Methionine analogs in ruminants: Data-driven insights

Isopropyl ester of the hydroxy analog of methionine (HMBi) stands out as the only reliable way to fulfill Met requirements in ruminant diets to encapsulated forms due to their stability against handling and aggressive thermo-physical processing.

Thermo-physical processing, such as pelleting and extrusion technologies, is still the most popular compound feed industry method. Due to high operating temperatures and tremendous pressure on the matrix during the processes, surface coating cannot provide enough coating capacity for encapsulated or coated methionine (Met) sources. Isopropyl ester of the hydroxy analog of methionine (HMBi) stands out as the only reliable way to fulfill Met requirements in ruminant diets to encapsulated forms due to their stability against handling and aggressive thermo-physical processing. To achieve expected performance from HMBi in sustainable and profitable ruminant nutrition, it is vital to center our attention on the journey of Met analogs from feed bunk to ultimate site in the body.

FATE OF HMBI IN THE RUMEN AND BEYOND
Methionine additive used in the animal industry (Table 1) can be categorized into three different sources as DL-Met, DL-Met sodium salt and Met analog-based. Currently, there are 5 major DL-Met-based products, one DL-Met sodium salt-based and three Met analog-based categories available in the market. The Met analog-based additives are the hydroxy analog of Met (HMBA), the calcium salt of the Met hydroxy analog (HMBA-Ca) and HMBi.

The HMBi escapes microbial degradation, unlike free methionine and 2-hydroxy-4-methylthiobutanoate (HMBA), due to rapid and effective absorption through the rumen wall (Graulet et al., 2004, 2005) and omasum (McCollum et al., 2000). In brief, absorbed HMBi is converted to HMBA and subsequently Met in peripheral tissues and transported to the mammary gland to contribute to milk protein yield (St-Pierre and Sylvester, 2005). Although omasum is accepted as an absorption site of HMBA, Noftsger et al. (2005) recovered only 2.3 percent of the ingested HMBi as HMBA in omasal digesta from rumen cannulated cows, while having a positive response in milk protein concentration of the same cows following HMBi supplementation during the same study. This data showed HMBi absorption occurred before the omasal site. Dietary HMBi intake in ruminants results in an increase of seven times higher plasma Met within two hours (Kihal et al., 2021). However, because of various testing techniques and limited data on ruminal escape, intestinal absorption and Met conversion site or rate, there is still no agreement on the final Met bioavailability value of HMBi (NRC, 2021), although there is sufficient scientific evidence to understand that it is a reasonable source of metabolizable Met.

At the rumen
Breves et al. (2010) observed the fate of HMBi via the in vitro using chamber system, which keeps tissues alive in buffer to monitor absorption kinetics. To better understand rumen wall absorption, they separated rumen side and serosal side (outside of the organ) and treated them separately with HMBi. They recovered a minimal amount of total HMBi from serosal buffer while obtaining greater HMBi from the rumen-side buffer. Evidence suggests that HMBi cannot pass the rumen wall intact, is hydrolyzed to HMB at tissue surface and carried over out of epithelium via passive diffusion or possibly at low rates of MCT-1 transport. Remarkably, the concentration gradient between the mucosal and serosal sides of rumen tissue serves as the driving force in both scenarios. This implies that a higher HMBi level in KESSENT^®MF (Kemin Europa, Belgium) may accelerate the rate of rumen absorption compared to any counterparts with lower levels of HMBi, even though ultimately available MP is estimated to be identical in the widely used nutritional models. On the other hand, there was also no Met available from HMBi in the artificial tissue system without any esterase enzymes. Earlier in vitro reports showed that HMBi remained intact up to 60% of total amount against microbial degradation (Robert et al., 2000). Although EU Commission (2003) concluded HMBi hydrolysis is a result of microbial degradation (non-specific microbial esterase activity), the incubation loss of HMBi (Breves et al., 2010) in both rumen tissue or Parafilm membrane (unless any enzyme or microflora) suggests that ruminal behavior of HMBi remains unclear.

Both recent in vitro and in vivo research showed that dietary HMBi had significant effects on rumen microbial environment and various final products of fermentation processes as well as the well-known systemic effects (Li et al., 2023, 2022; Qin et al., 2022). Evidence shows that “the remaining part of the HMBi in rumen” deserves more attention to better understand the mode of action of the molecule. For example, the flora can release isopropanol, which can be transformed into acetone by the enzyme alcohol dehydrogenase, and it is essential to note that this conversion is not permanent, as some of the generated acetone can be converted back into the initial form, the secondary alcohol (European Commission, 2003). Based on the previous data, Kemin is working on several product innovation projects to obtain scientific data and improve knowledge of scientific gaps in mode of action of the molecules described by different researchers in previous reports.

Beyond the rumen
The biological action of HMBi has still been discussed by researchers with various cow-side or in vitro approaches unless there is any current consensus. European Commission (2003) observed in vivo activity of labeled both [carbonyl-14C]-HMBi and [isopropyl-14C]-HMBi (per oral) in cows.

However, this is only a “tracer study,” and incorporating the labeled compound in the tissues does not indicate direct HMB or HMBi presence (European Commission, 2003). Therefore, this data should support other bioavailability data to understand biological fate of the HMBi.

A retrospective review of HMBi bioavailability trials was compiled by recent NRC (2021) committee in a separate section. In dairy cows, up to 50% of ingested dose was reported to flow from the rumen in earlier trials (Robert et al., 2000). This data was validated with “Met availability in the circulated blood of HMBi supplemented cows” perspective via nonlinear models (area under curve; AUC) (Graulet et al., 2005; Kihal et al., 2021), which is the accepted golden standard for nutritional models. The second study aimed to shed light on the current bioavailability of Kessent MF, sponsored by Kemin as a part of product innovation efforts.

In dairy cattle, Lapierre et al. (2011) infused labeled HMBA [1-13C] and L[methyl-2H3] Met to the jugular vein (neck vein) to determine fate of HMBA via continuous samples from arterial portal, hepatic portal (central liver vein), and mammary veins. Evidence shows that the infused HMBA provided directly 15% of the Met required for milk protein secretion, with 0.2 mmol/h synthesized within the mammary gland. Among this 15%, about 33% originates from the metabolism that occurs in splanchnic tissues, 15% takes place in the mammary gland, and the remaining 52% comes from conversions happening in peripheral tissues. The remaining 85% of HMBA is indirectly metabolized, wherein Met synthesized from HMBA within tissues is utilized for intracellular protein synthesis. This process allows the Met released from protein breakdown to be exported and used by the mammary gland. In vitro, (McCollum et al., 2000) and ovine (Lobley et al., 2006) research showed that L-Met synthesis from HMBA occurs in many tissues of ruminants. It is similar to both D- and L- isomers of HMBA. L-HMBA oxidase exists predominantly in the liver and kidney; however, dehydrogenase of D-HMBA was available in most of the tissue (NRC, 2021). This data can be considered similar in terms of post-absorptive HMBi behaviours due to conversion to HMBA.

The cow liver removed approximately 38% of the infused HMBA. However, this removed HMBA was not converted into Met in the liver, as there was a decrease in the net release of Met from the liver (Lapierre et al., 2011). The data mentioned above suggests that relying solely on plasma Met concentration may not be an appropriate method for estimating the availability of HMBi and its potential to supply Met (Ardalan et al., 2021; NRC, 2021), although the linear/nonlinear plasma responses of Met to HMBi supplementation have been considered the best approach.

CONCLUSION
Altogether, the cumulative scientific data based on isotope enrichments (Lapierre et al., 2011), nonlinear area under curve responses (Kihal et al., 2021), linear plasma-free AA responses (Ardalan et al., 2021), in vitro tissue batches (Breves et al., 2010), indirect milk responses (European Commission, 2003), and ruminal microbiota assays (Li et al., 2023) indicate that HMBi is a reasonable additive to meet Met requirements of ruminants and to balance individual dietary amino acids for sustainable production. However, science-driven sophisticated approaches and specific expertise are needed to enhance the maximum performance of the Met analogs in ruminant nutrition due to complexity of biological fate of the HMBi.

References
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Dr. Eyüp Eren Gültepe, DVM
Dr. Gültepe is a veterinarian and animal nutritionist, holding a PhD in the field. He earned both his DVM and PhD degrees from Afyon Kocatepe University, Türkiye, and expanded his expertise as a postdoctoral researcher at Kansas State University, KS, USA. With over a decade of experience as a dedicated university researcher, he now leads innovation projects in ruminant platform at Kemin Europa R&D team. Dr. Gültepe has an academic portfolio more than 30 peer-reviewed publications, including articles, reviews, and book chapters. He has also more than 40 oral/poster presentations and keynote speaks across more than 10 different countries around the world.

Dr. Diego Martinez del Olmo, DVM
Dr. Martínez del Olmo holds a degree in Veterinary and Research Sufficiency and Advanced Studies from León University. He received his PhD from Complutense University (Madrid) and a master’s degree in Dairy Farm Management from the Autonomous University of Barcelona, and in Strategic Management of Feed Companies from the Open University of Catalonia. He has an MBA from Isabel I University (Burgos). He started his career as a clinical veterinarian and subsequently held various positions in animal feed companies. He is currently the business manager for ruminants at Kemin EMENA. Diego is co-inventor of two European patents and has two published books and several peer-reviewed publications.