Celemin Sarmiento A, Bradford B J, Mamedova L K, Zhou G, Estes K A, Swartz T H
Department of Dairy and Food Science, South Dakota State University, Brookings, SD 57007.
Department of Animal Science, Michigan State University, East Lansing, MI 48824.
JDS Commun. 2025 Mar 12;6(3):333-338. doi: 10.3168/jdsc.2024-0697. eCollection 2025 May.
Choline is a methyl donor that may influence DNA methylation, gene expression, and cellular processes. Past studies have found an increase in milk yield when periparturient dairy cows were supplemented with dietary rumen-protected choline (RPC); however, the mechanism behind this response is unknown. Therefore, the objective of this study was to assess the effects of dietary RPC supplementation on mammary genome-wide DNA methylation. Parous Holstein cows were blocked by calving month and then randomly assigned within block to receive either 30 g/d of RPC (13.6 g/d of choline ions; CHOL30, n = 21) or no RPC (CON, n = 19) as a top-dress, starting 24 d before expected calving until 21 d postpartum. Mammary tissue was collected at d 17 postpartum and DNA was isolated. A subset of samples (n = 6 per group) was randomly selected and submitted for whole-genome bisulfite sequencing. Differentially methylated cytosines (DMC), regions, and genes (DMG) were determined using the 'genomation' R package. The cut-off values were set at false discovery rate-adjusted -value (q-value) <0.05 and absolute methylation difference >10%. There were 456 DMC by RPC; 241 were hypermethylated and 215 were hypomethylated in mammary tissue from CHOL30 cows as compared with CON. These DMC mapped to 109 genes, of which 51 genes had at least one hypomethylated CpG (cytosine-phosphate-guanine) site and 58 genes had at least one hypermethylated CpG site. The REACTOME pathway analysis did not identify any significantly enriched pathways with 3 or more DMG. Several genes relevant to our research question containing at least one hypermethylated CpG site included DNA polymerase α 1, catalytic subunit (), DNA primase subunit 2 (), thrombospondin 2 (), and sarcosine dehydrogenase (). Similarly, a few genes relevant to our research question containing at least one hypomethylated CpG site included methylmalonyl-CoA mutase (), isocitrate dehydrogenase [NAD(+)] 3 catalytic subunit α (), and pannexin 1 (). Our data suggest that dietary RPC supplementation alters DNA methylation in the mammary gland, potentially enhancing cellular proliferation ( and ) and metabolism ( and ). Nevertheless, the vast majority (95.6%) of the DMC were found in the intergenic regions, and very rarely found on the more critical regulatory elements such as the promoter regions, suggesting that dietary RPC supplementation may not exert a concerted hypomethylation or hypermethylation of genes in mammary tissue. Future studies are needed to determine if methylation status of these genes affects cellular phenotype and function in the mammary gland as a potential mechanism behind the milk production responses due to dietary RPC supplementation.
胆碱是一种甲基供体，可能影响DNA甲基化、基因表达和细胞过程。过去的研究发现，围产期奶牛日粮中添加瘤胃保护性胆碱（RPC）可提高产奶量；然而，这种反应背后的机制尚不清楚。因此，本研究的目的是评估日粮添加RPC对乳腺全基因组DNA甲基化的影响。经产荷斯坦奶牛按产犊月份进行分组，然后在组内随机分配，从预计产犊前24天开始至产后21天，一组接受30 g/d的RPC（13.6 g/d的胆碱离子；CHOL30，n = 21）作为顶层添加物，另一组不添加RPC（CON，n = 19）。在产后第17天采集乳腺组织并分离DNA。随机选择一部分样本（每组n = 6）进行全基因组亚硫酸氢盐测序。使用“genomation”R包确定差异甲基化的胞嘧啶（DMC）、区域和基因（DMG）。截断值设定为错误发现率调整后的P值（q值）<0.05且绝对甲基化差异>10%。RPC处理导致456个DMC；与CON组相比，CHOL30组奶牛乳腺组织中有241个发生高甲基化，215个发生低甲基化。这些DMC映射到109个基因，其中51个基因至少有一个低甲基化的CpG（胞嘧啶-磷酸-鸟嘌呤）位点，58个基因至少有一个高甲基化的CpG位点。REACTOME通路分析未发现任何有3个或更多DMG的显著富集通路。几个与我们的研究问题相关且至少有一个高甲基化CpG位点的基因包括DNA聚合酶α1催化亚基（）、DNA引物酶亚基2（）、血小板反应蛋白2（）和肌氨酸脱氢酶（）。同样，一些与我们的研究问题相关且至少有一个低甲基化CpG位点的基因包括甲基丙二酰辅酶A变位酶（）、异柠檬酸脱氢酶[NAD(+)]3催化亚基α（）和泛连接蛋白1（）。我们的数据表明，日粮添加RPC会改变乳腺中的DNA甲基化，可能增强细胞增殖（和）和代谢（和）。然而，绝大多数（95.6%）的DMC位于基因间区域，在更关键的调控元件如启动子区域很少发现，这表明日粮添加RPC可能不会对乳腺组织中的基因进行一致的低甲基化或高甲基化。未来需要进行研究，以确定这些基因的甲基化状态是否会影响乳腺中的细胞表型和功能，这可能是日粮添加RPC导致产奶量变化的潜在机制。
