University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2014 Absorption and Utilization of Choline and Vitamin B12 in Lactating Dairy Cows using Different Delivery Methods Virginia Maria Artegoitia Etcheverry University of Tennessee - Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Dairy Science Commons, Food Chemistry Commons, and the Large or Food Animal and Equine Medicine Commons Recommended Citation Artegoitia Etcheverry, Virginia Maria, "Absorption and Utilization of Choline and Vitamin B12 in Lactating Dairy Cows using Different Delivery Methods. " PhD diss., University of Tennessee, 2014. https://trace.tennessee.edu/utk_graddiss/3186 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Virginia Maria Artegoitia Etcheverry entitled "Absorption and Utilization of Choline and Vitamin B12 in Lactating Dairy Cows using Different Delivery Methods." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Food Science and Technology. Svetlana Zivanovic, Major Professor We have read this dissertation and recommend its acceptance: Michael O. Smith, Shawn Campagna, Michael de Veth, Federico Harte Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Absorption and Utilization of Choline and Vitamin B12 in Lactating Dairy Cows using Different Delivery Methods. A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Virginia Maria Artegoitia Etcheverry December 2014 DEDICATION Dedicated to the memory of my beloved mother Graciela M. Etcheverry Rossello ii ACKNOWLEDGEMENTS I am very thankful to Dr. Federico Harte for giving me the opportunity to pursue my PhD and gave me the freedom to choose and support through the subject of interest to me. I would like to express my deepest gratitude to my advisor, Dr. Michael de Veth, for his excellent guidance, caring, patience, and providing me with constant encouragement. His knowledge and commitment to the highest standards inspired and motivated me. I would like to thank Dr Shawn Campagna for allowed me to learn and use the LC-MS/MS, he has been very valuable support through all this experiments. I would also like to thank Dr. Christiane Girard and all her team at Agriculture Agri-Food Canada for allowed me to learn about dairy nutrition with a sophisticated technique and providing me with an excellent atmosphere doing my work. Her encouragement, insightful comments, and criticisms during the experiments and writing have been fundamentals during my studies. Special thanks go to Dr.Svetalana Zivanovic, who was willing to participate as main advisor in this few last months of my PhD, and also to Dr. Michael O. Smith who was willing to participate as a defense committee at the last moment. It was a pleasure and a great opportunity to work in a multi-disciplinary and across institutional settings. I very much enjoy this multidisciplinary approach as I believe ads to the relevance and meaning of my work (personally and professionally). Thanks to all the graduate students that I have had the privilege of working with during my time at FSCI. Many thanks to go to Jesse Middleton, Abigail Tester and Nicole Mooney from Chemistry Department and Maneesha Mohan, Vinay Mannam, Manpreet Cheema and Ray Trejo from Food Science and Technology Department. To my invaluable network of supportive, forgiving, generous and loving friends without whom I could not have survived the process: Ana Andino, Antonio Coello, Carolina Tovar, Andres iii Garcia, Diana Orozco, Nelly Castillo and Corina Fernandez. Special thanks to Omid Hosseinaei who was always there cheering me up and stood by me through the good and bad times. I would also like to thank my parents, two elder sisters, and elder brother. They were always supporting me and encouraging me with their best wishes. iv ABSTRACT Choline and vitamin B12 are essential nutrients for growth and performances of production animals. However, both nutrients are extensively degraded during digestion in the rumen. This thesis comprised three experiments. First, four cows equipped with a rumen cannula and catheters in the portal vein and a mesenteric artery received a post-ruminal bolus of: 1) cyanocobalamin (CN-CBL) alone (0.1 g) [gram], 2) CN-CBL (0.1 g) + casein (10 g) or 3) CN- CBL (0.1 g) + whey proteins (10 g). After the bolus, blood samples were taken until 24 h [hour] post-bolus. The intestinal absorption of CN-CBL was greater when the vitamin was given in solution with casein (4 μg [micro-gram]/h) compared with CN-CBL given alone or with whey protein (-25 μg/h and -19 μg/h, respectively). Second, a LC-MS/MS methodology was established for differentiation of choline metabolites in blood and milk, from different physiological states of the lactating cow. Total choline concentration in plasma, which was almost entirely phosphatidylcholine, increased 10-fold from early to late lactation (1,305 to 13,535 μmol/L [micro-mol per liter]). In milk, phosphocholine was the main metabolite in early lactation (492 μmol/L), but decreased exponentially through lactation to 43 μmol/L in late lactation. In contrast, phosphatidylcholine in milk was the main metabolite in mid and late lactation (188 μmol/L and 659 μmol/L, respectively). Third, the choline metabolites were measured in milk and blood after post-ruminal infusion (ABO) of choline chloride or dietary supplemented with rumen protected choline (RPC) in a low dose 12.5 g (L) and high dose 25 g (H) choline/d, respectively. Although lipid soluble metabolites or total choline in plasma were not affected by treatments, total choline was transfer into milk at a low level, 2% RPC-H and 5% ABO-H. For practical application of our findings, first, dietary formulation of CN-CBL with v addition of casein may improve CN-CBL absorption in dairy cows; second, milk from dairy cows at early lactation is higher in phosphocholine and thus might be used for infant formula to better match breast milk; third, prediction of choline supply may be possible based on betaine and phosphocholine yields in milk vi TABLE OF CONTENTS CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW ............................................... 1 Introduction ................................................................................................................................. 2 Vitamin B12 ................................................................................................................................. 3 Structure and synthesis of isomeric forms ............................................................................... 3 Distribution in food ................................................................................................................. 4 Digestion, Absorption and Metabolism ................................................................................... 4 Vitamin B12 in the ruminant .................................................................................................... 6 Choline ........................................................................................................................................ 8 Structure and Functions ........................................................................................................... 8 Distribution in food ................................................................................................................. 8 Digestion, Absorption and Metabolism ................................................................................... 9 Physiological Functions of Choline ....................................................................................... 11 Analytical methods for choline .............................................................................................. 14 Choline in the ruminant ......................................................................................................... 16 Choline in milk ...................................................................................................................... 19 Net flux of the nutrients across the gastrointestinal tract and the liver ..................................... 20 Portal drained viscera applications in choline and vitamin B12 studies ................................. 24 Summary and objectives ........................................................................................................... 26 References ................................................................................................................................. 27 Appendix ................................................................................................................................... 39 CHAPTER 2: CASEIN AND WHEY PROTEINS AS DELIVERY METHODS FOR CYANOCOBALAMIN TO INCREASE INTESTINAL ABSORPTION IN LACTATING DAIRY COWS ............................................................................................................................