplements of folic acid and vitamin B12 given from 3 wk before to 8 wk after calving on lactational performance and metabolism of 24 multiparous Holstein cows assigned to 6 blocks of 4 cows each according to their previous milk production. Supplementary folic acid at 0 or 2.6 g/d and vitamin B12 at 0 or 0.5 g/d were used in a 2 x 2 factorial arrangement. Supplementary folic acid increased milk production from 38.0 +/- 0.9 to 41.4 +/- 1.0 kg/d and milk crude protein yield from 1.17 +/- 0.02 to 1.25 +/- 0.03 kg/d. It also increased plasma Gly, Ser, Thr, and total sulfur AA, decreased Asp, and tended to increase plasma Met. Supplementary B12 decreased milk urea N, plasma Ile, and Leu and tended to decrease Val but increased homocysteine, Cys, and total sulfur AA. Liver concentration of phospholipids was higher in cows fed supplementary B12. Plasma and liver concentrations of folates and B12 were increased by their respective supplements, but the increase in plasma folates and plasma and liver B12 was smaller for cows fed the 2 vitamins together. In cows fed folic acid supplements, supplementary B12 increased plasma glucose and alanine, tended to decrease plasma biotin, and decreased Km of the methylmalonyl-coenzyme A mutase in hepatic tissues following addition of deoxyadenosylcobalamin, whereas it had no effect when cows were not fed folic acid supplements. There was no treatment effect on plasma nonesterified fatty acids as well as specific activity and gene expression of Met synthase and methylmalonyl-coenzyme A mutase in the liver. Ingestion of folic acid supplements by cows fed no supplementary B12 increased total lipid and triacylglycerols in liver, whereas these supplements had no effect in cows supplemented with B12. The increases in milk and milk protein yields due to folic acid supplements did not seem to be dependent on the vitamin B12 supply. However, when vitamin B12 was given in combination with folic acid, utilization of the 2 vitamins seems to be increased, probably more so in extrahepatic tissues. Metabolic efficiency seems also to be improved as suggested by similar lactational performance and dry matter intake for cows fed supplementary folic acid but increased plasma glucose and decreased hepatic lipids in cows fed folic acid and vitamin B12 together.

Effect of different forms of methionine on lactational performance of dairy cows.

Methionine is one of the first limiting AA in dairy cows. The use of rumen-protected Met to correct deficient diets is limited by the lack of a product that could be incorporated into a pelleted concentrate. The main objective of this trial was to test, at practical doses (approximately 10 g of absorbable Met), the efficacy of 2 forms of pelletable Met hydroxy analogs, D,L-2-hydroxy-4-(methylthio)-butanoic acid (HMB) and the isopropyl ester of HMB (HMBi), to provide Met to cows, especially for milk protein synthesis, compared with a negative control and to Smartamine M (SmM). These treatments were tested according to a 4 x 4 Latin square in 16 Holstein cows. Plasma Met concentrations were increased by 110 and 65% that of the control value after HMBi and SmM treatments, respectively. Milk protein yield increased by 32 and 41 g/d for HMBi and SmM, respectively. D,L-2-hydroxy-4-(methylthio)-butanoic acid supplementation did not improve Met availability to the cows for milk protein synthesis. The HMBi treatment induced an increase in 15:0 in milk at the expense of a general reduction in even-numbered short-and medium-chain fatty acids. Moreover, HMBi and SmM supplements led to an increase in the saturation level of C18 fatty acids consistent with the improvement of Met supply. It was concluded that HMBi is a new "rumen-protected" form of Met that can be supplied to cows integrated into pellets.

Methionine availability in plasma of dairy cows supplemented with methionine hydroxy analog isopropyl ester.

Adequate Met supply is especially important in the dairy cow for milk protein synthesis. Because of insufficient Met contents in the most frequently used feed-stuffs, Met becomes limiting in the diet of the dairy cow. To restore the amino acid balance of the diet and consequently to optimize lactation performance, Met must be supplied in a protected form because of its high degradability as a free amino acid by rumen microorganisms. A new chemical derivative of Met, the isopropyl ester of the 2-hydroxy-4-(methylthio)-butanoic acid (HMBi) was tested for its metabolic fate by following the evolution of plasma concentrations of its metabolites (2-hydroxy-4-(methylthio)-butanoic acid (HMB), Met, isopropyl alcohol, and acetone) after spot-dose supplementation (50 g Met equivalent) to 15 cows. Results indicated that HMBi would be quickly absorbed and hydrolyzed into HMB and isopropyl alcohol, and then converted to Met and acetone, respectively. In our experimental conditions, the Met availability for cows was estimated to be 48.34 +/- 2.05% using a calibration curve established by modeling the area under the curve response to increasing doses of Met supplied as Smartamine M, whose bioavailability (80%) is considered the reference value. Plasma kinetics and bioavailability of Met were compared between HMBi and Smartamine M in the same cows. Comparison of the kinetics suggests that HMBi would be absorbed through the rumen wall providing good protection against rumen microorganisms. It can thus be concluded that HMBi is a new source of Met for ruminants with an acceptable bioavailability.

Insulin-sensitive glucose transporter transcript levels in calf muscles assessed with a bovine GLUT4 cDNA fragment.

Previous studies have shown that the expression of the insulin-sensitive glucose transporter (GLUT4) is lower in oxidative muscles than in glycolytic muscles in bovines and goats in contrast to observations in rats. Additional experiments in this work provide very strong arguments that the immunoreactive band detected does represent GLUT4 protein, which further validates our previous results. Therefore, to determine the level of regulation, the main objective of the present study was to measure GLUT4 mRNA amounts in various bovine muscles. A 241-bp fragment of the bovine GLUT4 cDNA was cloned by polymerase chain reaction (PCR). It shares 80-90% sequence identity with related sequences in other species. This PCR-amplified bovine GLUT4 probe was used to determine the distribution of GLUT4 mRNA in bovine tissues in comparison with that of GLUT1 mRNA. Moreover, GLUT4 mRNA amounts were quantified by Northern-blot analysis in heart and seven skeletal muscles with various oxidative and glycolytic activities from seven ruminant calves. GLUT4 mRNA was detected by Northern-blot analysis only in calf insulin-sensitive tissues. In contrast, GLUT1 mRNA was detected in all tissues studied except liver. GLUT4 mRNA amount was the highest in masseter and heart, which are oxidative muscles (1.67 +/- 0.16 and 1.53 +/- 0.19 units/g wet tissue weight, respectively) and the lowest in glycolytic or oxido-glycolytic muscles (0.31 +/- 0.04 to 1.00 +/- 0.09 units/g wet tissue weight; SEM, n = 7). These data and our previous results provide evidence for translational and/or post-translational control mechanisms of bovine GLUT4 protein expression in a muscle type-specific manner.

Fatty acid metabolism and very low density lipoprotein secretion in liver slices from rats and preruminant calves.

The liver of bovine animals possesses a low ability to secrete triglycerides (TG) as part of the very low density lipoproteins (VLDL) compared with rat liver. We compared hepatic fatty acid (FA) metabolism between rat and calf in order to determine the limiting steps of TG-VLDL secretion in bovine animals. Liver slices from young Sprague-Dawley rats and preruminant Holstein x Friesian calves were incubated for 7 h with increasing concentrations (0.1, 0.2, 0.4, and 0.8 mM) of [14C]oleate. The oxidation of oleate to CO2 and acid-soluble products was 2- to 3-fold higher in rat than in calf liver slices. Since oleate uptake was 2-fold higher in rat than in calf, the oxidation rate represented 20-29% of oleate uptake in both animal species. Oleate was essentially incorporated into the neutral lipids (75-87% of total lipids) that were stored mainly in the cytosol in both animal species (81-90% of neutral lipids). The accumulation of neutral lipids in the cytosol was 3.4-fold higher while VLDL secretion was 6- to 18-fold more efficient in rat than in calf liver slices. Our results indicate that the slow rate of VLDL secretion by bovine liver is probably due to the limited availability of TG for VLDL packaging rather than to the preferential oxidation of FA.

Apolipoprotein B production and very low density lipoprotein secretion by calf liver slices.

Secretion of triglycerides by the liver in ruminants as components of very low density lipoproteins particles is low as compared with that in primates or rodents. The rate-limiting steps for the hepatic export of very low density lipoproteins have been studied in liver slices to determine the origin of the low lipotropic capacity of calf liver compared to that of rat liver. The rates of production of apolipoprotein B (apo B) and albumin as well as the rate of secretion of VLDL-apolipoproteins were measured during 12-h incubation of liver slices in organo-culture using [35S]methionine-cysteine labeling. Hepatic apo B production was similar in the two animal species but the VLDL-apolipoprotein secretion rate for calf liver slices amounted to only 20% of that observed for rat liver slices. Although calf and rat liver slices synthesized similar amounts of total protein, the hepatic production of albumin, measured in cells and media, was much higher in calf than rat liver slices (around 2.7-fold), whereas the rate secretion of albumin was similar in the two species. Our results showed that the slow rate of secretion of VLDL by calf liver cells was not consecutive to a low rate of synthesis of apo B but rather to a defect in VLDL assembly and/or secretion.

Lipoprotein lipase activity and mRNA levels in bovine tissues.

Lipoprotein lipase (LPL) in cattle has been extensively studied in adipose tissue, milk and mammary gland, but only to a limited extent in muscles. Therefore, we have adapted our in vitro LPL assay method for the measurement of LPL activity and describe, for the first time, sensitive procedures to quantify LPL activity and mRNA levels in bovine muscles. In vitro activation of bovine LPL activity is approximately 5-fold greater with rat than with bovine sera for heart and muscles, but not for adipose tissues. Values of LPL activity are in the upper range of those previously reported for rat or bovine tissues. With rat serum as activator, LPL activity in the heart of seven calves (662-832 mU g-1) is at least 3-fold lower than in the rat heart (2150-2950 mU g-1, P < 0.05). LPL activity is higher in bovine heart and oxidative muscles (412-972 mU g-1), except the diaphragm, than in mixed or glycolytic muscles (33-154 mU g-1, P < 0.05). The levels of LPL transcripts are positively related to LPL activity in bovine tissues, including muscles and adipose tissues.