Functional roles of folic acid in alleviating dexamethasone-induced fatty liver syndrome in laying hens

Abstract

Fatty liver syndrome (FLS) poses a threat to the poultry industry due to its high occurrence and mortality rate. Folic acid (FA) is a coenzyme crucial for one-carbon metabolism. However, the mechanism by which FA mitigates FLS in laying hens remains elusive. In this study, 60 21-week-old Hy-Line Brown layers were divided into three groups: the Control (Con) group, the dexamethasone (DXM) group, and the DXM + FA group. Results showed that liver index was significantly increased in the DXM group. H&E and oil red O staining showed the accumulation of lipid droplets in the liver was intensified, confirming the successful establishment of an early fatty liver model without inflammation. FA significantly reversed hepatic lipid deposition, and 57 differentially expressed genes affected by FA were identified in the transcriptome analysis. Their transcriptional and translational levels indicate that in the early FLS, insulin-like growth factor 2/phosphatidylinositol- 3-kinase/protein kinase B pathway related to lipid metabolism was activated; folate cycling was inhibited, while endoplasmic reticulum (ER) stress and apoptosis-related protein abundance were elevated. Dietary FA enhanced the folate circulation, reduced lipogenesis and ER stress, and apoptosis-related protein expression, thereby mitigating the lipid metabolism disturbance in FLS. Metabolomics identified 151 differential metabolites involved in early FLS occurrence, 34 of which were reversed by FA. Metabolites were also enriched in pathways related to lipid metabolism and hepatic damage. Collectively, these findings can be concluded that FA can alleviate early FLS by affecting lipogenesis, ER stress and apoptosis, which may be mediated by enhanced folate metabolism.

Keywords

fatty liver syndrome / folic acid / hepato-protective / laying hens / nutritional intervention

References

[1] Zhang, Y. H., Xie, H. L., Yang, Y. W., Wen, J., Liu, R. R., Zhao, G. P., Tan, X. D., Liu, Z., Zheng, Y., & Zhang, J. B. (2023). miR-375 upregulates lipid metabolism and inhibits cell proliferation involved in chicken fatty liver formation and inheritance via targeting recombination signal binding protein for immunoglobulin kappa J region (RBPJ). Poultry Sciense, 102(1), 102218-102229.

[2] Zaefarian, F., Abdollahi, M. R., Cowieson, A., & Ravindran, V. (2019). Avian liver: The forgotten organ. Animals, 9(2), 63-86.

[3] Dupont, J., Métayer-Coustard, S., Ji, B., Ramé C., Gespach, C., Voy, B., & Simon, J. (2012). Characterization of major elements of insulin signaling cascade in chicken adipose tissue: Apparent insulin refractoriness. General and Comparative Endocrinology, 176(1), 86-93.

[4] Yang, X., Li, D., Zhang, M., Feng, Y., Jin, X., Liu, D., Guo, Y., & Hu, Y. (2023). Ginkgo biloba extract alleviates fatty liver hemorrhagic syndrome in laying hens via reshaping gut microbiota. Journal of Animal Science and Biotechnology, 14(1), 97-115.

[5] Rozenboim, I., Mahato, J., Cohen, N. A., & Tirosh, O. (2016). Low protein and high-energy diet: A possible natural cause of fatty liver hemorrhagic syndrome in caged White Leghorn laying hens. Poultry Science, 95(3), 612-621.

[6] Shini, A., Shini, S., & Bryden, W. L. (2019). Fatty liver haemorrhagic syndrome occurrence in laying hens: Impact of production system. Avian Pathology, 48(1), 25-34.

[7] Hamid, H., Zhang, J. Y., Li, W. X., Liu, C., Li, M. L., Zhao, L. H., Ji, C., & Ma, Q. G. (2019). Interactions between the cecal microbiota and non-alcoholic steatohepatitis using laying hens as the model. Poultry Science, 98(6), 2509-2521.

[8] Tsai, M. T., Chen, Y. J., Chen, C. Y., Tsai, M. H., Han, C. L., Chen, Y. J., Mersmann, H. J., & Ding, S. T. (2017). Identification of potential plasma biomarkers for nonalcoholic fatty liver disease by integrating transcriptomics and proteomics in laying hens. Journal of Nutrition, 147(3), 293-303.

[9] Wu, Q., Tang, H., & Wang, H. (2019). The anti-oxidation and mechanism of essential oil of Paederia scandens in the NAFLD model of chicken. Animals, 9(10), 1547-1558.

[10] Zhuang, Y., Xing, C., Cao, H., Zhang, C., Luo, J., Guo, X., & Hu, G. (2019). Insulin resistance and metabonomics analysis of fatty liver haemorrhagic syndrome in laying hens induced by a high-energy low-protein diet. Scientific Reports, 9(1), 10141-10155.

[11] Qiu, K., Zhao, Q., Wang, J., Qi, G. H., Wu, S. G., & Zhang, H. J. (2021). Effects of pyrroloquinoline quinone on lipid metabolism and anti-oxidative capacity in a high-fat-diet metabolic dysfunction-associated fatty liver disease chick model. Journal of Molecular Science, 22(3), 1458-1474.

[12] Friedman, S. L., Neuschwander-Tetri, B. A., Rinella, M., & Sanyal, A. J. (2018). Mechanisms of NAFLD development and therapeutic strategies. Nature Medicine, 24(7), 908-922.

[13] Anstee, Q. M., Targher, G., & Day, C. P. (2013). Progression of NAFLD to diabetes mellitus, cardiovascular disease or cirrhosis. Nature Reviews Gastroenterology & Hepatology, 10(6), 330-344.

[14] Simon, T. G., Roelstraete, B., Khalili, H., Hagström, H., & Ludvigsson, J. F. (2021). Mortality in biopsy-confirmed nonalcoholic fatty liver disease: Results from a nationwide cohort. Gut, 70(7), 1375-1382.

[15] Stefan, N., Häring, H. U., & Cusi, K. (2019). Non-alcoholic fatty liver disease: Causes, diagnosis, cardiometabolic consequences, and treatment strategies. Lancet Diabetes & Endocrinology, 7(4), 313-324.

[16] Buzzetti, E., Pinzani, M., & Tsochatzis, E. A. (2016). The multiple-hit pathogenesis of non-alcoholic fatty liver disease (NAFLD). Metabolism, 65(8), 1038-1048.

[17] Stover, P. J., & Field, M. S. (2011). Trafficking of intracellular folates. Advances in Nutrition, 2(4), 325-331.

[18] Cui, L. H., Quan, Z. Y., Piao, J. M., Zhang, T. T., Jiang, M. H., Shin, M. H., & Choi, J. S. (2016). Plasma folate and vitamin B12 levels in patients with hepatocellular carcinoma. Journal of Molecular Science, 17(7), 1032-1042.

[19] Li, W., Ma, Y., Li, Z., Lv, X., Wang, X., Zhou, D., Luo, S., Wilson, J. X., & Huang, G. (2019). Folic acid decreases astrocyte apoptosis by preventing oxidative stress-induced telomere attrition. International Journal of Molecular Sciences, 21(1), 62-74.

[20] da Silva, R. P., Kelly, K. B., Al Rajabi, A., & Jacobs, R. L. (2014). Novel insights on interactions between folate and lipid metabolism. BioFactors, 40(3), 277-283.

[21] Pooya, S., Blaise, S., Moreno Garcia, M., Giudicelli, J., Alberto, J. M., Guéant-Rodriguez, R. M., Jeannesson, E., Gueguen, N., Bressenot, A., Nicolas, B., Malthiery, Y., Daval, J. L., Peyrin-Biroulet, L., Bronowicki, J. P., & Guéant, J. L. (2012). Methyl donor deficiency impairs fatty acid oxidation through PGC-1α hypomethylation and decreased ER-α ERR-α and HNF-4α in the rat liver. Journal of Hepatology, 57(2), 344-351.

[22] Pogribny, I. P., Kutanzi, K., Melnyk, S., de Conti, A., Tryndyak, V., Montgomery, B., Pogribna, M., Muskhelishvili, L., Latendresse, J. R., James, S. J., Beland, F. A., & Rusyn, I. (2013). Strain-dependent dysregulation of one-carbon metabolism in male mice is associated with choline- and folate-deficient diet-induced liver injury. Federation of American Societies for Experimental Biology Journal, 27(6), 2233-2243.

[23] Liu, Y., Shen, J., Yang, X., Sun, Q., & Yang, X. (2018). Folic acid reduced triglycerides deposition in primary chicken hepatocytes. Journal of Agricultural and Food Chemistry, 66(50), 13162-13172.

[24] Liu, Y., Liu, X., Zhou, J., Ren, Z., Yang, X., Cao, Y., & Yang, X. (2019). Folic acid perfusion administration reduced abdominal fat deposition in starter Arbor Acres broilers. Poultry Sciense, 98(12), 6816-6825.

[25] Liu, Y., Yang, J., Liu, X., Liu, R., Wang, Y., Huang, X., Li, Y., Liu, R., & Yang, X. (2023). Dietary folic acid addition reduces abdominal fat deposition mediated by alterations in gut microbiota and SCFA production in broilers. Animal Nutrition, 12, 54-62.

[26] Liu, Y., Zheng, Z., Wang, C., Wang, Y., Sun, X., Ren, Z., Yang, X., & Yang, X. (2024). Reorganization of 3D genome architecture provides insights into pathogenesis of early fatty liver disease in laying hens. Journal of Animal Science and Biotechnology, 15(1), 40.

[27] Li, Q., Tan, J. X., He, Y., Bai, F., Li, S. W., Hou, Y. W., Ji, L. S., Gao, Y. T., Zhang, X., Zhou, Z. H., Yu, Z., Fang, M., Gao, Y. Q., & Li, M. (2022). Atractylenolide III ameliorates non-alcoholic fatty liver disease by activating hepatic adiponectin receptor 1-mediated AMPK pathway. International Journal of Biological Sciences, 18(4), 1594-1611.

[28] Sun, X., Wang, Y., Wang, C., Wang, Y., Ren, Z., Yang, X., Yang, X., & Liu, Y. (2023). Genome analysis reveals hepatic transcriptional reprogramming changes mediated by enhancers during chick embryonic development. Poultry Science, 102(4), 102516-102527.

[29] Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC^T method. Methods, 25(4), 402-408.

[30] Li, L., Ren, W., Kong, H., Zhao, C., Zhao, X., Lin, X., Lu, X., & Xu, G. (2017). An alignment algorithm for LC-MS-based metabolomics dataset assisted by MS/MS information. Analytica Chimica Acta, 990, 96-102.

[31] Mete, A., Giannitti, F., Barr, B., Woods, L., & Anderson, M. (2013). Causes of mortality in backyard chickens in northern California: 2007-2011. Avian Diseases, 57(2), 311-315.

[32] Mardinoglu, A., Wu, H., Bjornson, E., Zhang, C., Hakkarainen, A., Räsänen, S. M., Lee, S., Mancina, R. M., Bergentall, M., Pietiläinen, K. H., Söderlund, S., Matikainen, N., Ståhlman, M., Bergh, P. O., Adiels, M., Piening, B. D., Granér, M., Lundbom, N., Williams, K. J., … Borén, J. (2018). An integrated understanding of the rapid metabolic benefits of a carbohydrate-restricted diet on hepatic steatosis in humans. Cell Metabolism, 27(3), 559-571.

[33] Wang, A. Q., Zhang, K. X., Fu, C. Y., Zhou, C. M., Yan, Z. G., & Liu, X. L. (2023). Alleviation effect of conjugated linoleic acid on estradiol benzoate induced fatty liver hemorrhage syndrome in Hy-line male chickens. Journal of Animal Science, 101, skad045.

[34] Wu, Y. L., Zhang, M. W., Meng, F. C., Ren, K. P., Li, D., Luo, X. G., & Hu, Y. (2024). Betaine supplementation alleviates corticosterone-induced hepatic cholesterol accumulation through epigenetic modulation of HMGCR and CYP7A1 genes in laying hens. Poultry Science, 103(3), 103435-103443.

[35] Rahimi, L., Rajpal, A., & Ismail-Beigi, F. (2020). Glucocorticoid-induced fatty liver disease. Diabetes, Metabolic Syndrome and Obesity, 13, 1133-1145.

[36] Chen, M., Bai, M., Yi, Y., Lu, S., Luo, J., Li, P., Zhang, H., Jiang, H., & Zhou, H. (2022). Upregulation of hepatic CD36 via glucocorticoid receptor activation contributes to dexamethasone-induced liver lipid metabolism disorder in mice. Toxicology Letters, 363, 1-10.

[37] Ojeda, M. L., Rua, R. M., Nogales, F., Díaz-Castro, J., Murillo, M. L., & Carreras, O. (2016). The benefits of administering folic acid in order to combat the oxidative damage caused by binge drinking in adolescent rats. Alcohol and Alcoholism, 51(3), 235-241.

[38] Sid, V., Shang, Y., Siow, Y. L., Hewage, S. M., House, J. D., & O, K. (2018). Folic acid supplementation attenuates chronic hepatic inflammation in high-fat diet fed mice. Lipids, 53(7), 709-716.

[39] Xin, F. Z., Zhao, Z. H., Zhang, R. N., Pan, Q., Gong, Z. Z., Sun, C., & Fan, J. G. (2020). Folic acid attenuates high-fat diet-induced steatohepatitis via deacetylase SIRT1-dependent restoration of PPARα. World Journal of Gastroenterology, 26(18), 2203-2220.

[40] Wang, X., Newkirk, R. F., Carre, W., Ghose, P., Igobudia, B., Townsel, J. G., & Cogburn, L. A. (2009). Regulation of ANKRD9 expression by lipid metabolic perturbations. BMB Reports, 42(9), 568-573.

[41] Martínez-García, A., Sastre, I., Recuero, M., Aldudo, J., Vilella, E., Mateo, I., Sánchez-Juan, P., Vargas, T., Carro, E., Bermejo-Pareja, F., Rodríguez-Rodríguez, E., Combarros, O., Rosich-Estrago, M., Frank, A., Valdivieso, F., & Bullido, M. J. (2010). PLA2G3, a gene involved in oxidative stress induced death, is associated with Alzheimer’s disease. Journal of Alzheimer’s Disease, 22(4), 1181-1187.

[42] Hrdinka, M., Dráber, P., Stepánek, O., Ormsby, T., Otáhal, P., Angelisová P., Brdicka, T., Paces, J., Horejsí V., & Drbal, K. (2011). PRR7 is a transmembrane adaptor protein expressed in activated T cells involved in regulation of T cell receptor signaling and apoptosis. Journal of Biological Chemistry, 286(22), 19617-19629.

[43] Sun, M., Ju, J., Ding, Y., Zhao, C., & Tian, C. (2022). The signaling pathways regulated by KRAB zinc-finger proteins in cancer. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 1877(3), 188731.

[44] Song, L., Wang, L., Hou, Y., Zhou, J., Chen, C., Ye, X., Dong, W., Gao, H., Liu, Y., Qiao, G., Pan, T., Chen, Q., Cao, Y., Hu, F., Rao, Z., Chen, Y., Han, Y., Zheng, M., Luo, Y., … Huang, Z. (2022). FGF4 protects the liver from nonalcoholic fatty liver disease by activating the AMP-activated protein kinase-Caspase 6 signal axis. Hepatology, 76(4), 1105-1120.

[45] Jin, L., Yang, R., Geng, L., & Xu, A. (2023). Fibroblast growth factor-based pharmacotherapies for the treatment of obesity-related metabolic complications. Annual Review of Pharmacology and Toxicology, 63(1), 359-382.

[46] Friedman, D. J., Crotts, S. B., Shapiro, M. J., Rajcula, M., McCue, S., Liu, X., Khazaie, K., Dong, H., & Shapiro, V. S. (2021). ST8Sia6 promotes tumor growth in mice by inhibiting immune responses. Cancer Immunology Research, 9(8), 952-966.

[47] Kuai, J., Zheng, L., Yi, X., Liu, Z., Qiu, B., Lu, Z., & Jiang, Y. (2021). ST8SIA6-AS1 promotes the development of hepatocellular carcinoma cells through miR-338-3p/NONO Axis. Digestive and Liver Disease, 53(9), 1192-1200.

[48] Ma, W. W., Ding, B. J., Yuan, L. H., Zhao, L., Yu, H. L., Xi, Y. D., & Xiao, R. (2017). Neurocalcin-delta: A potential memory-related factor in hippocampus of obese rats induced by high-fat diet. African Health Scienses, 17(4), 1211-1221.

[49] Zhang, Y. X., Zhang, X. T., Li, H. J., Zhou, T. F., Zhou, A. C., Zhong, Z. L., Liu, Y. H., Yuan, L. L., Zhu, H. Y., Luan, D., & Tong, J. C. (2021). Antidepressant-like effects of helicid on a chronic unpredictable mild stress-induced depression rat model: Inhibiting the IKK/IκBα/NF-κB pathway through NCALD to reduce inflammation. International Immunopharmacology, 93, 107165-107178.

[50] Wang, Y., Liu, L., Liu, X., Tan, X., Zhu, Y., Luo, N., Zhao, G., Cui, H., & Wen, J. (2022). SLC16A7 promotes triglyceride deposition by de novo lipogenesis in chicken muscle tissue. Biology, 11(11), 1547-1562.

[51] Yan, L., Li, Q., Yang, J., & Qiao, B. (2018). TPX2-p53-GLIPR1 regulatory circuitry in cell proliferation, invasion, and tumor growth of bladder cancer. Journal of Cellular Biochemistry, 119(2), 1791-1803.

[52] Xu, W., Zhang, Z., Yao, L., Xue, B., Xi, H., Wang, X., & Sun, S. (2022). Exploration of shared gene signatures and molecular mechanisms between periodontitis and nonalcoholic fatty liver disease. Frontiers in Genetics, 13, 939751.

[53] Leclerc, D., Christensen, K. E., Cauvi, O., Yang, E., Fournelle, F., Bahous, R. H., Malysheva, O. V., Deng, L., Wu, Q., Zhou, Z., Gao, Z. H., Chaurand, P., Caudill, M. A., & Rozen, R. (2019). Mild methylenetetrahydrofolate reductase deficiency alters inflammatory and lipid pathways in liver. Molecular Nutrition & Food Research, 63(3), e1801001.

[54] Jiang, Y., Cao, H., Chen, X., Yu, G., Song, C., Duan, H., Tian, F., Wan, H., & Shen, J. (2022). Associations of serum folate and vitamin C levels with metabolic dysfunction-associated fatty liver disease in US adults: A nationwide cross-sectional study. Frontiers in Public Health, 10, 1022928-1022938.

[55] Wang, P., Li, S., Wang, M., He, J., & Xi, S. (2017). Association of MTRR A66G polymorphism with cancer susceptibility: Evidence from 85 studies. Journal of Cancer, 8(2), 266-277.

[56] Zhang, J., Liu, G. C., Dai, X. L., Wang, J., Jin, M. H., Mi, N. N., & Wang, S. Q. (2020). The N-terminus of MTRR plays a role in MTR reactivation cycle beyond electron transfer. Bioorganic Chemistry, 100, 103836.

[57] Li, W. X., Lv, W. W., Dai, S. X., Pan, M. L., & Huang, J. F. (2015). Joint associations of folate, homocysteine and MTHFR, MTR and MTRR gene polymorphisms with dyslipidemia in a Chinese hypertensive population: A cross-sectional study. Lipids in Health and Disease, 14(1), 101-112.

[58] Badmus, O. O., Hillhouse, S. A., Anderson, C. D., Hinds, T. D., & Stec, D. E. (2022). Molecular mechanisms of metabolic associated fatty liver disease (MAFLD): Functional analysis of lipid metabolism pathways. Clinical Sciense (Lond), 136(18), 1347-1366.

[59] Kobayashi, M., Fujii, N., Narita, T., & Higami, Y. (2018). SREBP-1c-dependent metabolic remodeling of white adipose tissue by caloric restriction. International Journal of Molecular Sciences, 19(11), 3335-3346.

[60] Matsuzaka, T., Kuba, M., Koyasu, S., Yamamoto, Y., Motomura, K., Arulmozhiraja, S., Ohno, H., Sharma, R., Shimura, T., Okajima, Y., Han, S. I., Aita, Y., Mizunoe, Y., Osaki, Y., Iwasaki, H., Yatoh, S., Suzuki, H., Sone, H., Takeuchi, Y., … Shimano, H. (2020). Hepatocyte ELOVL fatty acid elongase 6 determines ceramide acyl-chain length and hepatic insulin sensitivity in mice. Hepatology, 71(5), 1609-1625.

[61] Su, Y. C., Feng, Y. H., Wu, H. T., Huang, Y. S., Tung, C. L., Wu, P., Chang, C. J., Shiau, A. L., & Wu, C. L. (2018). Elovl6 is a negative clinical predictor for liver cancer and knockdown of Elovl6 reduces murine liver cancer progression. Scientific Reports, 8(1), 6586-6594.

[62] Hinds, T. D., Jr., Burns, K. A., Hosick, P. A., McBeth, L., Nestor-Kalinoski, A., Drummond, H. A., AlAmodi, A. A., Hankins, M. W., Vanden Heuvel, J. P., & Stec, D. E. (2016). Biliverdin reductase a attenuates hepatic steatosis by inhibition of glycogen synthase kinase (GSK) 3β phosphorylation of serine 73 of peroxisome proliferator-activated receptor (PPAR) α. Journal of Biological Chemistry, 291(48), 25179-25191.

[63] Hinds, T. D., Jr., Hosick, P. A., Chen, S., Tukey, R. H., Hankins, M. W., Nestor-Kalinoski, A., & Stec, D. E. (2017). Mice with hyperbilirubinemia due to Gilbert’s syndrome polymorphism are resistant to hepatic steatosis by decreased serine 73 phosphorylation of PPARα. American Journal of Physiology Endocrinology and Metabolism, 312(4), E244-e252.

[64] Hinds, T. D., Jr., Creeden, J. F., Gordon, D. M., Stec, D. F., Donald, M. C., & Stec, D. E. (2020). Bilirubin nanoparticles reduce diet-induced hepatic steatosis, improve fat utilization, and increase plasma β-hydroxybutyrate. Frontiers in Pharmacology, 11, 594574-594585.

[65] Cheng, S., Liu, X., Gong, F., Ding, X., Zhou, X., Liu, C., Zhao, F., Li, X., & Shi, J. (2021). Dexamethasone promotes the endoplasmic reticulum stress response of bone marrow mesenchymal stem cells by activating the PERK-Nrf2 signaling pathway. Pharmacology Research & Perspectives, 9(3), e00791.

[66] Guo, Y., Hao, D., & Hu, H. (2021). High doses of dexamethasone induce endoplasmic reticulum stress-mediated apoptosis by promoting calcium ion influx-dependent CHOP expression in osteoblasts. Molecular Biology Reports, 48(12), 7841-7851.

[67] Al Zaid Siddiquee, K., & Turkson, J. (2008). STAT3 as a target for inducing apoptosis in solid and hematological tumors. Cell Research, 18(2), 254-267.

[68] Kondo, R., Ishino, K., Wada, R., Takata, H., Peng, W. X., Kudo, M., Kure, S., Kaneya, Y., Taniai, N., Yoshida, H., & Naito, Z. (2019). Downregulation of protein disulfide-isomerase A3 expression inhibits cell proliferation and induces apoptosis through STAT3 signaling in hepatocellular carcinoma. International Journal of Oncology, 54(4), 1409-1421.

[69] Lee, W. J., & Lee, S. H. (2022). Protocatechuic acid protects hepatocytes against hydrogen peroxide-induced oxidative stress. Current Research in Food Science, 5, 222-227.

[70] Sarna, L. K., Sid, V., Wang, P., Siow, Y. L., House, J. D., & O, K. (2016). Tyrosol attenuates high fat diet-induced hepatic oxidative stress: Potential involvement of cystathionine β-synthase and cystathionine γ-lyase. Lipids, 51(5), 583-590.

[71] Ghanim, A. M. H., Younis, N. S., & Metwaly, H. A. (2021). Vanillin augments liver regeneration effectively in Thioacetamide induced liver fibrosis rat model. Life Sciences, 286, 120036-120047.

[72] Guo, R., Zhao, B., Wang, Y., Wu, D., Wang, Y., Yu, Y., Yan, Y., Zhang, W., Liu, Z., & Liu, X. (2018). Cichoric acid prevents free-fatty-acid-induced lipid metabolism disorders via regulating Bmal1 in HepG2 cells. Journal of Agricultural and Food Chemistry, 66(37), 9667-9678.

RIGHTS & PERMISSIONS

2024 The Author(s). Animal Research and One Health published by John Wiley & Sons Australia, Ltd on behalf of Institute of Animal Science, Chinese Academy of Agricultural Sciences.