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).

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.