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Evonik Animal Nutrition

Supplemental dietary methionine sources have a neutral impact on oxidative status in broiler chickens

Published: September 17, 2018
By: Dr. Rose Whelan, Evonik Nutrition & Care GmbH, Animal Nutrition, Germany
Conclusions
  • Dietary methionine source (DL-Met, L-Met or DL-HMTBA at 65 % bioefficacy) did not affect the ratios of reduced to oxidized glutathione or reduced to total glutathione in intestinal tissue, liver or breast muscle
  • None of the dietary methionine sources (DL-Met, L-Met or DL-HMTBA) consistently affected biomarkers of antioxidant capacity (ferric reducing ability) or oxidative stress (TBARS, Protein Carbonyl) when comparing several tissues and two time points
  • Overall dietary methionine source did not alter the biomarkers of antioxidant capacity of broilers
Introduction
Methionine (Met) is an essential amino acid for poultry and the first limiting in broiler diets. Met is essential not only for protein synthesis and muscle deposition, but for several important biological functions related to oxidative stress. As a precursor for cysteine and subsequently glutathione (GSH), Met plays an important role in the antioxidant system.
Oxidants in the system naturally occur as part of the immune defense against pathogens and as a consequence of normal cellular metabolism. During inflammation or metabolic dysfunction in heat stress for example, oxidants in the system increase and cause tissue damage through the oxidation of lipids, proteins, carbohydrate and DNA structures of cells. The GSH system is essential to the preventative control of such oxidants and amelioration of oxidative stress (Gilbert 1984, 1985; Chai et al. 1994).
The GSH system is able to reduce oxidants through the peroxidation of reduced glutathione (rGSH). The oxidized disulfide form of glutathione (GSSG) that is produced in this reaction is then reduced back to rGSH by a reductase enzyme so it is further available as an antioxidant. This is apparent when looking at the ratios of rGSH to GSSG or rGSH to total GSH (TGSH). In normal conditions the ratio of rGSH to total GSH has been reported as high as 0.98 in some tissues; however, in stressful conditions as rGSH is reducing oxidants and GSSG is formed the ratios can fall by10 fold (Gilbert 1984, 1985; Chai et al. 1994).

As Met is converted through the transsulfuration pathway to cysteine and further GSH, it is not surprising that dietary Met supplementation has been found to enhance GSH production in broilers (Nemeth et al. 2004). However, there are several isoforms of dietary Met available that may affect the production of GSH and therefore the antioxidant capacity of tissues.
L-Met contains only the L-enantiomer of methionine, while DL-Met is a 50:50 mixture of L- and D-enantiomers of Met. A Methydroxy analogue is also available for dietary supplementation; DL-2-hydroxy-4-(methylthio) butanoic acid (DL-HMTBA) products contain 12 % water, 65 % monomers and 23 % oligomers of DL-HMTBA. The D-enantiomer of DL-Met as well as the D- and L-isomers of HMTBA must first be converted to L-Met before entering the transsulfuration pathway to produce glutathione. These differences in Met source metabolism have been hypothesized to affect the production of GSH. Therefore, the current trial was designed to test the effects of various dietary Met sources (DL-Met, L-Met or DL-HMTBA) on the GSH system and the balance between oxidative stress biomarkers and antioxidant capacity of tissues in broiler chickens. The trial was conducted in collaboration with the Virginia Polytechnic Institute and State University, USA.
Materials and Methods
Two-hundred and forty, day-old male chicks (Cobb 500) were randomly assigned to one of four dietary treatments with 6 pen replicates and 10 birds/pen. Corn, wheat and soybean meal based basal diets were formulated to meet Evonik amino acids recommendations (AMINOChick® 2.0) for starter (d 1-10), grower (d 11-21) and finisher (d 22-26) phases except for SID Met+Cys which was sub-optimal in the basal diet for each of the phases (0.78, 0.70, and 0.62 %, respectively). The basal diet composition is presented in Table 1, with analyzed nutrient composition presented in Table 2. The dietary treatments included: 1) a basal diet with sub-optimal Met+Cys (Control), 2) Control supplemented with 0.22 % analyzed DL-Methionine to meet Met+Cys recommendations (DL-Met), 3) Control supplemented with 0.22 % analyzed L-Methionine (L-Met) and 4) Control supplemented with 0.31 % analyzed DL-HMTBA (inclusion was formulated to be 0.34 % based on a 65 % bioefficacy of DL-HMTBA to DL-Met).
Body weight and feed intake were recorded on a pen basis, then calculated and reported as averages per bird. Feed conversion ratio (FCR) was calculated on a pen basis. At d 10 and d 26, 5 birds per treatment were euthanized by cervical dislocation and liver, breast muscle, duodenum, jejunum and ileum samples were collected for quantification of total glutathione (TGSH), reduced glutathione (rGSH), oxidized glutathione (GSSG), protein carbonyl, ferric reducing antioxidant power (FRAP) and thiobarbituric acid-reactive substances (TBARs).
Data were analyzed by ANOVA using JMP Pro version 12.0 (SAS Institute, Cary, NC). The overall tissue main effect (ages combined) was analyzed within each marker. To test the treatment effect, data were analyzed within each tissue (liver, breast muscle, duodenum, jejunum and ileum). The statistical model included the main effects of treatment, age and their interaction. All data were checked for normality and homogeneity of variances. No data were transformed prior to analysis. When significant differences were found, a 5 % of variance average was used, with Tukey’s multiple range test for comparisons among groups. Data are presented as least square means ± SEM and statistical significance assigned at P < 0.05.
Supplemental dietary methionine sources have a neutral impact on oxidative status in broiler chickens - Image 1
Supplemental dietary methionine sources have a neutral impact on oxidative status in broiler chickens - Image 2
Results and Discussion
The results of growth performance are presented in Table 3. Methionine supplementation significantly improved the live weight and feed conversion ratio of broilers compared to the methionine deficient basal diet; however, methionine source had no effect on growth performance.
Supplemental dietary methionine sources have a neutral impact on oxidative status in broiler chickens - Image 3
The results of the GSH profile are presented in Table 4. Met sources did not affect the GSH markers in any of the tissues with the exception of a higher concentration of TSGH and rGSH in breast tissues for birds fed L-Met compared to broilers fed the control and DL-Met diet. However, there were no differences between broilers fed DL-Met and DL-HMTBA. The ratio of reduced to total glutathione was not significantly affected by Met source and oxidized GSH was not detectable in breast tissue from any treatment group. There were a few significant interaction effects between Met source and broiler age observed (Figure 1). In the Jejunum the L-Met treatment group had higher oxidized glutathione (GSSG) than the control group or DL-Met treatment at d 26 but not at d 10 (Figure 1B). Conversely, in the ileum the L-Met treatment group had higher total and reduced glutathione than the DL-Met on d 26 but no differences were observed on d 10 (Figure 1D and E). However, none of the treatments affected the ratio of reduced to total glutathione in either jejunum or ileum at either time point (Figure 1C and 1F, respectively). Therefore, it can be concluded that dietary Met source did not affect the balance of the glutathione system in breast, liver, duodenum, jejunum or ileum in broilers at either d10 or d26.
Oxidative stress can lead to tissue damage due to oxidation of cell proteins, lipids and DNA. Therefore, biomarkers of protein oxidation (protein carbonyl) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS), were measured in various tissues (Table 5). Additionally, the antioxidant potential of the tissues was determined by measuring the ferric reducing antioxidant power (FRAP) of tissues from the broilers fed different dietary Met sources. Neither the lipid peroxidation marker TBARS, nor the antioxidant power FRAP were affected by dietary treatment. However, dietary treatment affected protein carbonyl levels in the jejunum and breast muscle. DL-HMTBA treatment decreased protein carbonyl in jejunum compared to DL-Met, but neither differed from control. In breast muscle there was a diet x age interaction effect for protein carbonyl showing no dietary effects at d 10 but significant effects of diet at d 26. L-Met increased protein carbonyl in breast muscle at d26 compared to Met deficient control and DL-HMTBA, but not DL-Met. These findings were not consistent over tissues and at different time points, as there were no effects of dietary treatment observed for liver, duodenum or ileum at any time point and no treatment effects observed at d10 in any tissue.
Overall, the results show that dietary methionine source (DL-Met, L-Met or DL-HMTBA) did not affect antioxidant status of broiler birds in physiologically normal conditions as there were no significant effects of dietary met source observed on antioxidant to oxidant ratios. Additionally, there were no consistent effects of dietary Met source on antioxidant or oxidative stress biomarkers when comparing different tissues or time points.
Unexpectedly, sub-optimal Met+Cys did not impact antioxidant status in the tissues of broilers in this trial. Although, a recently published study has shown that when Met supplementation is tested in a more sensitive dose response experimental design, sub-optimal dietary Met+Cys does increase oxidative stress in tissues (Zeitz et al. 2018). It can also be speculated that the lack of observable effects of the sub-optimal Met+Cys basal diet in this study could be partially attributed to the lack of oxidative stress challenge. If the broilers had been exposed to challenging conditions causing oxidative stress (such as heat stress or a disease challenge), the need for TGSH production would have been higher. Therefore, the negative impact of a deficiency in Met+Cys as a precursor for TGSH synthesis, on the GSH system and antioxidant capacity of the tissues may be more easily observed in oxidative stress conditions. Future research, will investigate the effect of Met dose and source in challenging conditions associated with oxidative stress.
In conclusion the study shows that DL-Met, L-Met and DL-HMTBA (formulated at 65 % BE) did not differentially affect growth performance or the oxidative status in the digestive, metabolic and meat tissues of healthy broilers.
Supplemental dietary methionine sources have a neutral impact on oxidative status in broiler chickens - Image 4
 
Supplemental dietary methionine sources have a neutral impact on oxidative status in broiler chickens - Image 5
 
Supplemental dietary methionine sources have a neutral impact on oxidative status in broiler chickens - Image 6

Chai et al. 1994: S-thiolation of individual human neutrophil proteins including actin by stimulation of the respiratory burst: evidence againsta a role for glutathione disulfide. Archives of Biochem and Biophys. 310: 273-281

Gilbert. 1984. Redox control of enzyme activities by thiol/disulfide exchange. Methods in Enzymology. 107: 330-351

Gilbert. 1985. Thiol/disulfide exchange equilibria and disulfide bond stability. Methods in Enzymolology. 251: 8-28

Nemeth et al. 2004.Effect of supplementation with methionine and different fat sources on the glutathione redox system of growing chickens. Acta Veterinaria Hungarica. 52: 369-378.

Zeitz et al. 2018. Tissue and plasma antioxidant status in response to dietary methionine concentration and source in broilers

Zhang et al. 2018. Supplemental methionine sources have a neutral impact on oxidative status in broiler chickens. Journal of Animal Physiology and Animal Nutrition. Ahead of print.  

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Authors:
Rose Whelan
Evonik Animal Nutrition
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Dr Valeriy Kryukov
27 de abril de 2020

Dr. Rose Whelan!
You've done some very interesting research!
I will not discuss interesting data on biochemistry, but table 3 is clear to everyone.it shows that the group with L-methionine was slightly better than the group with DL-methionine. It can be assumed that D-methionine from DL methionine still does not completely turn into L-methionine.
In passing, I note that you correctly consider the conversion rate of the hydroxy analog of methionine to methionine to be 0.65.

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