Abbreviations:
Met = Methionine
SAM = S-adenosylmethionine
AA = Amino acid
PtdChol = phosphatidylcholine
VLDL = very-low-density lipoprotein
5-mTHF = 5-methyltetrahydrofolate
HCY = homocysteine
CDP-choline = cytidine diphosphate-choline
PEMT = phosphatidylethanolamine N-methyltransferase
The transition period -defined as three weeks before calving and three weeks post-calving- is a critical phase of the cow’s life. Proper management of nutrition and health is vitally important during this period. Cows post-calving are susceptible to a plethora of disorders/diseases due to low nutrient intake, immunosuppression, and high metabolic stress. The nutrient requirements for milk production, especially for our modern cows, are exceptionally high. Cows during the early stage of lactation cannot consume enough energy to cover their requirements. This condition is referred to as “negative energy balance,” and as most lactating mammals, cows rely on body-reserve mobilization (fat, protein, ..) to meet this deficit. However, excessive fat mobilization triggered by low intake or imbalanced/deficient rations can cause fatty liver and jeopardize cows’ health and productivity. Secondary to energy imbalance, several imbalances in nutrients and metabolically active compounds may also occur during lactation. These include amino acids, vitamins, minerals, and compounds with labile methyl groups such as choline, betaine, and Met. Indeed, methyl group metabolism has received considerable attention from dairy scientists due to its essential role in metabolic health and productivity.
Unfortunately, the dietary supply of methyl donors during the onset of lactation is limited due to low feed intake and extensive microbial degradation of the methyl donors in the rumen. Whereas, the demand for such compounds is very high to support milk production (milk choline and milk protein), liver health, immune function, and mitigate oxidative stress.
When the supply of labile methyl groups is low (i.e. early lactation), methyl groups are synthesized by the interactions of two metabolic pathways: the folate (or B9) and the Met cycles (Figure 1: methylneogeneis is highlighted in blue, folate cycle in yellow, and Met cycle in green). Within the folate cycle, one-carbon sources such as serine and folate serve as a substrate for the formation of 5-mTHF. Met synthase within the Met cycle will utilize 5-mTHF to regenerate Met from HCY. In turn, Met is used to generate SAM, which is recognized as the universal methyl donor for various metabolic processes, including AA homeostasis and PtdChol synthesis. Other processes include supporting redox defence and DNA methylation (Figure 1, the transsulfuration pathway is highlighted in pink).
Figure 1. Folate cycle, Met cycle, and transsulfuration pathway (modified from McFadden et al. 2020). Methylneogenesis is highlighted in blue, folate pathway in yellow, Met pathway in green, and transsulfuration in pink. Compounds containing methyl groups are highlighted in blue and B-vitamins in red. An “S” denotes sulphur-containing compounds. DHF = dihydrofolate, SHMT = serine hydroxymethyltransferase, 5-mTHF = 5-methyltetrahydrofolate; MTHFR = methylenetetrahydrofolate reductase; MTR = methionine synthase, PEMT = phosphatidylethanolamine N-methyltransferase, PtdEth = phosphatidylethanolamine, PtdEth = phosphatidylethanolamine; SAH = S-adenosylhomocysteine; SAM = S-adenosylmethionine (other types of methyltransferases may utilize SAM; only PEMT is shown for simplicity). MATI/III = methionine adenosyltransferase I/III (liver-specific isoenzymes), HCY = homocysteine.
B-vitamins, such as folate, B6, and B12 (Figure 1, highlighted in red), play an essential role in methylneogenesis. For example, B12 plays a critical role in “coupling” the folate and Met pathways to form Met and SAM. In this process, the methyl group is transferred from 5-mTHF (folate cycle) to the cobalt group of the B12 vitamin and then transferred to the sulphur group of HCY in the Met cycle. Consequently, B-vitamins and HCY are the main limiting factors in the synthesis of labile methyl groups. It is important to note here that homocysteinyl moiety (i.e. HCY) cannot be synthesized in mammals and is derived from Met. Therefore, it is suggested that there is a “minimal absolute requirement” for Met to ensure the cycling of HCY in the methylneogenesis (McFadden et al., 2020).
Methyl groups generated via methylneogenesis (SAM) are transferred to over a hundred different pathways, one of which is the PEMT pathway. This pathway generates PtdChol, which is utilized in the liver to create VLDL and export lipid to various tissues for utilization. This pathway works in unison with the CDP-choline pathway to support phospholipid synthesis in the liver. The inability of the liver to export lipid can lead to lipid accumulation and fatty liver. Therefore, current research is focused on enhancing the PEMT and CDP-choline pathway activation.
As mentioned, methyl groups can be synthesized via the folate cycle when dietary supply is low. However, methyl groups necessary for SAM can be donated by exogenous sources (methyl donors) such as supplemental choline, betaine and Met (Figure 2, circled in orange). However, due to their extensive degradation in the rumen, it is suggested that the folate cycle remains the primary source for endogenous methyl groups for ruminants when these supplements are fed in an unprotected form (McFadden et al., 2020).
Figure 2. A simplification of Figure 1 showing the possible contributions of exogenous methyl donors to methylneogenesis (circled in orange). BAD = betaine aldehyde dehydrogenase; BHMT = betaine-homocysteine methyltransferase, CDH = choline dehydrogenase. (Additional abbreviations explained in Figure 1)
Supplementing rations with rumen-protected (RP) methyl donors such as RP-choline is an approach to enhance dairy cow liver health and milk production. In a recent meta-analysis, RP-choline feeding showed a consistent effect on metabolic health and performance during the onset of lactation (Arshad et al., 2020). Approximately half of the increase in milk production observed with RP-choline feeding is believed to be due to direct improvements in dry matter intake. The remaining half is likely due to the ability of choline to act as a methyl donor or enhance mammary epithelial cell proliferation. It is important to note that, in addition to being a methyl donor and supporting PEMT pathway activation, supplemental choline can be phosphorylated directly via the CDP-choline pathway to PtdChol. This pathway is energetically more feasible than the PEMT pathway and likely more relevant to liver health in lactating cows (McFadden et al., 2020). However, there is little or no information concerning RP-betaine. Available studies that fed unprotected betaine to cows have attributed its ability to enhance milk production to improved fibre digestibility (i.e. benefited the microbiota).
It was suggested that the status of methyl donors and nutritional practices should be considered when evaluating the impact of any supplementation strategy. For example, B-vitamin supplementation improved Met status -likely due to improved remethylation- but only when the diet was deficient with Met (no RP-Met provided). However, co-supplementing B vitamins with RP-Met increased protein synthesis, milk yield, milk protein, and lactose yield regardless of RP-Met supplementation (Preynat et al., 2009).
High levels of SAM and an increased supply of exogenous methyl groups can promote the activity of the transsulfuration pathway and, consequently, antioxidant production. On the other hand, oxidative conditions have been shown to downregulate Met synthesis and, therefore, the generation of SAM. This proposed mechanism was to divert the sulphur-containing compound HCY towards the transsulfuration pathway and generation of antioxidants (reviewed by McFadden et al., 2020). Hence, Met is not just an essential AA but a compound containing both methyl and sulphur groups and integrally situated on the intersection of methyl and sulphur transfer metabolism.
In conclusion, methyl donor nutrition plays an important role in the health and productivity of lactating cows. However, methyl donor requirements and status are not well defined, nor the specificity of each methyl donor. The ability to determine methyl-groups balance/status and best supplemental strategy can empower nutritionists and veterinarians to revise their guidelines to fit each herd’s requirements. This topic is a rather complex one, and this complexity is exacerbated if you were to consider the methyl-group requirement of the rumen microbiota, in addition to the cow’s requirements. For example, limited studies on unprotected betaine speculate an effect on microbial growth/efficiency, as evident by improvements in digestibility.
Furthermore, B-vitamins are synthesized by the ruminal microbiota to facilitate their various metabolic processes. The washed-out microbes are expected to meet the B-vitamin requirements of the host animal. However, recent findings suggested that B-vitamins production may become a limiting factor to the microbiota and the host animal under certain conditions. More studies are needed to uncover the underlying mechanisms and improve our understanding of methyl donor nutrition.
References:
Arshad, U., M. G. Zenobi, C. R. Staples, and J. E. P. Santos. 2020. Meta-analysis of the effects of supplemental rumen-protected choline during the transition period on performance and health of parous dairy cows. J. Dairy Sci. 103:282-300.
McFadden, J. W., C. L. Girard, S. Tao, Z. Zhou, J. K. Bernard, M. Duplessis, and H. M. White. 2020. Symposium review: One-carbon metabolism and methyl donor nutrition in the dairy cow. J. Dairy Sci. 103:5668-5683.
Preynat, A., H. Lapierre, M. C. Thivierge, M. F. Palin, J. J. Matte, A. Desrochers, and C. L. Girard. 2009. Effects of supplements of folic acid, vitamin B12, and rumen-protected methionine on whole body metabolism of methionine and glucose in lactating dairy cows. J. Dairy Sci. 92:677-689.