Broiler trial in the Netherlands confirms 2018 EFSA scientific opinion on methionine sources while validating the experimental approach

Published: September 6, 2018

By: Victor Naranjo. Andreas Lemme

Conclusion

The present study conducted at Schothorst Feed Research resulted in an average bioavailability of MHA-FA and diluted DLM65 of 65% and 61%, respectively compared to MetAMINO® considering the overall growth and carcass parameters.
These results demonstrate that the relative bioavailability of MHA-FA is significantly lower than its active content of 88% and consistent with the 2018 EFSA scientific opinion of 66% on product basis (75% on equimolar basis).
The average RBA of 61% for DLM65 was close to the expected value of 65% (because of dilution to 65% purity) and thus confirming the appropriateness of using simultaneous dose-response approach to determine the RBA of nutrient sources.

Introduction and objective

Commercial poultry diets are routinely supplemented with methionine (Met) sources to precisely meet their Met+Cys specifications. Around two thirds of the global market is covered with DL-methionine (DLM) and one third with DL-methionine hydroxy analogue products which are mainly in the liquid free acid form (MHA-FA). Despite the fact that DLM is the most used Met source globally, the question about the nutritive value being characterised by the relative biological availability (RBA) of liquid MHA-FA – or in other words, the replacement ratio of these products in feed –is always a critical factor for cost-effective purchasing and optimization of feed costs.

Various literature reviews have reported a RBA of about 65% for liquid MHA-FA compared to DLM in broilers on product-to-product basis (Jansman et al., 2003; Sauer et al., 2008; Lemme et al., 2012). More recently, the European Food Safety Authority (EFSA, 2018) released a scientific opinion on liquid MHA-FA and its calcium salt, confirming a RBA of 75% on equimolar basis which is equivalent to 66% on product-to-product basis for liquid MHA-FA. While this 65% RBA of MHA products compared to DLM has been challenged and confirmed in many field and university trials (Hoehler et al., 2005; Lemme et al., 2012; Sangali et al. 2014), the experimental setup to determine the RBA has been matter of recent discussion (Vazquez-Anon et al, 2006). In order to provide further evidence that the methodology of a simultaneous dose-response feeding trial is a valid approach and also the methodology of choice, a product can be included from which the RBA is known a priori. Diluting DLM with e.g. starch to a purity of 65% (65 parts DLM, 35 parts starch - DLM65) would be such a product in which the RBA would be assumed to be close to 65% because of its respective dilution. Although several experiments with this approach in poultry have already been published (Lemme et al., 2002; Hoehler et al., 2005, Elwert et al., 2008), data with modern broiler genetics are needed. Therefore, a study was conducted at Schothorst Feed Research, the Netherlands to determine the RBA of MHA-FA relative to MetAMINO® (DLM) for common performance parameters and to validate the suitability of the multi-exponential regression analysis for estimating the RBA by including 65%-diluted DLM (DLM65) as an internal standard.

Material and Methods

A total of 1,920 d-old male Ross 308 were allocated to 96 floor pens of 20 broilers each. Each pen (2m²) had one feeder, line of drinking nipples and wood shavings as bedding material. Ambient temperature was gradually decreased from 34°C at arrival of chicks to 20°C at 35 days of age. Light was continuously on for the first 24 hours to give birds the opportunity to readily find feed and water. After that, the light schedule was 22L:2D during one day, and then changed to 8L:4D:10L:2D during the remaining experimental period, complying the EU legislation of a minimum of six hours of darkness from the second day onwards. Diets were formulated to meet or exceed the ideal AA profile of AMINOChick® 2.0, except for digestible Met and Met+Cys. Main ingredients were analyzed by AMINONIR® and results were used for diet formulation. Broilers were fed starter (d 0 to 11), grower (d 11 to 28), and finisher diets (d 28 to 35). Each phase comprised16 treatments including a basal diet deficient in dig. Met+Cys without supplemental Met, and 5 increasing levels of either MetAMINO®, MHA-FA and DLM65. Starch was used to dilute MetAMINO® to a Met content of 65% in DLM65. Met sources were added in all phases on weight/weight basis at 0.40, 0.80, 1.20, 2.10 and 3.00 g/kg (Table 1). Starter feeds were produced in crumbles while grower and finisher diets (3.0 mm) were steam pelleted. Salinomycin was added to the starter and grower feeds as anticoccidial agent while no coccidiostat was added to the finisher diets. Feed and water were supplied ad libitum throughout the experimental period.

Broiler trial in the Netherlands confirms 2018 EFSA scientific opinion on methionine sources while validating the experimental approach - Image 1

The dig. Met+Cys levels of the starter, grower, and finisher basal diets were 0.62, 0.54 and 0.51%, respectively (Table 2). Growth performance parameters were evaluated for each phase. On day 35, two birds per pen which were close to the average BW of the pen, were selected for carcass evaluation and individually weighed. The carcasses were chilled for 4 hours before cutting up. Carcass weight (kg) was defined as plucked, bled and eviscerated carcasses without head, neck and feet. Carcass yield (CY) was expressed as % of BW at day 35. Breast meat weight included both musculus pectoralis major and minor (without skin). Breast meat yield was expressed as % of BW at day 35 (BMY-BW) and also as % of the carcass weight (BMY-CW). Growth performance and carcass data were subjected to multi-exponential regression analysis using the nonlinear-regression procedure described by Littell et al. (1997).

Results

Analyzed values of the experimental diets from all phases were in close agreement with the calculated values (Table 2). Recovery rates ranged from 97 to 109% for total Met+Cys and Lys. The high accuracy in the analytical results was accomplished by formulating diets based on the analyzed values of the main ingredients (AMINONIR®) and manufacturing feeds in a specialized research feed mill with confirmed dosing accuracy and mixing quality. Therefore, the calculated levels of Met product additions were used for RBA determinations.

Increasing levels of either MetAMINO^®, MHA-FA and DLM65 improved growth performance (Table 3) and carcass yields (Table 4) compared to the basal diet. Relative to the basal diet, the highest Met addition (3.00 g/kg) improved BWG by 70, 67, and 70% and reduced FCR by 22, 22, and 23% for MetAMINO®, MHA-FA, and DLM65 respectively. Similarly, CY was improved by 13, 11 and 13%, BMY-BW by 62, 60 and 63% and BMY-CY by 43, 43 and 45% for MetAMINO®, MHA-FA, and DLM65, respectively. These relative responses confirm that the main pre-requisites for an appropriate RBA determination were successfully met such as:1) a common starting point (basal) was clearly deficient in dietary Met+Cys, 2) three or more supplementation levels of each test product that had data points in the sensitive (curve-linear) range of the response curve and 3) all products showed a common plateau which was confirmed by the non-significant differences among Met sources for any of the performance parameters at the highest Met addition. The latter point also indicates that incorrect interpretations of the bioavailability of Met sources can occur if comparison of Met sources are done only at the asymptotic part of the curve. At this level no greater improvements to Met additions are expected (law of diminishing returns) and could lead to incorrect and costly conclusions. Using this approach, data from this trial will suggest that a diluted DLM to 65% purity is equally effective as MetAMINO® with 99% purity and certainly, this is not correct. To overcome this limitation, dose-response trials with subsequent multi-exponential regression analysis have been widely used to determine the bioavailability of not only Met sources but for different nutrient sources (eg. P, Se, Fe, Lys).

Relative bioavailability (RBA)

Responses to increasing levels of MetAMINO^®, MHA-FA and DLM65 are shown in Figures 1 and 2 for growth performance and carcass yield parameters, respectively. Multi-exponential regression analysis revealed that MHA-FA was 58, 66 and 62% as efficacious as MetAMINO® for BWG, FCR and EPEF (European production efficiency factor), respectively. Similarly, the estimated RBA of DLM65 were 56, 54 and 59 for BWG, FCR and EPEF, respectively. Based on carcass traits, MHA-FA was estimated to be 63, 65 and 73% as efficacious as MetAMINO® for CY, BMY-BW and BMY-CW. Correspondingly, the estimated RBA of DLM65 were 58, 65 and 73% for the previous parameters.

As summarized in Table 5, all RBA estimates were significantly (P < 0.05) lower than 88%, which is the active substance content in MHA-FA. Based on all the evaluated parameters, the average RBA of MHA-FA and DLM65 compared to MetAMINO® was 65% and 61% respectively. Moreover, the corresponding RBA estimates for liquid MHA-FA and DLM65 for the performance criteria did not differ suggesting similar nutritional value. The average RBA of 61% for DLM65 was close to the expected value of 65% (because of dilution to 65% purity) confirming the appropriateness of using simultaneous dose-response approach to determine the RBA of nutrient sources. Moreover, the results of this study are in line with earlier findings by Lemme et al. (2002), Hoehler et al. (2005), and Elwert et al. (2008) and therefore confirm the suitability of the experimental approach as well as the determined RBA of liquid MHA-FA.

Conclusions

These results demonstrate that the RBA of MHA-FA is significantly lower than its active content of 88% and close to 65% in agreement with previous publications. The inclusion of diluted DLM65 confirmed that the multi-exponential regression analysis is a valid approach to estimate the bioavailability of Met sources and resulted in a similar RBA (61 vs. 65%) as liquid MHA-FA.

Results from this experiments were presented at:

Naranjo, V., Lemme, A., Agostini, P., and P. van der Aar. 2018. Bioavailability of dl-methionine hydroxy analogue relative to dl-methionine and validation of the multi exponential regression approach by using 65%-diluted dl-methionine in broilers. Poult. Sci. 97(Suppl. 1):242(Abstr.)

References

European Food Safety Authority (EFSA), 2018: Safety and efficacy of hydroxy analogue of methionine and its calcium salt (ADRY+®) for all animal species, EFSA Journal 16(3): 5198 .

Jansman, A.J.M., C.A. Kan, and J. Wiegenga, 2003: Comparison of the biological efficacy of DL-methionine and hydroxy-4-methylthiobutanoic acid (HMB) in pigs and poultry, CVB documentation report nr. 29, Centraal Veevoederbureau (CVB, Lelystad, The Netherlands.

Hoehler, D., A. Lemme, K. Roberson and K. Turner, 2005a: Impact of methionine sources on performance in turkeys, Journal of Applied Poultry Research 14: 296-305.

Hoehler, D., A. Lemme, S.K. Jensen, and S.L. Vieira, 2005b: Relative effectiveness of methionine sources in diets for broiler chickens, Journal of Applied Poultry Research 14: 679-693.

Lemme, A., D. Hoehler, J.J. Brennan, and P.F. Mannion, 2002: Relative effectiveness of methionine hydroxy analog compared to DL-methionine in broiler chickens, Poultry Science 81: 838-845.

Lemme, A.. A. Helmbrecht, and S. Mack, 2012: Commercial methionine sources in poultry, AMINONews® Review: pp 39.

Littell, R.C., P.R. Henry, A.J. Lewis and C.B. Ammermann, 1997: Estimation of relative bioavailability of nutrients using SAS procedures, Journal of Animal Science 75: 2672-2683.

Sauer, N., K. Emrich, H.-P. Piepho, A. Lemme, M.S. Redshaw, and R. Mosenthin, 2008: Meta-analysis of the relative efficiency of methionine-hydroxy-analogue-free-acid compared with DL-methionine in broilers using nonlinear mixed models, Poultry Science 87: 2023-2031.

Sangali, C.P., Giusti Bruno L.D., Vianna Nunes, R., Oliveira Neto, A.R., Pozza, P.C., Oliveira, T.M., Frank, R. and Schone, R.A., 2014. Bioavailability of different methionine sources for growing broilers. Revista Brasileira de Zootecnia, 43: 140–145.

Vazquez-Anon, M., D. Kratzer, R. Gonzalez-Esquerra, I.G. Yi, and C.D. Knight, 2006: A multiple regression model approach to contrast the performance of 2-hydroxy-4-methylthio butanoic acid and DL-methionine supplementation tested in broiler experiments and reported in the literature, Poultry Science 85: 693-705.

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Comment

Victor Naranjo Haro

Evonik Animal Nutrition

8 de octubre de 2018

Dear Ms. Peris, Thank you for your comments. The first objective of this trial was to determine the relative bioavailability of Met sources in broilers. As stated by Jansman et al., (2003) proper studies are those that include a basal diet clearly deficient in Met, and at least 3 levels of supplementation of the test products. As in any dose-response trial, it is crucial that enough data points are included to properly describe the different sections of the response curve. Thus, it is important that the basal diet (starting point) is clearly deficient in the nutrient of interest. In this study, the basal diet was clearly deficient in Met confirmed by the significant reduction in growth performance. The 5 increasing levels of each Met source improved growth performance following - as expected - the law of diminishing returns and with data points well distributed along the complete response curve (deficiency and above plateau). Therefore, all criteria for a proper bioavailability study were met. Accurate “bioavailability values” cannot be determined or extrapolated by only comparing nutrient sources above the requirement. Using this approach, data from this trial will suggest that at the highest Met inclusion level a diluted DL-Met to 65% purity (DLM65) is equally effective as DL-Met with 99% purity! Certainly, this is not correct. As these points are in the plateau section of the response curve (requirement already met), no further responses are expected above this level (law of diminishing returns) which does not allow for any proper comparison of nutrient sources. Therefore, to properly determine the bioavailability of nutrient sources, trials need to include a basal diet clearly deficient in the nutrient of study with enough data points representing the different sections of the response curve.

The second objective of this trial was to validate the mathematical suitability of the multi-exponential regression analysis for estimating the relative bioavailability. For this, we used diluted DL-Met to a purity of 65% (DLM65) from which its bioavailability is known a priori (close to 65%). As DLM65 was included, equimolar comparison would have not been possible in this experiment (same inclusion as DLM). However, proper experiments designed either on equimolar or product basis led to the same conclusion (Jansman et al., 2003; Hoehler et al., 2005, EFSA, 2018). For example, based on an extensive literature study Jansman et al. 2003 reported an average bioefficacy of liquid HMTBA of 77% on equimolar basis compared to DL-Met in broilers. This translates to a bioefficacy of 68% on product basis compared with DL-Met (0.77 * 88). Similarly, the European Food Safety Authority (EFSA, 2018, Appendix A) released a scientific opinion on liquid HMTBA and its calcium salt, confirming a bioavailability of 75% on equimolar basis which is equivalent to 66% (0.75 * 88) on product-to-product basis for liquid MHA-FA. These previous results are in agreement with those obtained in the current study which resulted in an average bioavailability of HMTBA (MHA-FA) and DLM65 of 65 and 61%, respectively. Therefore, proper studies conducted either on equimolar or product basis will lead to the same conclusion.

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Silvia Peris

Novus

28 de septiembre de 2018

Dears Mr. Naranjo and Mr. Lemme, as you well mention in your study, the diets tested are deficient on Methionine (Met) and Methionine+Cystine, therefore not representative of the commercial conditions. It has already been demonstrated that along with the most rapid increase in plasma there is a more rapid feed intake with DL-Met over HMTBa (referred as MHA-FA in your study) at low levels of Met supplementation, such as the deficient diets you are showing in this design/model. In contrast, as level of Met supplementation increases, feed intake for HMTBa supplemented diets reaches that of DL-Met diets when both are supplemented at requirements and HMTBa even overtakes DL-Met, as described in the work of Gonzalez-Esquerra et al. (2007). In a similar way, Knight et al. (2006) using paired-feeding studies demonstrated that differences in performance between HMTBa and DL-Met were due to differences in intake and not inefficiency of conversion of HMTBa to L-Met. These studies have been done using 100% equivalence between the 2 on an equimolar basis. Equimolar means equal supplementation of molecules (1 gram of HMTBa results in 1 gram of DL-Met. MetAMINO equals 1.125 grams of Alimet for equal supplementation, as MetAMINO has 99% DL-Met and Alimet 88% HMTBa). Any feeding study that compares doses on an equal product basis instead of an equimolar basis is also statistically incorrect. That is because the initial assumption in an experiment is that two materials are EQUAL and the objective of the experiment is to determine if a statistically significant difference can be detected. If you start an experiment with DIFFERENT levels of supplementation for the two molecules being compared, lack of a significant difference becomes a conclusion that they are different, a major deviation from acceptable statistical experimental design and analysis. BOTTOMLINE: you can never prove that two things are the SAME, only that they are DIFFERENT or that you cannot prove that they are DIFFERENT, i.e. no significant difference. In your study, taking the data from Table 3, granting 100% efficacy of conversion to L-Met to both active substances (DL-Met and HMTBa), and plotting body weight, feed conversion and feed intake against the active substance, it again demonstrates and confirms what has been described in the works of Knight et al. (2006) and Gonzalez-Esquerra et al. (2007). It is a good exercise to do, as you clearly see the link between active substance intake and response in performance, expressed here as body weight and feed conversion, so nothing to do with conversion efficiency. Since HMTBa and DL-Met are different compounds, therefore showing differences in absorption and metabolism as it has widely been described in the scientific literature, there is not a unique bioequivalence factor, but it is mainly related to feed ingestion (thus active substance ingested), as explained above. Finally, I would just like to add that looking at the data from Agristats in US, mentioning for more than 700 billion broilers per year (representing more than 95% of broilers produced in that area), it is impossible that when splitting the data according to the methionine source used, animals supplemented with HMTBa are performing similar or even better than those supplemented with DL-Met while they are giving full credit to HMTBa conversion to L-Met in the animal (100% on equimolar basis meaning 88% for Alimet –liquid form- or 84% for MHA –powder form). References: Gonzalez-Esquerra R., Vázquez-Añón M., Hampton T., York T., Feine S., Wuelling C., and Knight C.D. 2007. Evidence of a different dose response in turkeys when fed 2-hydroxy-4(methylthio) butanoic acid versus DL-methionine. Poult. Sci., 86:517-524. Knight C.D., Dibner J.J., Gonzalez-Esquerra R., and Vázquez-Añón M. 2006. Differences in broiler growth rates when methionine (Met) sources are fed in deficiency or excess are equalized when feed consumption is equalized. Poult. Sci., 85 (Suppl. 1):P191 (Abstract).

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Broiler trial in the Netherlands confirms 2018 EFSA scientific opinion on methionine sources while validating the experimental approach

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