EFFECT OF OLEIC ACID AND EXOGENOUS EMULSIFIERS ON FATTY ACID ABSORPTION IN LACTATING DAIRY COWS By Crystal M. Prom A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Animal Science – Doctor of Philosophy 2020 ABSTRACT EFFECT OF OLEIC ACID AND EXOGENOUS EMULSIFIERS ON FATTY ACID ABSORPTION IN LACTATING DAIRY COWS By Crystal M. Prom As the milk yield and dry matter intake (DMI) of modern dairy cows continue to increase, so too does the amount of fatty acids (FA) reaching the small intestine. Furthermore, supplemental FA are often added to the diet in order to increase the energy density of the ration and support energy requirements of the cow. Thus, the combination of supplemental FA in the diet and increasing DMI causes a significantly higher amount of FA available for absorption in the small intestine. However, previous research has demonstrated that increasing the amount of FA flowing to the small intestine negatively impacts the absorption of FA and thus the amount of energy available to the cow. Our research examined potential strategies to improve FA absorption by utilizing oleic acid (OA) and exogenous emulsifiers. In the first research chapter, we evaluated the effects of varying the ratio of supplemental dietary stearic acid (SA; C18:0) and OA (cis-9 C18:1) on FA digestibility and milk production of post-peak dairy cows. We observed that overall inclusion of supplemental fat increased milk yield, milk fat yield, 3.5% fat-corrected milk, energy- corrected milk, and feed efficiency compared to a non-fat supplemented control. Increasing OA in the supplemental fat treatments increased the digestibility and absorption of FA but did not affect production responses. Our second research chapter examined the effects of increasing doses of OA infused into the abomasum. Increasing the amount of OA reaching the duodenum did not affect DMI but increased the digestibility and absorption of total, 16-carbon, and 18-carbon FA. Higher absorbed FA increased plasma insulin, but did not affect BW or BCS, and tended to increase milk yield, 3.5% FCM, and ECM. In the third research chapter, we abomasally infused 30 g/d of three exogenous emulsifiers (Tween) that differed in the FA attached to the polysorbate base. Compared to control, the overall effect of Tween did not affect DM intake or digestibility, but increased milk fat content and tended to increase milk fat yield and 3.5% FCM. The emulsifier containing OA, Tween80, improved digestibility of 16-carbon, 18-carbon, and total FA compared to control and to the two other emulsifier treatments. Tween80 also increased milk fat content and yield compared to control. Thus, Tween80 has the potential to improve nutrient digestibility and milk production, but it is unknown whether this is primarily due to the polysorbate or the attached OA. Our last research chapter directly compared abomasal infusions of OA and Tween80, as well as examined the interaction between the two. OA increased the absorption of total and 18-carbon FA but had negligible effects on production responses. Tween80 did not affect nutrient digestibility but increased milk yield, decreased milk fat content, and did not affect milk fat yield. These results contradict previous research utilizing Tween80, but the reason for this is unclear as energy status and FA intake was similar across studies. No interactions between OA and Tween80 were observed for nutrient digestibility but an interaction was observed for ECM, milk fat yield, feed efficiency, and BCS. Overall, there is evidence that both OA and Tween80 can improve FA digestibility. When directly compared, OA was more beneficial to total, 16-carbon, and 18-carbon FA digestibility than Tween80 and providing both had no additional benefits. Therefore, increasing the amount of OA reaching the small intestine is a viable strategy to improve FA digestibility and thus energy availability to the cow. Exogenous emulsifiers can also improve FA digestibility, but results are inconsistent and should be investigated further in the future. Furthermore, combinations of OA and various emulsifiers should be further examined for independent and interactive effects. ACKNOWLEDGEMENTS None of the work I have accomplished during my time at Michigan State would have been possible without the help and support of many people. The past few years have challenged me beyond my own capabilities, and I am forever grateful for both those who pushed me and those who helped encourage me through the tough days. First off, thank you to Dr. Lock for your mentorship throughout my time here. I appreciate your coaching me and helping me be a better scientist. I would like to also thank my committee members, Dr. Mike VandeHaar, Dr. Pamela Ruegg, Dr. Andres Contreras, and Dr. Adam Moeser for investing your time and expertise in me throughout my Ph.D. Thank you as well to Dr. Rob Tempelman for always being willing to answer my statistical questions. I would also like to thank Dr. John Newbold for his enlightening discussions regarding oleic acid and fatty acid digestibility. Additionally, I would like to thank Dr. Roger Thomson and Dr. Joe Domecq for all their advice and interesting anecdotes about the dairy industry. Special thanks to Lynn Worden for your friendship, expertise, and for being up for anything. I truly don’t know what I would have done without you and will miss you dearly. Thank you as well to Dave Main for teaching and assisting Lynn and I with new techniques over the course of my experiments. I owe a huge debt of gratitude to the staff at the MSU Dairy Teaching & Research Center for your assistance with my research and good attitudes; I will miss working with you all. An enormous thank you goes to my Lock lab mates— Dr. Yan Sun, Dr. Jonas de Souza, Dr. Arnulfo Pineda, Dr. Jose Dos Santos Neto, Marin Western, Alycia Burch, Ariana Negreiro, Jair Parales Giron, and all of our wonderful undergraduate students. I greatly enjoyed working, chatting, and laughing together and will always remember our times together. Thank you as well as to all of the other graduate students in our department for your friendship and encouragement. I should iv also acknowledge MSU Dairy Store ice cream, Red Bull, and Starbucks for their help and support over the last few years; I appreciate it. Thank you to my dear family for listening to and supporting me, even if you didn’t always understand what I was talking about. Thanks as well to Heather Ruddy, Jason Almerigi, and Enzo for adopting me into your family during my time here in Michigan. And lastly, thank you to Steve Byers for always cheering me up and pushing me onwards; I look forward to many more challenges and adventures with you. v TABLE OF CONTENTS CHAPTER 1: INTRODUCTION ................................................................................................ 1 REFERENCES .......................................................................................................................... 3 CHAPTER 2: LITERATURE REVIEW ................................................................................... 5 Fat Digestion and Metabolism in Ruminants ......................................................................... 5 Intake of fat ............................................................................................................................. 5 Fatty acids in the rumen .......................................................................................................... 5 Micelle formation in the small intestine ................................................................................. 7 Fatty acid uptake by enterocytes ........................................................................................... 10 Re-esterification within the enterocyte ................................................................................. 12 Circulation of fatty acids to peripheral tissues ...................................................................... 13 Effects of Fatty Acids on Digestibility ................................................................................... 13 Effects of fatty acid profile ................................................................................................... 13 Effects of fatty acid intake .................................................................................................... 15 Opportunities to Improve Fatty Acid Absorption ............................................................... 16 Importance of improving FA absorption .............................................................................. 16 Emulsifiers ............................................................................................................................ 16 Surfactants............................................................................................................................. 17 Natural surfactant production by the ruminant ..................................................................... 19 Exogenous lecithin supplementation .................................................................................... 21 Exogenous polysorbate supplementation .............................................................................. 22 Oleic acid supplementation ................................................................................................... 24 Conclusions and Dissertation Objective ............................................................................... 26 APPENDIX .............................................................................................................................. 27 REFERENCES ........................................................................................................................ 40 CHAPTER 3: EFFECTS OF ALTERING THE RATIO OF STEARIC AND OLEIC ACIDS IN SUPPLEMENTAL FAT BLENDS ON FATTY ACID DIGESTIBILITY AND PRODUCTION RESPONSES OF DAIRY COWS ................................................................. 49 Abstract .................................................................................................................................... 49 Introduction ............................................................................................................................. 50 Materials and Methods ........................................................................................................... 51 Design and Treatments ......................................................................................................... 51 Data and Sample Collection.................................................................................................. 52 Sample Analysis.................................................................................................................... 53 Statistical Analysis ................................................................................................................ 54 Results ...................................................................................................................................... 55 Nutrient Intake and Total-tract Digestibility......................................................................... 55 Production Responses ........................................................................................................... 55 Milk Fatty Acid Sources ....................................................................................................... 56 Plasma Hormones and Metabolites ....................................................................................... 56 Discussion ................................................................................................................................ 57 vi Conclusions .............................................................................................................................. 63 Acknowledgements ................................................................................................................. 63 APPENDIX .............................................................................................................................. 64 REFERENCES ........................................................................................................................ 73 CHAPTER 4: ABOMASAL INFUSION OF OLEIC ACID INCREASES FATTY ACID DIGESTIBILITY AND PLASMA INSULIN OF LACTATING DAIRY COWS ............... 84 Abstract .................................................................................................................................... 84 Introduction ............................................................................................................................. 85 Materials and Methods ........................................................................................................... 87 Design and Treatments ......................................................................................................... 87 Data and Sample Collection.................................................................................................. 88 Sample Analysis.................................................................................................................... 89 Statistical Analysis ................................................................................................................ 90 Results ...................................................................................................................................... 91 Nutrient Intake and Total-tract Digestibility......................................................................... 91 Production Responses ........................................................................................................... 91 Milk Fatty Acid Concentration and Yield ............................................................................ 91 Plasma Components .............................................................................................................. 92 Discussion ................................................................................................................................ 92 Conclusion ............................................................................................................................... 96 Acknowledgements ................................................................................................................. 97 APPENDIX .............................................................................................................................. 98 REFERENCES ...................................................................................................................... 110 CHAPTER 5: ABOMASAL INFUSION OF DIFFERENT EXOGENOUS EMULSIFIERS ALTERS FATTY ACID DIGESTIBILITY AND MILK FAT YIELD OF LACTATING DAIRY COWS .......................................................................................................................... 121 Abstract .................................................................................................................................. 121 Introduction ........................................................................................................................... 123 Materials and Methods ......................................................................................................... 125 Design and Treatments ....................................................................................................... 125 Data and Sample Collection................................................................................................ 126 Sample Analysis.................................................................................................................. 127 Statistical Analysis .............................................................................................................. 128 Results .................................................................................................................................... 128 Nutrient Intake and Total-tract Digestibility....................................................................... 128 Production Responses ......................................................................................................... 129 Milk Fatty Acid Concentrations and Yields ....................................................................... 130 Plasma Hormones and Metabolites ..................................................................................... 130 Discussion .............................................................................................................................. 130 Conclusion ............................................................................................................................. 136 Acknowledgements ............................................................................................................... 137 APPENDIX ............................................................................................................................ 138 REFERENCES ...................................................................................................................... 147 vii CHAPTER 6: ABOMASAL INFUSION OF OLEIC ACID AND EXOGENOUS EMULSIFIER ALTER FATTY ACID DIGESTIBILITY AND PRODUCTION RESPONSES OF LACTATING DAIRY COWS .................................................................. 158 Abstract .................................................................................................................................. 158 Introduction ........................................................................................................................... 160 Materials and Methods ......................................................................................................... 161 Design and Treatments ....................................................................................................... 161 Data and Sample Collection................................................................................................ 163 Sample Analysis.................................................................................................................. 163 Statistical Analysis .............................................................................................................. 164 Results .................................................................................................................................... 165 Nutrient Intake and Total-tract Digestibility....................................................................... 165 Production Responses ......................................................................................................... 165 Milk Fatty Acid Concentration and Yield .......................................................................... 166 Discussion .............................................................................................................................. 166 Conclusion ............................................................................................................................. 170 Acknowledgements ............................................................................................................... 171 APPENDIX ............................................................................................................................ 172 REFERENCES ...................................................................................................................... 180 FINAL CONCLUSIONS .......................................................................................................... 194 viii LIST OF TABLES Table 2.1. Amphiphilic properties of selected polar lipids. Adapted from Freeman (1969). ...... 28 Table 2.2. Critical micellar concentration and saturation ratio of fatty acids in sodium glycodeoxycholate solution at 37°C. Adapted from Freeman (1969). ......................................... 29 Table 3.1. Proportion of each fatty acid (FA) supplement for treatment blends and FA profile of FA blends. ..................................................................................................................................... 65 Table 3.2. Ingredient and nutrient composition of treatment diets. ............................................. 66 Table 3.3. Nutrient intake and apparent digestibility of cows fed increasing amounts of oleic acid (n = 8). ........................................................................................................................................... 67 Table 3.4. DMI, milk yield, milk composition, BW, and BCS of cows fed increasing amounts of oleic acid (n = 8). .......................................................................................................................... 68 Table 3.5. Summation of milk fatty acid (FA) concentration and yield for cows infused with treatments (n = 8). ......................................................................................................................... 69 Table 3.6. Milk fatty acid yield of cows fed increasing amounts of oleic acid (n = 8). ............... 70 Table 3.7. Milk fatty acid concentration of cows fed increasing amounts of oleic acid (n = 8). . 71 Table 3.8. Blood NEFA, BHBA, and insulin of cows fed increasing amounts of oleic acid (n = 8). .................................................................................................................................................. 72 Table 4.1. Fatty acid (FA) profile of dietary fat supplement and infused oleic acid. .................. 99 Table 4.2. Ingredient and nutrient composition of diet. ............................................................. 100 Table 4.3. Nutrient intake and apparent digestibility of cows infused with increasing amounts of oleic acid (n = 8). ........................................................................................................................ 101 Table 4.4. Milk yield, milk composition, BW, and BCS of cows infused with increasing amounts of oleic acid (n = 8). .................................................................................................................... 102 Table 4.5. Milk fatty acid yields of cows infused with increasing amounts of oleic acid (n = 8). ..................................................................................................................................................... 103 ix Table 4.6. Milk fatty acid concentrations of cows infused with increasing amounts of oleic acid (n = 8). ......................................................................................................................................... 105 Table 4.7. Plasma NEFA, BHBA, and insulin of cows infused with increasing amounts of oleic acid (n = 8). ................................................................................................................................. 107 Table 4.8. Fatty acid content of plasma triglycerides of cows infused with increasing amounts of oleic acid (n = 8). ........................................................................................................................ 108 Table 5.1. Fatty acid (FA) profile and total FA content of Tween supplements infused during treatment periods. ........................................................................................................................ 139 Table 5.2. Ingredient and nutrient composition of diet fed to all cows infused with treatments (n = 8). ............................................................................................................................................. 140 Table 5.3. Nutrient intake and total-tract nutrient digestibility of cows infused with treatments (n = 8). ............................................................................................................................................. 141 Table 5.4. Milk yield, milk composition, BW, and BCS of cows infused with treatments (n = 8). ..................................................................................................................................................... 142 Table 5.5. Summation of milk fatty acid (FA) concentration and yield for cows infused with treatments (n = 8). ....................................................................................................................... 143 Table 5.6. Milk fatty acid yield of cows infused with treatments (n = 8). ................................. 144 Table 5.7. Milk fatty acid concentration of cows infused with treatments (n = 8). ................... 145 Table 5.8. Plasma NEFA, insulin, and glucose of cows infused with treatments (n = 8). ......... 146 Table 6.1. Fatty acid (FA) profile and total FA content of oleic acid and Tween supplements infused during treatment periods. ............................................................................................... 173 Table 6.2. Ingredient and nutrient composition of diet fed to cows infused with treatments (n = 8). ................................................................................................................................................ 174 Table 6.3. Nutrient intake and total-tract nutrient digestibility of cows infused with treatments (n = 8). ............................................................................................................................................. 175 Table 6.4. Milk yield, milk composition, BW, and BCS of cows infused with treatments (n = 8). ..................................................................................................................................................... 176 x Table 6.5. Summation of milk fatty acid (FA) concentration and yield for cows infused with treatments (n = 8). ....................................................................................................................... 177 Table 6.6. Milk fatty acid yield of cows infused with treatments (n = 8). ................................. 178 Table 6.7. Milk fatty acid concentration of cows infused with treatments (n = 8). ................... 179 xi LIST OF FIGURES Figure 2.1. Glycerol phosphate pathway for de novo triacylglycerol (TAG) and glycerophos- pholipid synthesis.......................................................................................................................... 30 Figure 2.2. The tissue origins and postsecretory metabolic transfonnation of lipoproteins within the plasma compartment in ruminants .......................................................................................... 31 Figure 2.3. Relationship between study-adjusted C18:0 intestinal digestibility and duodenal flow of C18:0. ....................................................................................................................................... 32 Figure 2.4. Total FA digestibility for cows fed FA treatment diets (n = 24) ............................... 33 Figure 2.5. Relationship between study-adjusted total FA intestinal digestibility and total FA intake ............................................................................................................................................. 34 Figure 2.6. Relationship between study-adjusted total FA intestinal digestibility (%) and total FA duodenal flow (g/d) for 61 observations from 15 studies ....................................................... 35 Figure 2.7. Graphical representations of water-in-oil emulsions, bicontinuous emulsions, and oil- in-water emulsions ........................................................................................................................ 36 Figure 2.8. Differences in wetting behavior after two seconds of water droplet attachment ...... 37 Figure 2.9. Graphical representations of common polysorbate (Tween©) products ................... 38 Figure 2.10. Total FA digestibility for cows infused with Tween80 (n = 8) ............................... 39 Figure 4.1. Timeline of experimental periods. ........................................................................... 109 xii KEY TO ABBREVIATIONS AOAC Association of Official Agricultural Chemists BCS BH body condition score biohydrogenation BHBA -hydroxybutyrate BW C body weight carbon C16:0 palmitic acid C18:0 stearic acid cis-9 C18:1 oleic acid CM chylomicron(s) CMC critical micelle concentration CO2 CON CP d DIM DM DMI carbon dioxide control treatment crude protein day(s) days in milk dry matter dry matter intake ECM energy-corrected milk ER FA endoplasmic reticulum fatty acid(s) xiii FABP fatty acid binding protein(s) FATP fatty acid transport protein FCM FFA fat-corrected milk free fatty acid(s) FFAR free fatty acid receptor g GLC GPR h gram(s) gas-liquid chromatography G-protein coupled receptor hour(s) iNDF Indigestible NDF kg kilogram(s) LCFA long-chain fatty acid(s) min mL mm n minutes(s) milliliter(s) millimeter(s) number NDF neutral detergent fiber NEFA non-esterified fatty acid(s) NRC National Research Council OA PA PL oleic acid palmitic acid phospholipid(s) PUFA polyunsaturated fatty acid(s) xiv SA SAS SD SE stearic acid Statistical Analysis System standard deviation standard error SEM standard error of the mean T40 T60 T80 Tween®-40 Tween®-60 Tween®-80 TAG triacylglycerol TG triglyceride(s) TMR total mixed ration Trt VFA P-value associated with the treatment effect volatile fatty acid(s) VLCFA very long-chain fatty acid(s) VLDL very low-density lipoprotein(s) xv 1. CHAPTER 1: INTRODUCTION Agriculture industries are tasked with providing sufficient food for the global human population. Dairy products offer nutritious and healthful food options to the global population (Lawson et al., 2005; Álvarez-León et al., 2006; Aryana and Olson, 2017). As such, improving the quality and efficiency of dairy production, as well as the health and well-being of animals, is of utmost importance. Nutritionists, geneticists, and dairy producers continue to increase milk yield and feed efficiency of lactating dairy cows. Common feedstuffs in the diet of dairy cows are low in fatty acids (FA), but as milk yield and dry matter intake (DMI) increases, so too does the total intake of FA by the cow. In addition to the FA contained in the basal diet, supplemental FA are often added to the diet in order to increase the energy density of the ration and support nutrient requirements of the cow. Thus, the combination of supplemental FA in the diet and increasing DMI results in increasing amounts of FA available for absorption in the small intestine. However, it is established that increasing the amount of FA reaching the small intestine decreases the absorption of FA (Boerman et al., 2015). Because FA represent an important energy source, as well as possessing many other roles in the body, increasing the post-ruminal absorption of FA is important in order to maximize the FA available for use by the cow. Previous research suggests that increasing the amount of oleic acid (OA) reaching the small intestine improves digestibility and absorption of FA, especially those containing 18 carbons. OA likely improves FA digestibility due to natural amphiphilic characteristics that improves micelle formation and increases solubility of FA into the micelle (Freeman, 1969). These processes are critical to FA absorption as micelles transport FA across the unstirred water layer of the small intestine to the apical surface of the enterocyte (Noble, 1981). OA may also affect uptake and re- 1 esterification rates by the enterocyte (Ockner et al., 1972). Thus, increasing the amount of OA reaching the small intestine may increase overall FA absorption and improve milk yield in the cow. It is likely that the negative effects of increasing FA intake on FA absorption may be due to limitation in the natural capacity of the cow to produce lysolecithin and bile salts, which are both necessary emulsifiers for effective micelle formation (Harrison and Leat, 1972; Noble, 1981). As such, providing supplemental emulsifiers to the cow may aid in micelle formation and thus, FA absorption. Previous research has supplemented lecithin and lysolecithin in the diet, but results appear to be unsatisfactory due to hydrogenation in the rumen (Lee et al., 2018; Fontoura et al., 2019). However, an emulsifier commonly utilized in food and pharmaceutical industries called polysorbate (Tween80©) has been observed to increase FA absorption and production responses in dairy cows (de Souza et al., 2020). Many other emulsifiers are commercially available in food and pharmaceutical markets, including other products in the polysorbate family, and should be investigated for their potential to improve FA absorption in dairy cows. Exogenous emulsifiers and OA both appear to be viable strategies to increase FA absorption and should be further investigated, both independently and in combination. Thus, our overall objective for this dissertation was to examine OA and different types of Tween as potential strategies to improve FA absorption and production responses in lactating dairy cows. 2 REFERENCES 3 REFERENCES Álvarez-León, E.-E., B. Román-Viñas, and L. Serra-Majem. 2006. Dairy products and health: a review of the epidemiological evidence. Br. J. Nutr. 96:S94–S99. doi:10.1079/bjn20061709. Aryana, K.J., and D.W. Olson. 2017. A 100-Year Review: Yogurt and other cultured dairy products. J. Dairy Sci. 100:9987–10013. doi:10.3168/jds.2017-12981. Boerman, J.P., J.L. Firkins, N.R. St-Pierre, and A.L. Lock. 2015. Intestinal digestibility of long- chain fatty acids in lactating dairy cows: A meta-analysis and meta-regression. J. Dairy Sci. 98:8889–8903. doi:10.3168/jds.2015-9592. Fontoura, A.B.P., J.E. Rico, K.M. Keller, A.N. Davis, W.A. Myers, J.T. Siegel, R. Gervais, and J.W. McFadden. 2019. Effects of lecithin supplementation on milk production and circulating markers of metabolic health in Holstein cows. J Dairy Sci 102:427. Freeman, C.P. 1969. Properties of fatty acids in dispersions of emulsified lipid and bile salt and the significance of these properties in fat absorption in the pig and the sheep. Br. J. Nutr. 23:249. doi:10.1079/bjn19690032. Harrison, F.A., and W.M.F. Leat. 1972. Absorption of palmitic, stearic and oleic acids in the sheep in the presence or absence of bile and/or pancreatic juice. J. Physiol. 225:565–576. doi:10.1113/jphysiol.1972.sp009956. Lawson, R.E., A.R. Moss, and D. Ian Givens. 2005. The role of dairy products in supplying conjugated linoleic acid to man’s diet: a review. Nutr. Res. Rev. 14:153–172. doi:10.1079/095442201108729178. Lee, C., D. Morris, J. Copelin, J. Hettick, and I. Kwon. 2018. Effects of lysophospholipids on short-term production, nitrogen utilization, and rumen fermentation and bacterial population in lactating dairy cows. J. Dairy Sci. 96:128. doi:10.3168/jds.2018-15777. Noble, R.C. 1981. Digestion, absorption and transport of lipids in ruminant animals. Prog. Lipid Res. 17:55–91. doi:10.1016/b978-0-08-023789-3.50007-6. Ockner, R.K., J.P. Pittman, and J.L. Yager. 1972. Differences in the Intestinal Absorption of Saturated and Unsaturated Long Chain Fatty Acids. Gastroenterology 62:981–992. doi:10.1016/S0016-5085(72)80115-X. de Souza, J., M. Western, and A.L. Lock. 2020. Abomasal infusion of exogenous emulsifier improves fatty acid digestibility and milk fat yield of lactating dairy cows. J Dairy Sci in press. 4 2. CHAPTER 2: LITERATURE REVIEW Fat Digestion and Metabolism in Ruminants Intake of fat The typical feedstuffs fed to ruminant animals are low in FA content. Thus, the amount of FA consumed by ruminants relative to body weight is much lower than that of monogastrics (Noble, 1981). The majority of FA consumed by the cow are in the form of triglycerides (TG), which are found mainly in cereal grains, oilseeds, animal fats, and byproduct materials, or glycolipids, which are the predominant lipid type in forages. Phospholipids (PL) and free FA (FFA) are also present in feedstuffs in minor amounts. The major FA present in feedstuffs consumed by the cow are primarily the polyunsaturated FA linoleic (~20%) and linolenic (60-70%) acids (Noble, 1981). While monogastrics rely heavily on digestive enzymes to digest lipids, these enzymes only play minor roles in ruminant FA digestion. The intake of feedstuffs by the cow will trigger salivary lipase release from the vallate papillae of the tongue, the glossoepiglottis, and the pharyngeal end of the esophagus, as well as from submaxillary and sublingual glands (Sissons, 1981). The salivary lipase will begin breaking down a minor amount of lipids by releasing FA from triglycerides (Sissons, 1981; Keomanivong, 2016). Fatty acids in the rumen Once lipids enter the rumen, they will undergo the processes of lipolysis and biohydrogenation (BH). Lipolysis, which is the release of FA from esterified plant lipids, is a prerequisite for BH of unsaturated FA to occur (Harfoot, 1981; Jenkins, 1993). Microbes in the rumen produce extracellular lipases that efficiently hydrolyze the ester linkages in glycerol-based lipids to release FFA and glycerol (Jenkins, 1993). The hydrolysis of TG appears to be the limiting step as di- and monoglyceride intermediates are rarely detected (Garton et al., 1961; Harfoot, 1981). 5 The glycerol produced is rapidly fermented to volatile FA while unsaturated FA will then undergo the process of BH (Garton et al., 1961). Biohydrogenation, by which rumen microbes saturate double bonds in FA, is a process undertaken by microbes primarily to protect themselves from the bacteriostatic effects of unsaturated FA (Jenkins, 1993; Maia et al., 2010). Early work proposed the process was a way of disposing reducing power, but that has since been shown to be a negligible consequence of the process (Jenkins, 1993; Maia et al., 2010). The work by Maia and others (Maia et al., 2007, 2010) has aimed to understand the toxic effects of unsaturated FA on rumen microbes, but have yet to reach a final conclusion. Various possibilities are discussed by Maia et al. (2010). An obvious explanation is disruption of the lipid bilayer structure due to double bonds causing kinking, yet this does not explain clear differences of chain length on bacteria. Another possible explanation is a metabolic effect involving acyl CoA. However, butyrate-forming bacteria, such as the Butyrivibrio phylogenetic group have been shown to be the most sensitive to polyunsaturated FA (PUFA), suggesting the toxicity might be related to the butyrate kinase mechanism instead of the acyl CoA transferase (Paillard et al., 2007; Maia et al., 2010). It should also be noted that the BH process takes place on the surface of feed particles as FA are adsorbed from rumen fluid onto the feed (Palmquist and Jenkins, 2017). As such, feeding high-forage diets have demonstrated beneficial effects on rumen characteristics when supplemental fat is included in the diet (Weiss and Pinos-Rodríguez, 2009; Piantoni et al., 2015a). However, the interrelationships between microbiome, rumen pH, dietary nutrients, and FA are not well understood and need to be studied in further detail. The impact of these variables on alternate biohydrogenation pathways should also be considered. These pathways, and their effect on metabolism and milk FA synthesis, have been 6 reviewed in detail and will not be discussed further in this literature review (Moate et al., 2004; Bauman et al., 2008; Jenkins et al., 2008). Micelle formation in the small intestine Following lipolysis and BH, little to no long-chain FA (LCFA) are absorbed across the rumen epithelium or catabolized to volatile FA (VFA) or CO2 (Jenkins, 1993). Meanwhile, microbes will be synthesizing FA from carbohydrate sources. Thus, lipids leaving the abomasum and entering the duodenum of the small intestine are primarily in the form of saturated FFA (70- 80%) from either dietary or microbial origin, with the remainder being microbial PL or rumen inert TG (Moore and Christie, 1984; Bauchart, 1993). These lipids will be strongly adsorbed onto particulate matter due to the acidic conditions of the abomasum and duodenum (Noble, 1981). The intestinal lumen of the ruminant consists of an insoluble particulate phase and a soluble micellar phase (Harrison and Leat, 1975). In order to cross the aqueous environment from the insoluble, particulate phase to the apical surface of the enterocyte, the FFA, PL, and TG require transfer to the soluble, micellar phase. Micelles are bi-layer disks that are aggregates of bile salts, PL, TG, and FFA. The outer edge of the micelle is formed by the hydrophilic head groups with the hydrophobic tails facing towards the center. While similar to bilayer plasma membranes, micelles consist of only a single layer of lipids. Phospholipids and FA with amphipathic characteristics, such as oleic acid (OA), can be located in the formative aggregate layer of the micelle while FA with lower solubility, such as stearic acid, will be located in the hydrophobic inner core (Freeman, 1969; Hofmann and Mysels, 1987). Bile salts and PL are both critical for efficient FA absorption by the micelle. Work in sheep that isolated entry of bile and pancreatic juice to the small intestine observed malabsorption of FA in the absence of either bile or pancreatic juice (Harrison and Leat, 1972). Both bile salts and PL, 7 whether from ruminal or biliary origins, are characterized as soluble amphiphiles, meaning they have a polar hydrophilic head group and a non-polar hydrophobic tail, and will self-aggregate in aqueous environments to form micelles (Carey and Small, 1970; Hofmann and Mysels, 1987). Bile salts also play an important role in monogastrics to emulsify lipid droplets into smaller droplets to increase the surface area and thus access by pancreatic lipase; however, this action is negligible in ruminants as the majority of TG have been hydrolyzed prior to reaching the small intestine (Sissons, 1981). Cholesterol is utilized to synthesize bile acids in the liver, which are then conjugated with either glycine or taurine into bile salts. Ruminant animals primarily produce taurine-conjugated bile salts in order to remain ionized in acidic conditions as to remain in the micellar phase rather than adsorbing onto particulate matter (Harrison and Leat, 1975; Noble, 1981). Production of taurine-conjugated bile salts by ruminants differs from monogastrics, who primarily conjugate bile salts with glycine, due to the pH of the ruminant small intestine remaining low until the terminal jejunum (Noble, 1981). Following production in the liver, bile salts are stored in the gall bladder (Maldonado-Valderrama et al., 2011). The presence of LCFA in the intestinal lumen of mice has been shown to signal I-cells via G-protein coupled receptor 40 (GPR-40, also known as FFAR1) to secrete cholecystokinin (Liou et al., 2011). Cholecystokinin then stimulates the gall bladder to release bile into the intestinal lumen in order to promote absorption of FA. In addition to bile salts, bile contains lecithin and cholesterol (Carey et al., 1983). While bile salts are necessary to the process of micelle formation, pure bile salts have been shown to be poor emulsifiers of fat and require lecithin in order to properly absorb FA (Carey et al., 1983). A study in sheep utilizing just the bile salt sodium taurocholate found that solubilization of LCFA was greatly reduced compared to whole bile (Noble, 1981). Similarly, Lough and Smith (1976) observed that lysolecithin was a more effective solubilizer of FA than bile salts were on a molar basis. 8 Lecithin, and its derivative lysolecithin, is a term that can refer to several different PL. Lecithin specifically consists of a hydrophilic head group and two hydrophobic hydrocarbon tails (McFadden, 2019). It was first isolated from egg yolk and was thus named based on the Greek word ‘lekithos’, meaning egg yolk, by Theodore-Nicolas Gobley in 1850 (Sourkes, 2004). The main PL that make up lecithin in bile are phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol (McFadden, 2019). It was suggested 50 years ago that pancreatic juice is necessary for optimum fat absorption in sheep (Harrison and Leat, 1975). The authors correctly hypothesized that this is due to the need for phospholipases in the pancreatic juice to hydrolyze lecithin to lysolecithin. Lysolecithin action is negligible in monogastrics as mono-glyceride, derived from TG in the small intestine, acts as a potent emulsifier to support micelle formation (Harrison and Leat, 1975). Both phospholipase A1 and A2 are present in pancreatic juice; the former will hydrolyze FA in the sn-1 position, which are often saturated FA, while the latter will hydrolyze FA in the sn-2 position, which are often unsaturated FA (Noble, 1981). Phospholipase A2 is more stable in acidic environments and is thus likely to be more active in the upper small intestine while phospholipase A1 is generally inactive until the mid-jejunum (Noble, 1981). Once formed, lysolecithin serves as an emulsifying detergent to aid in micelle formation and solubilization of FA by the micelle. Transfer of lipid material from feed particles into the micelle occurs as the particulate matter passes through the small intestine. Research in sheep estimates that 5% of the transfer of FA occurs in the duodenum, 20% in the upper jejunum, 25% in the mid-lower jejunum, and 50% in the ileum (Leat and Harrison, 1975; Bauchart, 1993). Lipids entering the small intestine as TG will likely not be absorbed until further along the intestinal tract due to requiring hydrolysis by pancreatic lipase (Harrison and Leat, 1975; Sissons, 1981). Lysolecithin will be absorbed along 9 with the FA and will then be reacylated back into lecithin (Harrison and Leat, 1975; Noble, 1981). The majority of bile salts (95%) will be de-conjugated and the resulting bile acids will be re- absorbed in the ileum and recycled to the liver via enterohepatic circulation (Maldonado- Valderrama et al., 2011). Fatty acid uptake by enterocytes Little to no research examining the transfer of FA from the micelle to the enterocyte has been conducted in bovine models. Thus, the discussion on this process will be based on research conducted in human and murine models. There is not a consensus on how the process of FA adsorption from the micelle into enterocyte works in the human body. Hamilton (1998) breaks the process of FA transport into cells into three distinct steps: adsorption, transmembrane movement, and desorption. Meaning, the FA must first adsorb to the outer leaflet of the plasma membrane of the cell, then cross the membrane and re-orient the polar head group to the cytosolic face, then leave the inner leaflet. However, this process as described by Hamilton (1998) is for general transport of FA into cells and is not specific to enterocytes. Jay and Hamilton (2018) have also recently supported the theory that FA cross into cells via flip-flop passive diffusion. In contrast, Wang et al. (2013) disagrees with the passive diffusion model and hypothesizes that the transporters CD36, FATBP, and FABP play important roles. Potentially both theories are correct and protein-mediated uptake mechanisms become more important in the case of low extracellular FA concentration (Iqbal and Hussain, 2009; Hussain, 2014). CD36 (also known as SR-B2 or FA translocase) is a brush border membrane protein that has been shown to be important in facilitating FA uptake and processing within the enterocyte, both in humans and rodents (Cifarelli and Abumrad, 2018). Stahl et al. (2001) states that while CD36 doesn’t appear to be the primary FA transporter, it does seem to be required for FA uptake. Although Jay and Hamilton (2018) disagree 10 with the role CD36 plays in transport, they do support the hypothesis that CD36 is important for esterification within the cell. Another potential transporter is FA transport protein 4 (FATP4), which is the only FATP expressed in the intestines (Stahl et al., 2001). FATP family members are integral membrane proteins that have an affinity for LCFA and very long-chain FA (VLCFA; Pohl et al., 2004; Gimeno, 2007). As with CD36, the exact role of FATP4 is debated. It does not appear to directly translocate FA across the plasma membrane but affects absorption in other ways, possibly by increasing FA esterification at the endoplasmic reticulum (ER) level to drive FA uptake (Jia et al., 2007; Cifarelli and Abumrad, 2018). It is also possible that FATP4 facilitates FA transport by trapping CoA derivatives of FA that entered the cell via diffusion, thus lowering the concentration of FFA in the cytosol and lowering the concentration gradient (Stahl et al., 2001; Jia et al., 2007). Pohl et al. (2004) further proposes that FATP supports LCFA uptake by converting LCFA to their CoA derivatives, which are necessary to maintain lipid raft structures within cellular membranes. CD36, caveolin-1, and other proteins may use these lipid rafts to aid in LCFA transport (Pohl et al., 2004). Lastly, it has been proposed that FA binding protein (FABP) 1 and 2 may play roles in FA transport due to their high-affinity for binding LCFA within the intestine (Gajda and Storch, 2015). In support of this theory, a study fed FABP2-/- knockout mice a high fat diet and observed weight loss and decreased incorporation of FA in TG (Thumser et al., 2014). It has been hypothesized that FABP1 has a distinct role from FABP2 in the intestine and mediates the formation of prechylomicron transport vesicles by fusing FABP1 with the ER (Siddiqi and Mansbach, 2012; Thumser et al., 2014). Overall, there is clearly more research needed to understand how FA transfer occurs between the micelle and the enterocyte. Furthermore, it is still unknown how the chain length or amount of saturation of a FA affects the uptake rate (Ockner et 11 al., 1972; Wang et al., 2013). Thus, the FA present in the micelles of the small intestine of the cow may also affect FA absorption and should be studied further. Re-esterification within the enterocyte In addition to FA exerting different effects on FA uptake by the enterocyte, they may also affect re-esterification rates within the enterocyte. Following uptake by the enterocyte, FFA are transported to organelles for further processing via FABP1 and FABP2 (Iqbal and Hussain, 2009). One of the most important processes, re-esterification, will occur in the ER. Glycerol, sourced from absorbed glucose, and three FA will be esterified into TG via the α-glycerophosphate pathway in ruminant animals (Noble, 1981). The re-esterification process consists of three esterification reactions and one hydrolysis reaction as described by Takeuchi and Reue (2009; Figure 1) and Gruffat et al. (1996). First, glycerol-3 phosphate will be activated to acyl-glycerol- 3 phosphate (also known as lysophosphatidic acid) by glycerol-3 phosphate acyltransferate. Next, a second FA is added by 1-acylglycerol-3-phosphate acyltransferase, resulting in phosphatidic acid. Phosphatidic acid can then be used to synthesize other phospholipids or diacylglycerol (DAG). DAG is formed by the enzyme phosphatidate phosphate-1 then further converted to triacylglycerol (TAG) by diacylglycerol acyltransferase. Monogastrics will bypass the beginning of the α- glycerophosphate pathway due to the presence of monoacylglycerol (Noble, 1981; Iqbal and Hussain, 2009; Takeuchi and Reue, 2009). While not discussed in detail here, PL and cholesterol undergo similar esterification pathways within the enterocyte. As previously stated, FATP4 may function at the ER level to increase the rate of esterification, which would then decrease the intracellular FA concentration and increase the rate of FA uptake (Cifarelli and Abumrad, 2018). Thus, the re-esterification of FA within the enterocyte presents another opportunity to improve FA absorption in dairy cows and should be investigated. 12 Circulation of fatty acids to peripheral tissues Following re-esterification, TG, PL, and cholesterol will be incorporated into lipoproteins, either chylomicrons (CM) or very low-density lipoproteins (VLDL), to enter lymphatic circulation. CM are the largest and least dense lipoprotein. The primary role of CM is to transport TG to peripheral tissues for fat storage in adipose tissue, production of milk fat in mammary tissue, or oxidation in muscle (Bauchart, 1993). VLDL, which are smaller and less dense than CM, also function to transport TG to peripheral tissues. As discussed in detail by Bauchart (1993; Figure 2), there is evidence that unsaturated FA will stimulate CM secretion while saturated FA stimulate VLDL secretion. CM and VLDL will both exit the enterocyte by passing through the basal membrane into the lymphatic system via lacteals (Tso and Balint, 1986). The lymphatic system will circulate dietary FA to the peripheral tissues for storage, oxidation, or milk fat synthesis prior to remnants being transported to the liver to be utilized or re-circulated to the peripheral tissues. These processes will not be covered in further detail as the purpose of this review is to focus on digestibility and absorption of FA. Effects of Fatty Acids on Digestibility Effects of fatty acid profile Scientists have been studying the differing effects of individual FA on FA digestibility for many years. A study in sheep observed that the chain length of saturated FA was inversely related to FA digestibility (Steele and Moore, 1968). Another study in sheep supported this finding and also investigated the digestibility of unsaturated FA. They found that the digestibility coefficients of palmitic (PA; C16:0), stearic (SA; C18:0), and oleic (OA; cis-9 C18:1) acids were 87%, 82%, and 87%, respectively (Andrews and Lewis, 1970). Harrison and Leat (1972) followed up this work by duodenally infusing radio-labeled FA in sheep and observed that absorption was greatest 13 for OA, followed by PA, then SA. Similarly, in vitro experiments by Lough and Smith (1976) utilizing bile salt solutions demonstrated that the order of solubility was linoleic (C18:3) > OA > elaidic (trans-9 C18:1) > PA > SA. The characteristics of bile salt solutions appear to have minor effects on solubility as similar work that used a sodium glycodeoxycholate solution ranked the solubility of these FA as OA > linoleic > elaidic > PA > SA (Hofmann, 1963). These FA also differed in their critical micelle concentration (CMC). CMC, which will be discussed in more detail in a later section, is defined as the amount of a substance required for micelles to form. These distinctions in solubility and CMC between the FA cause clear differences in FA digestibility in the dairy cow. Borsting et al. (1992) fed different amounts of encapsulated vegetable oil, saturated fat, and fish oil and found that PA had higher true ileal digestibility than SA. A meta-analysis studying the effects of saturation on FA digestibility observed that increasing iodine value (increasing unsaturation) of dietary fats increased apparent FA digestibility and appeared to increase emulsification of fat in the small intestine (Firkins and Eastridge, 1994). A study feeding diets with no added fat or 5% added fat as either hydrogenated tallow, tallow, or animal-vegetable fat found that increasing the unsaturation of supplemental fat increased apparent and total-tract digestibilities of 16-carbon, 18-carbon, and total FA (Pantoja et al., 1996b). Another study supplementing 5% FA as either tallow, partially hydrogenated tallow, or hydrogenated tallow also observed that total-tract FA digestibility increased as unsaturation increased (Elliott et al., 1999). Harvatine and Allen (2006a) observed similar findings in a study altering amounts of saturated and unsaturated FA in a dietary fat supplement. They concluded that increasing the amount of saturated FA had detrimental effects on post-ruminal 18-carbon FA digestibility (Harvatine and Allen, 2006a). A meta-analysis by Boerman et al. (2015) confirmed a clear negative relationship between SA concentration and apparent 18-carbon FA digestibility (Figure 3). Recent 14 studies have continued to confirm that OA in FA supplements have the highest FA digestibility, followed by PA, then SA (Figure 4; Chamberlain and DePeters, 2017; de Souza et al., 2018, 2019). Effects of fatty acid intake In addition to the FA profile of fat supplements, researchers have investigated the effects amount of FA have on FA digestibility. While some authors have suggested that FA has no effect on FA digestibility (Chilliard et al., 1991; Börsting et al., 1992; Doreau and Ferlay, 1994), others have observed a relationship between the two (Jenkins and Jenny, 1989; Palmquist, 1991; Weisbjerg et al., 1992). A meta-analysis by Boerman et al. (2015) investigated these relationships in depth. They found that increasing the intake of FA, whether via FA supplementation or increased FA in the basal diet, negatively affected total FA digestibility (Figure 5). Logically, this relationship held true for duodenal flow of FA and total FA digestibility (Figure 6). When the effects on FA digestibility were examined by individual FA, it was clear that SA strongly depresses SA digestibility (Figure 3). Since the majority of FA reaching the duodenum are saturated FA (Bauchart, 1993), the negative relationship between SA flow and SA digestibility can have serious ramifications on total FA digestibility. SA has relatively low solubility in the aqueous environment of the small intestine (Hofmann, 1963; Freeman, 1969). Thus, increasing the amount of FA, especially SA, reaching the duodenum due to increased FA intake can stress the capacity of the cow to form micelles and solubilize FA. These limitations could be due to a finite amount of lecithin, pancreatic phospholipase, or bile salts produced by the cow, but more work should investigate the relationships between FA and these variables. Importantly, while there appears to be a limit to FA absorptive capacity in the dairy cow, there is potential to improve this capacity by altering the profile of FA reaching the small intestine. 15 Opportunities to Improve Fatty Acid Absorption Importance of improving FA absorption FA are important nutrients for ruminant animals for several reasons. Chiefly among these is the energy FA contain due to their chemical structure. FA contain approximately 9 calories/g as compared to 4 calories/g that proteins and carbohydrates contain. As such, FA provide an important energy source to meet the metabolic requirements and support increased milk yield potential of lactating dairy cattle. The natural FA content of a basal diet of a dairy cow is usually less than 5% due to the relatively low FA content of forages. However, researchers over the past 100 years have investigated the potential of adding supplemental FA to the diet to support milk and milk fat production, reproduction, and energy balance of the animal (Palmquist and Jenkins, 2017). While various combinations of types of FA supplements, FA profile of supplements, and amount of supplemental FA provided have been investigated over the years, detrimental effects are typically seen if more than 5% supplemental FA is fed (Palmquist and Jenkins, 1980). The upper limit of amount of supplemental FA tolerated by the animal is typically dictated by the effects of the supplemental FA in the rumen. As previously discussed, unsaturated FA are toxic to rumen microbes and thus need to undergo BH. At a certain point, the potential for BH by the microbes reaches maximal capacity and ruminal fermentation and digestion becomes disrupted (Jenkins, 1993). Due to this limitation on supplemented FA intake, it is critical to improve the absorption of FA reaching the small intestine in order to utilize dietary and supplemental FA. Emulsifiers There is potential to improve FA absorption by increasing the amount of micelle formation and FA solubility within the small intestine by utilizing supplemental emulsifiers. Emulsifiers are a sub-class of colloids, which refer to a normally immiscible substance dispersed in another 16 substance. A classic example of an emulsion is milk as lipid globules are suspended in water. The word “emulsion” is thus derived from the Latin word “emulgere” meaning “to milk out”. While the word has its root in dairy practices, it has now been applied to any suspension where both substances are liquids. Emulsions can either be oil-in-water or water-in oil (Figure 7), with the first word of the phrase referring to the dispersed phase and the second referring to the continuous phase. Besides milk, common examples of oil-in-water emulsions are lipoproteins in blood, cream foam in espresso, mayonnaise, and vinaigrette salad dressing. Water-in-oil emulsions are less common, but examples include butter and margarine. Some of these emulsions, such as milk, are naturally formed, but most require mechanical actions of centrifugation, homogenization, or mixing to form (Kralova and Sjöblom, 2009). These chemically formed emulsions are often unstable and quickly separate. However, surface active agents, referred to as surfactants, can be added to an emulsion in order to decrease the interfacial tension between the dispersed and continuous phases and thus increase the kinetic stability of the substance (Milić et al., 2017). Surfactants Surfactants are emulsifiers that are considered amphiphiles as they contain a polar, hydrophilic headgroup and a non-polar, hydrophobic tail (Carey and Small, 1970). While the headgroup will dictate the ionic charge of the surfactant, the tail length and saturation will dictate the hydrophobicity (Nagarajan, 2002; Rapp, 2017). Surfactants in aqueous phases aggregate together to form micelles, where the hydrophilic heads are in contact with the polar aqueous dispersed phase while the hydrophobic tails form the micelle core (Rapp, 2017). These micelles then allow for the transport of lipo-soluble molecules through aqueous environments (Reis et al., 2004). Surfactants in non-polar solutions will face the opposite direction so that the hydrophobic tail is facing outwards and the polar headgroup will be facing inwards. Thus, the outward face of 17 the surfactant will always match the polarity of the solution while the inward face will match the polarity of the suspended particles. Particles with similar polarity will naturally aggregate together and form larger droplets with less stable surface tension, so the addition of surfactants increases the stability of a suspension by masking the suspended particle and exposing the same polarity as that of the solvent. Thus, surfactants can reduce the amount of droplet coagulation and merging (Rapp, 2017). Surfactants also prevent the coalescence of newly formed droplets by repulsive electrostatic, steric, or electrosteric forces and thus stabilize the emulsion (Milić et al., 2017). Surfactants can be further categorized as detergents, wetting agents, foaming agents, nanoemulsions, dispersants, or combinations thereof. This review will discuss detergents as they are most relevant to the discussion at hand but will not further delve into the other classes of surfactants. All surfactants are generally characterized by their headgroup charge which then affects the action by which they maintain interface integrity. The four categories of surfactants are anionic, cationic, zwitterionic/amphoteric, and nonionic (Kume et al., 2008). Ionic surfactants mainly act through electrostatic repulsive forces. Anionic surfactants often contain a sulfur, sulfonate, or carboxylate attached to them and are commonly included in household cleaning products (Kume et al., 2008). Conjugated bile acids, such as taurocholic acid in the ruminant, are considered anionic surfactants and thus aid in lipid solubilization and digestion in the small intestine (Noble, 1981; Hofmann and Mysels, 1987). Cationic surfactants generally contain a quaternary ammonium functional group and are also commonly utilized in household cleaning products (Kume et al., 2008). Zwitterionic surfactants, also referred to as amphoteric surfactants, carry both a positive and negative charge but have a net charge of zero at certain pH values (Rapp, 2017). Phospholipids are an important subset of zwitterionic surfactants and contain a phosphate group head and two 18 hydrophobic fatty acid tails. Overall, they are the most important biological surfactant due to forming cellular membranes (Rapp, 2017). Nonionic surfactants, such as synthetic products Tween, Triton, and Brii, are often formed by reacting alcohols, alkylphenols, and amines with ethylene oxide or propylene oxide (Kume et al., 2008). Nonionic surfactants have a lower solubility than ionic surfactants but they may be a better choice in some situations due to their lack of effect on pH of the solution (Rapp, 2017). Surfactants can also be characterized by their CMC, which refers to the amount of surfactant required for micelle formation (Carey and Small, 1970). Once micelles are formed, insoluble lipids can be incorporated into the hydrophobic core of the micelle to form mixed micelles (Carey and Small, 1970). Thus, lipids with high solubility and low CMC characteristics can aid in the uptake of poorly soluble lipids into the micelle. The CMC will be lower for surfactants with large hydrophobic portions; thus CMC and chain size are generally inversely related (Carey and Small, 1970). Additionally, surfactants containing two tails, often phospholipids, will form bilayer membranes once CMC is reached while surfactants with one tail will form micelle structures (Rapp, 2017). Many of the differences observed between surfactants is due to their chemical structure and chain stiffness of the hydrophobic FA tail (Isailović et al., 2017). Fanun (2008) demonstrated that the chain length of a surfactant affects aggregation number, core radius, and interfacial area per surfactant molecule, which then affects the amount of surfactant and solubilized substances packed in micelle droplets. Natural surfactant production by the ruminant Ruminant animals naturally produce bile salts and lysolecithin that both act as surfactants in the small intestine to form micelles to transport lipids reaching the duodenum. Formation of 19 micelles allows for the solubilization of insoluble FA in the aqueous environment of the small intestine and is critical for absorption of dietary FA (Hofmann and Mysels, 1987). Interestingly, a study by Lough and Smith (1976) determined that lysolecithin is more effective than bile acids at solubilizing FA, but both are critical for maximal micelle formation. Bile acids are synthesized from cholesterol in the liver and concentrated in the gall bladder prior to secretion through the bile duct into the small intestine in response to gut hormonal signaling (Golding and Wooster, 2010). They are a distinct type of surfactant due to unusual properties due to their physical and chemical characteristics (Maldonado-Valderrama et al., 2011). While most surfactants have well defined head and tail groups, bile acids possess a planar polarity with a flat structure comprised of hydrophilic and hydrophobic faces with an aliphatic tail attached (Golding and Wooster, 2010). The unique structure of bile acids allows them to easily self-aggregate into micelles. Importantly, bile acids are differentiated by the number, position, and stereochemistry of the hydroxyl group and to the conjugation of the amino group to taurine, glycine, or other amino acids (Maldonado- Valderrama et al., 2011). The conjugation of bile acids with taurine in ruminants rather than glycine like in monogastrics is likely due to the need to remain ionized in acidic conditions in order to remain in a micellar phase rather than adsorbed onto particulate matter (Noble, 1981). Following adsorption of FA from micelles by the epithelial villi, bile acids will be reabsorbed in the ileum and recycled to the liver. Lysolecithin is formed in the small intestine when phospholipase A2 from the pancreas cleaves a FA from the sn-2 position of lecithin, which comes from microbial phospholipids produced in the rumen and secreted into the small intestine (Noble, 1981; Morgado et al., 1996). The term “lecithin” refers to multiple phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine in the bovine (Xu et al., 2011; McFadden, 2019). Each of these 20 phospholipids contain a positively charged headgroup (choline or ethanolamine, respectively) and negatively charged phosphate and carbonyl groups (Carey and Small, 1970). Thus, these molecules are charged but have a net neutral charge so are considered zwitterionic-type surfactants (Xu et al., 2011). Freeman (1969) established the importance of lysolecithin by demonstrating that the capacity of lysolecithin to solubilize stearic acid into micelles was twice as potent as 1- monoacylglycerol, which is the primacy amphiphile utilized in non-ruminant animals (Table 2.1). Exogenous lecithin supplementation Increasing amounts of FA in the diet and thus reaching the duodenum in high-producing dairy cows have led researchers to investigate the possibility of improving FA digestibility by providing supplemental lecithin and lysolecithin to the cow. In support of this theory, a study utilizing sheep observed that included soy lecithin in the diet at 5.2% DM tended to increase post- ruminal FA digestion (Jenkins and Fotouhi, 1990). While a study by Grummer et al. (1987) did not measure FA digestibility, increased plasma triglyceride-rich lipoprotein in response to soy lecithin infusion may indicate an enhanced capacity for the intestine to digest FA. Similarly, supplemental dietary lysolecithin increased plasma phosphatidylcholine, serum FA, and milk 18- carbon FA, suggesting improved intestinal FA absorption, yet no effect on FA digestibility was observed (Fontoura et al., 2019; Rico et al., 2019). Contradictory responses to lecithin supplementation could be due to ruminal digestion of unprotected lecithin (McFadden, 2019). A study by Jenkins (1990) that utilized dietary soy lecithin also had no effect on FA digestibility, but changes to rumen VFA indicates ruminal digestion of the lecithin. Duodenal infusion of lecithin would bypass the rumen and avoid potential ruminal digestion. However, infusion of 8.8 g/d soy lecithin had no effect on apparent FA digestibility (Chilliard et al., 1991). The authors postulated that the amount infused might not have been sufficient to determine if endogenous lecithin was 21 limiting FA digestion as 8.8 g/d is equal to only about 4% of endogenous lecithin production. They do not state why the dose of 8.8 g/d was chosen. Overall, the effects of supplemental lecithin and lysolecithin on FA digestibility are unclear due to potential ruminal effects in the majority of the studies. Further work with duodenally infused or ruminally protected lysolecithin should be conducted. Additionally, it should be noted that providing supplemental lecithin in the diet may be a practical challenge as it has been shown to be susceptible to hydrolytic and oxidative degradation as well as self-aggregation due to its zwitterionic characteristics (Isailović et al., 2017). Providing other exogenous emulsifiers, either independently or in combination with lecithin, may stabilize lecithin and impart synergistic benefits on micelle formation by reducing the CMC (Isailović et al., 2017; Rapp, 2017). Exogenous polysorbate supplementation Nonionic surfactants called polysorbates are often utilized in pharmaceutical and food industries (Kralova and Sjöblom, 2009; Isailović et al., 2017). These surfactants consist of a polyethoxylated sorbitan with a FA tail. A well-known brand of polysorbates, Tween, is often found in laboratory settings, as well as pharmaceutical and food industry settings, due to its detergent and wetting capabilities (Figure 8;Kralova and Sjöblom, 2009; Lallbeeharry et al., 2014). All Tween products consist of a core structure of a sorbitan sugar bound to three ethylene glycol side groups of varying lengths, which increases the hydrophilicity (Rapp, 2017). Tweens are differentiated by the hydrophobic FA tail attached to the polysorbate head group. Tween 20 contains lauric acid, Tween 40 contains palmitic acid, Tween 60 contains stearic acid, and Tween 80 contains oleic acid (Figure 9). While Tween surfactants are commonly used as a detergent in laboratory analyses to lyse cells and emulsify liquids, little work has been undertaken to examine the effects of Tween in a rumen model. 22 Past studies utilizing Tween both in vitro and in vivo bovine studies observed effects on microbial population, ruminal fermentation, and fiber digestion (Kamande et al., 2000; Goto et al., 2003; Lee and Ha, 2003; Lee et al., 2003; Cong et al., 2009). In vitro studies by Cong et al. (2009), Lee and Ha (2003), and Kamande et al. (2000) found that Tween positively affects digestion of maize stover, rice straw, wheat straw, orchardgrass, barley straw, and barley grain, but optimum fermentation may require co-surfactants such as sorbitan trioleate and alkyl polyglucoside. Other work has demonstrated increased growth rates of Fibrobacter succinogenes, Streptococcus bovis, Selenomonas ruminantium, Prevotella ruminicola, Megasphaera elsidenii, and Butyrivibrio fibrisolvens, as well as non-cellulolytic bacteria and fungi (Goto et al., 2003; Lee et al., 2003). Additionally, Tween may be harmful to some bacteria types (Minato and Suto, 1981), thus effectively altering the microbial population of the rumen. Other studies have utilized Tween to suspend fat soluble vitamins in liquid form to be given orally or intravenously (Eaton et al., 1951; Jenkins and Emmons, 1984; Drackley et al., 2007). To the best of our knowledge, no research examining the effects of Tween post-ruminally has been conducted outside of our research group. Our research group recently conducted a study that abomasally infused 0, 15, 30, and 60 g/d of Tween80 (Figure 10; de Souza et al., 2020). The dose of 30 g/d increased total FA digestibility by 10.2 percentage points compared to 0 g/d. Additionally, 16-carbon and 18-carbon digestibility were improved by 8.7 percentage points and 11.3 percentage points, respectively. Milk fat yield increased by 110 g/d in response to the improved digestibility. It should be noted that the majority of these past studies utilized Tween80 with no consideration given to the FA attached to the polysorbate; thus, differing effects may be observed when alternate Tween varieties are used. Overall, exogenous emulsifiers, or a combination thereof, possess the potential to improve FA digestibility and should be investigated further. 23 Oleic acid supplementation As reviewed earlier, increasing the amount of unsaturated FA reaching the small intestine either through dietary supplementation or abomasal infusion has been shown to improve FA digestibility in cattle. While BH creates uncertainty to the amount of each FA flowing to the duodenum, utilizing meta-analytical and abomasal infusate approaches has demonstrated the beneficial impact of OA on FA digestibility. The effect of OA on FA digestibility agrees with work that shows OA increasing the solubility of other FA (Freeman, 1969; Noble, 1981). The exact mechanism by which OA exerts the effect on FA digestibility is still under investigation. Possible mechanisms include: 1) improvements in micelle formation and function; 2) increased presence of proteins that aid in FA transport on the apical enterocyte membrane; and 3) increased re- esterification within the enterocyte. While OA possibly affects all three of these proposed mechanisms, it likely has the most beneficial effects on FA absorption by improving micelle formation and function. Freeman (1969) investigated the amphiphilic properties of various polar lipid solutes and developed an amphiphilic index, which was defined as the increase in stearic acid solubility in bile salt solution per unit increase in amphiphilic concentration. Results show that while lysolecithin was the most potent amphiphile, some FA, including OA, also have amphiphilic properties (Table 2.1). Due to the amphiphilic properties of OA, it was observed to have a much lower CMC and higher saturation ratio than most other FA reaching the duodenum (Table 2.2; Freeman, 1969). These characteristics allow OA to aid bile salts and lysolecithin in micelle formation, as well as increase the solubility of other FA into the micelle. Noble (1981) summarized that OA was as effective in solubilizing PA as lecithin was when included along with bile salts and lysolecithin. Thus, OA has clear and positive effects on micelle formation and the solubility of other FA. 24 Little work has been done to ascertain the effects of OA on FA transport into the enterocyte. While improvements in FA digestibility in response to OA (Burch et al., 2018; de Souza et al., 2018, 2019) is likely primarily due to improved micelle formation, it is possible OA is also affecting uptake of FA by the enterocyte. As discussed earlier, intestinal FA absorption is a complicated process that can be influenced by various factors in the lumen that affect FA access to the brush border as well as regulated at the enterocyte level (Wang et al., 2013). While some work indicates that the initial uptake rate of FA by the intestinal mucosa and other cells is similar (Ockner et al., 1972; Dokko et al., 1998), there is a paucity of literature describing how OA interacts with CD36, FATP4, FABP1, FATBP2, and other proteins that assist in cellular uptake of FA. However, it is likely that OA has effects on these transport proteins as it has been well demonstrated that OA interacts with various signaling pathways throughout the body (Itoh et al., 2003; Fujiwara et al., 2005; Hidalgo et al., 2011). More research should be undertaken in this area, especially utilizing bovine cell lines. Lastly, there is evidence that OA may affect re-esterification and packaging rates within cells. Multiple studies have suggested that OA increases intracellular TG as a means of protection against FA-induced lipotoxicity in various cell types (Cnop et al., 2001; Listenberger et al., 2003; Drosatos-Tampakaki et al., 2014). While increasing TG formation is potentially important in reducing the intracellular concentration of FFA and thus increasing uptake of FA, it is also critical for TG to be exported from the cell to avoid any negative feedback limitations (Börsting et al., 1992; Cifarelli and Abumrad, 2018). A study by Homan et al. (1991) comparing eicosapentaenoic acid and OA in HepG2 (human hepatocyte) cells reported no differences between the two with regards to TG synthesis, but they did observe that OA was more effective than eicosapentaenoic acid at promoting the uptake of TG by VLDL. Thus, there are indications that OA may exert effects 25 on esterification and lipid packaging in the enterocyte, but more research is needed in this area. Regardless of the exact mechanism, increasing the amount of OA reaching the small intestine has the potential to increase the digestibility of all dietary FA. Conclusions and Dissertation Objective The addition of supplemental FA sources to diets is a common practice in dairy nutrition to increase dietary energy density and to support milk production. The ability to understand and model FA, the effects of individual FA, and different FA supplements on nutrient digestibility and production parameters has direct impact on dairy industry recommendations and the usefulness of FA supplementation strategies. As milk production and DMI of dairy cows increases, so too does the intake of FA. While FA are important sources of energy for the cow, the digestibility of FA decreases as more FA reach the small intestine. Strategies to improve the digestibility and absorption of FA could therefore increase the available energy and benefit the lactating dairy cow. Previous research has demonstrated that OA possess’ amphiphilic characteristics that aid in micelle formation and thus FA absorption. Similarly, exogenous emulsifiers have been observed to overcome natural limitations to the emulsification capacity of the cow and aid in micelle formation and FA absorption. Therefore, the objective of my dissertation is to investigate the potential for OA and exogenous emulsifiers to improve post-ruminal absorption of FA. We hypothesize that both OA and exogenous emulsifiers will increase the absorption of dietary- derived FA. Closing the knowledge gap regarding the role of specific dietary FA and exogenous emulsifiers on the post-ruminal absorption of FA will aid researchers and nutritionists in including the optimal FA in the diet in order to positively impact dairy cow production, efficiency, health, and farm income. 26 APPENDIX 27 Table 2.1. Amphiphilic properties of selected polar lipids. Adapted from Freeman (1969). Amphiphile Amphiphilic Index 1 Oleic Acid Monoglyceride (1-Mono-olein) Linoleic Acid Lauric Acid Lysolecithin 0.138 0.138 0.154 0.164 0.280 1The amphiphilic index is defined as the increase in stearic acid solubility in bile salt solution per unit increase in amphiphilic concentration. 28 Table 2.2. Critical micellar concentration and saturation ratio of fatty acids in sodium glycodeoxycholate solution at 37°C. Adapted from Freeman (1969). Fatty Acid Palmitic acid Stearic acid Oleic acid Linoleic acid Lauric acid Elaidic acid (trans-9 C18:1) Vaccenic acid (trans-11 C18:1) CMC (mM) Saturation ratio 1.80 1.40 0.55 0.35 0.15 1.20 0.70 0.16 0.07 1.04 0.81 1.86 0.51 1.39 29 Figure 2.1. Glycerol phosphate pathway for de novo triacylglycerol (TAG) and glycerophos- pholipid synthesis. GPAT, glycerol-3-phosphate acyltransferase; LPA, lysophosphatidic acid; AGPAT, 1-acylglycerol-3-phosphate acyltransferase; PI, phosphatidylinositol; PG, phosphatidylglycerol; CL, cardiolipin; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; DAG, diacylglycerol; DGAT, diacylglycerol acyltransferase (Takeuchi and Reue, 2009). 30 Figure 2.2. The tissue origins and postsecretory metabolic transfonnation of lipoproteins within the plasma compartment in ruminants. HDL = High density lipoproteins, LDL =low- density lipoproteins, TG = triglyceride, CE = cholesterol ester, cetp =CE transfer protein, lcat =lecithin:cholesterol acyltransferase, CHYLO =chylomicron, VLDL = very low-density lipoprotein, HDL = high density lipoproteins, LDL =low-density lipoproteins, TG = triglyceride, CE = cholesterol esters, =lpl =lipoprotein lipase, hl =hepatic lipase, FA =fatty acid, FC =free cholesterol (Bauchart, 1993). 31 % , y t i l i b i t s e g i D 0 : 8 1 C 90 85 80 75 70 65 60 55 50 0 600 200 Duodenal Flow of C18:0, g/d 400 800 Figure 2.3. Relationship between study-adjusted C18:0 intestinal digestibility and duodenal flow of C18:0. Study-adjusted C18:0 digestibility (%) = 91.76 (SE = 1.98) – 0.0493 (SE = 0.0098) × duodenal flow of C18:0 (g/d); P < 0.001. Control treatments represented by ▲; animal-vegetable fat treatments represented by ◆; calcium salt treatments represented by ■; tallow treatments represented by ○; vegetable oil treatments represented by Δ; seed meal treatments represented by □; whole seed treatments represented by +; and other treatments represented by × (Boerman et al., 2015). 32 % , y t i l i b i t s e g i D A F l a t o T 90 85 80 75 70 65 60 55 50 ab b a c CON PA PA+SA PA+OA Figure 2.4. Total FA digestibility for cows fed FA treatment diets (n = 24). CON = control; PA = 1.5% of FA supplement blend to provide approximately 80% of C16:0; PA+SA = 1.5% of FA supplement blend to provide approximately 40% of C16:0 + 40% of C18:0; PA+OA = 1.5% of FA supplement blend to provide approximately 45% of C16:0 + 35% of C18:1 cis-9. Error bars represent SEM. For FA treatment effect, means within basal fat diets with different letters (a–c) differ (P < 0.05). Adapted from de Souza et al. (2018). 33 Figure 2.5. Relationship between study-adjusted total FA intestinal digestibility and total FA intake. Study-adjusted total FA digestibility (%) = 82.70 (SE = 2.03) – 0.00848 (SE = 0.00274) × total FA intake (g/d); P < 0.001. Control treatments represented by ▲; animal- vegetable fat treatments represented by ◆; calcium salt treatments represented by ■; tallow treatments represented by ○; vegetable oil treatments represented by Δ; seed meal treatments represented by □; whole seed treatments represented by +; and other treatments represented by × (Boerman et al., 2015). 34 ) % ( y t i l i b i t s e g i D A F l a t o T 90 85 80 75 70 65 60 55 50 0 1500 500 Total FA Duodenal Flow (g/d) 1000 2000 Figure 2.6. Relationship between study-adjusted total FA intestinal digestibility (%) and total FA duodenal flow (g/d) for 61 observations from 15 studies. Total FA digestibility (%) = 82.46 (SE = 2.15) − 0.0088 (SE = 0.0031) × total FA duodenal flow (g/d); P = 0.01. Control treatments represented by ▲; animal-vegetable fat treatments represented by ◆; calcium salt treatments represented by ■; tallow treatments represented by ○; vegetable oil treatments represented by Δ; seed meal treatments represented by □; whole seed treatments represented by +; and other treatments represented by × (Boerman et al., 2015). 35 Figure 2.7. Graphical representations of water-in-oil emulsions, bicontinuous emulsions, and oil-in-water emulsions (Isailović et al., 2017). 36 Figure 2.8. Differences in wetting behavior after two seconds of water droplet attachment of (a) a semi-dried pure whole milk particle and (b) a semi-dried whole milk particle with addition of 0.1% Tween 80 (Lallbeeharry et al., 2014). 37 Figure 2.9. Graphical representations of common polysorbate (Tween©) products. Images are public domain. 38 % , y t i l i b i t s e g i d A F l a t o T 75.0 70.0 65.0 60.0 55.0 50.0 CON 0 15 30 D-15 D-30 D-45 Figure 2.10. 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CHAPTER 3: EFFECTS OF ALTERING THE RATIO OF STEARIC AND OLEIC ACIDS IN SUPPLEMENTAL FAT BLENDS ON FATTY ACID DIGESTIBILITY AND PRODUCTION RESPONSES OF DAIRY COWS C.M. Prom and A.L. Lock Abstract The objective of our study was to determine the effects of altering the ratio of stearic (SA) and oleic (OA) acids in supplement fat blends on fatty acid (FA) digestibility and milk yield of dairy cows. Eight multiparous Holstein cows (157 DIM ± 11.8) were randomly assigned to treatment sequence in a replicated 4x4 Latin square design with 14-d periods. Production and digestibility data were collected during the last 4 d of each infusion period. The treatments were a non-FA supplemented control diet (CON), and 3 diets incorporating 1.4% DM FA supplement blends containing 50% SA and 10% OA (50:10), 40% SA and 20% OA (40:20), or 30% SA and 30% OA (30:30). FA blends were balanced to contain 33% palmitic, 5% linoleic, and <0.5% linolenic acids. FA supplements replaced soyhulls in the fat supplemented diets. There was no effect of treatment on DMI, but the replacement of soyhulls with FA in the diets decreased NDF intake. Compared to CON, FA treatments increased DM and NDF digestibility. As expected, FA inclusion increased the intake of total, 16- carbon (16-C), and 18-carbon (18-C) FA similarly compared to CON. Compared to CON, FA treatments decreased the digestibility of total and 18-C FA but increased absorption of total, 16-C, and 18-C due to increased FA intake. Within FA treatments, increasing OA increased the digestibility of total, 16-C, and 18-C FA, as well as the absorption of total, 16-C, and 18-C FA. Compared with CON, FA treatments increased milk yield, ECM, and fat yield and tended to increase milk protein yield. Compared to CON, FA treatments had no effect on the yield of de novo milk FA, decreased mixed milk FA, and increased preformed milk FA. The increase in preformed FA yield was predominantly due to FA treatments increasing the yield of OA in milk compared with CON. Within FA treatments, 49 increasing OA did not affect milk yield, milk fat yield, or milk protein yield. Increasing OA in FA treatments linearly decreased the yield of de novo and mixed milk FA but did not affect the yield of preformed FA. FA treatments increased plasma NEFA compared to CON. Overall, increasing OA within FA supplements increased total, C-16, and C-18 digestibility and absorption. Keywords: digestibility, fatty acid, stearic acid, oleic acid Introduction Fat supplements are commonly included in dairy cow diets in order to increase the energy density of the diet and to support metabolic requirements and milk production (Rabiee et al., 2012). Most commercially available fat supplements consist of mixtures of different fatty acids (FA). Thus, understanding the effects of various blends of FA on nutrient digestibility and production responses is important. The majority of feeding studies investigating the effects of FA on nutrient digestibility have focused on supplements primarily containing stearic acid (SA) and palmitic acid (PA; Rico et al., 2013; Chamberlain and DePeters, 2017; de Souza et al., 2020), since they are the predominant FA passing from the rumen to the duodenum (Doreau and Ferlay, 1994). However, some studies have shown that oleic acid (OA) may also have beneficial effects on nutrient digestibility and milk production (Elliott et al., 1996; de Souza et al., 2018, 2019). The digestibility of FA in the small intestine is of importance as the amount of specific FA absorbed can affect milk and milk component yields, as well as energy balance (Pantoja et al., 1996a; Moate et al., 2004; de Souza et al., 2018). Many studies have observed decreases in FA digestibility as the amount of SA in the diet increases (Weisbjerg et al., 1992; Boerman et al., 2017; Chamberlain and DePeters, 2017). These findings are supported by a meta-analysis that found that an increased flow of SA through the duodenum negatively affects FA digestibility (Boerman et al., 2015). Decreased absorption of SA is likely due to limited uptake by intestinal 50 micelles due to low solubility, ability to form insoluble salts with minerals in the intestine, and slower uptake and re-esterification within the enterocyte (Ockner et al., 1972; Brink et al., 1995; Glasser et al., 2008). Conversely, OA has the potential to improve FA digestibility due to greater micellar solubility, faster uptake and re-esterification by the enterocyte, and possible differences in affinity by FA transporters (Freeman, 1969; Ockner et al., 1972; Hamilton, 1998). These findings have been supported in recent in vivo studies utilizing bovine models. de Souza et al. (2018) observed that a fat supplement containing 45% PA and 35% OA had a total FA digestibility 11 percentage units higher than a supplement containing 40% PA and 40% SA. A similar study found that total FA digestibility increased as the amount of OA in a fat supplement increased and PA proportionately decreased (de Souza et al., 2019). Thus, while there are indications that OA improves FA digestibility compared to SA, no studies have directly compared changes in these two FA. Therefore, the objective of our study was to determine the effects of altering the ratio of SA and OA in supplement fat blends on FA digestibility and milk yield of dairy cows. We hypothesized that increasing OA would increase FA digestibility and consequently improve milk production. Design and Treatments Materials and Methods Experimental procedures were approved by the Institutional Animal Care and Use Committee at Michigan State University (East Lansing, MI). Eight multiparous Holstein cows averaging (mean ± SD) 157 ± 11.8 DIM, 47.2 ± 3.2 kg of milk/d, and 705 ± 33.9 kg of BW were randomly assigned to treatment sequences in a replicated 4x4 Latin square design. All animals received a common diet with no FA supplementation during a 14-d preliminary period to obtain baseline values. Cows were then blocked by milk yield and balanced for parity and BCS. Each treatment period was 51 14d with sampling during the last 4 d. Cows were fed a control diet or 3 combinations of FA supplements that were blended to achieve different ratios of SA and OA (Table 3.1). The treatments were (1) no FA supplementation (CON); (2) 50% SA + 10% OA (50:10); (3) 40% SA + 20% OA (40:20); and (4) 30% SA + 30% OA (30:30). The treatments were balanced to contain approximately 33% PA, 5% linoleic acid, and <0.5% linolenic acid. The FA supplement blends were fed at 1.4% FA (% of diet DM) and replaced soyhulls so that each FA diet had 2.0 to 2.7% less soyhulls than controls. Diets were formulated to meet the requirements of the animals as determined by NRC (2001; Table 3.2). Dry matter concentration of forages was determined twice weekly and diets were adjusted when necessary. Cows were housed in individual tie-stalls at the Michigan State University Dairy Cattle Teaching & Research Center throughout the experiment and milked twice daily (0400 and 1500 h). Access to feed was blocked from 0800 to 1000 h for collection of orts and offering of new feed. Feed intake was recorded and cows were offered 115% of expected intake at 1000 h daily. Water was available ad libitum in each stall and stalls were bedded with sawdust and cleaned twice daily. Data and Sample Collection Samples were collected during the last 4 d of each treatment period (d 11 to 14). Samples of all diet ingredients and orts from each cow were collected daily and composited by period for analysis. Fecal samples were collected every 9 h over the last 4 d of each period totaling 8 samples per cow per period. The 9 h interval over 4 d simulates sampling every 3 h over a 24-h period. Feces were stored in a sealed plastic cup at -20°C. Blood was collected once during the sampling period and stored on ice until centrifugation at 2,000 × g for 15 min at 4°C within 30 min of sample collection. Plasma was collected and stored at −20°C. Milk yield was recorded and two milk samples were collected at each milking. One aliquot was collected in a sealed tube with 52 preservative (Bronopol tablet; D&F Control Systems, San Ramon, CA) and stored at 4°C for milk component analysis. The second aliquot was stored without preservative at -20°C until analyzed for FA composition. BW measurements were taken daily during the sampling period following the afternoon milking. On the last day of the preliminary period and last day of each treatment period, three trained investigators determined BCS on a 5-point scale in 0.25-point increments (Wildman et al., 1982). Sample Analysis Diet ingredients, orts, and fecal samples were dried at 55°C in a forced-air oven for 72 h for DM determination. Dried samples were ground with a Wiley mill (1 mm-screen; Arthur H. Thomas, Philadelphia, PA). Feed ingredients, orts, and feces were analyzed for ash, NDF, indigestible NDF, CP, and starch by Cumberland Valley Analytical Services (Waynesboro, PA) as described by Boerman et al. (2017). FA concentrations of feed ingredients, orts, and feces were determined as described by Lock et al. (2013). Indigestible NDF was used as an internal marker to estimate fecal output to determine apparent total-tract digestibility of nutrients (Cochran et al., 1986). Milk samples were analyzed for fat, true protein, and lactose concentrations by mid-infrared spectroscopy (AOAC, 1990; method 972.160; NorthStar Michigan Lab, Grand Ledge, MI). Yields of 3.5% FCM, milk energy, and milk components were calculated using milk yield and component concentrations from each milking, summed for a daily total, and averaged for each collection period. Milk samples used for analysis of FA composition were composited based on milk fat yield (d 11-14 of each period). Milk lipids were extracted and FA-methyl esters prepared and quantified using GLC according to Lock et al. (2013). Yield of individual FA (g/d) in milk fat were calculated by using milk fat yield and FA concentration to determine yield on a mass basis using the 53 molecular weight of each FA while correcting for glycerol content and other milk lipid classes (Piantoni et al., 2013). Plasma samples from each cow were composited by period prior to analysis. All plasma samples were analyzed in duplicate with a coefficient of variation of <5% between duplicates. Plasma samples were analyzed using a Beckman Coulter AU series chemistry analyzer (Beckman Coulter, Brea, CA) at the Michigan State University Veterinary Diagnostic Laboratory (Lansing, MI) for NEFA and BHBA. Insulin was determined with a bovine insulin ELISA using a solid phase 2-site enzyme immunoassay (Mercodia, Uppsala, Sweden). Statistical Analysis All data were analyzed using the mixed model procedure of SAS (Version 9.4, SAS Institute, Cary, NC) according to the following model: Yijkl =  + Ci (Sj) +Pk + Tl + Pk x Tl + eijkl Where Yijkl = dependent variable,  = overall mean, Ci (Sj) = random effect of cow within square (j = 1 to 2), Pk = fixed effect of period (k = 1 to 4), Tl = fixed effect of treatment (l = 1 to 4), Pk x Tl = period by treatment interaction, and eijkl = residual error. Normality of the residuals was checked with normal probability and box plots and homogeneity of variances with plots of residuals vs. predicted values. Main effects were declared significant at P ≤ 0.05 and tendencies were declared at 0.05 < P ≤ 0.10. Period by treatment interactions were evaluated and removed from the statistical model when not significant (P > 0.20). Pre-planned contrasts included the comparison between CON and the average of the FA treatments (CON vs. FA) and the linear and quadratic effects of increasing OA within FA treatments. All data are expressed as least square means and standard error of the means. 54 Nutrient Intake and Total-tract Digestibility Results Overall, compared to CON, supplemental FA treatments did not affect DMI (P = 0.91), decreased NDF intake (P < 0.01), and increased intake of total (P < 0.01), 16-carbon FA (P < 0.01), and 18-carbon (P < 0.01) FA (Table 3.3). Overall, supplemental FA decreased the digestibility of total (P < 0.01) and 18-carbon (P < 0.01) FA but increased digestibility of DM (P = 0.02) and NDF (P = 0.03) compared to CON. Compared to CON, overall supplemental FA increased absorbed total (P < 0.01), 16-carbon (P < 0.01), and 18-carbon FA. Within FA treatments, increasing OA did not affect DMI (P = 0.13) or FA intake (P = 0.54) but tended to linearly decrease NDF intake (P = 0.07). Increasing OA within FA treatments quadratically increased digestibility of DM (P = 0.02) and NDF (P < 0.01). Similarly, increasing OA within FA treatments linearly increased digestibility of total (P < 0.01), 16-carbon (P < 0.01), and 18-carbon FA (P < 0.01) with the 30:30 treatment increasing digestibility by 11.5, 10.1, and 12.7 percentage points, respectively, compared to the 50:10 treatment. Within FA treatments, increasing OA increased absorbed total (P < 0.01), 16-carbon (P < 0.01), and 18-carbon FA (P < 0.01), with the 30:30 treatment increasing absorption by 97, 23, and 94 g/d, respectively, compared to the 50:10 treatment. Production Responses Overall, supplemental FA increased milk yield (P < 0.01), 3.5% FCM (P = 0.01), ECM (P = 0.02), and milk fat yield (P = 0.01), with no differences observed among FA treatments. Overall FA supplementation improved feed efficiency (ECM/DMI; P < 0.01; Table 3.4) due to increases in ECM while DMI remained unchanged. Within FA treatments, increasing OA linearly decreased fat (P = 0.02) and protein (P = 0.02) content but did not affect fat (P = 0.33) or protein (P = 0.61) 55 yield. Increasing OA quadratically affected BCS (P = 0.03) with it being lowest on the 40:20 treatment. Milk Fatty Acid Sources Milk FA are derived from either de novo synthesis in the mammary gland (<16 carbon FA) or preformed FA extracted from plasma (>16 carbon FA). Mixed-source FA (16 carbon) can be derived from either source. Overall, supplemental FA increased the yield of milk FA from mixed (P = 0.04) and preformed (P < 0.01) sources (Table 3.5). Within FA treatments, increasing OA decreased FA from de novo (P = 0.03) and mixed (P = 0.05) sources. Changes in yields were primarily due to decreases in FA with 10-16 carbons and increases in C18:0 and trans isomers of C18:1 (Table 3.6). As a proportion of total milk fat, overall FA supplementation increased milk FA from preformed sources (P < 0.01) and decreased FA from de novo (P < 0.01) and mixed sources (P = 0.07). Within FA treatments, increasing OA decreased the concentration of milk FA from de novo (P < 0.01) and mixed sources (P < 0.01) and increased FA from preformed sources (P < 0.01), primarily due to increases in C18:0 (P = 0.03), cis-9 C18:1 (P < 0.01), and trans isomers of C18:1 (all P < 0.01; Table 3.7). Plasma Hormones and Metabolites Compared to CON, supplemental FA increased NEFA (P < 0.01), but did not affect BHBA (P = 0.22) or insulin (P = 0.73; Table 3.8). Within FA treatments, increasing OA quadratically affected NEFA (P < 0.01), with 40:20 treatment resulting in the lowest NEFA. Increasing OA tended to linearly increase BHBA (P = 0.08) and to quadratically affect insulin (P = 0.08), with insulin being lowest on the 40:20 treatment. 56 Discussion Supplemental fat is often included in the diets of dairy cows in order to increase energy density. Generally, supplemental fat has been shown to improve milk production, but inconsistent effects have been observed on DMI and nutrient digestibility (Rabiee et al., 2012; Boerman et al., 2015; Palmquist and Jenkins, 2017). These differences are largely attributed to variation in the amount, profile, and esterification of the fat supplement, as well as fat content and composition of the basal diet. While past work has directly compared the proportion of PA and SA (Chamberlain and DePeters, 2017; de Souza et al., 2018) as well as PA and OA (de Souza et al., 2018, 2019) within fat supplements to examine the effects on FA digestibility, we are not aware of previous research that has directly compared proportions of SA and OA in fat supplements. Thus, our aim in our study was to investigate the effects of altering the ratios of SA and OA in a dietary FA supplement blend on FA digestibility and milk yield. In our study, we did not observe any treatment effects on DMI. In contrast, previous studies have observed increased DMI as the proportion of SA in a supplement increased (Piantoni et al., 2015b; Boerman et al., 2017; Chamberlain and DePeters, 2017). Meanwhile, older research utilizing unsaturated FA has shown depressed DMI (Firkins and Eastridge, 1994; Allen, 2000), but more recent work has shown no effect of supplements containing OA on DMI (He et al., 2012; Stoffel et al., 2015; de Souza et al., 2017). These inconsistencies could be due to total amount of unsaturated FA in the diet as de Souza et al. (2018) only observed an effect of OA on DMI when cottonseed was included in the basal diet to increase dietary FA content to 5% of diet DM compared to a diet containing FA at 3.5% of diet DM. As discussed by Allen (2000), other authors have ascribed the negative effects of unsaturated FA on DMI to ruminal distention caused by reduced fiber digestion, metabolic signaling due to increased FA absorption in the small intestine, 57 regulation by gut peptides, or hepatic FA oxidation. It should be noted that previous studies utilizing OA have differed in the amount of OA and type of FA supplement. In general, calcium salts of palm FA distillate have been shown to decrease DMI (Rabiee et al., 2012). However, none of the treatments in our study contained as much OA as typical calcium salts of palm FA distillate products. Thus, the effects of different FA and their proportions within FA supplements on feed intake are still not well understood and should be examined in future studies. While DMI was not affected by fat supplementation, NDF intake decreased as soyhulls were removed from the diet in order to include the FA blends. NDF digestibility increased with FA supplementation, either due to lower NDF intake, increased rumen retention rate due to increased cholecystokinin secretion (Harvatine and Allen, 2006a; Piantoni et al., 2013), increased hindgut digestion, or possible alterations of ruminal microbial populations by supplemental FA (Oldick and Firkins, 2000; Hristov et al., 2005). While a meta-analysis examining the effects of saturated fat or calcium salts of FA found either no or minor improvements on NDF digestibility (Weld and Armentano, 2017), abomasal infusions of OA have been shown to improve NDF digestibility (Romo et al., 1996). In contrast, dietary SA has been shown to have either negative or no impact on NDF digestibility (Boerman et al., 2017; Chamberlain and DePeters, 2017; de Souza et al., 2018). Supplemental PA has been shown to consistently increase NDF digestibility (de Souza et al., 2018; Western et al., 2020). However, the amount of PA in supplemental FA blends was held constant and thus was unlikely to be responsible for the impact on NDF digestibility observed across treatments. Thus, as with DMI, the response of NDF digestibility to dietary FA is variable and should be investigated further. Our study is novel in that we maintained a constant amount of PA in a FA supplement and only altered the composition of 18-carbon FA in order to examine whether OA can increase FA 58 digestibility compared to SA. We recognize that unsaturated 18-carbon FA can be biohydrogenated to SA in the rumen; thus, directly comparing FA supplements that differ in the ratio of SA and OA is important. Biohydrogenation is the process by which ruminal microbes hydrogenate a double bond in order to avoid toxic effects of unsaturated FA (Jenkins, 1993; Maia et al., 2010). While BH is necessary and beneficial for the microbial population, it makes it challenging to differentiate the amount of OA and SA reaching the small intestine as both are 18 carbon FA. The exact amount of OA and SA flowing to the small intestine in our study is unknown. However, we can investigate the effects of altering the proportions of OA and SA in a dietary FA supplement on FA digestibility. Overall, FA digestibility was quite high on our study relative to similar studies by de Souza et al. (2018), Rico et al. (2017), and Boerman et al. (2017). The differing effects between the studies is likely due to lower DMI and total FA intake on our study, which have been shown to positively affect FA digestibility (Palmquist, 1991; Boerman et al., 2015). Improved FA digestibility in response to increasing OA supplementation was observed in our study and agrees with previous work (Romo et al., 1996; Enjalbert et al., 2000; de Souza et al., 2019). Increasing OA from 10% to 30% of a fat supplement, with the remainder primarily being comprised of PA, improved total FA digestibility by 4.1 percentage units (de Souza et al., 2019). In comparison, increasing OA from 10% to 30% of a fat supplement in our current study, where the majority of the supplement was primarily SA, increased total FA digestibility by 11.5 percentage units. The larger order of magnitude change in our study is likely due to the strong negative effect of SA compared to PA on FA digestibility (Boerman et al., 2017). This decrease is likely due to the low solubility of SA hampering micelle formation and FA uptake by the enterocyte (Freeman, 1969; Harrison and Leat, 1972). 59 Multiple studies have demonstrated a reduction in FA digestibility when SA is included in the diet (Chamberlain and DePeters, 2017; de Souza et al., 2018; Western et al., 2020). For example, Boerman et al. (2017) fed increasing levels of a SA enriched supplement (93% C18:0) to dairy cows and observed no positive effect on milk yield, which was likely associated with the pronounced decrease in total FA digestibility as FA intake increased. A study that fed more commonly utilized ratios of PA, SA, and OA also observed that increasing the amount of SA in a FA supplement decreases total FA digestibility (de Souza et al., 2018). The negative effect of SA on FA digestibility is supported by a meta-analysis in which a negative relationship between the total flow and digestibility of FA was observed, with the decrease in total FA digestibility driven by the digestibility of SA (Boerman et al., 2015). In agreement with this effect of SA, a recent study by de Souza et al. (2020) observed decreased FA digestibility in response to SA in a dietary FA supplement when compared to a FA supplement containing 80% PA and a non-fat supplemented control diet. While the FA supplement containing the most OA (30:30) had the highest FA digestibility of the FA supplemented diets, all FA supplemented diets had lower total and 18-carbon FA than the non-FA supplemented control diet. The higher FA digestibility of the control diet is likely due a lower FA intake, which is thus expected to result in higher FA digestibility (Boerman et al., 2015). Interestingly, we observed an increase in 16-carbon FA digestibility with the 30:30 treatment compared to the non-fat supplemented control, which supports the conclusion of Börsting et al. (1992) that unsaturated FA have a synergistic relationship with the digestibility of saturated FA. Improvements of unsaturated FA on the digestibility of saturated FA may be due to the ability of OA to improve micelle formation in the intestine (Freeman, 1969) and esterification within the enterocyte (Ockner et al., 1972). Overall, the effects of increasing OA and decreasing 60 SA in a dietary fat supplement on FA digestibility demonstrates that some OA escaped ruminal biohydrogenation to improve micelle formation and absorption of FA. If assuming a rate of ruminal biohydrogenation of 40-80% (Jenkins and Bridges, 2007), approximately 20 to 60 g/d more of OA reached the small intestine on the 30:30 treatment compared to the 50:10 treatment. In comparison, abomasal infusion of 60 g/d of OA has been observed to increase total FA digestibility of 8.5 percentage units (Chapter 4). Thus, the amount of OA reaching the small intestine when biohydrogenation rates are accounted for still provides sufficient OA to improve FA digestibility. While period lengths and sample size for our study were designed to examine digestibility responses, some production responses were also observed. Overall, supplemental FA in our study increased milk yield, milk fat yield, 3.5% FCM, and ECM. The increase in milk and milk fat yield in response to fat supplementation has often been observed over the last 50 years (Palmquist and Jenkins, 2017), but the effects have differed depending on the FA profile of the supplement. Within FA treatments in the current study, increasing OA did not improve milk production despite improving FA absorption. One reason that milk yield did not increase could be that OA partitioned energy towards body reserves rather than milk yield (de Souza et al., 2018, 2019). Furthermore, cows producing ~45 kg/d of milk, which is similar to the production level in our study, have been shown to be more responsive to PA than OA in regards to partitioning energy towards milk (de Souza et al., 2019). Thus, a milk yield response to OA may have been observed in our study if the cows were higher producing animals. Furthermore, changes in BW, indicating partitioning energy towards body reserves, may have been observed if period lengths were longer. Recent research has highlighted the impact of altering dietary FA on milk fat yield (de Souza et al., 2018; Western et al., 2020). In our study, increasing OA linearly decreased milk fat 61 content, but there was no effect on milk fat yield. As such, the decrease in milk fat content is partially attributable to a dilution of milk fat as milk yield increased. It should also be noted that increasing OA increased trans-10 C18:1 in milk fat, which is an indicator of changes in rumen biohydrogenation that can result in milk fat depression (Bauman et al., 2011). Similar to our results, de Souza et al. (2018) demonstrated no difference between supplements enriched with OA and SA with respect to milk fat yield. Both SA and OA depress de novo milk FA synthesis in favor of utilizing preformed FA for milk fat (Palmquist, 2006). In contrast, multiple studies have demonstrated higher milk fat yield when PA is supplemented compared to either SA or OA (Dorea and Armentano, 2017; de Souza et al., 2018). PA likely increases milk fat yield compared to SA and OA by averting de novo FA depression due to available preformed PA (Palmquist, 2006) or preferentially utilizing PA during triglyceride synthesis (Kinsella and Gross, 1973). In our study, increasing OA decreased the amount of absorbed FA utilized for de novo milk fat synthesis, as indicated by decreased yield of milk FA from de novo sources (Banks et al., 1990). Compared to CON, supplemental FA increased absorbed FA by 180, 254, and 277 g/d for the 50:10, 40:20, and 30:10 treatments, respectively. However, milk fat yield for the respective treatments only increased by 90, 90, and 60 g/d. The fact that at least half of the increase in absorbed FA was not captured in milk indicates the importance of better understanding the mechanisms controlling energy partitioning. Conclusions of effects of FA supplementation on BW and BCS from our study should be interpreted with caution due to sample size and period lengths chosen in order to focus on effects on digestibility. FA profile can alter energy partitioning (de Souza et al., 2018), so we expected OA might have a linear effect on body tissue gain and plasma variables in our study. However, 62 our study was not designed to examine partitioning. Future work should aim to further elucidate the effects of FA profile and nutrient interactions on energy partitioning. Conclusions Supplemental FA blends containing different combinations of SA and OA did not affect DMI but increased milk yield, milk fat yield, 3.5% FCM, ECM, and feed efficiency. While all FA treatments decreased FA digestibility compared to the non-supplemented controls, increasing OA in the FA supplements increased the digestibility and absorption of total, 16-carbon, and 18-carbon FA. Acknowledgements We acknowledge L. Worden, J. de Souza, M. Western, J. Guy, H. Sharrard, E. Butler, A. Negreiro and A. Pineda (all in the Department of Animal Science, Michigan State University, East Lansing) and the staff of the Michigan State University Dairy Cattle Teaching & Research Center (East Lansing) for their assistance in this experiment. Crystal Prom was supported by a Pre- Doctoral Fellowship from USDA NIFA. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 63 APPENDIX Table 3.1. Proportion of each fatty acid (FA) supplement for treatment blends and FA profile of FA blends. Item Treatment1 50:10 40:20 30:30 Proportion of each fat supplement in treatment blends, % C18:0-enriched supplement2 C16:0-enriched supplement3 Ca salt of palm oil supplement4 Ca salt of palm oil and soy oil supplement5 FA profile of each fat blend, g/100g of FA C14:0 C16:0 C18:0 cis-9 C18:1 cis-9,cis-12 C18:2 cis-9, cis-12, cis-15 C18:3 53.0 9.0 25.0 13.0 0.3 33.0 50.5 10.0 5.0 0.3 41.0 40.0 12.0 7.0 0.5 33.1 40.0 19.8 5.4 0.2 28.5 71.5 0.0 0.0 0.7 33.9 29.1 29.5 5.4 0.2 1Treatments consisted of no fat supplementation (CON) or 50% stearic and 10% oleic, 40% stearic and 20% oleic, or 30% stearic and 30% oleic provided at 1.5% of diet DM. 2Stearic acid-enriched FA supplement (not commercially available; Wawasan Agrolipids, Johor, Malaysia). Contained (g/100 g of FA) 0.01 of C14:0, 6.93 of C16:0, 92.2 of C18:0, 0.07 of cis-9 C18:1, and 0.02 cis-9,cis-12 C18:2; 99.8% total FA. 3Palmitic acid-enriched FA supplement (Nutracor; Wawasan Agrolipids). Contained (g/100 g of FA) 0.70 of C14:0, 83.6 of C16:0, 2.54 of C18:0, 10.2 of cis-9 C18:1, and 2.29 cis-9,cis-12 C18:2; and 99.5% total FA. 4Calcium salts of palm FA supplement (Nutracal; Wawasan Agrolipids). Contained (g/100 g of FA) 1.05 of C14:0, 44.3 of C16:0, 4.10 of C18:0, and 40.6 of cis-9 C18:1, and 7.94 cis-9,cis-12 C18:2; 98.6% total FA . 5Calcium salts of palm and soy FA supplement (Essentiom; Arm and Hammer). Contained (g/100 g of FA) 0.62 of C14:0, 32.4 of C16:0, 4.20 of C18:0, 29.0 of cis-9 C18:1, and 29.3 cis-9,cis-12 C18:2; 96.1% total FA. Table 3.2. Ingredient and nutrient composition of treatment diets. Treatment1 CON 50:10 40:20 30:30 35.2 35.2 35.2 35.2 14.8 14.8 14.8 14.8 0.9 0.9 0.9 0.9 21.6 21.6 21.6 21.6 13.0 13.0 13.0 13.0 15.8 13.8 13.2 13.1 3.2 3.2 3.2 3.2 - 1.50 1.62 1.73 33.8 32.7 32.4 32.3 17.3 17.1 17.2 17.2 24.5 24.6 24.7 24.7 1.8 3.2 3.2 3.2 0.27 0.73 0.74 0.75 0.07 0.73 0.59 0.48 0.34 0.44 0.64 0.74 0.91 0.97 0.98 0.98 Item Ingredient, % DM Corn silage Alfalfa silage Wheat straw Ground corn Soybean meal Soyhulls Vitamin and mineral mix2 FA supplement Nutrient Composition, % DM NDF CP Starch FA C16:0 C18:0 cis-9 C18:1 cis-9, cis-12 C18:2 cis-9, cis-12, cis-15 C18:3 0.15 0.15 0.15 0.15 1Treatments consisted of no fat supplementation (CON), 50% stearic and 10% oleic, 40% stearic and 20% oleic, or 30% stearic and 30% oleic. 2Vitamin and mineral mix contained 34.1% dry ground shelled corn, 25.6% white salt, 21.8% calcium carbonate, 9.1% Biofos (The Mosaic Co., Plymouth, MN), 3.9% magnesium oxide, 2% soybean oil, and < 1% of each of the following: manganese sulfate, zinc sulfate, ferrous sulfate, copper sulfate, iodine, cobalt carbonate, vitamin E, vitamin A, vitamin D, and selenium. Table 3.3. Nutrient intake and apparent digestibility of cows fed increasing amounts of oleic acid (n = 8). Treatments1 Variable CON 50:10 40:20 30:30 Intake, kg/d DM NDF Intake, g/d Total FA 16-carbon 18-carbon Digestibility, % DM NDF Total FA 16-carbon FA 18-carbon FA Absorbed, g/d Total FA 16-carbon 18-carbon 30.5 10.5 567 86 469 68.8 52.4 80.8 74.1 83.7 457 63.6 392 30.6 10.2 980 223 718 69.0 51.9 64.9 70 63.4 637 155 454 30.9 10.2 996 226 749 70.5 54.4 71.5 75.6 71 711 171 531 29.7 9.8 965 223 723 69.9 53.9 76.4 80.1 76.1 734 178 548 SEM CON vs FA 0.91 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 1.00 0.29 34.2 8.3 23.6 0.37 0.76 1.55 1.13 1.64 25.6 6.67 21.4 Contrast2 Linear Quadratic 0.13 NS NS NS 0.05 NS 0.01 0.05 0.09 0.10 NS NS 0.09 0.16 <0.01 <0.01 <0.01 <0.01 0.02 0.03 <0.01 0.23 <0.01 <0.01 <0.01 <0.01 1Treatments consisted of no fat supplementation (CON), 50% stearic acid and 10% oleic acid, 40% stearic acid and 20% oleic acid, or 30% stearic acid and 30% oleic acid. 2P-values associated with contrasts: (1) the effect of fat supplementation (FA) compared to no fat supplementation (CON); (2) the linear effect of increasing oleic acid; and (3) the quadratic effect of increasing oleic acid. Table 3.4. DMI, milk yield, milk composition, BW, and BCS of cows fed increasing amounts of oleic acid (n = 8). Treatments1 Variable CON 50:10 40:20 30:30 SEM CON vs Yield, kg/d Milk 3.5% FCM3 ECM4 Fat Protein Lactose Milk Composition, % Fat Protein Lactose FCM/DMI Body Weight, kg BCS 43.0 42.8 43.4 1.49 1.41 2.10 3.47 3.30 4.87 1.42 723 3.33 44.7 45.1 45.5 1.58 1.45 2.19 3.57 3.25 4.88 1.48 725 3.40 45.7 45.4 45.8 1.58 1.47 2.23 3.46 3.21 4.87 1.48 722 3.29 45.2 44.5 44.9 1.55 1.44 2.20 3.44 3.18 4.88 1.50 724 3.37 1.75 1.51 1.58 0.05 0.06 0.09 0.10 0.05 0.02 0.05 6.92 0.10 Contrast Linear Quadratic 0.63 0.59 0.60 0.33 0.61 0.77 0.02 0.02 0.88 0.29 0.74 0.46 0.32 0.51 0.46 0.65 0.31 0.38 0.30 0.89 0.80 0.64 0.52 0.03 FA <0.01 0.01 0.02 0.01 0.10 0.01 0.72 <0.01 0.47 <0.01 0.99 0.61 1Treatments consisted of no fat supplementation (CON), 50% stearic acid and 10% oleic acid, 40% stearic acid and 20% oleic acid, or 30% stearic acid and 30% oleic acid. 2P-values associated with contrasts: (1) the effect of fat supplementation (FA) compared to no fat supplementation (CON); (2) the linear effect of increasing oleic acid; and (3) the quadratic effect of increasing oleic acid. 33.5% FCM = [(0.4324 × kg of milk) + (16.216 × kg of milk fat)]. 4ECM = [(0.327 × kg of milk) + (12.95 × kg of milk fat) + (7.20 × kg of milk protein)]. Table 3.5. Summation of milk fatty acid (FA) concentration and yield for cows infused with treatments (n = 8). Treatments1 CON 50:10 40:20 30:30 SEM Trt * Period CON vs FA Contrasts2 Linear Quadratic Variable Summation by Source3, g/d De Novo Both Preformed Summation by Source3, g/100 g FA De Novo Both Preformed 416 534 440 30.0 38.4 31.7 420 573 493 28.3 38.5 33.2 411 560 510 27.8 37.8 34.5 391 544 514 26.9 37.6 35.5 15.0 23.9 18.2 0.46 0.75 0.59 0.10 0.05 <0.01 <0.01 <0.01 <0.01 NS NS NS NS NS NS 0.35 0.04 <0.01 <0.01 0.07 <0.01 0.03 0.05 0.12 <0.01 <0.01 <0.01 1Treatments consisted of no fat supplementation (CON), 50% stearic acid and 10% oleic acid, 40% stearic acid and 20% oleic acid, or 30% stearic acid and 30% oleic acid. 2P-values associated with contrasts: (1) the effect of fat supplementation (FA) compared to no fat supplementation (CON); (2) the linear effect of increasing oleic acid; and (3) the quadratic effect of increasing oleic acid. 3De novo fatty acids originate from mammary de novo synthesis (<16 carbons), preformed fatty acids originate from extraction from plasma (>16 carbons), and mixed fatty acids originate from both sources (C16:0 plus cis-9 C16:1). Table 3.6. Milk fatty acid yield of cows fed increasing amounts of oleic acid (n = 8). Treatments1 Variable CON 50:10 40:20 30:30 SEM Trt Trt * Period CON vs FA Contrast Linear Quadratic Selected Individual FA5 , g/d C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 cis-9 C16:1 C18:0 trans-6 to 8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 cis-9 C18:1 cis-11 C18:1 cis-9, cis-12 C18:2 cis-9, trans-11 C18:2 cis-9, cis-12, cis-15 C18:3 1Treatments consisted of no fat supplementation (CON), 50% stearic acid and 10% oleic acid, 40% stearic acid and 20% oleic acid, or 30% stearic acid and 30% oleic acid. 2P-values associated with contrasts: (1) the effect of fat supplementation (FA) compared to no fat supplementation (CON); (2) the linear effect of increasing oleic acid; and (3) the quadratic effect of increasing oleic acid. 3De novo fatty acids originate from mammary de novo synthesis (<16 carbons), preformed fatty acids originate from extraction from plasma (>16 carbons), and mixed fatty acids originate from both sources (C16:0 plus cis-9 C16:1). 0.01 0.21 0.23 0.02 <0.01 0.15 <0.01 0.04 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.75 0.37 0.55 0.16 0.94 0.33 0.07 <0.01 <0.01 0.04 <0.01 0.06 <0.01 0.05 <0.01 <0.01 <0.01 <0.01 0.11 0.65 0.51 0.18 0.58 <0.01 0.09 0.69 0.09 <0.01 0.36 <0.01 0.02 0.02 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.43 0.13 0.91 0.08 1.52 1.03 0.66 2.10 3.03 7.85 1.36 22.2 1.99 3.61 0.13 0.14 0.37 0.57 12.0 0.53 1.65 0.24 0.39 36.9 27.4 17.8 54.4 68.5 200 15.0 550 23.4 109 2.84 2.11 4.49 7.2 250 7.25 34.5 5.28 4.16 37.7 27.6 17.7 52.8 64.8 196 14.4 537 22.7 111 3.19 2.43 5.00 7.67 264 7.06 33.7 5.11 4.52 37.0 26.5 16.7 49.1 59.8 189 13.1 523 21.1 116 3.62 2.67 5.52 7.85 264 7.13 33.9 5.07 4.29 0.47 0.37 0.38 0.53 0.75 0.78 0.50 0.93 0.43 0.71 0.72 0.60 0.99 0.20 0.31 0.57 0.57 0.61 0.18 NS NS NS NS NS NS NS NS NS 0.13 NS NS NS NS NS NS NS NS NS 33.5 25.9 17.2 54.7 70.8 199 15.8 510 23.9 89.9 2.27 1.73 3.94 6.37 218 6.98 32.8 5.17 3.95 70 Table 3.7. Milk fatty acid concentration of cows fed increasing amounts of oleic acid (n = 8). Treatments1 SEM Trt Trt * Period Contrast Variable CON 50:10 40:20 30:30 CON vs FA Linear Quadratic Selected Individual FA5 , g/100 g FA C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 cis-9 C16:1 C18:0 trans-6 to 8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 cis-9 C18:1 cis-11 C18:1 cis-9, cis-12 C18:2 cis-9, trans-11 C18:2 cis-9, cis-12, cis-15 C18:3 1Treatments consisted of no fat supplementation (CON), 50% stearic acid and 10% oleic acid, 40% stearic acid and 20% oleic acid, or 30% stearic acid and 30% oleic acid. 2P-values associated with contrasts: (1) the effect of fat supplementation (FA) compared to no fat supplementation (CON); (2) the linear effect of increasing oleic acid; and (3) the quadratic effect of increasing oleic acid. 3De novo fatty acids originate from mammary de novo synthesis (<16 carbons), preformed fatty acids originate from extraction from plasma (>16 carbons), and mixed fatty acids originate from both sources (C16:0 plus cis-9 C16). 0.02 0.09 0.67 0.05 0.04 <0.01 0.13 <0.01 0.18 <0.01 0.16 <0.01 0.08 <0.01 0.67 0.02 0.11 <0.01 0.37 <0.01 0.01 <0.01 0.01 <0.01 0.02 <0.01 0.04 <0.01 0.41 <0.01 0.44 0.03 0.09 0.49 0.01 <0.01 0.26 0.02 0.15 0.59 0.02 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.03 <0.01 <0.01 <0.01 <0.01 <0.01 0.71 0.63 0.70 0.39 <0.01 0.59 <0.01 <0.01 <0.01 <0.01 <0.01 0.29 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 0.17 0.38 <0.01 0.48 2.41 1.87 1.24 3.94 5.10 14.3 1.13 36.7 1.70 6.52 0.16 0.12 0.28 0.46 15.7 0.50 2.35 0.37 0.28 2.57 1.84 1.16 3.39 4.12 13.0 0.89 36.1 1.44 7.76 0.25 0.18 0.38 0.54 18.1 0.49 2.35 0.35 0.29 NS NS NS NS NS NS NS NS NS 0.16 NS NS NS NS NS NS NS NS NS 2.55 1.87 1.20 3.58 4.40 13.2 0.98 36.2 1.53 7.54 0.22 0.16 0.34 0.52 17.8 0.48 2.29 0.35 0.31 0.55 0.34 0.30 0.44 0.74 0.82 0.28 0.25 0.29 0.85 0.54 0.88 0.87 0.61 0.20 0.46 0.25 0.27 0.10 2.49 1.85 1.21 3.68 4.62 13.4 1.00 36.9 1.56 7.37 0.19 0.14 0.30 0.49 16.8 0.48 2.33 0.36 0.28 71 Table 3.8. Blood NEFA, BHBA, and insulin of cows fed increasing amounts of oleic acid (n = 8). Treatments1 Contrasts2 Variable CON 50:10 40:20 30:30 NEFA, mEq/L3 BHBA, mg/dL Insulin, μg/L 0.09 6.52 0.72 0.11 5.46 0.76 0.10 6.48 0.57 0.12 6.27 0.73 SEM 0.005 0.36 0.09 CON vs FA <0.01 0.08 0.33 Linear Quadratic NS NS NS <0.01 0.22 0.73 1Treatments consisted of no fat supplementation (CON), 50% stearic acid and 10% oleic acid, 40% stearic acid and 20% oleic acid, or 30% stearic acid and 30% oleic acid. 2P-values associated with contrasts: (1) the effect of fat supplementation (FA) compared to no fat supplementation (CON); (2) the linear effect of increasing oleic acid; and (3) the quadratic effect of increasing oleic acid. 72 REFERENCES 73 REFERENCES Agren, J.J., A. Julkunen, and I. Penttila. 1992. Rapid separation of serum lipids for fatty acid analysis by a single aminopropyl column. J. Lipid Res. 33:1871–1876. Ahn, G.C., J.H. Kim, E.K. Park, Y.K. Oh, G.Y. Lee, J. Il Lee, C.M. Kim, and K.K. 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Dairy Sci. 65:495–501. doi:10.3168/jds.S0022-0302(82)82223-6. 82 Wu, Z., O.A. Ohajuruka, and D.L. Palmquist. 1991. Ruminal Synthesis, Biohydrogenation, and Digestibility of Fatty Acids by Dairy Cows. J. Dairy Sci. 74:3025–3034. doi:10.3168/jds.S0022-0302(91)78488-9. 83 4. CHAPTER 4: ABOMASAL INFUSION OF OLEIC ACID INCREASES FATTY ACID DIGESTIBILITY AND PLASMA INSULIN OF LACTATING DAIRY COWS C. M. Prom, J. R. Newbold, and A. L. Lock Abstract Our objective was to determine if abomasal infusions of increasing doses of oleic acid (OA; cis-9 C18:1) improve fatty acid (FA) digestibility and milk production of lactating dairy cows. Eight rumen-cannulated multiparous Holstein cows (138 DIM±52) were randomly assigned to treatment sequence in a replicated 4x4 Latin square design with 18-d periods consisting of 7 d of washout and 11 d of infusion. Production and digestibility data were collected during the last 4 d of each infusion period. Treatments were 0, 20, 40, or 60 g/d of OA delivered at 6-h intervals. OA was dissolved in ethanol prior to infusions, which were delivered at 6-h intervals. Animals received the same diet throughout the study that contained (% diet DM) 28% NDF, 17% CP, 27% starch, and 3.3% FA (1.8% FA from a saturated FA supplement containing 32% C16:0 and 52% C18:0). OA infusion did not affect intake or digestibility of DM and NDF. Increasing OA linearly increased digestibility percentage of total FA, 16-carbon FA, and 18-carbon FA. Therefore, increasing OA linearly increased absorbed total FA, 16-carbon FA, and 18-carbon FA. Increasing OA tended to linearly increase milk yield, 3.5% fat-corrected milk, and energy-corrected milk. Increasing OA did not affect milk fat yield but tended to quadratically affect milk fat concentration. Increasing OA did not affect the yield of de novo or mixed milk FA but increased yield of preformed FA, predominantly through increased yield of OA. OA increased plasma insulin concentration but dose was not important. In conclusion, OA infusion increased FA digestibility, preformed milk FA yield, and circulating insulin without negatively affecting DMI. Keywords: digestibility, milk production, milk fatty acid, insulin 84 Introduction Energy requirements of modern dairy cows continue to increase in tandem with milk production. Supplemental fats are frequently used to help meet these energy requirements. Thus, the amount of fatty acids (FA) consumed by the cow continues to increase due to the use of supplemental fats and higher DMI. However, it has been demonstrated that digestibility of FA decreases as the amount of FA reaching the small intestine increases, indicating limitations to the natural capacity of the cow to absorb FA (Boerman et al., 2015). Lower FA digestibility can lead to a lack of milk production response as fewer FA are available for metabolic processes. Thus, understanding and improving FA digestibility is of both scientific and economic interest to the dairy industry. Incorporation of medium- and long-chain FA into micelles occurs in the duodenum in order to transfer FA adsorbed onto feed particles through the aqueous environment to intestinal epithelial cells (Noble, 1981). Absorption of FA from the micelle into the epithelial cells then occurs in the jejunum (Doreau and Ferlay, 1994). One potential strategy to improve FA digestibility may be to alter the composition of the FA reaching the small intestine as the digestibility of FA decreases as chain length and melting point of FA increases (Steele and Moore, 1968a; Andrews and Lewis, 1970; Pantoja et al., 1996a). There have also been indications that increasing the unsaturation of FA increases digestibility (Firkins and Eastridge, 1994; Boerman et al., 2015). The most common FA in bovine milk (Palmquist, 2006) and adipose tissue (Douglas et al., 2007) are oleic acid (OA; cis-9 C18:1), palmitic acid (PA; C16:0), and stearic acid (SA; C18:0). Recent studies supplementing these FA in dietary supplements have demonstrated that feeding a blend of OA and PA improves FA digestibility compared to PA alone (de Souza et al., 2019) and increasing the 85 amount of OA and decreasing the amount of SA in a fat supplement blend increased FA digestibility (Chapter 3). While there are indications that OA improves FA digestibility, the mechanism by which it does so is not well understood. Micelle uptake of FA is critical for transporting FA through the aqueous environment to the enterocyte surface. However, saturated FA are less likely to be integrated into micelles due to their low solubility and high melting point (Glasser et al., 2008). Freeman (1969) examined the amphiphilic properties of polar lipids in bile salt solutions and found OA had a positive effect on the micellar solubility of SA. Thus, increasing the flow of OA to the duodenum could have beneficial effects on saturated FA by improving micelle formation and uptake of FA. However, it has also been suggested that differing FA absorption rates are due to the faster uptake of unsaturated FA across the enterocyte plasma membrane and their faster re- esterification within the enterocyte (Ockner et al., 1972). These differences in uptake and re- esterification rates could be due to varying affinities of FA transporters for long-chain FA (Gimeno, 2007; Gajda and Storch, 2015; Cifarelli and Abumrad, 2018), but little work has been undertaken in bovine models. Factors affecting FA digestibility are most likely multifaceted. While the mechanism by which differences in FA digestibility occur needs to be further investigated, our current objective was to determine the effect of OA reaching the duodenum on FA digestibility. Understanding digestibility differences between FA is difficult in ruminant animals due to ruminal biohydrogenation of unsaturated FA. While there are indications from previous feeding studies that OA improves FA digestibility (de Souza et al., 2018, 2019; Chapter 3), the amount of individual 18-carbon FA flowing to the duodenum is uncertain. Abomasally infusing OA allows for direct determination on the effects of OA on FA digestibility. Therefore, the objective of our study was to determine the response of FA digestibility and milk production to abomasal infusion 86 of increasing doses of OA. We hypothesized that infusion of OA would increase total, 16-carbon, and 18-carbon FA digestibility, as well as increase the yield of milk and milk fat. Design and Treatments Materials and Methods All experimental procedures were approved by the Institutional Animal Care and Use Committee at Michigan State University (East Lansing, MI). Eight ruminally cannulated multiparous Holstein cows averaging (mean ± SD) 138 ± 52 DIM, 49.8 ± 4.6 kg of milk/d, and 690 ± 52 kg of BW were randomly assigned to treatment sequence in a replicated 4x4 Latin square design. Cows were blocked by milk yield and balanced for parity and BCS. Each 18-d treatment period consisted of a 7-d washout period and a 11-d infusion period, with sampling during the last 4 d (Figure 1). Treatments were 0, 20, 40, and 60 g/d of abomasally infused OA (O1008-1G, Sigma-Aldrich, St. Louis, MO; Table 4.1). Our dose range was based upon common DMI, supplemental FA feeding rates, inclusion of OA in FA supplements, and biohydrogenation estimates to model representative flows of OA from FA supplements to the abomasum. Daily doses of OA were suspended in 200 mL of ethanol in individual glass jars. The infusate solution was divided into four equal infusions per day occurring every six hours. Abomasal infusion devices were inserted into the abomasum five days before the beginning of the study. Infusion lines (0.5 cm diameter polyvinyl chloride tubing) passed through the rumen fistula and sulcus omasi into the abomasum (Lock et al., 2007). Lines were checked daily throughout the study to ensure proper placement. All animals received a common diet that was formulated to meet the requirements of the animals as determined by NRC (2001; Table 4.2). The diet included a commercially available saturated FA supplement (Energy Booster 100, Milk Specialties Global, Eden Prairie, MN; Table 4.1) at 1.8% diet DM during a 10-d preliminary period and throughout the experiment in order to increase the 87 amount of saturated FA flowing to the small intestine to allow for improvements in FA digestibility. Dry matter concentration of forages was determined twice weekly and diets were adjusted when necessary. Cows were housed in individual tie-stalls at the Michigan State University Dairy Cattle Teaching & Research Center throughout the experiment and milked twice daily (0400 and 1500 h). Access to feed was blocked from 0800 to 1000 h for collection of orts and offering of new feed. Feed intake was recorded and cows were offered 115% of expected intake at 1000 h daily. Water was available ad libitum in each stall and stalls were bedded with sawdust and cleaned twice daily. Data and Sample Collection Samples were collected during the last 4 d of each treatment period (d 15 to 18; Figure 1). Samples of all diet ingredients and orts from each cow were collected daily and composited by period for analysis. Milk yield was recorded and two milk samples were collected at each milking. One aliquot was collected in a sealed tube with preservative (Bronopol tablet; D&F Control Systems, San Ramon, CA) and stored at 4°C for milk component analysis. The second aliquot was stored without preservative at -20°C for FA composition analysis. Fecal (~400 g) and blood (~15 mL) samples were collected every 9 h over the last 4 d of each period totaling 8 samples per cow per period. The 9 h interval over 4 d simulates sampling every 3 h over a 24-h period to account for diurnal variation. Feces were stored in a sealed plastic cup at -20°C. Blood was stored on ice and centrifuged within 30 min at 2,000 × g for 15 min at 4°C. Plasma was transferred into microcentrifuge tubes and stored at −20°C. BW measurements were taken daily during the sampling period following the afternoon milking. On the last day of the preliminary period and last day of each treatment period, three trained investigators determined BCS on a 5-point scale in 0.25-point increments (Wildman et al., 1982). 88 Sample Analysis Diet ingredients, orts, and fecal samples were dried at 55°C in a forced-air oven for 72 h for DM determination. Dried fecal samples for each cow were then composited by period. Dried samples were ground with a Wiley mill (1 mm-screen; Arthur H. Thomas, Philadelphia, PA). Feed ingredients, orts, and feces were analyzed for ash, NDF, indigestible NDF, CP, and starch by Cumberland Valley Analytical Services (Waynesboro, PA) as described by Boerman et al. (2017). Indigestible NDF was used as an internal marker to estimate fecal output to determine apparent total-tract digestibility of nutrients (Cochran et al., 1986). FA concentrations of feed ingredients, orts, and feces were determined as described by Lock et al. (2013). Milk samples were analyzed for fat, true protein, and lactose concentrations by mid-infrared spectroscopy (AOAC, 1990; method 972.160; NorthStar Michigan Lab, Grand Ledge, MI). Yields of 3.5% FCM, ECM, and milk components were calculated using milk yield and component concentrations from each milking, summed for a daily total, and averaged for each collection period. Milk samples used for analysis of FA composition were composited based on milk fat yield (d 15-18 of each period). Milk lipids were extracted and FA-methyl esters prepared and quantified using GLC according to Lock et al. (2013). Yield of individual FA (g/d) in milk fat were calculated by using milk fat yield and FA concentration to determine yield on a mass basis using the molecular weight of each FA while correcting for glycerol content and other milk lipid classes (Piantoni et al., 2013). Plasma samples from each cow were composited by period prior to analysis. All plasma samples were analyzed in duplicate with a coefficient of variation of <5% between duplicates. Plasma samples were analyzed using a Beckman Coulter AU series chemistry analyzer (Beckman Coulter, Brea, CA) at the Michigan State University Veterinary Diagnostic Laboratory (Lansing, 89 MI) for NEFA and BHBA. Insulin was determined with a bovine insulin ELISA using a solid phase 2-site enzyme immunoassay (Mercodia, Uppsala, Sweden). Lipids were extracted from plasma as described by Folch et al. (1957) with minor modifications before undergoing solid phase extraction (HyperSep Aminopropyl SPE columns; Thermo Fisher Scientific, Waltham, MA) separation of lipid fractions based on the protocol by Agren et al. (1992). FA concentration of the triglyceride (TG) fraction was determined by the two step methylation procedure and quantified using GLC as described by Lock et al. (2013). Statistical Analysis All data were analyzed using the mixed model procedure of SAS (Version 9.4, SAS Institute, Cary, NC) according to the following model: Yijkl =  + Ci (Sj) +Pk + Tl + Pk x Tl + eijkl Where Yijkl = dependent variable,  = overall mean, Ci (Sj) = random effect of cow within square (j = 1 to 2), Pk = fixed effect of period (k = 1 to 4), Tl = fixed effect of treatment (l = 1 to 4), Pk x Tl = period by treatment interaction, and eijkl = residual error. Normality of the residuals was checked with normal probability and box plots and homogeneity of variances with plots of residuals vs. predicted values. Main effects were declared significant at P ≤ 0.05 and tendencies were declared at 0.05 < P ≤ 0.10. Interactions were declared significant at P ≤ 0.10 and tendencies were declared at 0.10 < P ≤ 0.15. Period by treatment interactions were evaluated and removed from the statistical model when P > 0.20. Pre-planned contrasts were the linear and quadratic effects of increasing OA and the comparison between the 0 g and 60 g doses. All data are expressed as least square means and standard error of the means. 90 Nutrient Intake and Total-tract Digestibility Results Infusing OA did not affect DMI (P = 0.41), NDF intake (P = 0.42), DM digestibility (P = 0.53), or NDF digestibility (P = 0.71; Table 4.3). Taking into account the amount of OA infused, increasing OA from 0, 20, 40, and 60 g/d linearly increased the intake of total (1086, 1118, 1117, and 1179 g/d; P < 0.01) and 18-carbon FA (774, 803, 807, and 856 g/d; P < 0.01), respectively, but did not affect the intake of 16-carbon FA (241, 244, 239, and 250 g/d; P > 0.20). Increasing OA linearly increased digestibility of total (P < 0.01), 16-carbon (P < 0.01), and 18-carbon FA (P < 0.01), with 60 g/d increasing FA digestibilities by 8.5, 8.3, and 8.6 percentage units, respectively. Absorption of total (P < 0.01), 16-carbon (P = 0.02), and 18-carbon (P < 0.01) FA were increased by 168, 27, and 132 g/d, respectively, when 60 g/d of OA were infused. Production Responses We observed a linear trend for increasing OA to increase milk yield (P = 0.09), 3.5% FCM (P = 0.08), and ECM (P = 0.09; Table 4.4). Compared to 0 g/d, 60 g/d of OA increased milk yield (P = 0.05) and tended to increase 3.5% FCM (P = 0.06) and ECM (P = 0.06). OA did not affect milk fat yield but tended to quadratically increase milk fat content (P = 0.06) with it being greatest at the 40 g/d dose. OA did not affect milk protein yield, protein content, or lactose content (all P > 0.20), but tended to increase lactose yield (P = 0.10). No treatment effects on BW or BCS were observed. Milk Fatty Acid Concentration and Yield Milk FA are derived from two sources: de novo synthesis in the mammary gland (<16 carbon FA) or preformed FA extracted from plasma (>16 carbon FA). Mixed-source FA (16 carbon) are sourced from either de novo synthesis or plasma extraction. OA infusion increased the 91 yield of preformed FA (P = 0.04) primarily due to increased cis-9 C18:1 (P < 0.01; Table 4.5). Yield of FA from de novo and mixed sources were not affected. On a content basis, OA infusion did not affect de novo FA (P = 0.98) but linearly decreased the content of FA from mixed sources (P < 0.01) and increased FA content from preformed sources (P < 0.01), primarily due to decreased concentration of C16:0 (P < 0.01) and increased cis-9 C18:1 (P < 0.01; Table 4.6). Plasma Components OA infusion increased plasma insulin (P < 0.01) but there were no differences between 20, 40, and 60 g/d doses (Table 4.7). An interaction between treatment and period was observed for non-esterified FA (NEFA; P = 0.02). NEFA content was highest during period 2 for 0 g/d and 60 g/d doses, but highest during period 1 for 20 g/d and 40 g/d doses. There no effects of OA infusion on β-hydroxybutyrate (P = 0.42). OA infusion linearly decreased C14:0 content (P < 0.01), linearly increased the content of cis-9 C18:1 (P < 0.01), and tended to increase trans-9 C18:1 (P < 0.01) within the plasma triglycerides (Table 4.8). Discussion Maximizing nutrient absorption is crucial to meet the increasing energy requirements of the dairy cow. While supplemental FA are often included in diets to increase the energy density of a ration, it has been shown that as the amount of FA flowing to the duodenum increases, the digestibility of FA decreases (Boerman et al., 2015). Therefore, understanding factors that influence and improve FA digestibility has direct impact on diet formulation strategies and dairy industry recommendations. The majority of FA supplements contain PA (C16:0), SA (C18:0), and OA (cis-9 C18:1) in varying proportions. Historically, fat supplements rich in SA have been heavily utilized as SA is the primary FA available for absorption in the small intestine due to ruminal biohydrogenation (Jenkins, 92 1993). However, multiple studies have demonstrated a reduction in FA digestibility when SA is included in the diet compared to other FA (Chamberlain and DePeters, 2017; de Souza et al., 2018, 2020). The meta-analysis by Boerman et al. (2015) suggested that OA had greater apparent FA digestibility than either SA or PA. We recently observed that FA digestibility increased in response to more OA in the diet (de Souza et al., 2018, 2019; Chapter 3. However, effects of OA on FA digestibility are difficult to determine in feeding studies due to biohydrogenation of unsaturated FA in the rumen (Jenkins, 1993). Therefore, our study abomasally infused OA in order to more accurately study the effects of OA on FA digestibility. As we expected, total FA digestibility of our control diet was low at 61% due to inclusion of a fat supplement that contained 52% SA and 32% PA in the diet in order to increase the amount of FA available for digestion. As hypothesized, abomasally infusing OA increased digestibility of total, 16- carbon, and 18-carbon digestibility. A quadratic response to increasing doses of OA was not observed; thus, FA digestibility might continue to increase with higher doses of OA. Improvements in 16-carbon digestibility indicate that OA has a beneficial effect on FA digestibility regardless of FA chain length. Similarly, the amount of total FA absorbed was more than the amount infused at each dose, clearly indicating the ability of OA to improve the absorption of other FA, even at a dose of 20 g/d. Recent feeding studies have also demonstrated increased FA digestibility when OA is included in a dietary fat supplement compared to other FA (de Souza et al., 2018, 2019; Chapter 3). Replacing either PA or SA with OA in a fat supplement fed at 1.5% DM increased total FA digestibility in a study by de Souza et al. (2018), with greater increases observed when comparing OA to SA. Based on the DMI and FA blend of the cows receiving OA in that study, we can estimate that 25-55 g/d of OA reached the small intestine if assuming biohydrogenation rates of 60-80%. Thus, as observed in our study, small amounts of OA have the potential to affect FA digestibility. 93 The effects of OA on FA digestibility may be due to the low critical micellar concentration of OA improving micellar formation (Freeman, 1969). Improvements in micelle formation would allow for improved absorption of all FA, not just OA. Uptake and re-esterification of long-chain FA by enterocytes are not well understood and may also be involved in the mode of action by which OA improves FA absorption (Ockner et al., 1972). While individual FA absorption cannot be determined from apparent total-tract digestibility analyses, we observed a linear increase in OA content of plasma triglycerides, indicating absorption of OA continued to increase as the amount of OA infused increased. We also observed a tendency for OA infusion to increase trans-9 C18:1 content of plasma triglycerides that was likely due to absorption of the OA supplement as it contained 1.62% of trans-9 C18:1. These linear increases in plasma OA and trans-9 C18:1 suggest the absorptive and esterification mechanisms were not limited and could potentially absorb more FA if the flow of OA to the small intestine continued to increase. Effects of FA supplementation on DMI are inconsistent and appear to vary by the amount, type of supplement, FA profile of the supplement, and FA profile of the basal diet. While some studies have shown decreased DMI when OA was incorporated into calcium salts (Rabiee et al., 2012), others have demonstrated no impact on DMI (Weld and Armentano, 2018; de Souza et al., 2019). Romo et al. (1996) demonstrated that abomasal infusions of ~60 g/d of either cis-C18:1 or trans-C18:1 had no effect on DMI, which agrees with the lack of response that we observed in our study. Accordingly, intake of NDF was not affected by OA infusion in our study. Although some studies have shown altered DM or NDF digestibility in response to OA (Romo et al., 1996; Hristov et al., 2011; de Souza et al., 2018), there was no such effect noted in our study, which is to be expected since we bypassed the rumen. 94 We observed few production responses in our study; however, this study was designed to investigate differences in FA digestibility and thus may not have had adequate power or period lengths to detect effects on production parameters. Even so, infused OA tended to linearly increase milk yield, 3.5% FCM, and ECM. These results emphasize the practical importance of improving FA digestibility. The dose of 60/d of OA increased milk yield by 3.7 kg/d which is greater than the 2.8 kg/d improvement seen in the study by Romo et al. (1996). Similarly, dietary inclusion at 1.5% diet DM of a FA supplement containing 45% PA and 35% OA increased milk yield by 1.9 kg/d (de Souza et al., 2018). These differences in magnitude between studies may be due to differences in DMI as cows consuming more DMI presumably had greater amounts of dietary FA reaching the small intestine. It has also been demonstrated that cows that differ in milk production levels respond differently to OA. De Souza et al. (2019) found that cows producing similar milk yield as the cows in our study (~50 kg/d) had no response to a decreased ratio of PA:OA in a dietary fat supplement, possibly due to partitioning energy towards body tissue accretion. However, OA did increase milk and component yield in cows that produced ~60 kg/d (de Souza et al., 2019); thus we might have observed greater increases in milk production responses in our current study if higher producing cows had been used. Infusing 60 g/d of OA tended to decrease milk fat content compared to 0 g/d, but milk fat yield was not affected. In contrast, a study that infused a similar amount of OA observed increased milk fat content and decreased milk protein content (Romo et al., 1996). OA linearly increased the yield of FA from preformed sources in our study, primarily due to increasing the yield of cis-9 C18:1, yet this increase was not large enough to affect overall milk fat yield. On a concentration basis, OA proportionally decreased FA from mixed sources due to the increased FA from preformed sources. The increase in milk FA from preformed sources when long-chain FA are 95 supplemented has been consistently observed in other studies (Steele and Moore, 1968b; He et al., 2012; de Souza et al., 2018). However, the lack of a decrease in milk de novo FA (16 carbons), and mixed fatty acids originate from both sources (C16:0 plus cis-9 C16:1). 4 A total of approximately 80 individual fatty acids were quantified. Only select fatty acids are reported in the table. 5Treatment by period interaction (P<0.10) 104 Table 4.6. Milk fatty acid concentrations of cows infused with increasing amounts of oleic acid (n = 8). Variable Summation by Source, g/100 g FA3 De novo5 Mixed Preformed Selected Individual FA4, g/100 g FA C4:0 C6:0 C8:0 C10:05 C12:05 C14:05 C16:0 cis-9 C16:15 C18:0 cis-9 C18:1 cis-11 C18:15 cis-9, cis-12 C18:2 cis-9, trans-11 C18:2 cis-9, cis-12, cis-15 C18:35 trans-6 to 8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 Treatments1 0 20 40 60 27.2 35.0 37.8 3.22 2.21 1.32 3.26 3.87 12.3 33.2 1.74 9.01 0.25 0.18 0.45 0.58 18.1 0.76 2.25 0.30 0.56 27.6 34.4 38.0 3.34 2.26 1.33 3.30 3.92 12.5 32.7 1.71 9.19 0.24 0.20 0.41 0.57 18.4 0.75 2.26 0.28 0.56 27.6 33.7 38.6 3.33 2.27 1.35 3.37 3.95 12.4 32.0 1.72 9.28 0.23 0.22 0.43 0.56 19.1 0.74 2.26 0.27 0.55 27.3 33.4 39.3 3.25 2.26 1.34 3.27 3.82 12.2 31.7 1.67 9.06 0.23 0.23 0.46 0.58 19.7 0.73 2.30 0.29 0.56 SEM 0.58 0.62 1.44 0.11 0.07 0.05 0.12 0.13 0.24 0.62 0.05 0.33 0.009 0.007 0.03 0.02 0.77 0.03 0.08 0.02 0.01 Contrast2 Linear Quadratic 0 vs. 60 0.98 <0.01 <0.01 0.70 0.35 0.49 0.72 0.77 0.60 <0.01 0.04 0.77 0.15 <0.01 0.70 0.84 <0.01 0.41 0.25 0.41 0.73 0.27 0.63 0.52 0.05 0.46 0.53 0.35 0.32 0.28 0.68 0.77 0.30 0.31 0.72 0.11 0.57 0.60 0.79 0.51 0.20 0.26 0.98 <0.01 <0.01 0.64 0.35 0.61 0.86 0.71 0.70 <0.01 0.03 0.86 0.16 <0.01 0.84 0.94 <0.01 0.43 0.21 0.47 0.89 1Treatments consisted of 0, 20, 40, or 60 g/d of abomasally-infused oleic acid. 2P-values associated with contrasts: (1) the linear effect of increasing oleic acid; (2) the quadratic effect of increasing oleic acid; and (3) the effect of 60 g/d of oleic acid compared to 0 g/d. 105 3De novo fatty acids originate from mammary de novo synthesis (<16 carbons), preformed fatty acids originate from extraction from plasma (>16 carbons), and mixed fatty acids originate from both sources (C16:0 plus cis-9 C16:1). 4 A total of approximately 80 individual fatty acids were quantified. Only select fatty acids are reported in the table. 5Treatment by period interaction (P<0.10) 106 Table 4.7. Plasma NEFA, BHBA, and insulin of cows infused with increasing amounts of oleic acid (n = 8). Variable NEFA, mEq/L BHBA, mg/dL Insulin, μg/L Treatments1 0 0.11 7.59 0.80 20 0.11 7.62 0.98 40 0.13 8.09 0.98 60 0.12 7.78 0.98 SEM 0.005 0.33 0.09 Trt * Period 0.02 NS NS Contrasts2 Linear Quadratic 0 vs. 60 0.02 0.42 <0.01 0.07 0.55 0.05 0.11 0.63 <0.01 1Treatments consisted of 0, 20, 40, or 60 g/d of abomasally-infused oleic acid. 2P-values associated with contrasts: (1) the linear effect of increasing oleic acid; (2) the quadratic effect of increasing oleic acid; and (3) the effect of 60 g/d of oleic acid compared to 0 g/d. 107 Table 4.8. Fatty acid content of plasma triglycerides of cows infused with increasing amounts of oleic acid (n = 8). Variable Selected Individual FA3, g/100 g FA C12:0 C14:0 C16:0 C18:0 cis-9 C18:1 cis-11 C18:1 cis-9, cis-12 C18:2 cis-9, cis-12, cis-15 C18:3 trans-6 to 8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 trans-12 C18:1 Treatments1 0 20 40 60 SEM Trt * Period Contrast2 Linear Quadratic 0 vs. 60 0.25 2.45 23.6 33.9 0.39 0.25 0.75 1.21 0.58 5.61 1.00 10.3 1.94 0.24 2.41 23.7 34.9 0.38 0.27 0.69 1.17 0.55 6.86 1.02 9.24 1.78 0.23 2.31 22.6 32.8 0.36 0.28 0.66 1.08 0.53 7.83 0.97 11.2 1.99 0.24 2.25 22.9 33.2 0.37 0.30 0.71 1.17 0.55 8.43 0.91 10.0 1.9 0.01 0.06 0.63 1.60 0.02 0.02 0.06 0.07 0.04 0.22 0.06 1.71 0.29 NS NS NS NS NS NS NS NS NS 0.08 NS NS NS 0.39 <0.01 0.25 0.55 0.51 0.09 0.40 0.69 0.47 <0.01 0.18 0.89 0.95 0.55 0.78 0.86 0.83 0.62 0.88 0.24 0.50 0.42 0.17 0.46 0.98 0.90 0.48 0.01 0.42 0.76 0.64 0.10 0.48 0.38 0.54 <0.01 0.23 0.90 0.92 1Treatments consisted of 0, 20, 40, or 60 g/d of abomasally-infused oleic acid. 2P-values associated with contrasts: (1) the linear effect of increasing oleic acid; (2) the quadratic effect of increasing oleic acid; and (3) the effect of 60 g/d of oleic acid compared to 0 g/d. 3A total of approximately 80 individual fatty acids were quantified. 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Eight rumen-cannulated multiparous cows (89±13 DIM) were assigned to a treatment sequence in 4×4 Latin squares with 18-d periods consisting of 7 d of washout and 11 d of infusion. Treatments were abomasal infusions of water only (CON) or 30 g/d of different emulsifiers: polysorbate-C16:0 (Tween®-40; T40), polysorbate-C18:0 (Tween®-60; T60), and polysorbate-C18:1 (Tween®-80; T80). T40 predominantly contains palmitic acid as the FA tail while T60 and T80 predominantly contain stearic and oleic acids, respectively. Emulsifiers were dissolved in water and delivered at 6 h intervals. Cows were fed the same diet which contained (% DM) 32% NDF, 16% CP, 26% starch, and 3.3% FA (1.9% DM from a FA supplement containing 34% C16:0 and 48% C18:0). The statistical model included the random effect of cow within square and the fixed effects of treatment, period, square, and their interactions. Pre-planned contrasts included CON vs. average of T40, T60, and T80 (TWEEN); CON vs. T80; and T80 vs. the average of T40 and T60 (T40+T60). Compared to CON, TWEEN did not affect DMI or digestibility, but increased milk fat content and tended to increase milk fat yield and 3.5% FCM. Compared to CON, T80 increased total (73.3% vs. 69.3%), 16-carbon (73.1% vs. 69.8%), and 18-carbon (73.0% vs. 68.8%) FA digestibility. T80 tended to decrease DMI (26.4 vs. 27.2 kg/d) and increase digestibility of DM and NDF compared to CON. T80 increased milk fat content (3.70% vs. 3.33%) and yield (1.66 vs. 1.49 kg/d), tended to increase 121 3.5% FCM (46.3 vs. 43.8 kg/d), and tended to reduce milk protein yield (1.34 vs. 1.40 kg/d) compared to CON. Compared to T40+T60, T80 increased total (73.3% vs. 69.4%), 16-carbon (73.1% vs. 70.1%), and 18-carbon (73.0% vs. 68.7%) FA digestibility and tended to increase DM digestibility, but did not affect DMI, NDF intake, or NDF digestibility. In conclusion, T80 infusion increased the digestibility of total, 16-carbon, and 18-carbon FA, compared to control and T40+T60, suggesting that the predominant FA attached to polysorbate impacts its ability to improve FA digestibility. Keywords: emulsifier, fatty acid digestibility, milk fat 122 Introduction It is well established that increasing the amount of FA reaching the duodenum has a negative impact on FA digestibility (Boerman et al., 2015). Similarly, differing FA have been observed to have different digestibilities and can impact the digestibility of other FA. Feeding studies have found that supplemental fat blends containing oleic acid typically have the highest total FA digestibility, followed by palmitic acid, then by stearic acid (Chapter 3; de Souza et al., 2019; Western et al., 2020). A study abomasally infusing oleic acid reported increases in total FA digestibility as the amount of oleic acid increased (Chapter 4). The effect of oleic acid on FA digestibility is likely due to its amphiphilic characteristics, with its polar, hydrophilic headgroup linked to a non-polar, hydrophobic tail, whereas stearic and palmitic acids only behave in a non- polar manner (Freeman, 1969). There is also potential to improve FA digestibility by supplying exogenous emulsifiers in the diet (de Souza et al., 2020). Surfactants are a class of emulsifiers with amphiphilic properties that disrupt surface tension and promote the formation and stabilization of an emulsion (Kralova and Sjöblom, 2009). This then allows two immiscible liquids, such as oil and water, to be thoroughly combined. Many surfactants will self-aggregate to form micelles with the polar head- groups facing outwards and the non-polar hydrophobic tails facing inwards (Carey and Small, 1970). Non-polar substances, such as stearic and palmitic acids, can then be contained within the micelle and suspended in aqueous environments. As such, micelles are critical to transport FA from feed particles in the lumen of the small intestine through the unstirred water layer to be absorbed by enterocytes. Both bile salts and lysolecithin, a natural emulsifier produced from the phospholipid lecithin in the small intestine, play critical roles in micelle formation and absorption of FA in 123 ruminant animals (Carey et al., 1983). While bile salts are efficiently recycled, the amount of lysolecithin produced by the cow may be a limiting factor to FA absorption, especially as the flow of FA to the small intestine increases (Drackley, 2004; Maldonado-Valderrama et al., 2011). Interestingly, recent work (de Souza et al., 2020) has shown potential for an exogenous emulsifier to improve FA absorption. As such, continued investigation of supplemental exogenous emulsifiers may be an important strategy to improve FA absorption. Polysorbates are nonionic surfactants consisting of a polyethoxylated sorbitan esterified with a FA. They are commonly utilized in the pharmaceutical (Kaur and Mehta, 2017) and food (Kralova and Sjöblom, 2009) industries to stabilize emulsions but have not been extensively studied in ruminant nutrition. Several studies have examined the effects of polysorbate, under the trade name Tween, on ruminal fermentation of fiber both in vitro and in vivo (Craig et al., 1984; Kamande et al., 2000; Ahn et al., 2009; Cong et al., 2009). However, only one study has investigated the use of polysorbate as an emulsifier to improve FA digestibility (de Souza et al., 2020). The authors observed that abomasal infusion of 30 g/d of Tween80 increased FA digestibility from 61% to 71%. It should be noted that polysorbates can be esterified with palmitic, stearic, and oleic acids, referred to as Tween40, Tween60, and Tween80, respectively. To our knowledge, no experiments have compared polysorbates containing different FA tails to examine effects of polysorbates with different FA. Differences in FA digestibility in response to these three FA may thus affect the ability of various Tweens in the small intestine to increase FA digestibility. Therefore, the objective of our study was to compare the effect of different exogenous emulsifiers (Tweens) on their ability to improve FA digestibility. We hypothesized that all Tweens would increase FA digestibility relative to a non-supplemented control. We further hypothesized 124 that the increase in FA digestibility will be greatest for Tween80 due to the oleic acid attached to the polyethoxylated sorbitan. Design and Treatments Materials and Methods All experimental procedures were approved by the Institutional Animal Care and Use Committee at Michigan State University (East Lansing, MI). Eight ruminally cannulated multiparous Holstein cows averaging (mean ± SD) 89 ± 13 DIM, 46.8 ± 4.7 kg of milk, and 625 ± 39 kg of BW were randomly assigned to treatment sequences in a replicated 4x4 Latin square design. Cows were blocked by milk yield and balanced for parity and BCS. Each 18-d treatment period consisted of a 7-d washout period and an 11-d infusion period, with sampling during the last 4 d. Treatments were abomasal infusions of water carrier only (CON) or 30 g/d of different emulsifiers enriched in different FA: polysorbate-C16:0 (Tween®-40; T40), polysorbate-C18:0 (Tween®-60; T60), and polysorbate-C18:1 (Tween®-80; T80; Sigma-Aldrich, St. Louis, MO; Table 5.1). Dose was chosen based on results from our dose response study using Tween80 (de Souza et al., 2020). Daily doses of emulsifiers were suspended in 200 mL of water in individual glass jars. The infusate solution was divided into four infusions per day occurring every six hours. Stainless steel abomasal infusion devices as described by Westreicher-Kristen and Susenbeth (2017), with the addition of a circular, flexible rubber flange, were inserted into the abomasum five days before beginning the study. Infusion lines attached to the infusion devices (0.5 cm diameter polyvinyl chloride tubing) passed through the rumen fistula and sulcus omasi into the abomasum (Lock et al., 2007). Lines were checked daily throughout the study to ensure proper placement. All animals received a common diet that was formulated to meet the requirements of the animals as determined by NRC (2001; Table 5.2). The diet included a commercially available saturated 125 FA supplement (Energy Booster 100, Milk Specialties Global, Eden Prairie, MN; Table 5.2) at 1.9% diet DM. The diet was fed during a 14-d preliminary period and throughout the experiment. Dry matter concentration of forages was determined twice weekly and diets were adjusted when necessary. Cows were housed in individual tie-stalls at the Michigan State University Dairy Cattle Teaching & Research Center throughout the experiment and milked twice daily (0400 and 1500 h). Access to feed was blocked from 0800 to 1000 h for collection of orts and offering of new feed. Feed intake was recorded and cows were offered 115% of expected intake at 1000 h daily. Water was available ad libitum in each stall and stalls were bedded with sawdust and cleaned twice daily. Data and Sample Collection Samples were collected during the last 4 d of each treatment period (d 15 to 18). Samples of all diet ingredients and orts from each cow were collected daily and composited by period for analysis. Milk yield was recorded and two milk samples were collected at each milking. One aliquot was collected in a sealed tube with preservative (Bronopol tablet; D&F Control Systems, San Ramon, CA) and stored at 4°C for milk component analysis. The second aliquot was stored without preservative at -20°C for FA composition analysis. Fecal (~400 g) and blood (~15 mL) samples were collected every 9 h over the last 4 d of each period totaling 8 samples per cow per period. The 9 h interval over 4 d simulates sampling every 3 h over a 24-h period to account for diurnal variation. Feces were stored in a sealed plastic cup at -20°C. Blood was stored on ice and centrifuged within 30 min at 2,000 × g for 15 min at 4°C. Plasma was transferred into microcentrifuge tubes and stored at −20°C. BW measurements were taken daily during the sampling period following the afternoon milking. On the last day of the preliminary period and last day of each treatment period, three 126 trained investigators determined BCS on a 5-point scale in 0.25-point increments (Wildman et al., 1982). Sample Analysis Diet ingredients, orts, and fecal samples were dried at 55°C in a forced-air oven for 72 h for DM determination. Dried fecal samples for each cow were then composited by period. Dried samples were ground with a Wiley mill (1 mm-screen; Arthur H. Thomas, Philadelphia, PA). Feed ingredients, orts, and feces were analyzed for ash, NDF, indigestible NDF, CP, and starch by Cumberland Valley Analytical Services (Waynesboro, PA) as described by Boerman et al. (2017). Indigestible NDF was used as an internal marker to estimate fecal output to determine apparent total-tract digestibility of nutrients (Cochran et al., 1986). FA concentrations of feed ingredients, orts, and feces were determined as described by Lock et al. (2013). Milk samples were analyzed for fat, true protein, and lactose concentrations by mid-infrared spectroscopy (AOAC, 1990; method 972.160; NorthStar Michigan Lab, Grand Ledge, MI). Yields of 3.5% FCM, ECM, and milk components were calculated using milk yield and component concentrations from each milking, summed for a daily total, and averaged for each collection period. Milk samples used for analysis of FA composition were composited based on milk fat yield (d 15-18 of each period). Milk lipids were extracted and FA-methyl esters prepared and quantified using GLC according to Lock et al. (2013). Yield of individual FA (g/d) in milk fat were calculated by using milk fat yield and FA concentration to determine yield on a mass basis using the molecular weight of each FA while correcting for glycerol content and other milk lipid classes (Piantoni et al., 2013). Plasma samples from each cow were composited by period prior to analysis. All plasma samples were analyzed in duplicate with a coefficient of variation of <5% between duplicates. 127 Plasma samples were analyzed using a Beckman Coulter AU series chemistry analyzer (Beckman Coulter, Brea, CA) at the Michigan State University Veterinary Diagnostic Laboratory (Lansing, MI) for NEFA and BHBA. Insulin was determined with a bovine insulin ELISA using a solid phase 2-site enzyme immunoassay (Mercodia, Uppsala, Sweden). Statistical Analysis All data were analyzed using the mixed model procedure of SAS (Version 9.4, SAS Institute, Cary, NC) according to the following model: Yijkl =  + Ci (Sj) +Pk + Tl + Pk x Tl + eijkl Where Yijkl = dependent variable,  = overall mean, Ci (Sj) = random effect of cow within square (i = 1 to 4; j = 1 to 2), Pk = fixed effect of period (k = 1 to 4), Tl = fixed effect of treatment (l = 1 to 4), Pk x Tl = period by treatment interaction, and eijkl = residual error. Normality of the residuals was checked with normal probability and box plots and homogeneity of variances with plots of residuals vs. predicted values. Period by treatment interactions were evaluated and removed from the statistical model when not significant (P > 0.20). Three pre-planned contrasts were used to evaluate (1) the overall effect of Tween treatments [control vs. TWEEN; (Tween40 + Tween60 + Tween80)/3]; (2) the effect of Tween80 compared to control (CON vs. T80); and (3) the effect of Tween80 compared to the other Tween treatments [T80 vs. ½ (T40 + T60)]. All data are expressed as least square means and standard error of the means. Contrasts were declared significant at P ≤ 0.05 and tendencies were declared at 0.05 < P ≤ 0.10. Results Nutrient Intake and Total-tract Digestibility Overall, TWEEN infusion did not affect the intake of DM, NDF, 16-carbon FA, 18-carbon FA, or total FA, compared to CON (Table 5.3). TWEEN infusions also did not affect the 128 digestibility or absorption of DM, NDF, 16-carbon FA, 18-carbon FA, or total FA compared to CON, primarily due to T60 decreasing the digestibility of these variables. Compared to CON, T80 tended to decrease intake of DM (P = 0.08) and 16-carbon FA (P = 0.10), but did not affect intake of other nutrients. T80 increased digestibility of 16-carbon (P = 0.04), 18-carbon (P = 0.02), and total FA (P = 0.02) and tended to increase digestibility of DM (P = 0.09) and NDF (P = 0.09) compared to CON. T80 tended to increase absorption of 18-carbon FA (P = 0.10) compared to CON. Compared with the other Tween treatments, T80 decreased 16-carbon FA intake (P = 0.04) but did not affect the intake of DM or other nutrients. T80 increased digestibility of 16-carbon (P = 0.04), 18-carbon (P = 0.01), and total FA (P = 0.01) and tended to increase digestibility of DM (P = 0.06) compared with the other Tween treatments. Improvements in FA digestibility in response to T80 resulted in an increased absorption of 18-carbon (P = 0.02) and total FA (P = 0.02) compared with the other Tween treatments. Production Responses Compared to CON, overall TWEEN infusions increased milk fat content (P = 0.04) and tended to increase milk fat yield (P = 0.06) and 3.5% FCM (P = 0.09; Table 5.4). Due to no difference in DMI, TWEEN also improved feed efficiency (P = 0.03; FCM/DMI). Compared to CON, T80 increased milk fat content (P = 0.03), milk fat yield (P = 0.04), and FCM/DMI (P = 0.01), and tended to increase 3.5% FCM (P = 0.06) and decrease milk protein yield (P = 0.06). Relative to the other Tween treatments, T80 decreased milk protein content (P = 0.02) and tended to decrease milk protein yield (P = 0.07). 129 Milk Fatty Acid Concentrations and Yields Milk FA are derived from either de novo synthesis in the mammary gland (<16 carbon FA) or preformed FA extracted from plasma (>16 carbon FA). Mixed-source FA (16-carbon) can be derived from either source. Compared to CON, overall TWEEN did not affect the yield of milk FA derived from de novo, mixed, or preformed sources (Table 5.5). However, T80 increased yield of milk FA from preformed sources compared to CON (P = 0.02) and tended to increase yield of milk FA from preformed sources compared with the other Tween treatments (P = 0.07). These increases were largely due to increased yield of OA in milk (Table 5.6). There was no effect of treatment on the concentration of FA from de novo, mixed, or preformed sources. Plasma Hormones and Metabolites There were no effects of treatments on plasma insulin or glucose (P > 0.10; Table 5.8). However, overall, TWEEN increased plasma NEFA compared to CON (P = 0.02). T80 increased plasma NEFA compared to CON (P = 0.04) but there was no difference compared with the other Tween treatments (P = 0.84). Discussion Emulsifiers are widely used in commercial applications to combine polar and non-polar substances into emulsions. Surfactants are a specific group of emulsifiers that lower the surface tension at the interface between the dispersed (solute) and continuous (solvent) phases and thus stabilize the droplets (Rosen and Kunjappu, 2012). Micelles are an aggregate of surfactant molecules where the hydrophilic heads are in contact with the surrounding aqueous phase while the hydrophobic tails form the core of the micelle (Kralova and Sjöblom, 2009). As such, micelles are utilized in the aqueous environment of the small intestine to transport lipids that are adsorbed onto feed particles to the apical surface of intestinal epithelium for absorption (Bauer et al., 2005). 130 In ruminants, lipids leaving the rumen are 70-80% saturated FA with the rest primarily comprised of microbial phospholipids (Noble, 1981; Moore and Christie, 1984). Due to the strongly hydrophobic nature of saturated FA, micelles are critical to the process of FA absorption. While monogastric animals rely on monoglyceride to act as the main surfactant to aid in micelle formation (Carey et al., 1983), ruminant animals completely hydrolyze triglycerides to FA in the rumen, thus causing an absence of monoglyceride in the duodenum of ruminant animals (Jenkins, 1993). As such, ruminants have developed a unique process to form micelles. In this process, the animal must produce two key factors: 1) bile in the liver and 2) phospholipase in the pancreas. Bile acids can directly act on lipid materials to form micelles, whereas phospholipase A2 indirectly acts by cleaving a FA from lecithin phospholipids, either from dietary, microbial, or biliary origins, to form lysolecithin (Morgado et al., 1996; Vasudevan et al., 2016). Lysolecithin then acts as a potent surfactant to increase and stabilize micelle formation. Interestingly, researchers have observed that as the amount of stearic acid reaching the duodenum increases, FA digestibility decreases (Boerman et al., 2015). The negative effects of stearic acid amount on FA digestibility suggests that the natural lysolecithin production capacity of the cow might not be sufficient to digest the higher amounts of FA reaching the duodenum in modern dairy cows due increased DMI. Thus, supplying exogenous emulsifiers may increase FA digestibility. While lysolecithin acts as an important surfactant in ruminants, providing supplemental lecithin in the diet may be a practical challenge as it has been shown to be susceptible to hydrolytic and oxidative degradation and self-aggregation in storage as well as BH in the rumen (Isailović et al., 2017; Fontoura et al., 2019). There are many other natural and synthetic emulsifiers used in food and biomedical industries, so supplying emulsifiers other than, or in addition to, lecithin may aid in FA absorption in the cow. Past work in ruminants has utilized a widely used, commercially 131 available nonionic surfactant known as Tween®, which is comprised of polyethoxylated sorbitan esterified with various FA (de Souza et al., 2020). Tween products have been used in bovine studies in the past, but often as a means to emulsify lipophilic treatments into solution to be abomasally or intravenously infused (e.g. Erickson et al., 1963; Drackley et al., 2007) or included in milk replacer (Jenkins and Emmons, 1984), rather than as a variable of interest. Several studies have been carried out in vitro and in vivo to examine the effects of Tween on rumen microbial populations, enzyme production, and fiber degradation (Kamande et al., 2000; Lee and Ha, 2003; Ahn et al., 2009). To our knowledge, the only previous study that has examined the effects of Tween on FA digestion in ruminants is our recent study by de Souza et al. (2020). These authors observed that 30 g/d of Tween80 infused abomasally increased total, 16-carbon, and 18-carbon FA digestibility by 10, 9, and 11 percentage units, respectively. Thus, Tween can act as a potent surfactant in the small intestine and we aimed to better understand the potential for synthetic emulsifiers to aid FA absorption in the cow. The Tween family of surfactants contains several variants that all contain the same base polyethoxylated sorbitan but differ by the FA esterified to it. Tween40 is esterified mostly with palmitic acid (~94%), Tween60 predominantly with stearic acid (~49%, ~43% palmitic acid), and Tween80 mostly with oleic acid (~84%). These differences in FA composition between the various Tweens should be considered as micelle formation and stability can be affected by the profile of FA in the small intestine (Hofmann, 1963). For example, non-polar FA, such as stearic and palmitic acids, have relatively low solubility in bile salt solution. In contrast, increasing the unsaturation of a FA imparts amphiphilic qualities to the FA by increasing the dissymmetry between polar and non-polar regions (Hofmann, 1963). Freeman (1969) solidified the basic mechanisms by which FA exert differing effects on micelle formation, and thus, FA absorption. 132 His work characterized factors that influence FA absorption in sheep and found that the critical micellar concentration for oleic acid is 60% lower than that for stearic acid, meaning micelles will begin to form at a lower concentration for oleic acid than for stearic acid. Furthermore, Freeman (1969) found that oleic acid was 6 and 14 times more soluble in a dilute bile salt solution than palmitic and stearic acids, respectively. Compared to lysolecithin, the predominant surfactant naturally produced by the cow from lecithin, oleic acid is roughly half as amphiphilic (Freeman, 1969). Differences in FA absorption in response to the profile of FA reaching the duodenum have been well demonstrated in the bovine. A recent study fed either a non-FA supplemented control diet or diets supplemented with proportions of FA at 1.5% diet DM that contained either 80% palmitic acid, 40% palmitic acid + 40% stearic acid, or 45% palmitic acid + 35% oleic acid. In this study, including oleic acid in the supplemental FA blend increased total FA digestibility whereas including stearic acid decreased total FA digestibility (de Souza et al., 2018). These results agree with work by Freeman (1969). Other studies have also observed that oleic acid increases total FA digestibility when compared directly to palmitic acid (de Souza et al., 2019) and to stearic acid (Chapter 3) or when infused directly into the abomasum (Chapter 4). Thus, we expected that total FA digestibility in this study would be highest for T80, followed by T40, T60, and CON. In our current study, overall, TWEEN had no effects on the digestibility of FA, largely due to opposing effects between the Tween treatments. T80 improved total, 16-carbon, and 18-carbon FA digestibility compared to both CON and to the other Tween treatments. The effects of T80 on FA digestibility support our hypothesis that T80 would have the greatest FA digestibility due to the esterified oleic acid. However, the results disagree with our hypothesis that all Tweens examined would improve FA digestibility, as T60 had numerically lower total, 16-carbon, and 18- 133 carbon FA digestibility than CON, likely due to the negative effects of the attached stearic acid. The strong effect of the attached FA was surprising as the infusate only contained 7, 7, and 6 g/d of FA for T40, T60, and T80, respectively. Thus, it is challenging to differentiate between the effects of the polysorbate and the attached FA. In our current study, the T80 treatment contained 5g/d of OA and increased total FA digestibility by 4 percentage units. In comparison, the smallest OA dose of 20 g/d in the study by Prom et al. (Chapter 4) increased total FA digestibility by 6 percentage units, suggesting the OA attached to T80 may be more potent at improving FA digestibility than the polysorbate per se. Future research should directly compare abomasal infusions of T80 and OA in order to better understand these differing effects. Compared to CON, absorption of total and 16-carbon FA in response to T80 were not affected due to minor decreases in DMI. Even so, we observed a tendency for an increase in 18- carbon FA absorption due to the large improvement in 18-carbon FA digestibility. Compared to the other Tweens, T80 did not affect DMI; thus, the improved FA digestibility translated into increased absorption of total and 18-carbon FA. Regardless of the attached FA, overall TWEEN increased plasma non-esterified FA compared to CON, which suggests that more FA may have been absorbed and entered circulation. Tween infusions had no overall effect on DMI, but T80 tended to decrease DMI compared to CON. In contrast, infusing 30 g/d of Tween80 had no effect on DMI in the study by de Souza et al. (2020). However, at a dose of 45 g/d, the authors did observe a tendency for a decrease in DMI. As the cows consumed more DM in that study (~29 kg/d) than in our study (~27 kg/d), the effect of T80 on DMI in our study could be dose related. While no work has examined the effects of exogenous emulsifiers on gut peptides, it is likely that the decreased DMI is related to satiety signaling (Choi and Palmquist, 1996). Furthermore, unsaturated FA, such as oleic acid in Tween80, 134 have been shown to affect cholecystokinin and GLP-1 (Relling and Reynolds, 2007; Bradford et al., 2008). Despite minor differences in DMI, there were no treatment effects on intake of NDF. Compared to CON, T80 tended to increase both DM and NDF digestibility. The effects of T80 on DM and NDF digestibility could be due to decreased DMI and increased rumen retention time caused by gut peptide signaling (Harvatine and Allen, 2006b). If Tween80 had been included in the diet instead of being abomasally infused, alterations to the microbial population in the rumen would also likely affect DM and NDF digestibility. Past studies utilizing Tween80 both in vitro and in vivo observed effects on microbial population, ruminal fermentation, and fiber digestion (Kamande et al., 2000; Goto et al., 2003; Lee and Ha, 2003; Cong et al., 2009). Since Tween has clear effects in the rumen, differences between abomasally and ruminally infused Tween should also be examined in the future. We observed no treatment effects on milk yield in this study, possibly due to the small decrease in DMI in response to T80. Similarly, abomasal infusion of Tween80 in the study by de Souza et al. (2020) did not affect milk yield. In the current study, Tween infusions increased milk fat content and tended to increase milk fat yield, mainly due to T80. These results agree with increased milk fat content and milk fat yield observed by de Souza et al. (2020), although the magnitude of increase was slightly larger in our current study. Accordingly, there was a tendency for T80 to increase 3.5% FCM. A study examining the effects of Tween80 on ruminal characteristics in a small number of Holstein cows in early lactation also observed increased milk yield, milk fat content, and 4% FCM, possibly due to improving FA absorption (Lee et al., 2003). T80 increased the yield of milk fat due to increasing the yield of certain FA. As T80 tended to increase 18-carbon FA absorption, it also increased the yield of most long-chain FA, which thus increased the yield of FA from preformed sources. Consistent with the increase in long-chain FA, 135 there were no treatment effects on yield of milk FA from de novo or mixed sources. In contrast, de Souza et al. (2020) observed increases in the yield of FA from de novo, mixed, and preformed sources. These differences could be related to energy signaling as the cows in the study by de Souza et al. (2020) had higher DMI and milk production than the cows in our present study. However, infusing 30 g/d of T80 did not affect BW, BCS, or plasma insulin in either our study or the study by de Souza et al. (2020) compared to CON. Tween infusions, as well as T80 specifically, improved feed efficiency (FCM/DMI) due to the increased milk fat yield and minimal effects on DMI. There were no treatment effects on BW or BCS, however studies designed with a larger sample size and longer period lengths are often necessary to see effects on these variables. There are indications from other studies that the profile of FA reaching the small intestine affects energy partitioning (de Souza et al., 2018, 2019) so differences between the Tween treatments could potentially affect body tissue reserves in studies designed to examine these variables. It should be noted that insulin, which is a key regulator of energy utilization, was increased by dietary oleic acid in previous studies. (de Souza et al., 2018, 2019; Chapter 4), but was not affected by any treatment in our study. Conclusion Overall, Tween infusions did not affect nutrient digestibility due to the negative impact of the Tween60 treatment. However, Tween80 increased the digestibility of total, 16-carbon, and 18- carbon FA, compared to control and to the average of the other two other Tween treatments. These results suggest that the predominant FA attached to polysorbate impacts its ability to improve FA digestibility. The impact of the attached FA may be greater than we anticipated and future work should directly compare abomasal infusions of Tween80 and oleic acid. Tween80 also increased milk fat content and yield compared to control. Therefore, Tween80 has the potential to improve 136 nutrient digestibility and milk production responses, but it is unknown whether these effects are primarily due to the polysorbate or the attached oleic acid. Acknowledgements Funding for this research was provided by the Michigan Alliance for Animal Agriculture. Crystal Prom was supported by a Pre-Doctoral Fellowship from USDA NIFA. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. We acknowledge L. Worden, J. de Souza, M. Western, J. Guy, H. Sharrard, E. Butler, A. Negreiro, A. Pineda, and A. Burch (all in the Department of Animal Science, Michigan State University, East Lansing) and the staff of the Michigan State University Dairy Cattle Teaching & Research Center (East Lansing) for their assistance in this experiment. 137 APPENDIX 138 Table 5.1. Fatty acid (FA) profile and total FA content of Tween supplements infused during treatment periods. Selected Individual FA, g/ 100g FA C14:0 C16:0 C18:0 trans C18:1 cis-9 C18:1 cis-11 C18:1 cis-9, cis-12 C18:2 cis-9, trans-11 C18:2 Total FA, % DM Tween401 Tween601 Tween801 1.55 43.4 48.5 0.32 2.27 0.11 0.32 - 0.26 4.66 1.41 5.23 83.6 2.35 0.06 - 0.38 94.7 4.63 - - - - - 24.0 22.6 18.3 1Sigma-Aldrich, St. Louis, MO 139 Table 5.2. Ingredient and nutrient composition of diet fed to all cows infused with treatments (n = 8). Ingredient, % DM Corn Silage Alfalfa Silage Wheat Straw Ground Corn High Moisture Corn Soybean Meal Soyhulls Vitamin Mineral Mix1 Fat Supplement2 Nutrient Composition, % DM DM3 NDF CP Starch FA C16:0 C18:0 cis-9 C18:1 cis-9, cis-12 C18:2 cis-9, cis-12, cis-15 C18:3 29.3 20.8 1.68 11.5 12.3 12.0 7.74 2.84 1.92 54.1 32.1 15.7 25.8 3.32 0.82 0.78 0.42 0.90 0.17 1Vitamin and mineral mix contained 34.1% dry ground shelled corn, 25.6% white salt, 21.8% calcium carbonate, 9.1% Biofos (The Mosaic Co., Plymouth, MN), 3.9% magnesium oxide, 2% soybean oil, and < 1% of each of the following: manganese sulfate, zinc sulfate, ferrous sulfate, copper sulfate, iodine, cobalt carbonate, vitamin E, vitamin A, vitamin D, and selenium. 2Energy Booster 100 (Milk Specialties Global, Eden Prairie, MN). Contained (g/100 g of FA) 4.09 of C14:0, 34.2 of C16:0, 47.7 of C18:0, 6.42 of cis-9 C18:1, and 0.89 cis-9,cis-12 C18:2; 80.5% total FA. 3Percent of as-fed diet. 140 Table 5.3. Nutrient intake and total-tract nutrient digestibility of cows infused with treatments (n = 8). Treatment1 Variable CON T40 T60 T80 16-carbon 18-carbon Intake, kg/d DM NDF Intake, g/d Total FA Digestibility, % DM NDF Total FA Absorbed, g/d Total FA 16-carbon FA 18-carbon FA 16-carbon 18-carbon 27.2 8.79 914 225 623 64.1 45.3 69.3 69.8 68.8 633 157 429 27.2 8.87 916 229 621 64.1 47.2 70.8 71.2 70.2 636 160 427 26.6 8.61 898 222 611 64.0 46.6 67.9 68.9 67.1 618 154 416 26.4 8.59 892 218 609 65.7 48.7 73.3 73.1 73.0 659 161 448 SEM 0.91 0.34 26.6 6.35 18.4 0.68 1.86 1.90 1.55 2.01 23.0 4.81 16.8 CON vs TWEEN 0.21 0.42 0.32 0.63 0.23 0.48 0.17 0.30 0.31 0.32 0.73 0.54 0.82 Contrasts2 CON vs T80 0.08 0.17 0.14 0.10 0.15 0.09 0.09 0.02 0.04 0.02 0.12 0.27 0.10 T80 vs T40+T60 0.19 0.22 0.23 0.04 0.41 0.06 0.29 0.01 0.04 0.01 0.04 0.29 0.02 1Treatments consisted of no fat supplementation (CON), Tween40 (T40; palmitic), Tween60 (T60; stearic), and Tween80 (T80; oleic). 2P-values associated with contrasts: (1) the effect of Tween supplementation (TWEEN) compared to no Tween supplementation (CON); (2) the effect of T80 supplementation compared to no Tween supplementation; and (3) the effect of T80 supplementation compared to the average of T40 and T60 supplementation. 141 Table 5.4. Milk yield, milk composition, BW, and BCS of cows infused with treatments (n = 8). Treatments1 Variable CON T40 T60 T80 SEM Trt * Period Contrasts2 CON vs TWEEN CON vs T80 vs T80 T40+T60 Yield, kg/d Milk 3.5% FCM3 ECM4 Fat Protein Lactose Milk Composition, % Fat Protein Lactose ECM/DMI Body Weight, kg BCS 45.4 43.8 44.0 1.49 1.40 2.26 3.33 3.09 4.97 1.63 649 3.07 45.4 45.7 45.6 1.62 1.40 2.25 3.60 3.10 4.95 1.68 651 3.08 44.8 44.5 44.5 1.55 1.38 2.23 3.50 3.10 4.96 1.68 649 3.08 44.3 46.3 45.7 1.66 1.34 2.20 3.70 3.04 4.95 1.72 652 3.05 1.63 1.98 1.81 0.12 0.04 0.09 0.31 0.11 0.03 0.06 8.54 0.07 NS 0.15 0.17 0.14 NS NS 0.12 NS NS 0.07 0.07 NS 0.35 0.09 0.14 0.06 0.31 0.30 0.04 0.82 0.39 0.04 0.56 0.97 0.17 0.06 0.12 0.04 0.06 0.15 0.03 0.11 0.34 0.02 0.61 0.53 0.29 0.30 0.17 0.28 0.07 0.29 0.32 0.02 0.68 0.21 0.76 0.26 1Treatments consisted of no fat supplementation (CON), Tween40 (T40; palmitic), Tween60 (T60; stearic), and Tween80 (T80; oleic). 2P-values associated with contrasts: (1) the effect of Tween supplementation (TWEEN) compared to no Tween supplementation (CON); (2) the effect of T80 supplementation compared to no Tween supplementation; and (3) the effect of T80 supplementation compared to the average of T40 and T60 supplementation. 33.5% FCM = [(0.4324 × kg of milk) + (16.216 × kg of milk fat)]. 4ECM = [(0.327 × kg of milk) + (12.95 × kg of milk fat) + (7.20 × kg of milk protein)]. 142 Table 5.5. Summation of milk fatty acid (FA) concentration and yield for cows infused with treatments (n = 8). Variable Summation by Source3, g/100 g FA CON Treatments1 T40 T60 T80 SEM Trt * Period CON vs TWEEN Contrasts2 CON vs T80 T80 vs T40+T60 De Novo Mixed Preformed Summation by Source3, g/d De Novo Mixed Preformed 27.1 38.7 34.2 378 542 475 25.7 38.6 35.6 374 550 491 26.6 38.5 34.9 388 562 500 27.0 38.1 34.9 421 586 537 0.67 0.77 1.31 31.4 47.5 25.7 0.04 NS NS NS NS 0.03 0.39 0.64 0.41 0.58 0.45 0.11 0.92 0.42 0.62 0.24 0.27 0.02 0.31 0.44 0.76 0.21 0.39 0.07 1Treatments consisted of no fat supplementation (CON), Tween40 (T40; palmitic), Tween60 (T60; stearic), and Tween80 (T80; oleic). 2P-values associated with contrasts: (1) the effect of Tween supplementation (TWEEN) compared to no Tween supplementation (CON); (2) the effect of T80 supplementation compared to no Tween supplementation; and (3) the effect of T80 supplementation compared to the average of T40 and T60 supplementation. 3De novo fatty acids originate from mammary de novo synthesis (<16 carbons), preformed fatty acids originate from extraction from plasma (>16 carbons), and mixed fatty acids originate from both sources (C16:0 plus cis-9 C16:1). 143 Table 5.6. Milk fatty acid yield of cows infused with treatments (n = 8). Treatments1 Variable CON T40 T60 T80 SEM Trt * Period Contrasts2 CON vs TWEEN CON vs T80 T80 vs T40+T60 Selected Individual FA3, g/d C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 cis-9 C16:1 C18:0 trans-6 to 8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 cis-9 C18:1 cis-11 C18:1 cis-9, cis-12 C18:2 cis-9, trans-11 C18:2 cis-9, cis-12, cis-15 C18:3 33.6 25.7 15.9 45.1 54.6 191 12.0 520 21.6 126 2.60 2.34 5.62 5.49 236 6.76 23.4 2.90 4.30 34.5 25.4 15.6 44.1 53.4 189 11.9 529 22.0 131 2.74 2.43 6.33 6.16 243 7.12 25.0 3.22 4.48 35.2 26.5 16.3 46.0 55.7 196 12.4 540 22.5 133 2.73 2.46 6.15 5.85 249 6.95 23.8 3.02 4.42 39.9 28.9 17.7 49.5 59.3 212 13.3 563 23.5 144 3.14 2.84 7.24 6.64 266 7.48 25.8 3.74 4.81 3.26 2.89 1.88 5.09 5.67 13.3 1.07 46.5 1.34 11.9 0.29 0.18 1.77 0.53 10.8 0.6 1.09 0.47 0.17 NS NS NS NS NS 0.14 NS NS 0.13 0.08 NS NS NS NS 0.01 0.13 0.04 NS 0.04 0.36 0.64 0.68 0.74 0.76 0.55 0.60 0.46 0.39 0.20 0.21 0.22 0.46 0.13 0.05 0.16 0.13 0.18 0.13 0.12 0.32 0.36 0.42 0.45 0.21 0.28 0.28 0.22 0.07 0.05 0.04 0.31 0.06 <0.01 0.06 0.05 0.04 0.03 0.14 0.29 0.31 0.35 0.39 0.17 0.26 0.39 0.35 0.16 0.08 0.06 0.46 0.21 0.03 0.16 0.18 0.08 0.06 1Treatments consisted of no fat supplementation (CON), Tween40 (T40; palmitic), Tween60 (T60; stearic), and Tween80 (T80; oleic). 2P-values associated with contrasts: (1) the effect of Tween supplementation (TWEEN) compared to no Tween supplementation (CON); (2) the effect of T80 supplementation compared to no Tween supplementation; and (3) the effect of T80 supplementation compared to the average of T40 and T60 supplementation. 3Approximately 80 individual fatty acids were quantified. Only select fatty acids are reported in the table. 144 Table 5.7. Milk fatty acid concentration of cows infused with treatments (n = 8). Treatments1 Variable CON T40 T60 T80 SEM Trt * Period CON vs TWEEN Selected Individual FA3, g/100 g FA C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C16:0 cis-9 C16:1 C18:0 trans-6 to 8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 cis-9 C18:1 cis-11 C18:1 cis-9, cis-12 C18:2 cis-9, trans-11 C18:2 cis-9, cis-12, cis-15 C18:3 2.41 1.84 1.14 3.23 3.91 13.8 0.86 37.1 1.55 8.98 0.19 0.17 0.42 0.40 17.0 0.49 1.7 0.21 0.31 2.41 1.75 1.06 2.99 3.63 13.1 0.81 37 1.60 9.41 0.20 0.17 0.47 0.45 17.8 0.54 1.84 0.24 0.33 2.41 1.8 1.11 3.12 3.79 13.5 0.86 36.9 1.57 9.12 0.20 0.18 0.48 0.42 17.5 0.50 1.69 0.22 0.31 2.55 1.82 1.11 3.12 3.78 13.8 0.88 36.5 1.59 9.00 0.21 0.19 0.53 0.45 17.5 0.51 1.72 0.26 0.32 0.07 0.07 0.05 0.13 0.15 0.40 0.05 0.79 0.08 0.67 0.03 0.02 0.16 0.05 0.69 0.07 0.13 0.05 0.02 NS NS 0.14 0.04 0.04 NS 0.09 NS 0.09 NS NS NS NS NS 0.08 NS NS NS NS 0.54 0.45 0.41 0.35 0.32 0.41 0.79 0.58 0.55 0.67 0.34 0.39 0.49 0.21 0.45 0.46 0.53 0.24 0.50 Contrasts2 CON vs T80 0.14 0.79 0.71 0.58 0.55 0.95 0.74 0.36 0.59 0.97 0.22 0.11 0.39 0.23 0.62 0.62 0.83 0.09 0.46 T80 vs T40+T60 0.09 0.54 0.60 0.69 0.71 0.21 0.35 0.42 0.92 0.59 0.42 0.11 0.62 0.76 0.82 0.86 0.60 0.20 0.75 1Treatments consisted of no fat supplementation (CON), Tween40 (T40; palmitic), Tween60 (T60; stearic), and Tween80 (T80; oleic). 2P-values associated with contrasts: (1) the effect of Tween supplementation (TWEEN) compared to no Tween supplementation (CON); (2) the effect of T80 supplementation compared to no Tween supplementation; and (3) the effect of T80 supplementation compared to the average of T40 and T60 supplementation. 3Approximately 80 individual fatty acids were quantified. 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Treatments were abomasal infusions at 6-h intervals of water carrier only (CON), 60 g/d oleic acid (OA), 30 g/d polysorbate-C18:1 (Tween®-80; T80), or both OA and T80 (BOTH). OA treatments were dissolved in ethanol and T80 treatments in water. Cows were fed a basal diet which contained (% DM) 30% NDF, 16% CP, 30% starch, and 3.2% fatty acids (FA), with 1.8% of diet DM from a FA supplement containing 29% C16:0 and 56% C18:0). OA increased 18-carbon FA digestibility by 6.3 percentage units and tended to increase digestibility of 16-carbon and total FA by 3.9 and 5.4 percentage units, respectively. T80 did not affect nutrient digestibility. Treatment did not alter DMI, body weight, BCS, or plasma insulin content. OA did not affect yield of milk or milk components but increased feed efficiency (FCM/DMI). T80 increased milk yield by 1.7 kg/d, decreased milk fat content, but did not affect milk fat yield. Interactions between OA and T80 were observed for milk fat yield and ECM. We observed an interaction between OA and T80 for the yield of milk FA from preformed sources with OA increasing preformed milk FA more than T80. The increase in the yield of milk FA from preformed sources was primarily due to increased OA. In conclusion, infusion of OA had minimal effects on milk production while infusion of an exogenous emulsifier increased milk yield and decreased milk fat content. Interactions between 158 OA and T80 were observed for milk fat yield and ECM due to the infusion of BOTH tending to decrease milk fat yield. OA increased digestibility of 18-carbon FA and tended to increase the digestibility of 16-carbon and total FA. Keywords: emulsifier, oleic acid, fatty acid digestibility 159 Introduction Improving nutrient utilization and efficiency continues to be an important goal of dairy producers. Fatty acids (FA) provide a valuable, energy dense nutrient for dairy cows. As milk production and DMI of dairy cows increase, so too does the amount of FA flowing to the small intestine. While these FA are a potential energy source for the cow, it is well established that FA digestibility decreases as more FA reach the small intestine (Boerman et al., 2015). Thus, it is critical to determine strategies to increase FA digestibility in the small intestine in order to maximize utilization of dietary FA. In ruminant animals, bile salts and lysolecithin are naturally produced emulsifiers that are both necessary to efficiently form micelles to transport FA through the aqueous environment of the small intestine in order to be absorbed by enterocytes (Lough and Smith, 1976; Noble, 1981; Reis et al., 2004). Importantly, micellar uptake of FA in the small intestine can potentially be limited by the amount of bile salts, lecithin, or pancreatic phospholipase as these are all necessary for effective micelle formation (Harrison and Leat, 1975). Thus, as more FA reach the small intestine, absorption of FA may be limited by the natural production capacity of lysolecithin by the cow and providing exogenous emulsifiers may aid FA absorption. Emulsifiers are substances with amphiphilic capabilities, meaning they possess a polar, hydrophilic headgroup and a non-polar, hydrophobic tail (Carey and Small, 1970). While many emulsifiers are commonly utilized in the pharmaceutical and food industries, one that possesses similar qualities to lysolecithin is polysorbate. Polysorbate-80 (Tween80) has been observed to increase total FA digestibility when infused into the abomasum of lactating dairy cows (de Souza et al., 2020). Polysorbates (Tween©) are nonionic surfactant emulsifiers consisting of a polyethoxylated sorbitan esterified with a FA (Kume et al., 2008). Various Tween products have been compared to assess the impact of the 160 esterified FA. Tween80 was found to have the most beneficial impact on total FA digestibility as it contained oleic acid (OA), which also possesses amphiphilic characteristics (Chapter 4). Freeman (1969) conducted a series of in vitro experiments defining the critical micellar concentration (CMC) and saturation ratio of various FA. CMC is defined as the amount of surfactant required for micelles to form (Carey and Small, 1970). Following micelle formation, insoluble lipids can be incorporated into the hydrophobic interior of the micelle; thus, FA with high solubility and low CMC can increase the uptake of poorly soluble FA into the micelle. Freeman (1969) observed that OA had a lower CMC than palmitic and stearic acids, as well as being about half as potent as lysolecithin at solubilizing stearic acid. Due to the potential for OA to improve micelle formation and solubilize other FA into micelles, we have consistently observed improvements in FA digestibility when OA is infused into the abomasum (Chapter 4) or included in the diet (Chapter 3; Burch et al., 2018). Therefore, the objective of our study was to determine if abomasal infusion of OA, Tween80, or both alters FA digestibility and milk production. We hypothesized that OA and Tween80 would independently increase total FA digestibility and have additive effects when infused together. Materials and Methods Design and Treatments All experimental procedures were approved by the Institutional Animal Care and Use Committee at Michigan State University (East Lansing, MI). Eight ruminally cannulated multiparous Holstein cows averaging (mean ± SD) 96 ± 23 DIM, 52.8 ± 3.8 kg of milk, and 695 ± 60 kg of BW were randomly assigned to treatment sequence in a replicated 4x4 Latin square design. Cows were blocked by milk yield and balanced for parity and BCS. Infusion periods were 11 d in length with sampling occurring during the last 4 d of infusions; 7-d rest periods were in between infusion periods. 161 Infusions were a 2x2 factorial arrangement of treatments that consisted of water carrier only (CON), 60 g/d oleic acid (OA; O1008-1G, Sigma-Aldrich, St. Louis, MO; Table 6.1), polysorbate-C18:1 (Tween®-80; T80; Sigma-Aldrich, St. Louis, MO), or both OA and T80 (BOTH). Doses of OA and T80 were chosen based on results from our dose response studies using OA (Chapter 4) and Tween- 80 (de Souza et al., 2020). Daily doses of OA were dissolved in 200 mL of ethanol and T80 in 200 mL of water in individual glass jars. The infusate solutions were divided into four infusions per day occurring every six hours. Stainless steel abomasal infusion devices as described by Westreicher- Kristen and Susenbeth (2017), with the addition of a circular, flexible rubber flange, were inserted into the abomasum five days before beginning the study. Infusion lines attached to the infusion devices (0.5 cm diameter polyvinyl chloride tubing) passed through the rumen fistula and sulcus omasi into the abomasum (Lock et al., 2007). Lines were checked daily throughout the study to ensure proper placement. All animals received a common diet that was formulated to meet the requirements of the animals as determined by NRC (2001; Table 6.2). The diet included a commercially available saturated FA supplement (Energy Booster 100, Milk Specialties Global, Eden Prairie, MN; Table 6.2) at 1.8% diet DM. The diet was fed during a 7-d preliminary period and throughout the experiment. Dry matter concentrations of forages were determined twice weekly, and diets were adjusted when necessary. Cows were housed in individual tie-stalls at the Michigan State University Dairy Cattle Teaching & Research Center throughout the experiment and milked twice daily (0400 and 1500 h). Access to feed was blocked from 0800 to 1000 h for collection of orts and offering of new feed. Feed intake was recorded and cows were offered 115% of expected intake at 1000 h daily. Water was available ad libitum in each stall and stalls were bedded with sawdust and cleaned twice daily. 162 Data and Sample Collection Samples were collected during the last 4 d of each treatment period (d 15 to 18). Samples of all diet ingredients and orts from each cow were collected daily and composited by period for analysis. Milk yield was recorded and two milk samples were collected at each milking. One aliquot was collected in a sealed tube with preservative (Bronopol tablet; D&F Control Systems, San Ramon, CA) and stored at 4°C for milk component analysis. The second aliquot was stored without preservative at -20°C for FA composition analysis. Fecal (~400 g) and blood (~15 mL) samples were collected every 9 h over the last 4 d of each period totaling 8 samples per cow per period. The 9-h interval over 4 d simulates sampling every 3 h over a 24-h period to account for diurnal variation. Feces were stored in a sealed plastic cup at -20°C. Blood was stored on ice and centrifuged within 30 min at 2,000 × g for 15 min at 4°C. Plasma was transferred into microcentrifuge tubes and stored at −20°C. BW measurements were taken daily during the sampling period following the afternoon milking. On the last day of the preliminary period and last day of each treatment period, three trained investigators determined BCS on a 5-point scale in 0.25-point increments (Wildman et al., 1982). Sample Analysis Diet ingredients, orts, and fecal samples were dried at 55°C in a forced-air oven for 72 h for DM determination. Dried fecal samples for each cow were then composited by period. Dried samples were ground with a Wiley mill (1 mm-screen; Arthur H. Thomas, Philadelphia, PA). Feed ingredients, orts, and feces were analyzed for ash, NDF, indigestible NDF, CP, and starch by Cumberland Valley Analytical Services (Waynesboro, PA) as described by Boerman et al. (Boerman et al., 2017). Indigestible NDF was used as an internal marker to estimate fecal output 163 to determine apparent total-tract digestibility of nutrients (Cochran et al., 1986). FA concentrations of feed ingredients, orts, and feces were determined as described by Lock et al. (2013). Milk samples were analyzed for fat, true protein, and lactose concentrations by mid-infrared spectroscopy (AOAC, 1990; method 972.160; NorthStar Michigan Lab, Grand Ledge, MI). Yields of 3.5% FCM, ECM, and milk components were calculated using milk yield and component concentrations from each milking, summed for a daily total, and averaged for each collection period. Milk samples used for analysis of FA composition were composited based on milk fat yield (d 15-18 of each period). Milk lipids were extracted and FA-methyl esters prepared and quantified using GLC according to Lock et al. (2013). Yield of individual FA (g/d) in milk fat were calculated by using milk fat yield and FA concentration to determine yield on a mass basis using the molecular weight of each FA while correcting for glycerol content and other milk lipid classes (Piantoni et al., 2013). Plasma samples from each cow were composited by period prior to analysis. All plasma samples were analyzed in duplicate with a coefficient of variation of <5% between duplicates. Insulin was determined with a bovine insulin ELISA using a solid phase 2-site enzyme immunoassay (Mercodia, Uppsala, Sweden). Statistical Analysis All data were analyzed using the mixed model procedure of SAS (Version 9.4, SAS Institute, Cary, NC) according to the following model: Yijklm =  + Ci (Sj) +Pk + Ol + Tm + Ol x Tm + Pk x Ol x Tm + eijkl Where Yijklm= dependent variable,  = overall mean, Ci (Sj) = random effect of cow within square (i = 1 to 4; j = 1 to 2), Pk = fixed effect of period (k = 1 to 4), Ol = fixed effect of oleic acid treatment (l = 1 to 2), Tm= fixed effect of Tween80 treatment (m = 1 to 2), Ol x Tm = interaction 164 between oleic acid andTween80, Pk x Ol x Tm = interaction between period, oleic acid, and Tween80, and eijkl = residual error. Normality of the residuals was checked with normal probability and box plots and homogeneity of variances with plots of residuals vs. predicted values. Period by treatment interactions were evaluated and removed from the statistical model when not significant (P > 0.20). All data are expressed as least square means and standard error of the means. Significance was declared at P ≤ 0.05 for main effects and P ≤ 0.10 for interactions. Tendencies were declared at P ≤ 0.10 for main effects and P ≤ 0.15 for interactions. Nutrient Intake and Total-tract Digestibility Results Treatment did not alter intake of DM (P > 0.84) or NDF (P > 0.75; Table 6.3). OA infusion increased intake of total (P < 0.01) and 18-carbon FA (P < 0.01) but did not affect intake of 16- carbon FA (P = 0.66). Infusion of T80 did not alter intake of total, 16-carbon, or 18-carbon (all P > 0.78). The interaction between OA and T80 did not affect nutrient intake. Treatment did not alter digestibility of DM (P > 0.23) and NDF (P > 0.36). OA increased digestibility of 18-carbon FA (P = 0.05) and tended to increase the digestibility of total (P = 0.06) and 16-carbon FA (P = 0.10). Neither T80 nor the combination of OA and T80 altered digestibility of total, 16-carbon, or 18-carbon FA (all P > 0.21). OA increased absorption of total (P < 0.01) and 18-carbon FA (P < 0.01) but did not affect absorption of 16-carbon FA (P = 0.23). Production Responses OA infusion did not affect production of milk or milk components (Table 6.4). T80 infusion increased yield of milk (P = 0.02) and lactose (P = 0.03) and decreased milk fat content (P = 0.05) but did not affect milk fat yield (P = 0.87). We observed interactions between OA and T80 for ECM (P = 0.08), milk fat yield (P = 0.10), feed efficiency (ECM/DMI; P = 0.05), and 165 BCS (P = 0.08). Due to minor numerical effects on DMI and milk yield, OA infusion resulted in improved feed efficiency (ECM/DMI; P = 0.03). We did not observe any effect of treatment on plasma insulin (P > 0.77). Milk Fatty Acid Concentration and Yield Milk FA are derived from either de novo synthesis in the mammary gland (<16 carbon FA) or preformed FA extracted from plasma (>16 carbon FA). Mixed-source FA (16 carbon) can be derived from either source. Both OA (P < 0.01) and T80 (P = 0.05) increased yield of FA from preformed sources (Table 6.5). We also observed an interaction between OA and T80 on yield of FA from preformed sources (P = 0.03) as BOTH had an intermediate response between OA and T80 rather than the expected additive effect. Neither OA nor T80 significantly altered the yield of milk FA from de novo sources but their interaction was significant (P = 0.06), with BOTH decreasing de novo FA yield compared to the expected additive effect. With regards to OA infusion, the increase in yield of preformed FA was due to increased cis-9 C18:1 (P < 0.01), trans-9 C18:1 (P < 0.01), trans-11 C18:1 (P = 0.05), cis-9, trans-11 C18:2 (P < 0.01), and cis-9, cis-12, cis-15 C18:3 (P < 0.01; Table 6.6). Infusion of OA and T80 each increased the concentration of FA from preformed sources (P < 0.01) and decreased the concentration of FA from mixed sources (P < 0.01), but there were no interactions observed. Both OA and T80 increased the concentration of cis-9 C18:1 (P < 0.01) and trans-9 C18:1 (P < 0.01) and decreased the concentration of C16:0 (P < 0.01; Table 6.7). There was no effect of the interaction of OA and T80 on the concentration of milk FA. Discussion 166 Improving FA absorption in lactating dairy cows is important to supply energy to the cow and support milk yield and metabolic requirements. The cow naturally produces bile salts and lysolecithin, which are both required for adequate micelle formation to transport FA through the aqueous lumen of the small intestine and are thus essential for maximal digestion and absorption of FA (Noble, 1981). Bile salts and lysolecithin form micelles due to their emulsifying potential. Emulsifiers are defined as amphiphiles, meaning they contain both a polar, hydrophilic head and a non-polar hydrophobic tail (Carey and Small, 1970). As DMI and FA density of a diet increase, the likelihood that bile salts or phospholipase are limiting for FA absorption also increase. In an analysis of available reports at the time, Boerman et al. (2015) demonstrated conclusively that FA digestibility decreases as the flow of FA to the small intestine increases, especially for increases in stearic acid flow. Based on that analysis, we suggest that bile salts or lysolecithin may be inadequate for optimal absorption of FA when fats are supplemented to high-producing cows and that providing exogenous emulsifiers may increase FA digestion and absorption by the cow. Previous research has examined supplying exogenous lecithin to dairy cows. Results to supplemental lecithin have been inconsistent, possibly due to differences in microbial digestion of the products used (Jenkins, 1990; Jenkins and Fotouhi, 1990; Fontoura et al., 2019). Protection from ruminal degradation may increase the utility of lecithin to improve FA absorption in the dairy cow but providing other exogenous emulsifiers in place of or in addition to lecithin may be beneficial. Recent research has observed that an emulsifier called polysorbate80 (Tween80©), which consists of polyoxyethylene sorbitan with an attached OA tail, can increase total FA digestibility when abomasally infused (de Souza et al., 2020; Chapter 5). Furthermore, OA itself has been shown to increase the solubility of other FA into micelles due to its own amphiphilic characteristics (Freeman, 1969; Chapter 4). Therefore, the objective of our study was to determine 167 possible interactive effects between abomasal infusions of OA and Tween80 on FA digestibility and milk production. We hypothesized that OA and Tween80 would independently increase total FA digestibility with the increase being higher when both were infused together. As expected, OA increased 18-carbon FA digestibility and tended to increase total and 16- carbon FA digestibility. Total, 16-carbon, and 18-carbon FA digestibility were increased by 5, 4, and 6 percentage units, respectively. These increases are slightly less than what we observed in our previous study (Chapter 4); in that study, we found that abomasally infusing the same dose of OA improved total, 16-carbon, and 18-carbon FA digestibility by 9, 8, and 9 percentage units, respectively. Cows in the previous study were consuming a similar DMI but producing 7 kg/d more milk than the cows in our current study. Perhaps the differences in magnitude of FA digestibility could be due to energy signaling and partitioning by cows producing different amounts of milk (de Souza et al., 2018, 2019). Interestingly, a study that abomasally infused 630 g/d of a fat mixture high in cis-C18:1 isomers which contained approximately 60 g OA increased total FA digestibility by 5 percentage units, yet the cows had much lower DMI and milk yield than on our study (Romo et al., 1996). Contrary to our hypothesis, T80 did not affect FA digestibility in our current study. In previous studies, abomasally infused T80 at 30 g/d increased total FA digestibility by 10.2 percentage units (de Souza et al., 2020) and 4.0 percentage units (Chapter 5). DMI, DIM, and milk yield were similar between our study and the study by de Souza et al. (2020), so the lack of effect of T80 on FA digestibility in our study is unclear. Total FA intake was slightly higher (45 g/d) in the study by de Souza et al. (2020) than in our study yet the ratio of 18-carbon to 16-carbon FA was higher in our study (3.3 vs. 2.9). Neither DM nor NDF intake were affected by abomasal infusion of OA, T80, or an interaction between the two. Some studies have observed decreased DMI in response to dietary 168 OA when it is incorporated into calcium salts (Rabiee et al., 2012), likely due to more OA reaching the small intestine, but others have not observed an effect of dietary OA on DMI when OA is not within a calcium salt (Chapter 3; Weld and Armentano, 2018). Similarly, abomasal infusion of OA has not affected DMI, possibly due to bypassing the rumen and thus not triggering the release of gut peptides (Romo et al., 1996; Bradford et al. 2008; Chapter 4). NDF intake was not affected by OA due to no basal diet differences between treatments and lack of effect of OA on DMI. Digestibility of DM and NDF were not affected by OA due to bypassing the rumen and maintaining DMI. T80 also did not affect digestibility of DM and NDF although past work with T80 observed minor, yet contradicting, effects on these variables (de Souza et al., 2020; Chapter 5). We observed few production responses in our study; however, this study was designed to investigate differences in FA digestibility and thus may not have had adequate power or period lengths to detect effects on production parameters. While OA did not affect DMI or milk yield, minor numerical improvements in these variables increased feed efficiency, as defined as ECM/DMI. As stated earlier, improving the efficiency of food production is important and should be considered when altering composition of the diet. An interaction between OA and T80 was observed for ECM/DMI, as the addition of T80 decreased feed efficiency. Interestingly, T80 increased milk yield, likely due to minor numerical increases in FA digestibility. In contrast, OA increased FA digestibility but did not improve milk yield. However, other studies increasing the amount of OA reaching the small intestine increased milk yield (Chapter 4), likely due to improved FA digestibility, but these affects may differ by production level of the cows as demonstrated by Western et al. (2018) and de Souza et al. (2019) . No differences in plasma insulin were observed in this study but have been observed in other studies in response to OA (de Souza et al., 2018, 2019; Chapter 4). These differing results indicate that there are likely some energy partitioning or 169 cell signaling differences in response to OA that need to be better understood (Itoh et al., 2003; Fujiwara et al., 2005; de Souza et al., 2018). Milk FA either arise from the uptake of preformed long-chain from dietary FA or from de novo synthesis of short- and medium-chain FA in the mammary gland. OA did not affect milk fat content or yield but increased the yield of FA from preformed sources, primarily due to increased yield of cis-9 C18:1 in milk fat, which agrees with past work (Chapter 4). T80 decreased milk fat content, likely due to a dilution effect as milk yield was increased while milk fat yield was unaffected. In comparison, both de Souza et al. (2020) and Prom and Lock (Chapter 5) observed that 30 g/d of T80 increased milk fat content and yield, but did not affect milk yield. As with OA, T80 increased yield of milk FA from preformed sources due to increasing absorbed dietary FA. We observed an interaction between OA and T80 for the yield of FA from de novo and preformed sources. Rather than having an additive effect, OA and T80 interacted to moderate the increase of preformed FA via cis-9 C18:1, trans-9 C18:1, cis-9,trans-11 C18:2, and cis-9,cis-12,cis-15 C18:3. Interestingly, OA and T80 interacted to decrease milk FA from de novo sources, although neither did independently. It has been well demonstrated that increasing the amount of preformed FA available for use by the mammary gland will decrease de novo milk FA synthesis (He et al., 2012; Dorea and Armentano, 2017). However, the interaction between OA and T80 did not increase preformed FA in milk more than OA independently, so we are unsure why there was an interaction to decrease de novo synthesis when OA did not also decrease de novo FA. Conclusion Abomasal infusion of OA increased the digestibility of 18-carbon FA and tended to increase the digestibility of 16-carbon and total FA. Thus, OA increased the absorbed total and 18- carbon FA. While OA had negligible effects on milk production responses, it improved feed 170 efficiency (ECM/DMI) and increased the yield of milk FA from preformed sources. Abomasal infusion of an exogenous emulsifier, Tween80, did not affect nutrient digestibility but increased milk yield, decreased milk fat content, and did not affect milk fat yield. These results contradict previous research utilizing Tween80, but the reason for these differences are unclear as energy status and FA intake was similar between studies. No interactions between OA and Tween80 were observed for nutrient digestibility but an interaction was observed for ECM, milk fat yield, feed efficiency, and BCS. Overall, OA was more beneficial to FA digestibility than an exogenous emulsifier and providing both had no additional benefits. Acknowledgements We acknowledge L. Worden, A. Burch, A. Negreiro, A. Pineda, U. Abou Rjeily, M. Machiela, and M. Kloboves (all in the Department of Animal Science, Michigan State University, East Lansing) and the staff of the Michigan State University Dairy Cattle Teaching & Research Center (East Lansing) for their assistance in this experiment. Crystal Prom was supported by a Pre- Doctoral Fellowship from USDA NIFA. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. 171 APPENDIX 172 Table 6.1. Fatty acid (FA) profile and total FA content of oleic acid and Tween supplements infused during treatment periods. Selected Individual FA, g/ 100g FA C14:0 C16:0 C18:0 trans C18:1 cis-9 C18:1 cis-11 C18:1 cis-9, cis-12 C18:2 cis-9, trans-11 C18:2 Total FA, % DM 1O1008-1G, Sigma-Aldrich, St. Louis, MO 2P1754, Sigma-Aldrich, St. Louis, MO Oleic Acid1 - 0.80 1.90 0.99 92.9 - 3.08 0.01 99.9 Tween802 0.14 3.63 1.06 2.43 85.3 1.97 0.09 0.02 17.0 173 Table 6.2. Ingredient and nutrient composition of diet fed to cows infused with treatments (n = 8). Ingredient, % DM Corn Silage Alfalfa Silage Ground Corn Soybean Meal High Moisture Corn Soyhulls Vitamin Mineral Base Mix1 High Cow Mix2 Fat Supplement3 Nutrient Composition, % DM DM4 NDF CP Starch FA C16:0 C18:0 cis-9 C18:1 cis-9, cis-12 C18:2 cis-9, cis-12, cis-15 C18:3 37.6 18.0 12.8 8.04 7.24 7.01 5.79 1.79 1.78 49.7 30.3 16.3 30.0 3.20 0.72 0.90 0.44 0.89 0.09 1Vitamin and mineral base mix contained 22.0% dry ground corn, 20.5% dolomitic lime, 20.0% calcium carbonate, 19.1% dicalcium phosphate, 10.0% white salt, 4.6% sodium sesquinate, 2.0% selenium, and <1% of each of the following: tallow, IntelliBond vital 5 (Micronutrients, Indianapolis, IN), vitamin E, vitamin A, and vitamin D. 2High cow mix contained 39.5% AminoPlus (Ag Processing Inc, Omaha, NE), 18.4% MetAAtein (Perdue Agribusiness, Salisbury, MD), 15.8% sodium sesquinate, 12.8% calcium carbonate, 9.7% dry ground corn, 2.6% urea, and 1.1% Smartamine (Adisseo, Alpharetta, GA). 3Energy Booster 100 (Milk Specialties Global, Eden Prairie, MN). Contained (g/100 g of FA) 4.09 of C14:0, 34.2 of C16:0, 47.7 of C18:0, 6.42 of cis-9 C18:1, and 0.89 cis-9,cis-12 C18:2; 80.5% total FA. 4Percent of as-fed diet. 174 Table 6.3. Nutrient intake and total-tract nutrient digestibility of cows infused with treatments (n = 8). Variable 16-carbon 18-carbon Intake, kg/d DM NDF Intake, g/d Total FA Digestibility, % DM NDF Total FA Absorbed, g/d Total FA 16-carbon FA 18-carbon FA 16-carbon 18-carbon Treatment1 OA 31.2 9.42 1057 221 780 63.0 38.2 67.1 66.4 67.1 708 147 523 T80 31.3 9.42 1005 222 727 64.8 41.2 65.2 65.4 64.6 655 145 570 BOTH 31.1 9.39 1051 219 777 63.9 38.0 66.4 66.0 66.2 701 145 516 SEM 0.82 0.26 25.3 5.68 18.4 1.10 1.93 2.65 1.97 2.95 30.80 5.48 24.3 CON 31.3 9.46 1005 223 726 63.3 39.0 61.7 62.5 60.8 624 140 445 P-values2 Oleic Tween Interaction 0.85 0.76 <0.01 0.66 <0.01 0.82 0.75 0.06 0.10 0.05 <0.01 0.23 <0.01 0.92 0.78 0.99 0.79 0.93 0.23 0.34 0.22 0.23 0.22 0.26 0.32 0.25 0.86 0.96 0.82 0.82 0.81 0.74 0.47 0.31 0.34 0.29 0.34 0.36 0.32 1Treatments consisted of no fat supplementation (CON), 60 g/d oleic acid (OA), 30 g/d Tween80 (T80), and OA+T80 (BOTH). 2P-values refer to the ANOVA results for the main effect of oleic acid treatment (OA), the main effect of Tween80 treatment (T80), and the interaction between OA and T80. 175 Table 6.4. Milk yield, milk composition, BW, and BCS of cows infused with treatments (n = 8). Variable Yield, kg/d Milk 3.5% FCM3 ECM4 Fat Protein Lactose Milk Composition, % Fat Protein Lactose ECM/DMI Body Weight, kg BCS Treatment1 P-values2 CON OA T80 BOTH 45.3 48.5 48.6 1.79 1.50 2.17 4.00 3.33 4.82 1.56 728 3.30 46.2 50.0 50.0 1.85 1.54 2.23 4.00 3.33 4.82 1.62 729 3.33 47.0 49.2 49.4 1.78 1.55 2.26 3.81 3.29 4.79 1.59 734 3.31 47.1 48.3 48.6 1.72 1.53 2.28 3.71 3.27 4.85 1.57 726 3.27 SEM 1.62 1.29 1.29 0.07 0.05 0.08 0.20 0.06 0.04 0.05 20.6 0.08 OA 0.18 0.12 0.12 0.24 0.21 0.11 0.98 0.96 0.79 0.03 0.77 0.27 T80 Interaction 0.02 0.45 0.34 0.87 0.14 0.03 0.05 0.34 0.39 0.26 0.21 0.71 0.34 0.08 0.08 0.10 0.23 0.50 0.41 0.61 0.19 0.05 0.18 0.08 1Treatments consisted of no fat supplementation (CON), 60 g/d oleic acid (OA), 30 g/d Tween80 (T80), and OA+T80 (BOTH). 2P-values refer to the ANOVA results for the main effect of oleic acid treatment (OA), the main effect of Tween80 treatment (T80), and the interaction between OA and T80. 33.5% FCM = [(0.4324 × kg of milk) + (16.216 × kg of milk fat)]. 4ECM = [(0.327 × kg of milk) + (12.95 × kg of milk fat) + (7.20 × kg of milk protein)]. 176 Table 6.5. Summation of milk fatty acid (FA) concentration and yield for cows infused with treatments (n = 8). Treatments1 P-values2 SEM OA T80 BOTH OA T80 Interaction Variable Summation by Source3, g/100 g FA CON De Novo Mixed Preformed Summation by Source3, g/d De Novo Mixed Preformed 29.3 40.2 30.5 491 673 511 29.3 37.7 33.0 508 654 567 29.2 38.4 32.3 487 644 536 29.0 36.7 34.3 468 594 551 0.94 <0.01 <0.01 0.19 0.42 <0.01 0.42 0.66 0.68 21.7 32.0 17.2 0.82 <0.01 <0.01 0.77 0.22 0.05 0.52 0.23 0.43 0.06 0.33 0.03 1Treatments consisted of no fat supplementation (CON), 60 g/d oleic acid (OA), 30 g/d Tween80 (T80), and OA+T80 (BOTH). 2P-values refer to the ANOVA results for the main effect of oleic acid treatment (OA), the main effect of Tween80 treatment (T80), and the interaction between OA and T80. 3De novo fatty acids originate from mammary de novo synthesis (<16 carbons), preformed fatty acids originate from extraction from plasma (>16 carbons), and mixed fatty acids originate from both sources (C16:0 plus cis-9 C16:1). 177 Table 6.6. Milk fatty acid yield of cows infused with treatments (n = 8). Treatments1 P-values2 Variable Selected Individual FA3, g/d C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 cis-9 C14:1 C16:0 cis-9 C16:1 C18:0 trans-6 to 8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 cis-9 C18:1 cis-11 C18:1 cis-9, cis-12 C18:2 cis-9, trans-11 C18:2 cis-9, cis-12, cis-15 C18:3 CON 50.3 37.0 22.9 62.0 74.9 226 17.8 644 28.7 122 3.14 3.06 5.53 7.14 250 7.86 29.4 3.71 4.67 OA 52.9 39.3 24.5 65.6 77.5 231 16.9 626 27.4 131 3.24 3.78 5.76 7.99 289 8.02 32.0 4.06 5.07 T80 51.0 36.7 22.6 60.6 72.2 226 17.7 615 28.5 125 3.34 3.30 5.74 7.36 266 8.43 29.5 4.11 4.69 BOTH 49.6 35.5 22.1 59.0 69.6 216 16.1 568 25.8 124 3.26 3.67 5.99 7.54 285 8.42 30.8 4.00 4.73 SEM 1.96 1.75 1.23 3.88 4.75 9.14 1.27 31.0 1.48 6.90 0.19 0.09 0.60 0.39 9.13 0.60 0.69 0.38 0.17 OA 0.13 0.07 0.03 0.07 0.22 0.40 0.18 0.12 0.16 0.06 0.32 <0.01 0.40 0.05 <0.01 0.61 <0.01 0.07 <0.01 T80 0.68 0.79 0.70 0.45 0.22 0.97 0.97 0.20 0.86 0.53 0.07 <0.01 0.48 0.58 0.02 0.07 0.87 0.04 0.61 Interaction 0.11 0.06 0.04 0.06 0.09 0.08 0.36 0.35 0.27 0.15 0.23 <0.01 0.96 0.24 0.05 0.70 0.21 0.10 0.02 1Treatments consisted of no fat supplementation (CON), 60 g/d oleic acid (OA), 30 g/d Tween80 (T80), and OA+T80 (BOTH). 2P-values refer to the ANOVA results for the main effect of oleic acid treatment (OA), the main effect of Tween80 treatment (T80), and the interaction between OA and T80. 3Approximately 80 individual fatty acids were quantified. Only select fatty acids are reported in the table. 178 Table 6.7. Milk fatty acid concentration of cows infused with treatments (n = 8). Treatments1 P-values2 SEM OA T80 Interaction Variable Selected Individual FA3, g/100 g FA C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 cis-9 C14:1 C16:0 cis-9 C16:1 C18:0 trans-6 to 8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 cis-9 C18:1 cis-11 C18:1 cis-9, cis-12 C18:2 cis-9, trans-11 C18:2 cis-9, cis-12, cis-15 C18:3 CON OA T80 BOTH 3.01 2.21 1.36 3.69 4.46 13.5 1.06 38.5 1.72 7.26 0.19 0.18 0.34 0.43 14.9 0.47 1.76 0.22 0.28 3.06 2.27 1.41 3.77 4.46 13.4 1.02 36.1 1.62 7.64 0.19 0.22 0.34 0.47 16.8 0.47 1.90 0.24 0.29 3.07 2.20 1.35 3.61 4.31 13.7 1.08 36.7 1.71 7.48 0.21 0.20 0.34 0.42 16.1 0.51 1.83 0.23 0.28 3.08 2.20 1.37 3.63 4.29 13.4 1.01 35.1 1.61 7.67 0.21 0.23 0.38 0.48 17.8 0.53 1.92 0.24 0.29 0.08 0.04 0.03 0.13 0.17 0.21 0.08 0.69 0.07 0.30 0.02 0.009 0.05 0.03 0.41 0.04 0.05 0.02 0.006 0.49 0.20 0.05 0.19 0.97 0.42 0.15 <0.01 <0.01 0.08 0.91 <0.01 0.84 0.13 <0.01 0.72 <0.01 0.08 0.07 0.40 0.75 0.54 0.17 0.03 0.25 0.56 <0.01 0.97 0.29 0.09 <0.01 0.62 0.70 <0.01 0.04 0.11 0.26 0.94 0.67 0.32 0.33 0.49 0.84 0.82 0.49 0.22 0.96 0.52 0.87 0.42 0.33 0.58 0.76 0.32 0.40 0.61 0.86 1Treatments consisted of no fat supplementation (CON), 60 g/d oleic acid (OA), 30 g/d Tween80 (T80), and OA+T80 (BOTH). 2P-values refer to the ANOVA results for the main effect of oleic acid treatment (OA), the main effect of Tween80 treatment (T80), and the interaction between OA and T80. 3Approximately 80 individual fatty acids were quantified. 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Oleic acid (OA) and an exogenous emulsifier, Tween80, have both been observed to improve FA absorption and were chosen to be the focus of our work. Our overall objective was to understand the role of OA and exogenous emulsifiers on post-ruminal absorption of FA. We hypothesized that both OA and exogenous emulsifiers will increase the absorption of dietary-derived FA. In Chapter 3, we altered the ratios of stearic acid and OA in supplemental dietary fat blends. While all FA treatments decreased FA digestibility compared to the non-supplemented control treatment, increasing OA in the FA supplements increased the digestibility and absorption of FA. Improvements in FA digestibility in response to increasing OA had no effect on yield of milk, milk fat, or milk protein. FA supplementation did not affect DMI but overall inclusion of FA increased milk yield, milk fat yield, 3.5% FCM, ECM, and feed efficiency. Compared to control, FA treatments had no effect on the yield of de novo milk FA, decreased mixed milk FA, and increased preformed milk FA. The increase in preformed FA yield was predominantly due to FA treatments increasing the yield of OA in milk compared with control. Chapter 4 further examined the effects of OA on FA digestibility by infusing increasing doses into the abomasum of lactating dairy cows for 11 days per period. Increasing the amount of OA reaching the duodenum did not affect DM or FA but increased the digestibility and absorption of FA. The increase in absorbed FA due to OA infusion tended to increase milk yield, 3.5% FCM, and ECM, although there was no effect on the yield of milk fat or protein. The yield of FA in milk fat from preformed sources was increased, primarily due to an increase in OA. Infusion of OA did not increase BW or BCS; however, plasma insulin concentration was increased, indicating OA 194 might have increased BW if our treatment period had been longer. Chapter 5 investigated the efficacy of three different exogenous emulsifiers to improve FA absorption in the small intestine. The emulsifiers all consisted of a similar polysorbitol base but varied in the FA attached. Tween40 contains palmitic acid, Tween60 contains stearic acid, and Tween80 contains OA. Overall, Tween infusions did not affect nutrient digestibility due to the negative impact of the Tween60 treatment, likely due to the attached stearic acid. However, Tween80, increased FA digestibility and absorption compared to control and to the average of the other 2 Tween treatments. Tween80 also increased milk fat content and yield compared to control. These results show that the predominant FA attached to polysorbate impacts its ability to improve FA digestibility and milk production. However, it was unclear from this study whether the effects of Tween80 were due to the polysorbate or the attached OA. In Chapter 6 we directly compared abomasal infusions of OA and Tween80, as well as the interaction between the two. OA increased the digestibility and absorption of FA. While it had negligible effects on production responses, OA improved feed efficiency and increased the yield of preformed milk FA. Contrary to previous studies, Tween80 did not affect nutrient digestibility. Reasons for these contradicting responses are unclear as energy status and FA intake were similar across studies. However, Tween80 increased milk yield, decreased milk fat content, and did not affect milk fat yield. No interactions between OA and Tween80 were observed for nutrient digestibility but an interaction was observed for ECM, milk fat yield, feed efficiency, and BCS. Overall, OA was more beneficial to FA digestibility than Tween80 and providing both had no additional benefits. Altogether, these studies increased our understanding of potential strategies to improve FA absorption in lactating dairy cows, which often led to beneficial production responses. Our results 195 indicate that increasing the amount of OA reaching the duodenum, either by dietary supplementation or abomasal infusion, increases FA absorption. Similarly, results indicate that providing an exogenous emulsifier may help cows overcome limitations in their natural emulsification capacity and improve FA absorption, but the type of emulsifier utilized will affect the response. In these studies, we found no benefit of supplementing both OA and Tween80, but perhaps this lack of benefit was related to the specific doses of these studies. Thus, we suggest that other doses of these two supplements and other types of emulsifiers should be investigated. Following examination of other emulsifiers, development of a rumen-protected product containing OA, synthetic emulsifiers, and lecithin will be the likely next step to aid nutritionists and dairy producers in maximizing FA absorption. A single product containing multiple substances proven to improve FA absorption will be more practical and cost-effective than producing individual products with OA and emulsifiers. However, other effects of OA supplementation should be considered when formulating a product to aid FA absorption. Research suggests OA influences energy partitioning in lactating dairy cows and thus supplemental OA may not be appropriate or ideal for all groups of cows. Furthermore, nutritionists may desire an ideal dietary ratio of OA to other FA and would thus need to consider the amount of OA contained in OA-emulsifier product. As development of a product containing protected OA and emulsifiers to improve FA absorption may take several years, nutritionists and dairy producers can take immediate steps to improve FA absorption in their herds by altering the ratios of their FA supplements to increase dietary OA and decrease dietary stearic acid. However, the effects of OA on production and energy partitioning at varying production levels should be considered when making these decisions. Overall, OA and exogenous emulsifiers are viable options to improve FA absorption. Our results will help 196 nutritionists and dairy producers increase the absorbed FA and thus, energy available to the cow. 197