Progress 01/01/16 to 09/30/20
Outputs
Target Audience:Target audiences would include any segments of the U.S. population that would be at risk for mild-to-moderate deficiencies ofspecific micronutrients. Moreover, the target population would include gestating women and those breastfeeding newborns,and the focus of this project is the risk of perinatal micronutrient deficiency to cause subsequent abnormalities in braindevelopment and cognitive processing (i.e., learning and memory). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Research conducted under this project was either a major or minor focus for twograduate students. Each of these students gained experience conducting animal experiments, summarizing/analyzing the data, and creating publications/presentations to disseminate the findings. Working directly with me as their academic advisor, each student learned academic prowess and the ability to communicate their science to not only the scientific community, but also to the lay public through outreach efforts my lab participates in at the departmental, college, and university levels. Moreover, all students had the chance to participate in oral and/or poster presentation competitions, which served to hone their communication skills, and the graduate students also attended the American Society for Nutrition annual meeting held in conjunction with Experimental Biology. In this large meeting format, the students developed professional skills in terms of networking and presentations. How have the results been disseminated to communities of interest?Research outputs stemming from this research have been disseminated via peer-reviewed publications, multiple theses and dissertations, and through public outreach projects.My lab routinely participates in outreach activities that showcase our use of the pig as a preclinical and translational model to study pediatric nutrition and neurodevelopment. As such, we put on multiple workshops each year where 50-100 members of the public, extending from kindergartners to elderly members, gain first-hand and hands-on knowledge about studying neurodevelopment using the pig. Demonstrations include basic nutritional concepts regarding the importance of iron for brain developmentand include brain dissection demonstrations and multi-media presentations of how we can use magnetic resonance imaging to study the brain. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. Determine how prenatal choline status alters cognition in an age-dependent manner. A) Assess the longitudinal performance of pigs in complementary behavioral paradigms of cognitive function. B) Quantify changes in profiles of brain metabolites that directly relate to cognitive processing. • Major activities completed/experiments conducted: One major study and multiple pilot studies have were conducted in our translational pig model to provide direct evidence of neurodevelopmental trajectories over time. • Data collected: Data generated as part of this objective included the creation of validated behavioral techniques for testing cognitive performance in the pig. This includes the novel object recognition (NOR) task of learning and memory, eye-blink conditioning task of cognitive processing efficiency, and development of robust testing infrastructure in creating operant testing chambers for pigs (i.e., touch-sensitive video screens to perform a multitude of associative learning tasks). • Summary statistics and discussion of results: Building upon previous research from our laboratory that confirmed the importance of choline in neurodevelopment, new findings highlight the importance of additional nutrients [e.g., iron, arachidonic acid (ARA), and docosahexaenoic acid (DHA)] and nutritional technologies (e.g., prebiotics and probiotics) in altering the trajectory of brain development as related to choline metabolism. These findings confirm that nutrient delivery to the pig cannot be properly interpreted without also considering influences of the intestinal microbiota, as evidenced by alteration of brain growth, structure, and function (i.e., learning and memory) by the early-life provision of iron and prebiotic substances in recent publications. Additional evidence directly implicating the importance of choline in neurodevelopment comes from research in pigs where dams received diets without or with whole egg products, which represent a rich source of choline and related nutrients, especially fatty acids. Publications involving this research are currently being prepared for submission. • Key outcomes: We experienced a change in knowledge as our data suggested that perinatal status of choline/iron/fatty acid intake has profound implications on brain growth, composition, and function. Moreover, we discovered important links between the microbiota and host in terms of intestinal health, immunity, metabolism, and brain structure/function, which all have direct implications in the human clinical realm. Finally, these studies have caused a change in action in that greater focus should be placed on a wider variety of nutrients important for early-life development,and finding interactions between diet and microbiota has emerged as a quickly-evolving factor warranting further study. Findings from this objective resulted in the generation of a Ph.D. thesis and 7+ peer-reviewed publications. 2. Determine how prenatal choline status influences structural brain development in an age-dependent manner. A) Assess longitudinal brain development using high-resolution neuroimaging techniques to quantify brain structure, composition, and angiogenesis. B) Quantify changes in cellular and molecular targets involved in structural brain organization and development. • Major activities completed/experiments conducted: A total of onemajor study was conducted in our translational pig model to provide direct evidence of early-life nutrient status on the trajectory of brain growth, composition, and structure. • Data collected: Validated magnetic resonance imaging (MRI) procedures adapted from human sequences and optimized for pigs as part of this project were used to quantify brain development of pigs. Neuroimaging data included volumes of the whole brain and 19 discrete sub-regions, voxel-based morphometric (VBM) analysis of how these regions differed in shape, quantification of brain microstructure using diffusion tensor imaging (DTI), quantification of brain neurochemicals by magnetic resonance spectroscopy (MRS), and quantification of myelination using myelin water fraction (MWF). Neuroimaging procedures were applied to a large cohort of pigs at seventime-points (1, 2, 3, 4, 8, 12, 18, and 24 weeks of age) to permit modeling of longitudinal brain development for the domestic pig. • Summary statistics and discussion of results: In this thrust, we have generated an updated MRI brain atlas for the domestic pig, which will be made freely available for public consumption once the peer-reviewed publication is accepted. Additionally, we are in the process of finalizing manuscripts involving age-dependent models of brain development in pigs exposed to various nutritional backgrounds as we have come to learn the importance of quantifying long-term outcomes. We have learned that there are generally no differences in the developmental models between sow-reared and artificially-reared pigs, which lends credence to the animal model that we have been using. Additionally, we have identified that changes in the early-life source of protein had minimal effects on microbiota, brain structure, and behavioral outcomes, which suggests that greater emphasis on early-life micronutrient status may be more important once macronutrient requirements have been met. • Key outcomes: Collectively, these studies have caused us to experience a change in knowledge as the data provide clear evidence that a number of postnatal nutritional interventions elicited neurodevelopmental effects. Moreover, we have experienced a change in knowledge in that for certain micronutrients, like iron, peripheral outcomes include the microbiota and hematological outcomes, could be rescued by subsequent repletion, yet detriments in structural brain outcomes were permanently and negatively affected (i.e., could not be corrected by nutritional adequacy). Findings related to this second experimental objective resulted in the generation of an M.S. thesis and five pluspeer-reviewed publications that are either published or in preparation for submission.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Fleming, S.A., A.T. Mudd, J. Hauser, J. Yan, S. Metairon, P. Steiner, S.M. Donovan and R.N. Dilger. 2020. Dietary oligofructose alone or in combination with 2'-fucosyllactose differentially improves recognition memory and hippocampal mRNA expression. Nutrients 12:2131. doi:10.3390/nu12072131.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Fleming, S.A., A.T. Mudd, J. Hauser, J. Yan, S. Metairon, P. Steiner, S.M. Donovan and R.N. Dilger. 2020. Human and bovine milk oligosaccharides elicit improved recognition memory concurrent with alterations in regional brain volumes and hippocampal mRNA expression. Front. Neurosci. 14:770. doi:10.3389/fnins.2020.00770.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2020
Citation:
Hahn, K.E., I. Dahms, C.M. Butt, N. Salem Jr., V. Grimshaw, E. Bailey, S.A. Fleming, B.N. Smith and R.N. Dilger. 2020. Impact of arachidonic and docosahexaenoic acid supplementation on neural and immune development in the young pig. Front. Nutr.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Jena, A., C.A. Montoya, J.A. Mullaney, R.N. Dilger, W. Young, W.C. McNabb and N.C. Roy. 2020. Gut-brain axis in the early postnatal years of life: A developmental perspective. Frontiers in Integrative Neuroscience 14 doi:10.3389/fnint.2020.00044.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2019
Citation:
Fleming, S.A. 2019. The role of oligosaccharides in cognitive development. (http://hdl.handle.net/2142/105769). Doctoral dissertation, University of Illinois, Urbana-Champaign.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Target audiences would include any segments of the U.S. population that would be at risk for mild-to-moderate deficiencies of specific micronutrients. Moreover, the target population would include gestating women and those breastfeeding newborns, and the focus of this project is the risk of perinatal micronutrient deficiency to cause subsequent abnormalities in brain development and cognitive processing (i.e., learning and memory). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The work described in this annual report was predominantly conducted by one graduate student who was mentored by the PI on this proposal. Joanne is a fourthyear PhD in the Neuroscience Program at the University of Illinois at Urbana-Champaign, and as part of her dissertation research, Joanne has improved our neuroimaging pipeline for the pig. Additionally, she has created a high-resolution brain atlas for the domestic pig and is currently completing a modeling project to directly compare rates of human and pig brain development to expand the translational power of this animal model. How have the results been disseminated to communities of interest?In total, work on this project has been incorporated into at least 20 invited talks given by the PI and 8 peer-reviewed publications. As micronutrients, choline and iron are the primary nutritional interventions that have been used to highlight the sensitivity of the behavioral and MRI assays that have been used to elucidate structural and functional analyses of brain development using the domestic pig. What do you plan to do during the next reporting period to accomplish the goals?We plan to make additional progress on validating behavioral, neuroimaging, and cellular and molecular techniques pertinent to this project over the next year. We recently developed operant conditioning chambers for the young pig that will allow sensitive and flexible assessment of finite behaviors in the pig. Over the next year, we expect to validate multiple behavioral assays using operant conditioning and preference testing in these chambers. Moreover, we have collected data on structural brain development of pigs from birth through 24 weeks of age (i.e., sexual maturity), and we will model responses of the pig and compare those models to early-life human MRI data from colleagues. This modeling approach will allow direct chronological comparison of brain development rates between pigs and humans to extend the translational power of this animal model.
Impacts
What was accomplished under these goals?
Progress made over the past year included the publication of a series of research projects involving post-natal iron deficiency and changes in peripheral organs, brain, and microbiota profiles during periods of deficiency and subsequent repletion of dietary iron. Moreover, we have improved the animal model (especially cognitive assessment and neuroimaging procedures) and data analysis pipelines we use to test effects of early-life nutrition on cognitive development. Additionally, we have created a high-resolution brain atlas for the domestic pig and begun to compare rates of pig and human brain development using a modeling approach, which will serve to elevate the pig as a dual-purpose model. These findings have direct implications in both the agricultural and human clinical realms.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Zimmerman, B.J., A.T. Mudd, J.E. Fil, R.N. Dilger and B.P. Sutton. 2018. Noninvasive imaging of cerebral blood volume in piglets with vascular occupancy MR imaging and inflow vascular space occupancy with dynamic subtraction. Magn. Reson. Imaging 50:54-60. doi:https://doi.org/10.1016/j.mri.2018.03.009.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Knight, L.C., M. Wang, S.M. Donovan and R.N. Dilger. 2019. Early-life iron deficiency and subsequent repletion alters development of the colonic microbiota in the pig. Front. Nutr. 6 doi:10.3389/fnut.2019.00120.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2019
Citation:
Fil, J.E., S. Joung, B.J. Zimmerman, B.P. Sutton and R.N. Dilger. 2019. An updated magnetic resonance imaging-based atlas of the young domesticated pig (Sus scrofa). Journal of Magnetic Resonance Imaging (Under Review).
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Target audiences would include any segments of the U.S. population that would be at risk for mild-to-moderate deficiencies of specific micronutrients. Moreover, the target population would include gestating women and those breastfeeding newborns, and the focus of this project is the risk of perinatal micronutrient deficiency to cause subsequent abnormalities in brain development and cognitive processing (i.e., learning and memory). Changes/Problems:While not a challenge, the focus of this project has taken a small step back to encompass not only choline but also iron as an important micronutrient that influences brain development when provided during the perinatal period. Therefore, the focus of this project is beginning to broaden as we allow the science and interpretive findings to drive the study design. What opportunities for training and professional development has the project provided?The work described in this annual report was predominantly conducted by one graduate student who was mentored by the PI on this proposal. Austin Mudd completed his PhD student in the Neuroscience Program in May 2018, and while leading this project, he was responsible for all animal work and all data collection, processing, and summary. In total, Austin served as lead author on 12 of 15 publications on which he contributed, and he brought to fruition both peer-reviewed articles involving early-life choline and iron status and brain development in the pig. How have the results been disseminated to communities of interest?Work on this project has been incorporated into at least fifteen invited talks given by the PI and five peer-reviewed publications. As micronutrients, choline and iron are the primary nutritional interventions that have been used to highlight the sensitivity of the behavioral and MRI assays that have been used to elucidate structural and functional analyses of brain development using the domestic pig. What do you plan to do during the next reporting period to accomplish the goals?We plan to make additional progress on validating behavioral, neuroimaging, and cellular and molecular techniques pertinent to this project over the next year. A recently concluded study in our lab, which involved gestational supply of choline-enriched egg products, will be analyzed, summarized, and published as part of the PhD dissertation of another student (Stephen Fleming). This research will also be directly related to the current proposal and will provide valuable insights as including the role of prenatal intake by the dam on postnatal brain development of the offspring.
Impacts
What was accomplished under these goals?
Progress made over the past year included improving the animal model (especially cognitive assessment and neuroimaging procedures) and data analysis pipelines we use to test effects of early-life nutrition on cognitive development. Additionally, our focus expanded beyond choline and also included early-life iron status on longitudinal brain development, which was found to also influence choline metabolism. As such, we published a peer-reviewed article highlighting how early-life choline status influences white and grey matter development in pigs (i.e., structural effects of micronutrient status; see Mudd et al., 2018a). Additionally, we identified that insufficient early-life dietary iron reduced brain iron content and caused untoward structural and physiological changes (including to grey and white matter volumes and subcortical white matter integrity), despite a subsequent period where subjects were provided adequate iron intake (see Mudd et al., 2018b). Finally, despite iron repletion later in life, inadequate postnatal iron ingestion negatively affected both volumetric growth and connectivity within the pig brain (see Mudd et al., 2018c), which has direct implications in both the agricultural and clinical realms. In summary, with Austin Mudd's dissertation, we published a number of peer-reviewed manuscripts emphasizing the importance of early-life micronutrient status that involved both choline and iron.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Mudd, A.T., Getty, C.M. and Dilger, R.N. 2018. Maternal dietary choline status influences brain grey and white matter development in young pigs. Curr. Develop. Nutr. 2:nzy015. doi:10.1093/cdn/nzy015.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Mudd, A., Fil, J., Knight, L., Lam, F., Liang, Z.-P. and Dilger, R.N. 2018. Early-life iron deficiency reduces brain iron content and alters brain tissue composition despite iron repletion: A neuroimaging assessment. Nutrients 10:135. doi:10.3390/nu10020135.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Mudd, A.T., Fil, J.E., Knight, L.C. and Dilger, R.N. 2018. Dietary iron repletion following early-life dietary iron deficiency does not correct regional volumetric or diffusion tensor changes in the developing pig brain. Front. Neurol. 8:735. doi:10.3389/fneur.2017.00735.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2018
Citation:
http://hdl.handle.net/2142/101125
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Target audiences would include any segments of the U.S. population that would be at risk for mild-to-moderate deficiencies of specific micronutrients, in this case, choline. Moreover, the target population would include gestating women and thosebreastfeeding newborns, and the focus of this project is the risk of perinatal choline deficiency to cause subsequentabnormalities in cognitive processing (i.e., learning and memory). Changes/Problems:While only a minor change, our approach in studying early-life choline provision is starting to shift toward using foods that contain appreciable concentrations of choline, like the egg. This approach will position our research in a more balanced position between animal and human nutrition, and allow findings to be easily extended into either realm. What opportunities for training and professional development has the project provided?The work described in this annual report is being conducted by one graduate student who is being mentored by the PI on this proposal. Austin Mudd is a fifthyear Ph.D. student in the Neuroscience Program, and he is responsible for all animal work andall data collection, processing, and summary. Austin has served as lead author on eight of the tenpublications on which he contributed,and he was instrumental in conducting, summarizing, presenting, and publishing peer-reviewed articles related to early-life choline status and brain development in the pig over the last year. How have the results been disseminated to communities of interest?Scientific findings generated as part of this project have been disseminated in at least teninvited talks given by the PI and fourpeer-reviewed publications. In particular, the PI was invited to a Federation of the American Society for Experimental Biology (FASEB) Science Research Conference (SRC) entitled 'Origins and Benefits of Biologically Active Components in Human Milk' that was held in Lisbon, Portugal in July 2017. This conference brought together scientists from academia, industry, and government organizations and Dr. Dilger's grouplead the choline session. Overall, choline insufficiency is the primary nutritional intervention that has been used to highlight the sensitivity of the behavioral and MRI assays that have been used in Dr. Dilger's laboratory to elucidate structural and functional analyses of brain development using the domestic pig. What do you plan to do during the next reporting period to accomplish the goals?We plan to make additional progress on validating behavioral, neuroimaging, and cellular and molecular techniques pertinentto this project over the next year. We are about to initiate a long-term study involving gestational effects of ingesting egg (of which choline is a major constituent) on longitudinal implications of brain development in offspring. Moving from feeding pure choline to providing this nutrient in a 'whole food' approach will extend the usefulness of findings toward application in the human nutrition realm.
Impacts
What was accomplished under these goals?
The bulk of the progress made over the past year related to improving the animal model we use to test effects of early-life nutrition on cognitive development. First, we completed development and validation of the novel object recognition task of learning and memory for the young pig. While not directly supported by Hatch funds, this behavioral paradigm will definitely be used to test the effects of early-life choline status in the near future and this publication has been included in the current progress report (see Fleming and Dilger, 2017). Second, we published a literature review on using the domestic pig as an animal model to study the effects of early-life nutrition on cognitive development (see Mudd and Dilger, 2017). Again, while this effort was not directly supported by Hatch funds, the review itself discussed effects of 'one-carbon metabolites', which includes choline, and references previous publications generated as part of this Hatch project. In addition to publications, we made significant improvements in our neuroimaging sequences, including iVASO, iron quantification, whole-brain magnetic resonance spectroscopy, and we are also close to finalizing a more sensitive pig brain atlas that increases resolution seven-fold over the current version that was developed by our laboratory. In terms of behavioral outcomes, the eyeblink conditioning (EBC) data acquisition and analysis pipeline is nearing completion and will be entering the publication phase soon. In terms of molecular techniques, we also confirmed the accuracy of quantifying neuronal spine density and arborization using ballistic labelingtechniques in conjunction with a research partner. Collectively, the last year was about making improvements in our animal model, such that we are poised to apply these techniques in planned studies aimed at investigating the role of early-life choline in brain development over the next year.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Mudd, A.T. and Dilger, R.N. 2017. Early-life nutrition and neurodevelopment: Use of the piglet as a translational model. Adv. Nutr. 8:92-104. doi 10.3945/an.116.013243.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Fleming, S.A. and Dilger, R.N. 2017. Young pigs exhibit differential exploratory behavior during novelty preference tasks in response to age, sex, and delay. Behav. Brain Res. 321:50-60. doi 10.1016/j.bbr.2016.12.027.
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Progress 01/01/16 to 09/30/16
Outputs
Target Audience:Target audiences would include any segments of the US population that would be at risk for mild-to-moderate deficiencies of specific micronutrients, in this case, choline. Moreover, the target population would include gestating women and those breastfeeding newborns, and the focus of this project is the risk of perinatal choline deficiency to cause subsequent abnormalities in cognitive processing (i.e., learning and memory). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The work described in this annual report is being conducted by one graduate student who is being mentored by the PI on this proposal. Austin Mudd is a 4th year PhD student in the Neuroscience Program, and he is responsible for all animal work and all data collection, processing, and summary. Austin has served as lead author on 5 of 7 publications on which he contributed, and he brought to fruition both peer-reviewed articles involving early-life choline status and brain development in the pig that were published in 2016. How have the results been disseminated to communities of interest?Work on this project has been incorporated into at least ten invited talks given by the PI and two peer-reviewed publications. Choline insufficiency is the primary nutritional intervention that has been used to highlight the sensitivity of the behavioral and MRI assays that have been used to elucidate structural and functional analyses of brain development using the domestic pig. What do you plan to do during the next reporting period to accomplish the goals?We plan to make additional progress on validating behavioral, neuroimaging, and cellular and molecular techniques pertinent to this project over the next year. Once we are in a position to initiate another round of animal studies, we will induce differential choline status and track offspring longitudinally using the broad spectrum of outcomes that our lab has developed for the pig. We plan to initiate at least one cohort of pigs over the next year.
Impacts
What was accomplished under these goals?
In the past year, we have improved upon the pig brain atlas and post-acquisition sequence of MRI data processing methods for analyzing and interpreting neuroimaging datasets. Combined, these efforts continue to deepen our knowledge about short- and long-term effects of early-life choline sufficiency. By making use of the Piglet Nutrition and Cognition Laboratory research facility that was dedicated in April 2015, we have made significant progress toward developing, validating, and optimizing behavioral tasks pertinent to the present Hatch proposal, including the back test (a measure of anxiety in pigs), eye-blink conditioning (a measure of associative learning and memory), and novel object recognition (a measure of short-term memory formation). These behavioral tests will ultimately be used to test the effects of choline status on cognitive development, and will complement the neuroimaging techniques that continue to be improved. In addition to the outcomes described above, our laboratory has developed cellular and molecular techniques for quantifying neurodevelopment in the pig, including Western blot quantification of pre- and post-synaptic proteins and immunohistochemical staining and semi-quantitative procedures for protein targets in the brain. Additionally, we are in the process of working with an external laboratory to enable quantification of neuronal spine density and arborization complexity in the pig. Combined with our recently-developed methods to quantify brain catecholamines using high-performance liquid chromatography methods, we now have a broad set of validated laboratory techniques to enable characterization of the developing pig brain, and we are now well-positioned to determine how early-life choline status affects these outcomes. As a result of the work that was conducted over the past year, we have experienced both a change in knowledge and actions. The information gathered thus far was worthy oftwo publications, in that our development of a choline-sensitive behavioral paradigm of learning and memory in the pig provides clear evidence that sensitive periods exists during cognitive development. Moreover, the development of MRI sequences for the domestic pig has generated a plethora of additional research questions that can be best tested using the translational nature of the pig brain to humans. A new shift in action will occur in 2016 upon final implementation of the eye-blink conditioning paradigm to provide another measure of learning and memory. The second shift in action is implementation ofnew MRI sequences, including improvements in diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) sequences, which quantify neural connectivity/complexity and neurochemical concentrations in live pigs, respectively. Development and refinement of these techniques represent outcomes for assessing cognitive development, and we anticipate that use of these procedures will reveal quantitative changes in brain development induced by differential early-life choline status.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Mudd, A. T., C. M. Getty, B. P. Sutton, and R. N. Dilger. 2016. Perinatal choline deficiency delays brain development and alters metabolite concentrations in the young pig. Nutr. Neurosci. doi:10.1179/1476830515Y.0000000031
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Mudd, A.T., L.S. Alexander, S.K. Johnson, C.M. Getty, O.V. Malysheva, M.A. Caudill and R.N. Dilger. 2016. Perinatal dietary choline deficiency in sows influences concentrations of choline metabolites, fatty acids, and amino acids in milk throughout lactation. J. Nutr. 146:2216-2223. doi:10.3945/jn.116.238832.
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