Yves Mercier

Effects of dietary supplementation of methionine and its hydroxy analog DL-2-hydroxy-4-methylthiobutanoic acid on growth performance, plasma hormone levels, and the redox status of broiler chickens exposed to high temperatures.

Heat stress is known to impair performance and to induce oxidative stress in poultry. The aim of the present study was to compare the effects of dietary supplementation of dl-methionine (dl-M) or the synthetic analog 2-hydroxy-4-methylthiobutanoic acid (dl-HMTBA) on broiler growth performance, plasma hormone levels, and some oxidative stress-related parameters under conditions of chronic exposure to high temperatures (HT). From 2 to 6 wk of age, male broiler chickens were reared under either a constant temperature of 32°C until 6 wk of age or a normal temperature scheme (gradual decrease to 18°C at 5 wk of age). Chicks in both the normal and HT treatments were provided with a commercial grower diet supplemented with either 1.0 or 1.2 g/kg of dl-M or 1.0 or 1.2 g/kg of dl-HMTBA. Because there were no effects of supplement dose, data were pooled over both doses within each temperature treatment. The chronic HT treatment impaired feed intake and BW gain, but these negative effects were less pronounced when the chickens received dl-HMTBA. Exposure to HT was also associated with decreased (P < 0.001) plasma thyroid hormones and increased (P < 0.0001) plasma corticosterone levels. At 4 wk of age, and irrespective of the supplemental source, chickens subjected to HT were characterized by significantly lower plasma TBA-reactive substance levels. In contrast, at 6 wk of age, plasma TBA-reactive substance levels were significantly increased by HT, but this effect was observed only for the chickens receiving dl-M and not for those receiving dl-HMTBA. High temperatures induced a significant increase in hepatic total glutathione (GSH) and oxidized GSH levels, regardless of the supplemental source. However, the hepatic ratios of reduced GSH to total GSH and reduced GSH to oxidized GSH were highest in chickens supplemented with dl-HMTBA. In conclusion, dl-HMTBA supplementation partially prevented the growth-depressing effects of chronic heat exposure compared with dl-M supplementation. It can be inferred that dl-HMTBA is more efficient in alleviating HT-induced oxidative damage because of a more favorable reduced GSH-to-total GSH ratio.

Comparative study of a new organic selenium source v. seleno-yeast and mineral selenium sources on muscle selenium enrichment and selenium digestibility in broiler chickens.

Two experiments were conducted on broiler chickens to compare the effect of a new organic Se source, 2-hydroxy-4-methylselenobutanoic acid (HMSeBA; SO), with two practical Se additives, sodium selenite (SS) and Se yeast (SY). The relative bioavailability of the different Se sources was compared on muscle (pectoralis major) total Se, selenomethionine (SeMet) and selenocysteine (SeCys) concentrations and apparent digestibility of total Se (ADSe). In the first experiment, from day (d) 0 to d21, Se sources were tested at different supplied levels and compared with an unsupplemented diet (NC). No significant effects were observed on growth performance during the experimental period. However, the different Se sources and levels improved muscle Se concentration compared with the NC, with a significant source effect in the following order: SS < SY < SO (P<0·05). Seleno-amino acids speciation results for NC, SY and SO at 0·3 mg Se/kg feed indicated that muscle Se was only present as SeMet or SeCys, showing a full conversion of Se by the bird. The second experiment (d0-d24) compared SS, SY or SO at 0·3 mg Se/kg feed. The ADSe measurements carried out between d20 and d23 were 24, 46 and 49% for SS, SY and SO, respectively, with significant differences between the organic and mineral Se sources (P<0·05). These results confirmed the higher bioavailability of organic Se sources compared with the mineral source and demonstrated a significantly better efficiency of HMSeBA compared with SY for muscle Se enrichment.

Effects of dietary protein content and 2-hydroxy-4-methylthiobutanoic acid or DL-methionine supplementation on performance and oxidative status of broiler chickens.

Besides its typical role as an amino acid in protein synthesis, methionine is an important intermediate in methylation reactions. In addition, it can also be converted to cysteine and hence plays a role in the defence against oxidative stress. The present study was conducted to investigate further the role of DL-methionine (DLM) and its hydroxy analogue, DL-2-hydroxy-4-methylthiobutanoic acid (DL-HMTBA), on zootechnical performance and oxidative status of broiler chickens. Male broiler chickens were reared on two diets differing in crude protein (CP) content (low-protein, 18·3 % v. high-protein, 23·2 % CP) and were supplemented either with 0·25 % DLM or 0·25 % DL-HMTBA. Reducing the dietary protein content resulted in an impaired body weight gain (P < 0·0001). However, supplementation of DL-HMTBA to the low-protein diet partially alleviated these negative effects (P = 0·0003). This latter phenomenon could be explained by the fact that chickens fed DL-HMTBA-supplemented diets displayed a better antioxidant status as reflected in lower lipid peroxidation probably as a consequence of their higher hepatic concentrations of total and reduced glutathione compared with their DLM counterparts. On the other hand, within the high protein levels, uric acid might be an important antioxidant to explain the lower lipid peroxidation of high-protein DL-HMTBA-supplemented chickens. Hepatic methionine sulfoxide reductase-A gene expression was not significantly affected by the dietary treatments. In conclusion, the present study indicates that there are interactions between dietary protein content and supplementation of methionine analogues with respect to broiler performance and antioxidant status, also suggesting a causal link between these traits.

Changes in plasma amino acid profiles, growth performance and intestinal antioxidant capacity of piglets following increased consumption of methionine as its hydroxy analogue

The aim of the present study was to determine whether early weaning-induced growth retardation could be attenuated by increased consumption of methionine as DL-methionine (DLM) or DL-2-hydroxy-4-methylthiobutyrate (HMTBA) in both lactating sows and weaned piglets. Therefore, diets containing DLM and HMTBA at 25% of the total sulphur-containing amino acids (AA) present in the control (CON) diet were fed to lactating sows and weaned piglets and their responses were evaluated. Compared with the CON diet-fed sows, the HMTBA diet-fed sows exhibited a tendency (P<0·10) towards higher plasma taurine concentrations and the DLM diet-fed sows had higher (P<0·05) plasma taurine concentrations, but lower (P<0·05) isoleucine concentrations. Suckling piglets in the HMTBA treatment group had higher (P<0·05) intestinal reduced glutathione (GSH) content, lower (P<0·05) oxidised glutathione (GSSG):GSH ratio, and higher (P<0·05) plasma cysteine and glutathione peroxidase (GPx) activity than those in the CON and DLM treatment groups. The feed intake (P<0·05) and body weight of piglets averaged across post-weaning (PW) days were higher (P< 0·05) in the HMTBA treatment group than in the DLM treatment group and were higher (P<0·05) and tended (P<0·10) to be higher, respectively, in the HMTBA treatment group than in the CON treatment group. Increased (P<0·05) GSSG content and GSSG:GSH ratio and down-regulated (P<0·05) expression of nutrient transport genes were observed in the jejunum of piglets on PW day 7 than on PW day 0. On PW day 14, the HMTBA diet-fed piglets had higher (P<0·05) intestinal GSH content than the CON diet-fed piglets and higher (P<0·05) plasma GPx activity, villus height and goblet cell numbers than the CON diet- and DLM diet-fed piglets. In conclusion, early weaning-induced growth retardation appears to be attenuated through changes in plasma AA profiles and elevation of growth performance and intestinal antioxidant capacity in piglets following increased consumption of methionine as HMTBA.

Changes in body composition in broilers by a sulfur amino acid deficiency during growth

In the factorial approach, amino acid (AA) requirements are determined using the AA composition of retained protein, which is assumed to be constant. However, this hypothesis may not be valid because the AA composition of body protein can be affected by the diet. The objective of this study was to quantify the changes in chemical body composition of broilers receiving diets either deficient (TSAA-) or sufficient (TSAA+) in TSAA. Diet TSAA+ was formulated according to the Ross recommendation. Diet TSAA- provided 36% true digestible Met:Lys and 64% true digestible TSAA:Lys, which were, respectively, 34 and 22% lower compared with diet TSAA+. Performance and tissue weight gain between 7 and 42 d of age were not affected by the TSAA supply. In TSAA- chickens, protein gain was lower in the carcass (P < 0.01) and tended to be lower in the empty body (P = 0.06) and pectoralis major muscle (P = 0.10). Compared with TSAA+ chickens, lipid gain in TSAA- chickens was 78% greater in the pectoralis muscle (P < 0.001), 28% greater in abdominal fat (P < 0.05), and 10% greater in the carcass (P = 0.10). In the pectoralis muscle, there was a tendency for an increase in the redness value (a*; P = 0.10). The TSAA supply affected the AA composition of tissues and tissue gain, but the Met and Cys concentrations were changed only in the offal (P = 0.08). The deficient TSAA supply resulted in an increase in the Ser concentration in the empty body, carcass, and pectoralis muscle (P < 0.05). In contrast, it resulted in a decrease in the concentrations of Lys and Glu in the empty body, of Phe, Tyr, Gly, and Glu in the pectoralis muscle, and of Ala in the offal (P < 0.05). This indicates that although chickens cope with a TSAA deficiency predominantly by changing the protein and lipid concentration in the body, the AA composition is also affected. This calls into question the use of a constant ideal AA profile in poultry nutrition.

Effect of pH on l- and d-methionine uptake across the apical membrane of Caco-2 cells

The transport systems involved in intestinal methionine (Met) absorption are described as Na(+)-dependent and Na(+)-independent mechanisms. However, since recent studies have suggested the importance of the H(+) gradient as a driving force for intestinal nutrient absorption, the aim of the present work was to test whether Met transport across the apical membrane of Caco-2 cells is affected by extracellular pH. The results show that l- and d-Met uptake was increased by lowering extracellular pH from 7.4 to 5.5, in both the presence and absence of Na(+). Cis-inhibition experiments revealed that inhibition of l-Met transport by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) or l-lysine (l-Lys) was higher at a pH of 5.5. Moreover, the BCH-insensitive component was not affected by pH, whereas the l-Lys-insensitive component was increased by lowering extracellular pH, thus suggesting the participation of system L. The contribution of another mechanism, sensitive to both BCH and l-Lys, was also considered. The inhibition obtained with taurine (Tau) was also higher at a pH of 5.5, thus suggesting the involvement of system B(0,+) on pH-stimulated component. As for d-Met uptake, the results showed higher inhibition with l-Lys and Tau at a pH of 5.5 and no effect on the l-Lys- or Tau-insensitive component. In conclusion, Met transport across the apical membrane of Caco-2 cells is increased by low extracellular pH as the result of the stimulation of two transport systems functionally identified with systems L and B(0,+) for l-Met and with system B(0,+) for d-Met.

2-Hydroxy-4-methylselenobutanoic acid induces additional tissue selenium enrichment in broiler chickens compared with other selenium sources

Two experiments were conducted in broiler chickens to compare the effect of different Se sources on Se tissue enrichment: sodium selenite (SS), seleno-yeast (SY), and a new organic Se source (SO) containing 2-hydroxy-4-methylselenobutanoic acid (HMSeBA) as an active substance. For each experiment, treatments differed only in source or dose of Se additive. Relative efficiency was compared by plasma and tissue [muscle (pectoralis major) and liver] total Se concentrations. The first experiment compared Se sources (SS, SY, and SO) at different concentrations (mg of Se/kg of feed; SS-0.3; SY-0.1 and -0.3; SO-0.1 and -0.3; and a negative control, 0) in broilers between 0 and 42 d of age. Plasma, liver, and muscle Se concentrations were improved by all Se sources at both d 21 and 42 compared with the negative control group. Between Se sources, minor differences were observed for plasma and liver results, whereas a significant dose effect was observed from 0.1 to 0.3 mg of Se/kg of feed (P < 0.05) for each source. Muscle Se concentrations were improved such as SO > SY > SS (P < 0.05). Moreover, the relative muscle Se enrichment comparison, using linear regression slope ratio, indicated an average of 1.48-fold (95% CI 1.38, 1.58) higher Se deposition in muscle for SO compared with SY. In the second experiment, excessive dietary doses of 5 mg of Se/kg of feed from SS and SO showed a lower deleterious effect of SO on BW and feed intake in comparison with standard Se doses (P < 0.05). Seleno amino acid measurements conducted on different tissues of animals fed SO at 0.5 mg/kg of feed showed that HMSeBA is fully converted into selenomethionine and selenocysteine. These results of both experiments demonstrate the higher relative bioavailability of SO compared with SS and SY as determined through tissue Se enrichment.

A Methionine Deficient Diet Enhances Adipose Tissue Lipid Metabolism and Alters Anti-Oxidant Pathways in Young Growing Pigs.

Methionine is a rate-limiting amino-acid for protein synthesis but non-proteinogenic roles on lipid metabolism and oxidative stress have been demonstrated. Contrary to rodents where a dietary methionine deficiency led to a lower adiposity, an increased lipid accretion rate has been reported in growing pigs fed a methionine deficient diet. This study aimed to clarify the effects of a dietary methionine deficiency on different aspects of tissue lipid metabolism and anti-oxidant pathways in young pigs. Post-weaned pigs (9.8 kg initial body weight) were restrictively-fed diets providing either an adequate (CTRL) or a deficient methionine supply (MD) during 10 days (n=6 per group). At the end of the feeding trial, pigs fed the MD diet had higher lipid content in subcutaneous adipose tissue. Expression levels of genes involved in glucose uptake, lipogenesis but also lipolysis, and activities of NADPH enzyme suppliers were generally higher in subcutaneous and perirenal adipose tissues of MD pigs, suggesting an increased lipid turnover in those pigs. Activities of the anti-oxidant enzymes superoxide dismutase, catalase and glutathione reductase were increased in adipose tissues and muscle of MD pigs. Expression level and activity of the glutathione peroxidase were also higher in liver of MD pigs, but hepatic contents in the reduced and oxidized forms of glutathione and glutathione reductase activity were lower compared with control pigs. In plasma, superoxide dismutase activity was higher but total anti-oxidant power was lower in MD pigs. These results show that a dietary methionine deficiency resulted in increased levels of lipogenesis and lipolytic indicators in porcine adipose tissues. Decreased glutathione content in the liver and coordinated increase of enzymatic antioxidant activities in adipose tissues altered the cellular redox status of young pigs fed a methionine-deficient diet. These findings illustrate that a rapidly growing animal differently adapts tissue metabolisms when facing an insufficient methionine supply.

The methionine precursor DL-2-hydroxy-(4-methylthio)butanoic acid protects intestinal epithelial barrier function.

DL-2-hydroxy-(4-methylthio)butanoic acid (HMTBA) is a source of dietary methionine (Met) that is widely used in poultry nutrition. We have previously shown that HMTBA is preferentially diverted to the transsulfuration pathway, which gives antioxidant metabolites such as taurine and glutathione. Therefore, here we hypothesize that this Met source can protect epithelial barrier function in an in vitro model of intestinal inflammation of Caco-2 cells. The results show that HMTBA prevents the increase in paracellular permeability induced by H2O2 or tumour necrosis factor-α. This effect can be attributed to the increased production of taurine and reduced glutathione. Similar results were obtained for DL-Met, although the protective role of the amino acid was less pronounced than that of the hydroxy analogue. In conclusion, the diversion to the transsulfuration pathway means that this Met precursor is of greater value than previously thought, due to its capacity to improve intestinal homeostasis and the quality of poultry products destined for human consumption.

Intestinal cell conversion of DL-2-hydroxy-(4-methylthio)butanoic acid in vitro: dietary up-regulation by this methionine precursor.

DL-2-Hydroxy-(4-methylthio)butanoic acid (HMTBA) is a synthetic source of dietary methionine (Met) widely used in poultry nutrition. HMTBA is transported in the intestinal epithelium by the monocarboxylate transporter 1, after which its biological utilisation relies on its conversion to L-Met. This process involves stereospecific HMTBA oxidation to 2-keto-(4-methylthio)butanoic acid (KMB) and transamination to L-Met. In the present study, we examined HMTBA conversion to L-Met, further incorporation into cellular proteins and the regulation of both processes by HMTBA supplementation in differentiated intestinal Caco-2 cells. The results showed D- and L-HMTBA oxidation in the enterocytes, this process being up-regulated by HMTBA. The data also revealed that KMB transamination is not linked to a specific amino group donor. However, the branched-chain amino acid L-leucine is the preferred amino group donor. Furthermore, transamination was not affected by HMTBA availability. The incorporation of radioactivity from HMTBA into cellular proteins was not significantly different from that of L-Met and was not affected by HMTBA supplementation. In conclusion, the results reveal the capacity of Caco-2 cells to convert HMTBA to L-Met and the up-regulation of conversion by nutritional HMTBA supplementation, thus highlighting the contribution of the intestinal epithelium in the utilisation of HMTBA as a dietary source of Met.