PLACE IN REI‘URN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 1/98 www.mu PERIPARTURIENT RESPONSES OF COWS FED VARYING DIETARY CATION-ANION DIFFERENCES AND CALCIUM CONTENTS PREPARTUM BY Luis Alberto Rodriguez-Suarez A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1 998 ABSTRACT PERIPARTURIENT RESPONSES OF COWS FED VARYING DIETARY CATION-ANION DIFFERENCES AND CALCIUM CONTENTS PREPARTUM By Luis Alberto Rodriguez-Suarez Effects on acid-base and Ca status of Holstein cows of several treatments with different dietary cation-anion differences (DCAD; meq: [(Na + K) - (Cl + S)]I100 g of dietary DM) and Ca concentrations were investigated in four experiments. In Experiment 1,117 nonlactating pregnant cows and heifers were fed prepartum dietary treatments with DCAD and Ca% of +11 and 0.63 (Treatment 1), -11 and 0.95 (Treatment 2) and -26 meql100 g of DM and 1.17% (Treatment 3). Dietary treatments did not affect milk fever incidence; however, lowering DCAD while increasing dietary Ca, decreased urine pH and reduced the incidence of postpartum subclinical hypocalcemia. The incidence of displaced abomasum was greater in cows fed Treatments 2 and 3 compared with those fed Treatment 1. In Experiment 2, 32 nonlactating, nonpregnant cows were fed dietary treatments of Control (no anion source added, +16.9 meql100 g of dietary DM), Bio-Chlorm, an anionic salts mixture designed to match the anionic and cationic contributions of Bio-Chlorm, and H'Cl; DCAD with supplemental anions averaged -7.7 meql100 g of dietary DM. Factored across anion sources were dietary Ca concentrations of 0.49 or 2.10%. Cows fed Control had 7.5% greater DMI than cows fed anion sources. Urine pH of cows fed Control remained at 8.0 to 8.2 across the treatment period (21 d), but urine pH in those fed anion sources declined from 8.0 to 5.7 on average d 2 after commencement of feeding anion sources, and remained below 6.0. In Experiment 3, 31 nonlactating, nonpregnant cows were assigned in a randomized incomplete block reversal design with three 21-d periods. Dietary treatments were similar to those in Experiment 2. Cows fed Control had 3.6% greater DMI than cows fed anion sources. Cows fed anion sources had lower urine pH (6.0) compared with cows fed Control (8.1). Supplemental anions from all sources were acidogenic. In Experiment 4, 22 nonlactating, pregnant multiparous cows were assigned randomly to two dietary treatments 0.48% or 1.98% Ca (supplemental CaCOa). The DCAD of both treatment diets was -11.2 meq/100 g of DM with inclusion of extruded heat-treated soybean meal treated with HCl. Dry matter intake decreased 30% from wk 3 prepartum through wk 1 prepartum (pooled across both treatments). Cows had similar plasma ionized Ca concentrations prepartum regardless of dietary Ca concentration; however, on the day of and the day after parturition, cows fed 1.98% Ca had lower plasma ionized Ca concentrations than cows fed 0.48% Ca. Feeding 1.98% Ca with negative DCAD prepartum increased incidence of subclinical hypocalcemia at parturition and affected Ca metabolism reducing apparent bone Ca mobilization during the periparturient period. General conclusions from this research include the following. Lowering DCAD, while increasing dietary Ca concentration, decreased urine pH and reduced postpartum subclinical hypocalcemia. Supplemental anions from all sources were acidogenic. Feeding 1.98% Ca with negative DCAD prepartum increased incidence of subclinical and clinical hypocalcemia and reduced bone Ca mobilization during the periparturient period. I dedicate this dissertation to my wife Carolina, my son Diego and my daughter Cristina, who gave the inspiration and support to achieve this goal. To my parents who taught me to be consistent and work hard in life. To God that was always with us as a family in the good and the hard times. iv ACKNOWLEDGMENTS I would like to express special gratitude to Dr. David K. Beede, my major advisor, for trusting me and giving me the opportunity to work with him, and to achieve this goal. Dr. Beede your guidance, friendship, support and workmanship will be remembered always. I felt very good working with you, especially the last two years of my program when we were able to establish many interesting scientific discussions. I thank my committee members Dr. Michael Allen, Dr. Margaret Benson, Dr. Tom Herdt and Dr. Michael VandeHaar for their guidance and honest advice. To Dr. Benson and Dr. VandeHaar, special thanks for giving me the opportunity to teach in your undergraduate courses. It was a great experience to interact with you and your students. Special thanks Dr. Gretchen Hill, Dr. Pao Ku, and Jane Link for their guidance in the mineral nutrition laboratory and to Marcia Carlson, Josep Garcia, and Suzanne Hoover for their help in analyzing mineral elements. Your help was invaluable, especially when I broke my leg. Acknowledgments are extended to Jim Liesman, David Main, Cara Ianni, Corey Risch, Tom Pilbeam and graduate students Sara Scheurer, Jill Davidson, Doug Mashek, Masahito Oba, Jing Xu, Yun Ying and Richard Longuski for their technical assistance in sampling and laboratory analyses. I also thank the personnel of the Michigan State University Dairy Teaching V and Research Center, and the Kellogg Biological Station Dairy for their cooperation in managing and feeding cows, and the Michigan State University Office of Radiation, Chemical and Biological Safety personnel for their help handling and mixing the HCI. I thank Dr. Jesse Goff at the National Disease Center, Ames, Iowa for analyzing the parathyroid hormone, vitamin D, and hydroxyproline in plasma, and for his invaluable recommendations. Special thanks to my wife Carolina, my son Diego and my daughter Cristina for giving me the time and encouraging me to keep working for this goal. To my parents who from far away always supported me morally and last but not least, to God who kept us together as a family and inspired us to keep going. vi TABLE OF CONTENTS LIST OF TABLES ............................................... xi LIST OF FIGURES ............................................. xiv LIST OF ABBREVIATIONS ..................................... xxi CHAPTER 1 INTRODUCTION ............................................... 1 CHAPTER 2 REVIEW OF LITERATURE ....................................... 5 Effects of Anion Supplementation on Dry Matter Intake and Acid- Base Status ......................................... 5 Absorption of Ca from the Digestive Tract ....................... 7 Transcellular (Active) Transport of Ca ..................... 7 Paracellular (Passive) Transport of Ca ................... 10 Effects of Dietary Cation-Anion Difference and Dietary Ca on the Absorption of Ca from the Digestive Tract ................ 11 Resorption of Ca from Bone ................................ 18 Effects of Dietary Cation-Anion Difference and Dietary Ca on the Resorption of Ca from Bone ........................... 22 Excretion of Ca ........................................... 29 Effects of Dietary Cation-Anion Difference and Dietary Ca on the vii Excretion of Ca ...................................... 30 Focus of Research ........................................ 33 CHAPTER 3 LOWERING DIETARY CATION-ANION DIFFERENCE WHILE RAISING CALCIUM CONTENT REDUCES PERIPARTUM SUBCLINICAL HYPOCALCEMIA Abstract ................................................. 36 Introduction .............................................. 37 Materials and Methods ..................................... 39 Results and Discussion ..................................... 43 Conclusions .............................................. 51 Tables ................................... - ............... 53 Figures .................................................. 60 CHAPTER 4 EFFECTS OF DIETARY ANION SOURCES AND CALCIUM CONCENTRATION ON TEMPORAL CHANGES IN FEED INTAKE AND URINE pH Abstract ................................................. 61 Introduction .............................................. 62 Materials and Methods ...................................... 63 Results and Discussion .............. ' . . . . . ................. 66 Conclusions .............................................. 71 Tables .................................................. 73 Figures .................................................. 77 viii CHAPTER 5 ANION SOURCES AND DIETARY CALCIUM CONCENTRATION REDUCED DRY MATTER INTAKE AND CHANGED ACID-BASE STATUS OF NONLACTATING NONPREGNANT HOLSTEIN COWS Abstract ................................................. 84 Introduction .............................................. 86 Materials and Methods ...................................... 88 Results and Discussion ..................................... 94 Conclusions ............................................. 1 14 Tables ................................................. 1 16 Figures ................................................. 129 CHAPTER 6 HIGH (1.98%) VERSUS LOW (0.48%) CALCIUM IN PREPARTUM DIETS WITH HYDROCHLORIC ACID AFFECTS ACID-BASE STATUS AND CALCIUM HOMEOSTASIS Abstract ............................................... 130 Introduction ............................................ 1 32 Materials and Methods .................................... 134 Results and Discussion ................................... 141 Conclusions ............... 163 Tables ................................................. 165 Figures ................................................. 170 CHAPTER 7 SUMMARY AND RECOMMENDATIONS FOR FUTURE RESEARCH . . . . 220 APPENDIX A TABLES OF STATISTICAL ANALYSIS FOR CHAPTER 3 ........ 228 B TABLES OF STATISTICAL ANALYSIS FOR CHAPTER 4 ........ 233 C TABLES OF STATISTICAL ANALYSIS FOR CHAPTER 5 ........ 234 D TABLES OF STATISTICAL ANALYSIS FOR CHAPTER 6 ........ 245 LIST OF REFERENCES ........................................ 248 BIOGRAPHICAL SKETCH ...................................... 262 LIST OF TABLES CHAPTER 3 Table 1. Dietary ingredients and chemical composition from laboratory analysis of the diets with different dietary cation-anion differences and Ca concentrations, Experiment 1 .................................. 53 Table 2. Dietary ingredients and chemical composition from laboratory analysis of the diets with different dietary cation-anion differences and Ca concentrations, Experiment 2 ................................... 54 Table 3. Least square means of urine and plasma variables of cows fed different dietary cation-anion differences and Ca concentrations pooled across the 8 d experimental period, Experiment 1 ............... 55 Table 4. Measurements in urine and plasma before and after calving of cows fed different dietary cation-anion differences and Ca concentrations by treatment, Experiment 2 .......................... 56 Table 5. Measurements in urine and plasma before calving of cows fed different dietary cation-anion differences and Ca concentrations by parity and season, Experiment 2 ................................ 57 Table 6. Measurements in plasma after calving of cows fed different dietary cation-anion differences and Ca concentrations by parity and season, Experiment 2 ....................................... 58 Table 7. Incidence of milk fever and abomasum displacement of cows fed different dietary cation-anion differences and Ca concentrations by treatment and parity, Experiment 2 ................................. 59 CHAPTER 4 Table 1. Ingredient composition of dietary treatments with different anion sources and Ca concentrations ............................... 73 Table 2. Analyzed chemical composition of dietary treatments with different anion sources and Ca concentrations ........................ 74 xi Table 3. Dry matter and water intake, and urine pH of cows fed different anion sources and Ca concentrations, pooled across Ca concentrations and day of experiment ............................... 75 Table 4. Dry matter and water intake, and urine pH of cows fed different anion sources and Ca concentrations, pooled across anion sources and day of experiment ........................................... 76 CHAPTER 5 Table 1. Dietary ingredients (% of dietary DM) of treatments with different anion sources and Ca concentrations .............................. 116 Table 2. Analyzed chemical composition (% of dietary DM) of treatments with different anion sources and Ca concentrations .................... 117 Table 3. Least-squares means of dry matter intake, apparent DM digestibility, water intake, and apparent absorption of water of cows fed different anion sources and dietary Ca concentrations .......... 118 Table 4. Least-squares means urine volume and urine acid-base measurements of cows fed different anion sources and dietary Ca concentrations ................................................ 1 19 Table 5. Least-squares means of urine glomerular filtration rate and fractional excretion of mineral elements in cows fed different anion sources and dietary Ca concentrations ............................. 120 Table 6. Least-squares means of blood plasma pH, pCOz, bicarbonate, strong ion difference, Na, K, and CI of cows fed different anion sources and dietary Ca concentrations .................................... 121 Table 7. Least-squares means of blood plasma Ca, P, Mg, parathyroid hormone, and 1,25(OH)2 vitamin 03 concentrations of cows fed different anion sources and dietary Ca concentrations ......................... 122 Table 8. Least-squares means of apparent balance of Ca for cows fed different anion sources and dietary Ca concentrations .................. 123 Table 9. Least-squares means of apparent balance of P for cows fed different anion source and dietary Ca concentrations .................. 124 Table 10. Least-squares means of apparent balance of Mg for cows fed different anion sources and dietary Ca concentrations ............... 125 xii Table 11. Least—squares means of apparent balance of Na for cows fed different anion sources and dietary Ca concentrations ............... 126 Table 12. Least-squares means of as apparent balance of K for cows fed different anion sources and dietary Ca concentrations ............... 127 Table 13. Least-squares means of apparent balance of CI for cows fed different anion sources and dietary Ca concentrations ............... 128 CHAPTER 6 Table 1. Dietary ingredients (% of dietary DM) of prepartum and postpartum dietary treatments .................................... 165 Table 2. Analyzed chemical composition (% of dietary DM) of the prepartum and postpartum dietary treatments ........................ 166 Table 3. Incidence of metabolic diseases by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +30.8 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ..................................... 167 Table 4. Mineral element balances pooled across days by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum . . . . 168 Table 5. Correlations for plasma parameters at d -10, and from d -4 through d 0 of the experimental period .............................. 169 xiii LIST OF FIGURES CHAPTER 3 Figure 1. Urine pH pattern of cows fed diets with different dietary cation-anion differences and Ca concentrations, Experiment 1 ........... 60 CHAPTER 4 Figure 1. Dry matter intake across time of cows fed dietary treatments with different anion sources, pooled across dietary Ca concentrations ...... 77 Figure 2. Dry matter intake across time of cows fed dietary treatments with different Ca concentrations, pooled across anion sources ............ 78 Figure 3. Water intake across time of cows fed dietary treatments with different anion sources, pooled across dietary Ca concentrations .......... 79 Figure 4. Water intake across time of cows fed dietary treatments with different Ca concentrations, pooled across anion sources ................ 80 Figure 5. Urine pH across time of cows fed dietary treatments with different anion sources, pooled across dietary Ca concentrations .......... 81 Figure 6. Urine pH across time of cows fed dietary treatments with different Ca concentrations, pooled across anion sources ................ 82 Figure 7. Effect of dietary cation-anion difference intake (meq/d/kg of BW) on urine pH of cows fed dietary treatments with different anion sources, pooled across dietary Ca concentrations ...................... 83 CHAPTER 5 Figure 1. Effect of dietary cation-anion difference intake (meq/d per kg of BW) on urine pH of cows fed dietary treatments with different anion sources, pooled across dietary Ca concentrations ..................... 129 CHAPTER 6 Figure 1. Peripartum DMI of cows fed different dietary Ca concentrations xiv with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ................................. 170 Figure 2. Postpartum milk yield of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum .............................. 171 Figure 3. Peripartum plasma pH of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum .............................. 172 Figure 4. Peripartum plasma pCO2 concentrations of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ..................... 173 Figure 5. Peripartum plasma bicarbonate concentrations of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ........ 174 Figure 6. Peripartum plasma Na concentrations of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 175 Figure 7. Peripartum plasma K conCentrations of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 176 Figure 8. Peripartum plasma Cl concentrations of cowsfed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 177 Figure 9. Peripartum plasma strong ion difference concentrations (meq [Na + K - CI]IL) of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ................................................ 178 Figure 10 Peripartum plasma total Ca concentrations of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 179 Figure 11. Peripartum plasma ionized Ca concentrations of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 180 Figure 12. Peripartum ratio of plasma ionized Ca to total Ca concentration of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum. . . . . 181 Figure 13. Peripartum plasma P concentrations of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum .............................. 182 Figure 14. Peripartum plasma Mg concentrations of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 183 Figure 15. Peripartum plasma parathyroid hormone of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 184 Figure 16. Peripartum plasma 1,25(OH)2 vitamin D3 of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 185 Figure 17. Peripartum plasma hydroxyproline of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 186 Figure 18. Peripartum plasma osteocalcin concentration of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 187 Figure 19. Peripartum urine deoxypyridinoline concentration of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ........ 188 Figure 20 Peripartum urine pH of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ..................................... 189 Figure 21. Peripartum urine Na concentrations of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 190 Figure 22. Peripartum fractional excretion of Na of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl 4- S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 191 Figure 23. Peripartum urine K of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ..................................... 192 Figure 24. Peripartum fractional excretion of K of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 193 Figure 25. Peripartum urine CI of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum. .................................... 194 Figure 26. Peripartum fractional excretion of CI of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 195 Figure 27. Peripartum urine Ca of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ..................................... 196 xvii Figure 28. Peripartum fractional excretion of Ca of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 197 Figure 29. Peripartum urine P of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ..................................... 198 Figure 30. Peripartum fractional excretion of P of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 199 Figure 31. Peripartum urine Mg of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ..................................... 200 Figure 32. Peripartum fractional excretion of Mg of cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 201 Figure 33. Postpartum intake of Ca by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 202 Figure 34. Postpartum fecal excretion of Ca by cows fed different dietary Ca concentrations with ~11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 203 Figure 35. Postpartum apparent absorption of Ca by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ..................... 204 Figure 36. Postpartum secretion of Ca in milk by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 205 xviii Figure 37. Postpartum urine excretion of Ca by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 206 Figure 38. Postpartum apparent balance of Ca by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 207 Figure 39. Postpartum intake of P by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ........................ 208 Figure 40 Postpartum fecal excretion of P by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 209 Figure 41. Postpartum apparent absorption of P by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 210 Figure 42. Postpartum secretion of P in milk by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 211 Figure 43. Postpartum urine excretion of P by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 212 Figure 44. Postpartum apparent balance of P cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 213 Figure 45. Postpartum intake of Mg by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 214 xix Figure 46. Postpartum fecal excretion of Mg by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]II 00 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 215 Figure 47. Postpartum apparent absorption of Mg by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 81/100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 216 Figure 48. Postpartum secretion of Mg in milk by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] — [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 217 Figure 49. Postpartum urine excretion of Mg by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [CI + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 218 Figure 50. Postpartum apparent balance of Mg by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]I100 g of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum ...................... 219 ADF AS BW CP DCAD DM DMI iCa iCalCa IV NAE NDF PTH SEM SID VDR LIST OF ABBREVIATIONS apparent absorption acid detergent fiber anionic salts Bio-ChlorTM body weight control crude protein dietary cation-anion difference dry matter dry matter intake ionized Ca the ratio of ionized Ca to total plasma Ca intravenous net acid excretion neutral detergent fiber parathyroid hormone standard error of the mean strong ion difference vitamin D receptor xxi VIII water intake 1 ,25(OH),D3 1,25-dihydroxyvitamin 03 xxii CHAPTER 1 INTRODUCTION The incidence of metabolic disorders increases in dairy cows near and after parturition. Reinhardt et al. (1988) reported that Ca homeostasis in vertebrates is maintained with remarkable precision, except in the aged parturient dairy cow that can develop milk fever. Homeostatic control of Ca results from a complicated balance of input, output, and recycling of Ca (Reinhardt et al., 1988). The blood plasma Ca pool (2.5 to 3 g) is maintained using different mechanisms such as, absorption of Ca from digestive tract, bone resorption, and secretion of Ca in milk, and endogenous fecal and urine losses (Horst et al., 1994). Milk fever, a hypocalcemic disorder, is associated with parturition and the initiation of lactation in dairy cows. Its incidence is approximately 6% for all cows, but incidence in multiparous cows was 13.5% in Michigan (Dyk, 1995). It has been estimated recently that the average milk fever case costs about $334 (Guard, 1996). This is the direct cost associated with clinical treatments and production losses; however, cows with milk fever are susceptible to other metabolic diseases that are not accounted for in this cost. Calcium is required for normal functions of a variety of tissues and physiologic processes (Goff et al., 1991 b). Beede (1995) proposed that low concentrations of Ca in blood peripartum create an early postpartum hypocalcemic cascade. Subclinical and clinical hypocalcemia can reduce smooth muscle function, which can cause digestive tract stasis leading to abomasum displacement and (or) low feed intake, and poor reproductive performance, including reduced uterine motility, increased retained placenta, and slow uterine involution. Manipulation of dietary cation-anion difference (DCAD) meq [(Na + K) - (Cl + S)]l100 g of dietary DM in diets of cows in late pregnancy has been successful in reducing the incidence of hypocalcemia in several studies (Block, 1984; Goff et al., 1989; Goff et al., 1991a; Oetzel et al., 1988; Wang, 1990) and other postpartum disorders (Joyce et al., 1997; Lema et al., 1992; Wang, 1990). The DCAD has direct impact on blood acid-base status of the cow. Block (1994) reported that diets with low DCAD affected one or more of the following variables in the blood: increased H‘ and decreased bicarbonate concentrations and pH. Changes in acid-base status can alter Ca metabolism by increasing mobilization of Ca from bone (Block, 1984, Gaynor et al., 1989), and possibly enhancing absorption of Ca from the gut (Freeden et al., 1988b, Goff et al., 1991a) thus, increasing the ability of the cow to maintain near normal blood Ca concentrations during the early postpartum period. Reducing the DCAD by anion supplementation in the field has not always 2 improved Ca status and other peripartum problems. Possible reasons include: the actual DCAD was unknown or likely more positive than presumed from diet formulation; unpalatability inherent with some anion sources; and, improper ration mixing. Practically, it is very difficult to make changes in dietary ingredients through time and know the DCAD, monitor uncontrollable changes in DCAD (e.g., when forage base of the ration is changed), and acquire needed and accurate nutrient analysis in a timely fashion. Having supplemental anion sources as a separate mix with a carrier allows alteration of the anion inclusion rate through time without changing the basic ration formulation. Also, urine pH can be used as a measure of the cow's acid-base status when feeding anion sources, helping access whether the ration is having the desired effects or not. However, varying the DCAD fed to cows before calving can reduce DMI (Joyce et al., 1997; Schoenbaum et al., 1994; Moore et al., 1997) and energy balance (Moore et al., 1997). The advantages of lowering DCAD in prepartum diets to control or reduce hypocalcemia may be offset by the depression in DMI sometimes observed in cows fed anionic diets. So, it is important to find sources of anions that effectively alter acid-base status and have minimal effects on DMI. Several experiments have been conducted to study varying DCAD and dietary Ca. But, in each study the two factors were confounded (Moore et al., 1997; Wang, 1990), or negative DCAD was fed with high dietary Ca (Goff et al., 1991a), or negative DCAD was fed with low dietary Ca (Block, 1984). Results from some of these studies suggest that it may be beneficial to feed higher dietary Ca prepartum than is recommended currently (NRC, 1989), especially 3 with low or negative DCAD. However, the literature is lacking of the studies where varying DCAD is factored with varying dietary Ca in periparturient cows to delineate the optimal dietary Ca concentration and characterize the cow's metabolic and physiological responses. Four experiments are conducted in the current research. The overall objective was to determine how varying dietary Ca concentrations and DCAD affected Ca metabolism, renal physiology, and physiological performance of nonlactating nonpregnant and pregnant dairy cows. CHAPTER 2 REVIEW OF LITERATURE The following review of literature covers the most important areas related to Ca metabolism in the periparturient dairy cow. lnforrnation on how the cow regulates Ca to maintain Ca status, and how the DCAD can affect DMI, Ca status, and Ca metabolism are presented. EFFECTS OF ANION SUPPLEMENTATION ON DRY MATTER INTAKE AND ACID-BASE STATUS The DCAD concept is based on the strong ion difference theory (Stewart, 1983). This theory suggests that influx of any mineral anion into the animal's body results in perturbation of acid-base status. Therefore, the cow's acid-base status is determined by the amount of anion and cations absorbed. If the diet has more absorbable cations than anions, the cow's acid-base status will be alkalotic, however, if the diet has more absorbable anions it will be acidotic. The most often used equation to predict DCAD is meq [(Na + K) - (Cl + S)]I100 g of dietary DM (Ender et al., 1973). This equation does not take into consideration other cations and anions such as Ca, Mg and P04, but because they are not absorbed to the same extent as Na, K, and CI do not greatly influence the cow's acid-base status. The most used sources of supplemental anions have been anionic salts such as, MgCIz, MgSO,., NH4CI, (NH4)ZSO4, CaClz, Ca 80,, however, recently there has been some interest in using the acid forms of Cl and S (Goff and Horst, 1997a). Tucker et al. (1991) fed lactating Holstein cows DCAD of 0 and +30 meq/100 g of dietary DM using double sulfate of K and Mg and CaClz. Cows fed 0 meq/100 g of dietary DM had similar acid-base measurements when fed CI or S; however, cows fed supplemental Cl had numerically lower urine pH compared with cows fed 8 (6.83 and 7.39, respectively). Oetzel et al. (1991) fed nonlactating, nonpregnant Holstein cows DCAD of -17 meq/100 g of dietary DM using six anionic salts [MgCl2-6H20, MgSO4-7H20, CaCl2-2H20, CaSO,-2H20, NH4CI, and, (NH,)ZSO4 ]. Dry matter intake and blood pH were similar among anionic salts. Ammonium chloride resulted in the greatest reduction of urine pH (7.39), whereas MgSO,-7H20 caused the lowest reduction (7.96). Oetzel and Bannore (1993) fed nonlactating, pregnant multiparous cows DCAD of + 31 and +11 meq/100 g of dietary DM using anionic salts in a concentrate mixture [MgSO,-7H20, CaCl2-2HZO, CaSO4-2H20, and NH,CI]. Intake of the concentrate mixture was 48% lower for cows fed anionic salts compared with cows fed control. The concentrate mixture containing MgSO4-7H20 resulted in the least depression of DMI. This study did not characterized acid-base status of the cows fed different anionic sources. It is difficult to compared the intake data with other studies because it is unknown if 6 these diets changed acid-base status at +11 meq/100 g of dietary DM. More recently, Goff and Horst (1996) reported similar DMI and acid-base status in nonlactating, nonpregnant Jersey cows fed Cl anion from CaCl2 or HCI. Goff et al. (1997) also evaluated the acidifying activity of 6 different anion sources in nonlactating, nonpregnant Jersey cows fed 2 qud. Urine pH were 6.2, 7.1, 7.0, 7.6, 7.9 and 8.2 for HCI, CaClz, NH4CI, CaSO,, M9804, and elemental S, respectively. Authors concluded that sulfate salts are less acidogenic than chloride salts. ABSORPTION OF Ca FROM THE DIGESTIVE TRACT In the dairy cow, Ca homeostasis is achieved using different honnonally- mediated processes. Horst et al. (1994) proposed a model of Ca homeostasis in which inputs of Ca to the Ca pool are: absorption from the intestine, enhanced by 1.25 dihydroxyvitamin D3 (1,25(OH),D3), and bone resorption, enhanced by 1,25(OH),,D3 and parathyroid hormone (PTH). Outputs of Ca from this model are: milk, fetal bone, endogenous fecal and urinary Ca losses. Transcellular (active) transport of Ca Bronner (1992) reported Ca is absorbed from the digestive tract by a transcellular and a paracellular pathway. The transcellular pathway is dominant in the proximal intestine, mainly the duodenum, whereas uptake of Ca in a paracellular fashion takes place throughout the length of the intestine. Three steps are involved in the transcellular pathway: 1) entry of Ca into the enterocyte across the brush border membrane, 2) movement through the cytosol, and 3) extrusion across the basolateral membrane. Calcium concentrations found in the intestinal lumen are in the millimclar range, whereas inside the cell concentrations are in the nanomolar range (Stein, 1992). The entry of Ca into the intestinal cell does not require energy because Ca moves down a steep electrochemical gradient (Fullmer, 1992). Bronner (1991) proposed that Ca enters the enterocyte using Ca channels in the brush border, however, existence of these channels still awaits experimental proof (Bronner, 1992). Movement of Ca across the cytosol of the enterocyte has been proposed to be facilitated by the Ca-binding protein complex, which ferries Ca from the brush border to the basclateral membrane (Bronner, 1987; Stein, 1992). Also, Nemere (1992) proposed that lysosomes may play a role in the facilitated transport of Ca through the cell. The last step in the transcellular pathway is the exit of Ca from the cell. Wasserman et al. (1992) reported that different Ca extrusion processes occurred at the basolateral membrane, which included the Ca channels, the operation of an ATP-dependent Ca pump, and a Na‘lCa2+ exchanger. The primary regulator of transcellular intestinal Ca transport is 1,25(OH)2D3 (Fullmer, 1992). Favus et al. (1989) demonstrated 1,25(OH)203 has stimulatory effects on ATP-dependent movement of at the basclateral membrane into the body, however, the Na‘lCa" exchanger was not affected 8 when 1,25(OH)2D3 was given to vitamin D-deficient rats (Ghijsen et al., 1983). Additionally, Bronner (1992) reported that vitamin D enhanced Ca entry into the cell by 20 to 40%, binding to fixed organelles by up to 100%, intracellular diffusion in direct proportion of the calbindin cellular content, and Ca-ATPase activity by 200 to 300%. As mentioned previously, 1,25(OH)203 regulates Ca homeostasis and enhances Ca absorption from the digestive tract. Horst et al. (1994) reported this hormone accumulates only in tissues that have intracellular receptors for 1,25(OH).‘,D3 (vitamin D receptor, VDR). Intestinal VDR numbers decline as the cow ages (Horst et al., 1990). However, pregnancy and lactation increase VDR number in dairy cows (Horst et al., 1994). Goff et al. (1995) measured VDR concentration in the colon of Jersey cows 6 to 10 d before parturition, at parturition, and 7 d into lactation. Precalving colon VDR concentrations were 90 fmollmg of protein, but declined to 66 fmollmg of protein at parturition and increased to 94 fmollmg of protein 7 d into lactation. Decline in intestinal VDR number as the cows age may explain why hypocalcemia is seen rarely in cows beginning their first lactation. Increased intestinal VDR during pregnancy and lactation may explain increased efficiency of Ca absorption during these stages (Halloran and DeLuca, 1980). Intestinal VDR increased two-fold when 1,25(OH).‘,D3 was infused continuously into Jersey cows (Naito et al., 1989). Horst et al. (1994) suggested this finding may explain partially the mechanism to prevent milk fever when cows are injected with exogenous vitamin D compounds. Paracellular (passive) transport of Ca Paracellular transport is another mechanism used in the absorption of Ca from the digestive tract. Karbach (1992) reported that paracellular transport was responsible for 60 to 70% of the mucosa-to-serosa Ca flux measured across rat duodenum, jejunum, and ileum; whereas, transcellular transport was responsible for 30 to 40% of the Ca flux. The paracellular pathway of Ca absorption consists in three sequential regions: 1) the tight junction, 2) the intermediate junction, and 3) a much wider basclateral space (Bronner, 1987). Bronner (1992) indicated that paracellular Ca movement was necessarily down a chemical gradient because the concentration of Ca in the intestinal lumen is higher than at the serosal pole of the intestinal cell. However, Karbach (1992) proposed an alternate mechanism that may involve a Na+ osmotic gradient. This gradient would cause water to move from the lumen into the intercellular space. The hydrostatic gradient would create pressure in the intercellular space driving water and Ca through the basolateral membrane. This mechanism may be responsible for the net absorption and secretion of Ca in the digestive tract. Karbach (1992) also reported 1,25(OH)203 enhanced paracellular Ca flux in both directions in the rat duodenum, jejunum and ileum, suggesting these observations may be due to changes in paracellular Ca permeability. Paracellular transport of Ca has not been demonstrated in other mammalian species than the rat. 10 EFFECTS OF DIETARY CATION-ANION DIFFERENCE AND DIETARY Ca ON ABSORPTION OF Ca FROM THE DIGESTIVE TRACT In this review, DCAD is defined as the meq [(Na + K) - (Cl + S)]I100 g of dietary DM, otherwise the formula will be indicated. Lomba et al. (1978) summarized data from nonpregnant nonlactating and lactating cows fed 55 different rations. Because no differences in Ca balance were found between lactating and nonlactating cows, data from the two groups were analyzed together. Dietary Ca concentrations were not reported, however, ranges and means of daily intake of Ca in g/cow were from 8.2 to 143.4 and 37.5 in nonlactating cows, and from 26.2 to 109.8 and 62.9 in lactating cows. A negative correlation existed between digestible Ca and [(Na + K) - (Cl + S + P)] meq/d for all the rations. When the rations were divided into two groups according to Ca balance, digestible Ca and [(Na + K) - (Cl + S + P)] meq/d were highly correlated in rations maintaining a positive Ca balance, but not in those with a negative Ca balance. These conclusions must be evaluated carefully because of the narrow range of DCAD in the diets (-11 to -1 meq/d, including P) and daily intakes of Ca (8.2 to 143.4 gld). It's difficult to make conclusions about the digestible Ca from research that included a wide variety of rations and combined data from lactating and nonlactating cows. Braithwaite (1982) measured endogenous fecal loss of Ca in sheep of various ages and physiological states. The majority of endogenous loss of Ca is in feces. He reported a highly significant linear relationship between endogenous fecal loss of 11 Ca and food intake. For this reason I consider that apparent digestibility data for Ca from lactating and nonlactating cows should not be combined in analyses. Freeden et al.(1988b) fed diets with DCAD of -2 and +71 meq/100 g of DM (Na + K - CI) to nonpregnant nonlactating goats. Although not significant, true Ca absorption (using Ca45 marker) expressed as a percentage of Ca intake increased in goats fed negative DCAD (43.3%) compared with those fed positive DCAD (33.1%). The non-significant response to negative DCAD may be due partially to the low dietary Ca concentration fed (0.78% of DM). However, when the Ca chelator ethylene glycol-bis (B-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA) was infused to simulate removal of Ca during lactation, true Ca absorption decreased to 17.7% in goats fed positive DCAD, whereas in those fed negative DCAD true Ca absorption was maintained at 44.8%. A similar study was conducted in sheep fed different DCAD with 0.95% dietary Ca and infused with Ca‘5(Takagi and Block, 1991c). The DCADs were +34, +4 and -13 meq/100 g of DM during the eucalcemic period and +43, +7 and -15 meq/100 g of DM during the EGTA infusion. Calcium absorption was not different among sheep fed different DCAD during the eucalcemic period. However, during EGTA infusion sheep fed negative DCAD had a greater Ca absorption. Also, Freeden and Van Kessel (1990) found that nonlactating nonpregnant ewes fed DCAD of +27 meq/100 g of DM (Na + K - Cl), with 0.35% dietary Ca, and challenged with EGTA, did not increase true Ca absorption from the digestive tract. However, Freeden (1990) reported increased Ca absorption 12 expressed as a percentage of Ca intake, when sheep infused with EGTA were fed a higher dietary Ca concentration (0.55%) and +63 meq/100 g of DM (Na + K - CI), but not with low dietary Ca concentration (0.33%) and +15 meq/100 g of DM. Gaynor et al. (1989) reported that cows fed anionic diets had greater plasma concentrations of 1,25(OH)2D3 compared with cows fed a cationic diet. Also, Goff et al. (1995) reported similar numbers of colon VDR in cows fed anionic and cationic diets precalving and at parturition, however, cows fed the anionic diet had greater colon VDR numbers 7 d into lactation. The transcellular transport of Ca may be the dominant mechanism when cows are fed negative DCAD and low dietary Ca, because increased concentrations of plasma 1,25(OH)2D:, can enhance uptake of Ca by this pathway (Bronner, 1992; Fullmer, 1992). Based on these results, negative DCAD and high dietary Ca may change the capacity of the digestive tract to respond to sudden Ca loss from the exchangeable blood Ca pool. Sheep fed three different DCAD (+34, +22, +6 and +28, +6 and -3 meq/100 g of DM) with low (0.47%) and high (0.82%) dietary Ca concentrations, respectively, had no difference in apparent absorption of Ca from the digestive tract (T akagi and Block, 1991a). Lack of differences in Ca absorption among DCAD may be due partially to DCAD that was not low enough to increase active mechanisms of Ca absorption. Urine pH can be used as a tool to monitor acid- base status of dairy cows fed anion sources (Goff and Horst, 1997a). The lowest urine pH observed in these sheep was 7.69 for the -3 meq/100 g of DM 1 3 diet. However, when high Ca diets were fed a greater apparent absorption of Ca was observed; passive absorption of Ca in the digestive tract may be the mechanism responsible for the increased absorption of Ca from these diets. Also, Leclerc and Block (1989) reported no differences in apparent Ca absorption from the digestive tract from d 24 to d 21 prepartum in nonlactating cows fed +39 (control), +12, +10 and +6 meq/100 g of DM. However, when apparent absorption of Ca was measured from d 7 prepartum to d 1 postpartum low DCAD diets reduced apparent Ca absorption from the digestive tract. Although dietary Ca was high, differences in Ca absorption among DCAD may be due to lower Ca concentrations in the treatment diets (1.3%) compared with the control diet (1.5%). Additionally, Schonewille et al. (1994) found no difference in apparent Ca absorption of cows fed two concentrations of Ca (0.55 and 0.95%) with negative DCAD -11 meq/100 of DM. Lack of response in this study may be due partially to the few number of cows used (5), low dietary concentration of Ca and to the low requirement of Ca of nonlactating nonpregnant cows. Ender et al. (1971) conducted Ca balance studies 9 d before and 9 d after parturition, in cows fed +27 and -2 meq/100 g of DM with 0.34 or 1.27% dietary Ca. Before parturition, regardless of the diet fed all cows had a positive Ca balance. After parturition, cows fed positive DCAD were in a marked negative Ca balance, whereas, Ca balance of cows fed negative DCAD was near zero, slightly positive or negative. Statistical analyses of the responses to dietary concentration of Ca were not reported. However, inferences from their tables 14 indicated that cows fed positive DCAD and high dietary Ca had a more pronounced negative Ca balance the first 2 d after parturition compared with cows fed positive DCAD and low dietary Ca. Contrary to this, cows fed negative DCAD and high dietary Ca had a slightly negative or positive Ca balance the first 4 d after parturition. Whereas, cows fed negative DCAD and low dietary Ca had negative Ca balance. Ender et al. (1971) suggested that diets with high amounts of chlorine and sulfur reduced intestinal pH and promote Ca absorption. Also, although not significant, Takagi and Block (1991a) found 25% increase in apparent Ca absorption when sheep were fed negative DCAD diets. Differences between the apparent and true absorption measurements are likely due to increased endogenous Ca secretions when negative DCAD or low Ca diets are fed, which can not be accounted by the apparent absorption method. Moore et al. (1997) fed Holstein cows diets containing +14, 0 and -15 meq/100 g of DM with 0.44, 0.97 and 1.5% dietary Ca, respectively. Decreasing DCAD while increasing dietary Ca increased plasma iCa concentrations at parturition in multiparous cows, but not in cows calving for the first time. Treatments had no effect on plasma hydroxyproline (indicator of bone Ca mobilization) concentrations before or after calving, however, plasma 1 ,25(OH)203 increased in multiparous but not in primiparous cows when DCAD was reduced at the same time dietary Ca was increased. Reduction in plasma 1 ,25(OH).‘,D3 concentrations may be due to the increased dietary Ca, however, plasma iCa concentrations increased at the same time plasma 1,25(OH)ZD was reduced. This suggests that the mechanism used by these cows at parturition to 15 maintain Ca status was less dependent on plasma 1,25(OH)2D:,. Cows fed high dietary Ca and negative DCAD may have had increased Ca absorption, mainly by the paracellular pathway, because plasma 1,25(OH)2D3 was lower and plasma hydroxyproline was not different among treatments. Oetzel (1991) determined the nutritional risk factors for milk fever, using meta-analysis techniques, and analyzing data from 75 published trials (1165 cows, of which 214 developed milk fever). Results indicated that extremes in concentrations of dietary Ca, high and low, reduced the incidence of milk fever. Dietary Ca concentrations of about 1.16% were associated with the greatest milk fever incidence. Responses of cows and the mechanisms used to maintain Ca status when fed low Ca diets are understood (Jorgensen, 1974; Green et al., 1981). However, the potential ability of high dietary Ca to prevent milk fever has not been explained. It is possible that the paracellular absorption of Ca plays a role, because this mechanism is non-saturable, independent of nutritional and physiological regulation, and concentration-dependent (Bronner, 1987). Khorasani and Armstrong (1992) reported that level of Ca intake had a pronounced effect on net absorption and secretion of Ca in the digestive tract of ruminants. Their results showed that the major site of net Ca absorption of animals fed high Ca diets was prior to the small intestine, probably in the reticulo-rumen compartment. The net flux of Ca across the rumen wall of sheep was studied by Holler et al. (1988b), using the temporarily isolated washed rumen, filled with buffer solutions with five different Ca concentrations (0.2 to 3.6 mmollL). Increasing Ca concentration of the rumen buffer increased net flux of 16 Ca linearly, showing Ca net secretion into the lumen at the lowest Ca concentration (0.2 mmollL) and net absorption from the lumen at Ca concentrations of 1.7 mmollL and above. They postulated that for paracelullar transport of Ca to occur, Ca concentration in the rumen must be at least 6 mmollL, otherwise, Ca absorption is mediated by a primary or secondary intracellular mechanism, may be active transport. Holler et al. (1988b) indicated that mucosal-to-serosal net flux of Ca was abolished when the Na*,K*-dependent ATPase was blocked using ouabain. This may indicate that active transfer of Na across the basclateral membrane is in some way essential for the observed net flux of Ca. A reduction of Ca absorption when Na was absent from the medium was observed in everted sacs of rat duodenum (Martin and DeLuca, 1969). Also, Holler et al. (1988a) reported that net flux of Ca was abolished after addition of ouabain. or when Na-free buffer solutions were used in isolated mucosal tissue from the sheep omasum. These observations might explain why O'Connor (1987) reported decreased ruminal concentrations of Ca when high Na (0.60 vs 0.22%) diets were fed to late lactation cows. There has been interest in using oral administrations of Ca to improve Ca status around parturition. Sanchez et al. (1995) reported 24% increase in Plasma iCa 30 min after administration of 110 g of Ca as Ca propionate and PrOpylene glycol gel. Goff and Horst (1995) assessed absorption of Ca by the increase of plasma Ca observed in the cows administered Ca propionate Slurries. Administration of 95 g of Ca increased plasma Ca concentration by 17 18% above pretreatment. However, when NaCl was added to the drench plasma Ca concentration increased 29% above pretreatment. They stipulated that the addition of Na might stimulate closure of the esophageal groove. However, it actually might be that the increased Na concentration increased the Ca absorption from the rumen. Based on these studies, it is speculated that negative DCAD may not affect Ca absorption in the nonlactating pregnant cow. However, near parturition when greater amounts of Ca are needed in the Ca pool, it seems that negative DCAD may stimulate the active transport mechanism(s) of Ca absorption from the digestive tract. Paracellular transport of Ca may play an important role in the cows' Ca absorption from the digestive tract. Increasing dietary Ca concentrations in prepartum diets may be beneficial to increase absorption of Ca around parturition when DM intake is low and Ca demand is high. However, increasing Ca intake will lead to down-regulation of the transcellular pathway, whereas the amount of Ca moved by the paracellular pathway will be in proportion to the Ca intake (Pansu, et al. 1981). Effects of high dietary Ca concentrations and different DCAD on cow's acid-base and Ca status needs to be researched. RESORPTION OF Ca FROM BONE As stated earlier, Ca homeostasis is achieved by absorption of Ca from the digestive tract and bone Ca mobilization. The mobilization of Ca from the 18 ‘3 L? C? (I) bone is stimulated by a concerted effort of PTH and 1,25(OH)2D3 (Horst et al., 1994). The primary regulator of1,25(OH)2D3 production is PTH (Reinhardt et al., 1988). Horst (1986) characterized the Ca adaptation mechanisms used by the dairy cow to achieve Ca homeostasis. Calcium status is "sensed" by the parathyroid gland, and in response to low blood Ca, PTH is secreted. Production of 1,25(OH)1,D3 results from the binding of PTH to the membrane receptors in kidney cells (Reinhardt et al., 1988). Increased plasma concentrations of 1,25(OH)203 increase Ca absorption from the digestive tract. Also, increased concentrations of PTH and 1,25(OH)2D3 result in increased bone Ca resorption and increased concentrations of plasma Ca. High concentration of plasma Ca cause down regulation of the same mechanisms that increase plasma Ca concentration. It decreases PTH, which decreases kidney 1-or-hydroxylase and 1,25(OH)203 production, and lowers absorbed Ca from the digestive tract and mobilized Ca from the bone, resulting in lower plasma Ca concentrations. Bone resorption involves release of bone mineral and degradation of the bone matrix. Most bone resorption is probably cell-mediated (Mundy and Raisz, 1981). Four different types of cells are associated with bone. Osteoclasts, bone lining cells and osteoblasts are abundant in bone surfaces, whereas osteocytes are entrapped within the mineralized matrix (Wasserrnan, et al. 1993). The large multinucleated osteoclast is responsible for most if not all bone resorption. However, other factors such as collagenase, lysosomal enzymes, and pH may be involved in the bone resorbing process (Mundy and Raisz, 1981; Wasserman et a., 1993). Osteoclastic activity is increased by PTH (Raisz, 1965), the active 1 9 metabolites of vitamin D (T rummel et al., 1969), but is inhibited by calcitonin (Raisz and Niemman, 1967). Osteoblasts are the only bone cell to express the VDR (Horst et al., 1994). Horst et al. (1990) reported bone from older rats contains fewer VDR compared with bone from young rats. Also, Malluche and Faugere (1986) reported bone osteoblast numbers declined with age, and this may be the reason why VDRs in bone are reduced also with age. So in the aged animal, capacity for bone resorption may be delayed because fewer osteoblasts exist to respond to PTH and 1,25(OH)2D3 stimulation. Calcium homeostasis of dairy cows undergoes an adaptation process around parturition (Horst et al., 1994). Barton et al. (1981) characterized concentrations of Ca, P and 1,25(OH)203 during the lactation cycle of young, nonparetic aged and paretic aged cows. The period of greatest variation in plasma Ca and 1,25(OH)ZD was from 7 d prepartum to 7 d postpartum. All cows reported some degree of hypocalcemia the first days after calving as reflected by decreased plasma Ca and increased in plasma 1,25(OH).‘,D3 concentrations. However, paretic cows had the lowest plasma Ca concentrations and the highest 1,25(OH)2D3 concentrations after parturition. Also, increased concentrations of PTH were observed after parturition in paretic cows (Goff et al., 1986). Ramberg et al. (1984) reported bone resorption plays a minor role in Ca homeostasis the first 2 wk postpartum because the mechanisms used are relatively inactive during the dry period. Reinhardt et al. (1988) reported bone resorptive response is somewhat refractory to stimulus of PTH and 1,25(OH)2D3. However, this scenario changes when cows are fed low Ca diets prepartum. 20 Increased concentrations of plasma Ca, 1,25(OH).‘,D3 and hydroxyproline were found during the peripartum period in cows fed low Ca (8 gld) compared with high Ca (80 gld) prepartum (Green et al., 1981). Low Ca intake places the cow in a negative Ca balance, stimulates the production of PTH and 1,25(OH),D3, and therefore, the Ca homeostatic mechanisms prepartum. Goff et al. (1986) measured the responses of pregnant Jersey cows infused with 146 ug/PTH per h for 48 and 96 h. When cows were infused for 48 h, plasma 1,25(OH)203 concentrations increased after 8 h and plasma Ca concentrations increased after 32 h of infusion. However, no changes were observed in plasma PTH and hydroxyproline concentrations. When cows were infused for 96 h responses were similar to cows infused 48 h; however, increased plasma concentrations of hydroxyproline were observed 72 h after infusion. Increased plasma Ca concentrations observed 32 h after the infusion may be due to the increased concentrations of plasma 1,25(OH)2D3, which stimulated Ca transport from the digestive tract because bone mobilization did not occur until 72 h after infusion. Also, injections of 1-or-hydroxyvitamin 03 and 25-hydroxyvitamin 03 (Hodnett et al., 1992; Zepperitz and Grun, 1993) and subcutaneous implants of 24F-1,25 dihydroxyvitamin D3 (Goff and Horst, 1990) increased plasma 1,25(OH)203, Ca and P concentrations at parturition and reduced incidence of milk fever. Increased plasma Ca concentrations when vitamin D compounds are inl'ected is primarily due to their ability to increase intestinal absorption of Ca before calving (Hove, 1984). This is supported by the fact that bone resorption is 21 not affected before parturition because plasma hydroxyproline concentrations did not change (Bar et al., 1985; Goff et al., 1988; Goff and Horst, 1990). However, there are some potential problems with using exogenous vitamin D. Vitamin D may be toxic under certain conditions (Littledike and Horst, 1982), requires precise timing of administration of the injection relative to parturition (Goff and Horst, 1990), and treatment with exogenous 1-or-OHD3 causes a reduction in kidney 1-or hydrolase activity (Littledike et al., 1986). Also, prolonged inhibition of 1-or-hydrolase activity due to exogenous vitamin D, can make the cows extremely susceptible to hypocalcemia once the exogenous vitamin D source is removed. EFFECTS OF DIETARY CATION-ANION DIFFERENCE AND DIETARY Ca ON RESORPTION OF Ca FROM BONE The periparturient period is associated with a high secretion of Ca in colostrum (Horst, 1986). Freeden and Van Kessel (1990) challenged nonlactating nonpregnant ewes fed 0.35% dietary Ca with EGTA to simulate drop of blood Ca similar to that occurring with colostrum formation. Increase in bone Ca resorption was the short term metabolic response to the induced blood Ca deficit. Similar results were reported by Freeden (1990). Contrary to this, Takagi and Block (1991c) reported no difference in mobilization of Ca from bone When sheep were challenged with Na2*-EDTA. However, a 48% reduction in bone accretion was observed. 22 Green et al. (1981) fed high (80 gld) or low Ca (8 gld) to Jersey cows prepartum. Cows fed low Ca tended to have low plasma Ca concentration prepartum, but greater concentrations during the peripartum period. Increased peripartum plasma Ca concentrations in this study may be due to a combination of increased absorption from the digestive tract and bone resorption because concentrations of plasma 1,25(OH).‘,D3 and hydroxyproline were greater also. Kichura et al. (1982) fed high (86 gld) or low (9.5 gld) dietary Ca with high (82 gld) or low (10 gld) dietary P to Jerseys cows prepartum. Similarly, cows fed low dietary Ca increased peripartum plasma Ca, hydroxyproline and 1,25(OH)203 concentrations compared with cows fed high dietary Ca (Green et al., 1981). However, cows fed the high-Ca-low-P diet had similar peripartum plasma Ca concentrations as cows fed low Ca diets; even though lower plasma hydroxyproline and 1,25(OH)2D3 concentrations were found prepartum. Takagi and Block (19913) reported increased plasma hydroxyproline concentrations in sheep fed a normal Ca (0.47%) diet compared with sheep fed a high Ca (0.82%) diet. Although all sheep were in positive Ca balance, greater plasma hydroxyproline concentrations may indicate some bone mobilization in the sheep fed normal Ca diet, because apparent absorption of Ca was greater with the high Ca diet. Also, sheep fed similar dietary Ca concentrations (0.45%) and challenged with Na*-EDTA infusions had greater rates and amounts of Ca mobilized from the bone than sheep fed 0.74% dietary Ca (T akagi and Block, 1991b) Jonsson et al. (1980) also found that plasma PTH concentrations 23 decreased inversely with increasing amounts of dietary Ca prepartum and peripartum (37, 75 and 150 gld). However, no changes in plasma Ca and hydroxyproline were observed during the prepartum and peripartum periods due to dietary Ca. Saeki and Hayashi (1981) challenged with EDTA Holstein calves fed high (115 gld) and low (28 gld) dietary Ca for 36 d. Parathyroid function was altered in some calves 9 d after high and low Ca feeding. High dietary Ca reduced PTH function, whereas, low dietary Ca increased PTH function. Also, increased plasma PTH concentrations were observed within 4 d after feeding a Ca-deficient diet (8.2 gld) prepartum (Goings et al., 1974). Results from these studies indicate that high dietary Ca may down-regulate Ca mobilization from bone. Markedly lower concentrations of Ca and P and elevated concentrations of PTH and hydroxyproline were observed in both paretic and non-paretic cows during the peripartum period (d -2 to 5) compared with the prepartum period (Jonsson et al., 1980). Also, Horst et al. (1978) reported higher concentrations of plasma PTH and 1,25(OH)ZD3 in blood of cows with milk fever compared with cows without milk fever. Nonexistent or delayed production of 1,25(OH)203 was reported in cows with milk fever fed high (125 gld) dietary Ca and positive DCAD (+48 meq/100 g of dietary DM) prepartum (Goff et al., 1989). Regardless of high concentrations of plasma PTH observed in relapsing cows (treated for hypocalcemia more than once), concentrations of plasma 1,25(OH)2D3 were not increased in these cows. Authors suggested that kidneys of cows with milk fever are temporarily refractory to PTH stimulation. Horst et al. (1994) also suggested 24 that intestine, bone and kidney (target tissues of PTH and 1,25(OH)2D) of cows with milk fever have lost the ability to respond to the Ca-regulating hormones. Research done with dogs (Bumell, 1971) and rats (Beck and Webster, 1976) may indicate that in an alkaline acid-base state, bone and perhaps renal tissues are refractory to the effects of PTH. However, during metabolic acidosis these tissues may be more responsive to PTH. Gaynor et al. (1989) fed high DCAD (+125 meq/100 g of dietary DM) and low DCAD (+22 fed meq/100 g of dietary DM; Na + K - Cl) diets with 1.2% dietary Ca to Jersey cows prepartum. Cows fed the high DCAD had lower concentrations of plasma Ca after parturition, lower plasma concentrations of 1,25(OH)203 3 d before calving, and tended to have lower concentrations of plasma hydroxyproline prior to parturition than cows fed the low DCAD. Phillipo et al. (1994) fed anionic and cationic diets with 1.16% dietary Ca to Holstein cows during the last 28 d of pregnancy. Plasma PTH concentrations were similar for cows fed each diet. However, the concentrations of plasma 1,25(OH)2D3 tended to be higher for cows fed the anionic compared with those fed the cationic diet. Also, nonnocalcaemic cows fed the cationic diet tended to have higher prepartum 1,25(OH)203 than cows that developed milk fever. In a similar study, Goff et al. (1991a) fed anionic (-23 meq/100 g of DM) and cationic (+98 meq/100 g of DM) diets with 1.7% dietary Ca to Jersey cows prepartum. Cows fed the cationic diet had lower plasma Ca concentrations at parturition and 2 d after parturition regardless of similar plasma PTH concentrations peripartum. From regression analysis of the slope of the line of plasma 1,25(OH)2D3 on 25 plasma PTH, concentrations of plasma 1,25(OH)2D3 were greater in cows fed the anionic compared with those fed the cationic diet. This suggests the kidneys were temporarily refractory to PTH stimulation. Also, in cows fed the cationic diet, plasma hydroxyproline concentrations tended to decrease just prior to parturition, despite increasing plasma PTH concentrations. Authors suggested that, osteoclasts may be refractory to PTH stimulation in cows with metabolic alkalosis. F urthennore, Horst et al. (1994) proposed that metabolic alkalosis may have somehow disrupted the integrity of PTH receptors on target tissues. A study conducted by Goff and Horst (1997) supported this proposed effect. They fed diets containing 1.1, 2.1 and 3.1% K with 0.5 or 1.5% Ca to Jerseys cows. Diets had DCAD of -8, +21 and +43 meq/100 g of dietary DM. Cows fed 2.1 and 3.1% K had the greatest plasma PTH concentrations, but the lowest plasma hydroxyproline concentrations. This suggests that bones of cows consuming high K diets were refractory to PTH stimulation. Greater bone mobilization occurred during the peripartal period when cows were fed diets with reduced DCAD prepartum as indicated by hydroxyproline concentrations in blood (Block, 1984). Leclerc and Block (1989) fed diets with DCAD of +39, +12, +10 and +6 meq/100 g of DM with 1.38% dietary Ca to Holsteins cows prepartum. Cows fed low DCAD reduced the severity of the plasma Ca decline during the periparturient period. Also, cows fed +10 and +6 meq/100 g of DM had greater concentrations of plasma hydroxyproline from 2 d prepartum to 1 d postpartum compared with cows fed high DCAD. 26 Contrary to this, no significant changes in urinary hydroxyproline were observed when cows were fed a cationic (+57 meq/100 g of dietary DM) or an anionic (0 meq/100 g of dietary DM) diet with 75 g/d dietary Ca (Van Mosel et al., 1994). Bone biopsies were taken from the tuber coxae at parturition. In cows with 3 or more parities, the percentage of osteoid volume and the ratio of osteoid volume to percentage of osteoid surface were greater in cows fed the anionic diet. In a separate study, the acid-base status of these cows was characterized (Van Mosel et al., 1993). Plasma Ca of cows fed the two diets did not differ, however, increased urinary Ca and reduced urinary pH of cows fed the anionic diet indicated changed acid-base status of these cows. This study indicates that a DCAD of 0 meq/100 g of dietary DM, affected some measurements of bone formation, but not bone resorption at parturition. Changes in acid-base status may have increased Ca absorption from the digestive tract at this DCAD. Bone breaking strength for the 7th and 9th rib is another approach to measure bone Ca and P status (Jackson and Hemken, 1994). They used male Holstein calves fed diets containing DCAD of -18 and +13 meq/100 g of dietary DM with 0.42 and 0.52% dietary Ca. Bone breaking strength for the 7th rib was higher for calves fed +13 compared with -18 meq/100 g of DM, and also higher for calves fed 0.52 compared with 0.42% dietary Ca. These results suggest anionic diets and dietary Ca changed Ca metabolism and therefore bone density. Additionally, Takagi and Block (1991b) found increased amounts of Ca mobilized and increased rates of Ca mobilization when sheep were challenged With EDTA and fed low DCAD (+13 and +3 meq/100 g of DM) compared with 27 q. 0‘ AA. only, 41$" t‘, .: VIU ten: ~ I‘ S. -o I 4.... I“. I. E'I‘; l‘ (I) high DCAD (+35 meq/100 g of DM). Some studies have been conducted using Ca“ isotopes to measure the response of feeding anionic salts on Ca kinetics. Braithwaite (1972) measured the effects of ammonium chloride (0.2 glkg of BW) on Ca metabolism of sheep. Calcium intakes ranged from 5.2 to 5.7 gld. Addition of ammonium chloride did not change bone accretion or resorption. However, Ca absorption from the digestive tract increased with the addition of ammonium chloride. Takagi and Block (1991c) found decreased bone accretion in sheep fed -13 meq/100 g of dietary DM and challenged with EDTA compared with sheep fed +34 meq! 100 g of DM and challenged with EDTA. Also, although not significant bone resorption tended to be greater in sheep fed negative diets compared to those fed positive diets. Freeden et al. (1988) measured Ca kinetics (using Ca”) in nonpregnant nonlactating does fed diets with DCAD of -2 and +71 meq/100 g of DM (Na + K - CI). Although not significant, Ca resorption expressed as a percentage of Ca intake was greater in goats fed negative DCAD (40.6%) compared with those fed positive DCAD (34.5%). However, when EGTA was infused, Ca accretion was decreased in goats fed positive DCAD, whereas it was increased in those fed negative DCAD. Also, during EGTA infusion Ca resorption increased in goats fed both diets. However, goats fed the anionic diet had a greater bone Ca resorption expressed as a percentage of Ca intake (93.3%) compared with goats fed the cationic diet (66.6%). Different responses in mobilization of Ca from the bone may be due to different experimental DCAD used. It is important to determine at 28 which DCAD mobilization of Ca from the bone is increased. EXCRETION OF Ca There are four major routes of loss of Ca from the dairy cow's body (Horst et al., 1994). In the pregnant, nonlactating cow the major routes are the Ca going to fetal bone (2 to 7 gld) and endogenous fecal Ca loss (5 to 8 gld); whereas, urinary Ca loss is minimal (0.2 to 1 gld). However, the major drain of Ca from the cow's body is at onset of lactation, when it reaches 20 to 80 gld. About 2.5 g of Calkg of colostrum are secreted. This indicates that a cow producing 25 kg of colostrum secretes 62.5 g of Ca in the first day of lactation, 25 times the amount of Ca present in the blood plasma Ca pool at one time. Braithwaite (1982) examined the results from 374 sheep used in Ca metabolism studies. A linear relationship existed between endogenous fecal loss of Ca and amount of feed intake; endogenous loss increasing by about 0.64 mg/d per kg of BW. The NRC (1989) reported an endogenous Ca fecal loss of 1.54 g/100 kg of live weight for maintenance requirements. Values reported by NRC (1989) may not be accurate because as concluded by Braithwaite (1982) endogenous fecal Ca loss in highly correlated with amount of feed intake. Urinary Ca and P04 excretion are controlled by plasma PTH and calcitonin concentrations (Dickson ,1993). Urinary Ca excretion is enhanced by calcitonin and depressed by PTH and 1,25(OH)203. Increased concentrations of plasma PTH enhanced renal retention of Ca through the distal convoluted 29 tubules and the collecting ducts of the kidney. Also, increased concentrations of plasma PTH enhanced excretion of P04 through the distal convoluted tubules, and this excretion is accompanied by increased excretion of Na and bicarbonate. Nonlactating pregnant cows infused with 146 uglh of PTH for 48 h had a rapid decrease of urinary Ca and increase of urinary P within 24 h of the start of the infusion (Goff et al., 1986). Dickson (1993) also reported calcitonin had similar effects of those of PTH enhancing the excretion of P0,, but calcitonin increases the renal excretion of Ca. EFFECTS OF DIETARY CATION-ANION DIFFERENCE AND DIETARY Ca ON EXCRETION OF Ca Reducing DCAD reduced the incidence of hypocalcemia, due to the influence of the anionic salts on the acid-base status of the cow by causing a mild acidosis (Block, 1984; Goff et al., 1989; Goff et al., 1991; Wang, 1990). Compensation for this mild metabolic acidosis is evident by increased urinary excretion of acid (Oetzel et al., 1991; Goff and Horst, 1993) and reduced blood pH (Wang and Beede, 1992). The use of urine pH to monitor anionic salt feeding has been proposed (Beede, 1995; Goff 'and Horst, 1997a; Vagnoni and Oetzel, 1997). Also, reduced urinary pH was observed when anionic salts were fed to cats (Pastoor, et al. 1994) and rats (Whitting and Cole, 1986). Beside changes in urinary pH, anionic salt feeding affects the way the kidney handles some other metabolites. Reduced urinary bicarbonate excretion 30 was observed in cows (Van Mosel et al., 1993) and goats (Freeden et al., 1988a) fed anionic diets. Beede and Wang (1992) reported increased urinary Ca excretion in cows fed ammonium chloride and ammonium sulfate. Additionally, Gaynor et al. (1989) found increased urinary excretion of Ca and Mg and reduced Na excretion when cows were fed diets with +22 meq compared with those fed +126 meq/100 g of dietary DM (Na + K - Cl). However, no differences were observed in urinary excretion of K. Takagi and Block (1991a) reported increased urinary Ca excretion in sheep fed negative compared with those fed positive DCAD. However, dietary Ca had no effect on urinary excretion of Ca. Also, decreasing the DCAD increased urinary Mg and S excretion, but decreased Na urinary excretion. Whitting and Cole (1986) measured the effect of replacing dietary carbonate by sulfate or chloride on urinary Ca excretion in rats. Feeding either Cl or SO, increased urinary Ca excretion to a similar extent when equivalent changes in acid-base status were induced. Also, greater excretion of SO4 or Cl were observed when rats were fed the 804 or Cl diet. Based on these results, excretion of mineral elements in the urine can be changed according to the amount of mineral element fed and the amount of anions fed. Braithwaite (1972) measured the effect of feeding ammonium chloride on Ca metabolism of sheep using Ca‘s. Inclusion of ammonium chloride in the diet decreased urinary pH and increased the rate of excretion of Ca. Takagi and Block (1991c), also using Ca“5 observed increased urinary excretion of Ca as a percentage of Ca intake in eucalcemic and EGTA-infused weathers when fed anionic diets. 31 4_.__\ ‘ (.J k? Freeden and Van Kessel (1990) evaluated the effects of sudden Ca loss (induced by EGTA infusion) on Ca metabolism in ewes. The infusion increased urinary Ca excretion from 0.025 to 1.04 gld. Similar responses were observed in goats infused with EGTA regardless of whether they were fed an anionic or cationic diet (Freeden et al., 1988b). Also, Freeden (1990) observed increased urinary excretion of Ca when nonlactating nonpregnant ewes were infused with EGTA. Increased Ca excretion in the urine of ruminants challenged with EGTA may be a secondary effect of increased plasma Ca concentration, due to stimulation of Ca bone resorption, absorption from the digestive tract, or both. It is well documented that feeding anionic salts to lower DCAD produces metabolic acidosis, which increases urinary excretion of Ca. Sutton et al. (1979) proposed the mechanism by which metabolic acidosis induces hypercalciuria. He suggested that it was a decrease in renal tubular resorption of Ca. This might be corroborated by the work of Jacob et al. (1983), who found that the addition of up to 8 meq/kg of BW of acidogenic salt (as ammonium chloride) in rats increased net acid excretion in the urine, but did not change urinary Ca excretion. However, only when the acid load exceeded 16 meq/kg of BW was urinary Ca excretion increased. Higher rates of Ca excretion may decrease Ca retention, thus inducing secretion of PTH and production of1,25(OH)2D3 increasing bone resorption and Ca absorption from the digestive tract. Block (1994) proposed another possible mechanism of how anionic salt feeding can affect kidney function. Production of renal 1,25(OH)203 is an enzyme-dependent process (1 or-hydroxylase) that is pH 32 sensitive. Changes of blood pH by feeding anionic salts could alter the efficiency of conversion of 1,25(OH).‘,D3 in the kidney. FOCUS OF RESEARCH As described previously, the overall objective of this research was to determine how varying dietary Ca concentrations and DCAD affect Ca metabolism, renal physiology, and physiological performance of nonlactating nonpregnant and pregnant dairy cows. Anion supplementation in the field has not always improved Ca status and other peripartum problems. Possible reasons include: the actual DCAD was unknown; unpalatability inherent with anion sources; and, improper ration mixing. Practically, it is very difficult to make changes in dietary ingredients through time and know the DCAD. Having supplemental anion sources as a separate mix with a carrier allows alteration of the anion inclusion rate. Also, urine pH may be a useful measure of the cow's acid-base status when feeding anion sources. Therefore, the objectives of the first experiment were to characterize the temporal pattern of changes in urine pH and blood variables in relation to time of feeding of non-lactating pregnant multiparous Holstein cows fed varying amounts of an anion-Ca supplement, and to evaluate acid-base and Ca status, and Peripartum health of Holstein cows fed one of three different dietary treatments varying in DCAD and Ca content. Varying the DCAD fed to cows before calving can reduce DMI (Joyce et 33 al., 1997; Schoenbaum et al., 1994; Moore et al., 1997) and energy balance (Moore et al., 1997). The advantages of lowering DCAD in prepartum diets to control or reduce hypocalcemia may be offset by the depression in DMI sometimes observed in cows fed anionic diets. It is important to find sources of anions that effectively alter acid-base status and have minimal effects on DMI. Therefore, the objective of the second experiment was to determine if the addition of dietary anions from different anion sources and varying dietary Ca from CaCO3 affected feed intake and urine pH across time in nonpregnant, nonlactating Holstein dairy cows which had completed at least one lactation. The objective of the third experiment was to determine if the addition of dietary anions from different anion sources and dietary Ca from CaCO3 affected feed intake, acid-base status, and macromineral metabolism and utilization using nonlactating nonpregnant Holstein dairy cows as an experimental model. Several experiments were conducted to study varying DCAD and dietary Ca, but in each study, the two factors were confounded (Moore et al., 1997; Wang, 1990), or negative DCAD was fed with high dietary Ca (Goff et al., 1991a), or negative DCAD was fed with low dietary Ca (Block, 1984). Results from some of these studies suggest that it may be beneficial to feed higher dietary Ca prepartum than current recommendation (NRC, 1989), especially with low or negative DCAD. However, the literature is lacking studies where DCAD is factored with dietary Ca in periparturient cows. Therefore, the objective of the fourth experiment was to determine if increasing dietary Ca concentration using CaCO3 in prepartum diets with 34 negative DCAD resulted in improved periparturient Ca status and utilization of periparturient multiparous Holstein dairy cows. 35 CHAPTER 3 LOWERING DIETARY CATION-ANION DIFFERENCE WHILE RAISING CALCIUM CONTENT REDUCES PERIPARTUM SUBCLINICAL HYPOCALCEMIA ABSTRACT In Experiment 1, six pregnant, multiparous non-lactating Holstein cows were fed an anion-Ca supplement and assigned randomly to a repeated measures design to determine the temporal pattern of urine pH and blood variables. Treatments had dietary cation-anion differences (DCAD; meq [(Na + K) - (Cl + S)]I100 g of DM) of +17, -11 and -26 meq/100 g of DM with dietary Ca concentrations of 0.61, 1.01, and 1.86%, respectively. Urine pH averaged 8.1 during the first 4 (I when cows were fed +17 meq. On d 5 two cows each were switched to -11 and -26 meq/100 g of DM, with two remaining on +17 meq/100 g of DM. Mean urine pH of cows fed -11 and -26 meq decreased from 8.1 to 5.8 by d 7. In Experiment 2, 117 Holstein cows were fed dietary treatments for 21 d before eXpected calving in a randomized block (parity) design to investigate effects of changing DCAD and dietary Ca content on periparturient Ca and health status. Dietary treatments had DCAD and Ca% of +11 and 0.63 36 (Treatment 1), -11 and 0.95 (Treatment 2) and -26 meq/100 g of DM and 1.17% (T reatment 3). Prepartum cows fed Treatment 1 had higher mean urine pH than cows fed Treatments 2 and 3, and cows fed Treatment 2 had higher mean urine pH than cows fed Treatment 3. After calving, cows fed Treatment 1 had lower mean plasma ionized Ca than cows fed Treatments 2 and 3. Treatments had no effect on incidence of milk fever. Incidence of abomasum displacement was greater in cows fed Treatments 2 and 3, and most cases were in first parity cows. Lowering DCAD, while increasing dietary Ca concentration, decreased urine pH and reduced postpartum subclinical hypocalcemia (plasma ionized Ca < 4mgldl). Abbreviation key: DCAD = dietary cation-anion difference; iCa = ionized Ca; PTH = parathyroid hormone; VDR = vitamin D receptor; SID = strong ion difference. INTRODUCTION Transition from the pregnant nonlactating state to the lactating state occurs with a series of physiological and hormonal changes. Most of the metabolic diseases in dairy cows occur during the first 2 wk of lactation (Goff and Horst, 1997b). Proper nutritional management during the transition period can help reduce the risk of metabolic diseases and improve early lactation performance. 37 In dairy cattle nutrition the dietary cation-anion difference (DCAD) can be defined as: meq [(Na + K) - (CI + S)]l100 g of DM (Ender et al., 1971). Typical feedstuffs and diets for dairy cows have a positive DCAD due to relatively higher K and Na concentrations compared with Cl and S concentrations. A negative DCAD can be formulated by addition of anion sources such as MgSO4o7H20, CaSO402HZO, (NH,)ZSO,, NH4CI, CaCIzoZHZO, and MgCIzo6H20 (Oetzel et al., 1991). It has been shown that decreasing the DCAD affects systemic acid-base status (Oetzel et al., 1991), Ca metabolism (Block, 1984; Goff et al., 1991), peripartum health (Block, 1984; Dishington, 1975; Goff and Horst, 1997a), and postpartum productive and reproductive performance (Wang, 1990). A negative DCAD causes mild metabolic acidosis, increases mobilization of Ca from bone (Block, 1984, Gaynor et al., 1989), and possibly enhances absorption of Ca from the gut (Freeden et al., 1988b, Goff et al., 1991), thus increasing the ability of the cow to maintain blood Ca concentration and reducing the risk of milk fever and other problems related to hypocalcemia in the early postpartum period. Reducing the DCAD by anion supplementation in the field has not always improved Ca status and other peripartum problems. Possible reasons include: the actual DCAD was unknown or likely more positive (eg. with higher K or Na concentrations) than presumed from diet formulation; possibly dietary Ca concentration was too low; unpalatability inherent with anion sources; and, improper ration mixing. Also due to varying DCAD of the forage base, the practice of using a standard anion package in rations of prepartum cows in several different dairy farms may not necessarily result in the desired changes in 38 acid-base and Ca status of cows in all farms. Practically, it is very difficult to make changes in dietary ingredients through time and know the DCAD, monitor uncontrollable changes in DCAD (e.g., when forage base of the ration is changed), and acquire needed and accurate nutrient analysis in a timely fashion. Practically, having supplemental anion sources as a separate mix with a carrier allows alteration of the anion inclusion rate through time without changing the basic ration formulation. Recently, Goff and Horst (1997a) suggested that urine pH be used as a measure of the cow's acid-base status when feeding anion sources; this could help assess whether the ration is having the desired effects or not. Therefore, the objectives of this study were to characterize the temporal pattern of changes in urine pH and blood variables in relation to time of feeding of non-lactating pregnant multiparous Holstein cows fed varying amounts of an anion-Ca supplement and to evaluate acid-base and Ca status, and peripartum health of Holstein cows fed one of three dietary treatments varying in DCAD and Ca content. These three dietary treatments could represent different prepartum diets resulting from adjustment of the inclusion rate of an anion-Ca supplement in commercial dairies. MATERIALS AND METHODS Experiment 1 Six pregnant non-lactating multiparous Holstein cows beginning their 39 second lactation, averaging 32 d before expected calving (range 18 to 46 d) were blocked by expected calving date and assigned randomly to treatment in a repeated measures design. Diets had similar dietary concentrations of DM from forages and concentrates (T able 1). Dietary treatments were control (no anion- Ca supplement) or 5.0% or 7.9% of the total diet DM was replaced with an anion- Ca supplement. The anion-Ca supplement composition was 51.2% ground corn, 4.3% CaCOa, 14.1% Ca80402H20, 12.3% MgSO,o7HZO and 18.1% CaClzoZHZO, DM basis. Cows were fed dietary treatments as a TMR at 1000 h daily. Before the experiment started all cows were fed a diet with a 65:35 forage to concentrate ratio, dry basis. Individual feeds were sampled before the experiment, and one TMR sample during the experiment and sent to the Northeast DHIA Forage Laboratory (Ithaca, NY) for analyses. From d 1 through 4 all cows were fed Treatment 1 (Trt 1), then from d 5 through 8, two cows remained on this diet, and two cows each were assigned to Treatment 2 (Trt 2) and Treatment 3 (T rt 3). Cows were housed in maternity pens, fed individually, and feed refusals were weighed once daily. Urine and blood samples were collected at 0800, 1200, 1400 and 1600 h on d 1 through 8. Urine samples were collected into a plastic cup by manually stimulating the area around the vulva. Urine pH was measured immediately using a hand held pH tester (Hach Company, Loveland, Co). Blood samples (10 ml) were taken immediately after the urine sample from the tail vein using Li-heparin coated tubes (Fisher Scientific, Chicago, IL). 40 Samples were centrifuged following collection at 2800 x g for 10 min, plasma harvested, refrigerated and transported to the laboratory for analyses. Plasma samples were analyzed between 2 to 10 h after centrifugation. A blood gas and mineral element analyzer (Stat Profile 4, Nova Biomedical, Walthman, MA) was used to determine plasma pH, HCO; , ionized Ca (iCa), Na, K, and Cl concentrations. Data were analyzed by method of least squares ANOVA using the general linear model procedures (PROC GLM) of SAS (1996). The statistical model consisted of treatment, cow within treatment, day, treatment by day, cow within treatment by day, hour, day by hour, treatment by hour and treatment by day by hour. Results are presented as least square means. Statistical significance for treatments effects was tested using cow within treatment as the error term; day and treatment by day interactions were tested using cow within treatment by day as the error term; and hour, day by hour, and treatment by hour interactions were tested using cow within treatment by day by hour as the error term. The effects of treatments (T rt 1 vs. Trt 2 and Trt 3, Trt 2 vs. Trt 3), day (linear, quadratic, cubic and quartic) and treatment by day interactions were compared using orthogonal contrasts. For the day effect, the highest order function which was significant was evaluated. Experiment 2 Fifty-five primiparous and 62 multiparous Holstein cows were assigned in 41 a randomized block (parity) design and fed dietary treatments 21 d before expected day of calving. Totally mixed diets contained approximately 25% alfalfa silage, 16% corn silage, and 59% concentrates, DM basis (Table 2). Forage samples were taken every 3 mo and sent to the Northeast DHIA Forage Laboratory (Ithaca, NY) for analyses. Each time forages were analyzed inclusion rate of the anion-Ca supplement was adjusted to maintain the same DCAD. Dietary treatments were: Treatment 1 (Trt 1, no anion-Ca supplement), and for Treatments 2 (T rt 2) and Treatment 3 (T rt 3) the inclusion rate of the anion-Ca supplement (same as Experiment 1) was adjusted through time to maintain DCAD of -11 and -26 meq/100 g of DM, respectively. With the inclusion of the anion supplement in Trt 2 and Trt 3, total dietary Ca increased from 0.63% (T rt 1) to 0.95 and 1.17% in Trt 2 and Trt 3, respectively. Urine samples were collected into plastic cups by manually stimulating the area around the vulva 14 d after cows started on treatment diets . Urine pH was measured immediately using a hand held pH tester (Hach Company, Loveland, Co). Blood samples were taken from the tail vein using Li-heparin coated tubes (Fisher Scientific, Chicago, IL) 14 d after cows were on treatments and within 24 h after parturition. Blood samples were processed and analyzed in. the same manner as in Experiment 1. Data were analyzed by method of least squares ANOVA using the general linear model procedures (PROC GLM) of SAS (1996). The statistical model consisted of treatment, season, parity, treatment by season, treatment by parity, 42 season by parity and treatment by season by parity. Results are presented as least square means. The study was conducted for 17 mo beginning in February 1995 and ending in May 1996. Calvings occurring from October through April were grouped as winter calvings and those occurring from May through September as summer calvings. Cows were grouped into first parity (Parity 1), second parity (Parity 2) and third and greater parity (Parity 3+). Statistical significance for treatment, parity, season, and their interactions were tested using cow within treatment by parity by season as the error term. The effect of treatment 1 vs. 2 and 3, treatment 2 vs. 3, winter vs. summer, parity 1 vs. 2 and 3+, and parity 2 vs. 3+ were compared using orthogonal contrasts. Categorical variables such as milk fever, abomasum displacement, retained placenta, metritis, and dead calf were analyzed using contingency table analysis. Blood and urine pH data were analyzed also as H” concentration. RESULTS AND DISCUSSION Experiment 1 Results from mineral element analyses of individual feeds, sampled at the beginning of the experiment, indicated that actual DCAD values were +17, -11 and -26 meq/100 g of DM with Ca concentrations of 0.61, 1.01, and 1.86% of DM for Trt 1, Trt 2 and Trt 3, respectively. Chemical composition of dietary treatments were similar (Table 1). However, Ca, CI and 8 concentrations were higher for Trt 2 and Trt 3 due to inclusion of the anion-Ca supplement. Dry 43 urn—a matter intake was not different among treatments over the 8 d experimental period (Table 3); however, it tended to decline for cows fed Trt 3 after d 4 (data not shown). Urine pH pooled across the 8 d experimental period was lower for cows fed Trt 2 and Trt 3 compared with cows fed Trt 1. Over the 8 d period urine pH responded in a quartic fashion for Trt 1 vs. Trt 2 and Trt 3 (P < 0.05). Urine pH during the first 4 d when all cows were fed Trt 1 averaged 8.1 1 0.29, however, beginning on d 5 when the anion-Ca supplement was fed urine pH of cows fed Trt 2 and Trt 3 decreased to a nadir of 5.8 :I: 0.24 by d 7 (Figure 1). During the same time period urine pH of cows fed Trt 1 remained at about 8.0. Urine pH pooled across d 5 to 8 was higher in cows fed Trt 1 compared with cows fed Trt 2 and 3 (data not shown). Similarly, Sanchez et al. (1995) observed a drop in urine pH 4 d after cows were fed a negative DCAD. Also, Goff and Horst (1997a) observed lower urine pH in late gestation Jersey cows fed negative DCAD. In that study (Goff and Horst, 1997a), cows fed -10 meq/100 g of DM had urine pH of 5.8, similar to the urine pH observed 2 d after cows were fed Trt 2 and Trt 3 in the current study. Over the 8 d experimental period, mean plasma pH of cows fed Trt 2 was higher than cows fed Trt 3. Several have reported that feeding of a negative DCAD created a mild metabolic acidosis (Goff and Horst, 1994; Wang and Beede, 1992a). The pH of extracellular fluid is highly regulated in the body (Houpt, 1993). Plasma pH of cows fed Trt 1 was not different from that of cows fed Trt 2 and Trt 3 across the 8 d period and when pooled from d 5 to 8, 44 l however, inclusion of the anion-Ca supplement (T rt 2 and Trt 3) affected the cows' acid-base status as evidenced by the increased acidity of the urine. Contrary to this study, Sanchez et al. (1995) found a reduced blood pH 4 d after cows were fed negative DCAD. There was a linear increase (P < 0.07) in plasma iCa concentrations during the 8 d experimental period for cows fed Trt 2 and Trt 3 compared with those fed Trt 1. The major changes in plasma iCa concentrations occurred after the anion-Ca supplementation was started (d 5 through 8). Improved Ca status rapidly after anion-Ca supplementation started may be partially due to greater Ca absorption because parathyroid hormone (PTH) stimulation does not increase bone Ca resorption until after about 48 h (Horst et al., 1994). Also, increased plasma iCa concentrations were reported after cows were fed negative DCAD for 4 d (Sanchez et al., 1995), and 25 d (Wang and Beede, 1992a). A linear decrease in plasma Na concentrations over days of experiment (d 1 through 8 ) was detected for cows fed Trt 2 and Trt 3 comparedwith cows fed Trt 1 (P < 0.02). The major changes in plasma Na concentrations occurred after the anion-Ca supplement was added to each dietary treatment (d 5 through 8). A linear increase (P < 0.04) of plasma K concentrations .over the experimental period was observed for cows fed Trt 2 and Trt 3 compared with cows fed Trt 1. From d 5 to 8, cows fed Trt 2 and Trt 3 increased plasma K concentrations whereas cows fed Trt 1 maintained similar plasma K concentrations to concentrations before anion-Ca supplementation (data not shown). Similarly, 45 increased plasma K concentrations were observed when cows were fed negative DCAD (Block, 1984). Normally, plasma K concentrations are elevated during metabolic acidosis because K exchanges for extracellular H” (Scott and Buchan, 1981). Overall, no differences in plasma Cl concentrations were detected due to treatment diets. However, plasma Cl concentrations of cows fed Trt 2 increased linearly (P < 0.02) over the 8 d period compared with cows fed Trt 3. Changes in plasma Cl concentrations occurred particularly from d 5 to 8 of the experimental period for Trt 2 and Trt 3. Also, Block (1984) and Freeden et al. (1988a) observed increased plasma Cl concentrations when negative DCAD were fed. Increased plasma Cl concentrations can be expected especially when chloride- based salts are fed. Although cows fed Trt 2 had a numerically greater DMI than cows fed Trt 3, total CI intake was greater for cows fed Trt 3, making it difficult to explain the higher plasma Cl concentrations observed in cows fed Trt 2. Although, mineral element concentrations in urine were not measured and there was not a decrease in plasma pH (mild metabolic acidosis) pooled across the last 4 d of the experimental period, it was evident that acid-base status was affected, as detected by decreased urine pH. Based on results of this experiment, changes in urine and blood plasma measurements related to acid- base status could be expected to occur within 2 to 4 d of adding the anion supplement to achieve DCAD of -11 to -26 meq/100 g of DM. 46 Experiment 2 Ingredient and chemical composition of dietary treatments is in Table 2. The DCAD were +11, -11 and -26 meq/100 g of DM and Ca concentrations were 0.63, 0.95 and 1.17% for Trt 1, Trt 2 and Trt 3, respectively. Cows averaged 20.3 d on treatment before calving. Cows fed Trt 1 had higher urine pH before calving than cows fed Trt 2 and Trt 3; cows fed Trt 2 had higher urine pH before calving than cows fed Trt 3 (Table 4). However, when analyzed as H“ concentrations only the first contrast (T rt 1 vs. Trt 2, Trt 3) was different (P < 0.01). Reduced urinary pH was also observed when negative DCAD diets were fed with more than 1.2% dietary Ca (Goff and Horst, 1997a; Wang and Beede, 1992a; Wang and Beede, 1992b). Similarly, Moore et al. (1997) reported urine pH of 6.2 in cows fed diets with -15 meq/100 g of DM and 1.5% dietary Ca before calving. Reduced urine pH is an indication that cows fed Trt 2 and Trt 3 were in a mild state of metabolic acidosis even though cows were fed greater Ca concentrations. Urine pH can be a practical tool to evaluate whether reducing DCAD has caused mild metabolic acidosis. Plasma pH and H” concentrations from samples taken about 14 d after cows started on treatments (before calving) were not affected by treatment, parity or season. Also, Wang (1990) reported no difference in blood serum pH of cows fed diets containing - 25 meq/100 g of DM compared with cows fed + 5 meq/100 g of DM. Contrary to this, other studies have found reduced serum or plasma pH in cows fed negative DCAD diets (Goff and Horst, 1997a; Wang and 47 ”ET-”’1 Beede, 1992a). Discrepancies in plasma pH in response to DCAD in studies may be due to time of sampling relative to time of feeding or to time of sampling relative to when analysis is done (Davidson et al., 1996). Pooled across treatments and seasons, cows in Parity 1 had higher plasma pH after calving than cows in Parity 2 and 3+ (Table 6). Also, Parity 3+ cows had lower plasma pH than Parity 2 cows after calving. Similar results were observed when the plasma pH was analyzed as H+ concentration. Differences may not be physiologically important, but were significantly different due primarily to the lower pH observed in Parity 3+ cows. Plasma bicarbonate concentrations before calving pooled across season and parity were higher for cows fed Trt 1 compared with cows fed Trt 2 and Trt 3 (Table 4). Reduced bicarbonate concentrations also were observed when low DCAD diets were fed to cows before calving (Wang, 1990), during early lactation (Delaquis and Block, 1995b) and in mid lactation (West et al ,1991). Although plasma pH was not significantly affected by dietary treatments, lower urine pH and plasma bicarbonate indicated a mild metabolic acidosis in cows fed the anion-Ca supplement. There was season by parity interaction before calving for plasma bicarbonate (data not shown, P < 0.03). Parity 1 cows had a lower plasma bicarbonate concentrations during the winter compared with summer, whereas, plasma bicarbonate concentrations of Parity 2 and 3+ cows did not change between seasons. Differences in plasma bicarbonate concentration among parities and seasons can not be explained. Plasma iCa concentrations pooled across season and parity were higher 48 before calving for cows fed Trt 2 (4.53 mgldl) and Trt 3 (4.57 mgldl) compared with cows fed Trt 1 (4.31 mgldl). After calving, cows fed Trt 1 had lower plasma iCa (3.56 mgldl) than cows fed Trt 2 (3.81 mgldl) and Trt 3 (4.11 mgldl). Thus, postpartum hypocalcemia (plasma iCa < 4.0 mgldl; Oetzel et al., 1988) was evident in cows fed Trt 1 or Trt 2, but not Trt 3. Other experiments also have reported improved iCa status with negative DCAD (Oetzel et al., 1988; Sanchez and Joyce, 1995; Wang,1990). There is evidence suggesting reduced responsiveness of bone and kidney to PTH in cows fed positive DCAD (Goff et al., 1991; Goff and Horst, 1997a). Cows fed negative DCAD had greater plasma hydroxyproline concentration than cows fed positive DCAD (Goff and Horst, 1997a). Cows fed negative DCAD have increased concentrations of plasma 1,25(OH)1,D3 (Goff et al, 1991) which may result in stimulation of active transport mechanism of Ca in the gastrointestinal tract (Goff et al., 1986). Greater bone mobilization due to lower DCAD, or greater Ca absorption from the gastrointestinal tract due to lower DCAD and greater dietary Ca may account for the improved Ca status observed in cows fed Trt 2 and Trt 3. After calving, Parity 1 cows had greater plasma iCa concentrations than cows in Parity 2 and 3+ (Table 6). Also, Parity 2 cows had greater plasma iCa concentrations than Parity 3+ cows. Similarly, Wang (1990) found reduced plasma iCa concentrations as parity increased. Tissue vitamin D receptor (VDR) number regulates tissue responsiveness to a 1,25(OH)2Da stimulus (Horst et al., 1994). The number of VDR in the intestine and the ability to mobilize Ca from bone declines as the cow ages (Horst et al., 1990). Differences among parities 49 -flil in,» d in ability to maintain .Ca status around parturition may be due to different VDR number in target tissues. There also was an effect of season with cows having greater plasma iCa concentrations after calving during the summer compared with winter (Table 6, pooled across parity and treatment). Goff et al., (1991) reported greater incidence of milk fever in Jersey cows calving from November through January compared with the rest of the year. After calving, plasma Cl concentrations pooled across parity and season were lower for cows fed Trt 1 compared with cows fed Trt 2 and Trt 3 (Table 4). Increased plasma Cl concentrations also were observed when negative DCAD diets were fed to cows before calving (Block, 1984; Wang, 1990) and this is likely due to the higher concentration of Cl provided in these diets. The strong ion difference (SID) is defined as meq ([Na + K - CI]IL, Stewart, 1983) and the normal extracellular SID is about 42 meq/L (Eicker, 1990). Cows fed Trt 1 had higher plasma SID (42.1 meq/L) before calving compared with cows fed Trt 2 (40.7 meq/L) and Trt 3 (38.2 meq/L, Table 4). Also, cows fed Trt 2 had a higher plasma SID before calving compared with cows fed Trt 3. However, these differences were not observed after calving. Although plasma pH was not affected by dietary treatments, lower concentrations of plasma bicarbonate, lower plasma SID and urine pH, and increased concentrations of plasma Cl when cows were fed the anion-Ca supplement indicate cows were in mild metabolic acidosis. The overall incidence of milk fever was low (3.4%, Table 7). Normal incidence of milk fever varies from herd to herd but nationwide is approximately 50 (J 5% of all milking cows (Horst, 1986). The low incidence of milk fever observed in this study may be due partially to the great number of Parity 1 cows in the experiment (47%). All milk fever cases occurred in Parity 3+ cows with 2 cases for cows fed Trt 1, and 1 case in each of the cows fed Trt 2 or Trt 3. Dietary treatments had no effect on milk fever incidence, however, cows fed Trt 2 and Trt 3 had improved plasma iCa status compared with cows fed Trt 1. The incidence of displaced abomasum was greater in cows fed Trt 2 and Trt 3 compared with those fed Trt 1 (Table 7; P < 0.08) The overall incidence was 7%, but 5 of the 8 cases occurred in Parity 1 cows. Cows fed negative DCAD can have reduced DMI prepartum (Joyce et al., 1997; Moore et al., 1997; Oetzel and Barrnore, 1993; Schoenbaum et al., 1994). Although DMI was not measured in this experiment, greater incidence of displaced abomasum may be due to lower DMI before calving in cows fed negative DCAD diets with anion supplementation from CaSO4.2H20, MgSO,.7H20 and CaCl2.2H20. More than 60% of cases of abomasal displacement occurred in Parity 1 cows which did not have subclinical hypocalcemia. This indicates it is not beneficial to feed negative DCAD to Parity 1 cows before calving. CONCLUSIONS Lowering DCAD, while increasing dietary Ca concentration, decreased urine pH (Experiment 1 and 2) and reduced postpartum subclinical hypocalcemia (Experiment 2). Changes in urine pH and blood plasma measurements related to acid-base status could be expected to appear within 2 to 4 d after reducing 51 DCAD. Urine pH can be used as a tool to monitor changes in DCAD in diets before calving. Anion-Ca supplement inclusion rate can be adjusted to control DCAD and to achieve a targeted urine pH through time reducing periparturient hypocalcemia. Diets with DCAD of -11 or -26 meq/100 g of DM increased incidence of displaced abomasum around parturition, especially in first parity cows. Because first parity cows did not have plasma iCa below 4 mgldl in any of the dietary treatments, feeding negative DCAD to first parity cows should be avoided if possible. 52 Table 1. Dietary ingredients and chemical composition from laboratory analysis of the diets with different dietary cation-anion differences and Ca concentrations, Experiment 1. Item Treatments 1 2 3 Ingredients % of DM Alfalfa silage 25.39 25.39 25.39 Corn silage 15.68 15.68 15.68 Corn, high moisture 20.91 20.91 20.91 Corn, cracked 22.83 17.83 14.89 Protein-TM pellets‘ 7.72 7.72 7.72 Cottonseed, whole 7.46 7.46 7.46 Anion-Ca supplement2 0.00 5.00 7.94 Chemical composition’ CF 14.7 14.5 15.2 ADF 16.4 13.4 14.9 NDF 24.2 22.5 22.3 Ca 0.61 1.01 1.86 P 0.48 0.45 0.60 Mg 0.28 0.34 0.39 K 1.26 1.24 1.09 Na 0.31 0.30 0.53 CI 0.65 1.23 1.74 S 0.17 0.34 0.45 DCAD‘ +17.0 -11.0 -26.0 ‘ Composition: 48.5% CP, 5% ADF, 8% NDF, 2.9% lipid, 15% ash, 4.1% Ca, 1.7% P, 1.2% Mg, 1% K, 1.15% Na, 1.0% CI, 0.55% S, 1.3 ppm Co, 115 ppm Cu, 576 ppm Fe, 5.8 ppm l, 461 ppm Mn, 3 ppm Se, 461 ppm Zn, 55 KlUIkg vitamin A, 13 KlUIkg vitamin D, 330 KlUIkg vitamin E. 2 Composition: 51.2% ground corn, 4.3% CaCO3, 14.1% CaSO,.2H20, 12.3% MgSOJHzO and 18.1% CaCl2.2H20, DM basis. 3 Total mixed ration samples were taken once during the 8 d experimental period. ‘ Dietary cation-anion difference, meq [(Na+K) - (CI+S)]I100 g of DM. 53 Table 2. Dietary ingredients and chemical composition from laboratory analysis of the diets with different dietary cation-anion differences and Ca concentrations, Experiment 2. Item Treatments 1 2 3 Ingredients % of DM Alfalfa silage 25.39 25.58 25.58 Corn silage 15.68 15.82 15.82 Corn, high moisture 20.91 21.07 21.06 Corn, cracked 22.83 18.0 15.11 Protein-TM pellets‘ 7.72 7.72 7.72 Cottonseed, whole 7.46 7.46 7.46 Anion-Ca supplement’ 0.00 4.34 7.24 Chemical composition’ CP 15.3 15.3 15.2 ADF 18.42 18.45 18.35 NDF 27.96 28.04 28.0 Ca 0.63 0.95 1.17 P 0.39 0.38 0.38 Mg 0.23 0.28 0.31 K 0.98 0.98 0.98 Na 0.13 0.14 0.15 CI 0.29 0.74 1.04 S 0.18 0.34 0.45 DCAD‘ +11.0 -11.0 -26.0 ‘ Composition: 48.5% CF, 5% ADF, 8% NDF, 2.9% lipid, 15% ash, 4.1% Ca, 1.7% P, 1.2% Mg, 1% K, 1.15% Na, 1.0% CI, 0.55% S, 1.3 ppm Co, 115 ppm Cu, 576 ppm Fe, 5.8 ppm I, 461 ppm Mn, 3 ppm Se, 461 ppm Zn, 55 KlU/kg vitamin A, 13 KlUIkg vitamin D, 330 KlUIkg vitamin E. 2 Composition: 51.2% ground corn, 4.3% CaCOa, 14.1% CaSO4.2H20, 12.3% MgSO,.7HZO and 18.1% CaCl2.2H20. DM basis. 3 Forage samples were taken every 3 months and anion-Ca supplement inclusion rate adjusted to maintain DCAD. ‘ Dietary cation-anion difference, meq [(Na+K) - (CI+S)]I100 g of DM. 54 do 00~_:o_ 0.50.0 . ovoxmn $0000 .5 u 0.00:0 0:0 .0 u 05:0 .0 n 0000000 r. n 50:: 05 :2 00:0:Eo0 0:03 >00 0:0 E0500: :0 5:00:95 05 ”00:00 _E:0E:00x0 0 m 05 :06 m .m> N €0,500; 0:0 .m 0:0 N .m> F E2500; :0 0:005:00 .mcomocto N .8 $03 2.... .2: a 8:85 8. u m 06.58: 05 50 $6.. 05 so a 850:. :- u N 2258: ”m0 $5.0 Em .20 m 8.85 2.. n F 06.58: . ._ .80 02 02 02 man 38 0.8... +08 .202 ._0 028.: mz ._ .30 02 02 02.0 00.8 3.2 3.2 .202 .0. «Emma 02 ._ .85 02 02 $0 :08 0.08 $8 .29: .mz 058.: mz ._ .Bd 02 02 So a: «I. 21‘ .205 ..m0_ 058.: 02 02 5o 02 08.0 8a 03 84. 92 x ..1 «Emma 02 02 5o 02 mood mums mom: #3 In name: 02 ._ .80 02 02 os 32 SK 3 we x ..I 055 02 5 .86 02 80 80 Re N: 80 I: 0E: 02 02 02 .02 was 0.2 :3 «.9 29. .3: a .2, u an .2, a n .2, a «a .2, F 00 n u . «>00 3 «5.502... 20:300.... 320.500.... 0.00:; ._. 2080096 00:00 62080096 0 o 05 00900 00.00: 2800:0050 00 0:0 03:20.50 520.5000 >520 0:20.50 00.. 0260 .0 00.0008, «Emma 0:0 0:0: :0 0:00.: 06:00 .0000 .n 030... 55 Table 4. Measurements in urine and plasma before and after calving of cows fed different dietary cation-anion differences and Ca concentrations by treatment, Experiment 2. Variable Treatmente‘ Treatment Contrasts 1 2 3 SE 1 vs. 2,3 2 vs. 3 Urine before calving P < pH 8.12 6.88 6.36 0.14 0.01 0.01 H‘, x 10'7 0.72 6.24 9.04 1.58 0.01 NS Plasma before calving pH 7.629 7.624 7.579 0.018 NS NS H‘, x 10'8 2.391 2.442 2.666 0.099 NS NS Na, mgldl 335.6 333.6 333.1 1.1 NS NS K, mgldl 16.58 16.86 17.07 0.28 NS NS CI, mgldl 382.7 385.9 393.1 3.3 NS NS ionized Ca, mgldl 4.31 4.53 4.57 0.07 0.01 NS HCOs', mmollL 24.68 22.96 21.83 0.5 0.01 NS SIDZ, meq/L 42.1 40.7 38.2 0.89 0.02 0.06 Plasma after calving pH 7.620 7.604 7.571 0.018 NS NS H’, x 10“8 2.435 2.556 2.732 0.100 NS NS Na, mgldl 337.2 336.8 336.9 1.1 NS NS K, mgldl 16.13 17.61 17.41 0.23 0.01 NS CI, mgldl 381.4 385.9 390.6 2.4 0.02 NS ionized Ca, mgldl 3.56 3.81 4.11 0.1 ' 0.01 0.05 HCO3', mmollL 25.10 23.68 23.48 0.8 NS NS SID, meq/L 42.8 42.0 40.9 0.75 NS NS ‘ Treatment 1 = +11 meq/100 g DM and 0.63% Ca; Treatment 2 = -11 meq/100 g DM and 0.95% Ca; and Treatment 3 = -26 meq/100 g DM and 1.17% Ca. 2 Strong ion difference = meq (Na + K - Cl)/L. 56 1:20 - v. + 020 00:. u 00:90:20 :2 0:95 a 009068 3:95 >05. 90: @5200 n .0993 ”.004. 3:95 0:900 E9.— mm:_>_00 u .952, 0 :0090019005 :0 9.5 055600 0260 u +m 3:00 . 3.: 02 02 9.: 0:: v. 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Incidence of milk fever and abomasum displacement of cows fed different dietary cation- anion differences and Ca concentrations by treatment and parity. Experiment 2. Contrast Variable Treatments‘ Treatments Parity Milk Fever 1 2 3 Total 1 vs. 2.3 2 vs. 3 1 vs. 2.3+ 2 vs. 3+ Parlty’ 1 0/18 0/18 0/18 0/54 NS3 NS NS 0.02 2 0/14 0/10 0/10 0/34 3+ 2/9 1/9 1/1 1 4/29 Total 2/41 1/37 1/39 4/1 17 Abomasum Displacement 1 0/18 2/18 3/18 5/54 0.05 NS 0.08 NS 2 0/14 2/10 0/10 2/34 3+ 0/9 1/9 0/1 1 1/29 Total 0/41 5l37 3/39 8/1 17 ‘Treatment 1 = +11 meq/100 g DM and 0.63% Ca; Treatment 2 = -11 meq/100 g DM and 0.95% Ca; and Treatment 3 = -26 meq/100 g DM and 1.17% Ca. 2 Parity 3+ = cows beginning third or greater lactation. 3P>0.10 59 .w c 29.25 F u Ea: P E953; ufi So; 258 __m ”mo $8; new .20 ho a co :cmE mm. .m EmEfimF u . ”mo $3... us... .29 co m 8:82 5. .~ 5958: u I ”8 $56 .25 so a a 8:35 2+ .c Ewes»: u e .. Emezmaxm .mcozgcoocoo no can. mmocmcmEu :oEméozmu ESQ“. 255:6 5:5 Ego no. 958 he Emtmn In mat: ._. 0.52... Each—3.: 332.9 :0 gun a h o n v m N —. rm..." rad umd .95 the ,3 l----/ sq» 1m.» -06 _._..o n 2mm Hd euun 60 CHAPTER 4 EFFECTS OF DIETARY ANION SOURCES AND CALCIUM CONCENTRATION ON TEMPORAL CHANGES IN FEED INTAKE AND URINE pH ABSTRACT Objective was to determine if the addition of dietary anions from different anion sources and varying dietary Ca affect feed intake and urine pH across a 21 d feeding period. Thirty-two nonlactating, nonpregnant Holstein cows were assigned randomly to treatments. Totally mixed diets were 20% alfalfa silage, 40% corn silage, and concentrates, dry basis. Dietary treatments were: Control (no anion source added) with a dietary cation-anion difference (DCAD; meq [(Na+K)-(CI+S)]I100 g of DM) = +16.9 meq; Bio-Chlorm; an Anionic Salts mixture designed to match the anionic and cationic contributions of Bio-Chlorm; and HCI. The DCAD of treatments with supplemental anions averaged -7.7 meq/100 g of dietary DM (40 to -10.9 meq/100 g of dietary DM). Factored across Control and treatments with anion sources were dietary Ca concentrations of 0.49 or 2.10% (dry basis) with supplemental CaCO3 (4 x 2 factorial arrangement of treatments). Pooled across dietary Ca concentrations and day, cows fed Control had 7.5% 61 greater DMI than cows fed anion sources. Dry matter and water intakes of cows fed HCl responded differently across time compared with cows fed anionic salts. Pooled across anion sources and day, cows fed low dietary Ca had greater dry matter and water intakes than cows fed high dietary Ca. Urine pH of cows fed Control remained at 8.0 to 8.2 across the treatment period. Urine pH of cows fed anion sources dropped from 8.0 (before feeding anion sources) to 5.7 on average d 2 after commencement of feeding anion sources, and remained below 6.0 through d 7. On d 14 and 21, urine pH remained below 6.0 for cows fed HCl, rose to about 6.0 for cows fed Anionic Salts, and increased to 6.3 on d 14 and to 6.7 on d 21, for cows fed Bio-Chlorm. Dietary Ca content did not affect un'ne pH. Abbreviation key: DCAD = dietary cation-anion difference; C = control; B = Bio- Chlorm; AS = anionic salts; Wl = water intake. INTRODUCTION Dietary cation-anion difference (DCAD) is defined as the sum of meq [(Na + K) - (Cl + S)]I100 g of dietary DM. The strong ion difference theory in acid- base physiology suggests that increasing the concentration of anions in body fluid compartments reduces the pH in that compartment (Stewart, 1983). Manipulation of DCAD has a direct impact on blood acid-base physiology of the cow. Block (1994) reported diets with low or negative DCAD affected one or more of the following variables in the blood: increased H“ concentration, 62 decreased HCO; concentration, and decreased pH. However, the ability of the anion sources to change acid-base status depends on the anion source used (Goff et al., 1997). For example, Bigner et al. (1997) influenced the acid-base status of cows under metabolic acidosis by drenching them with Na propionate and NaHCOa, but not with NaCl. Furthermore, it was suggested that relative absorption of cations and anions has an important influence on the cow's acid- base status. Besides changes in acid-base status, addition of anions to rations of prepartum cows can affect Ca metabolism (Block, 1984; Goff et al., 1991), peripartum health (Block, 1984; Dishington, 1975; Goff and Horst, 1997a; Oetzel et al., 1988), and postpartum productive and reproductive performance (Block, 1984; Wang, 1990). However, some anion sources are not very palatable (Oetzel and Bannore, 1993) and sometimes can reduce DMI when fed to prepartum cows (Joyce et al., 1997; Schoenbaum et al., 1994). Therefore, the objective of this experiment was to determine if the addition of dietary anions from Bio-Chlorm, anionic salts and HCI and varying dietary Ca from CaCO3 affected feed intake and urine pH across time in non-pregnant, non-lactating Holstein dairy cows which had completed at least one lactation. MATERIALS AND METHODS Design, Cows and Treatments Thirty-two non-lactating non-pregnant Holstein cows were assigned 63 randomly to one of eight dietary treatments for the 21 d experiment. Four anion sources were factored with two dietary Ca concentrations. Dietary treatments were Control (C, no anion source added); Bio-ChlorT'“I (B, an organic supplement, Biovance, Omaha, NE); anionic salts (AS, a mixture of MgCl2-6HZO, NH4CI, (NH4),SO4 and NaOH, designed to match the anionic and cationic profile of B); and, hydrochloric acid (HCI). Anions treatments were factored with or without addition of CaCO3 to give total dietary Ca concentrations of 0.49% or 2.1% (Table 1). Dietary treatments were formulated to have approximately 1.6 Mcal MEL/kg of dietary DM and 15% CP. All diets had the same amount of forage, consisting of corn and alfalfa silage in a ratio of 2:1 (DM basis). The forage-to-concentrate ratio of the TMR was 60:40. The concentrate portion of the basal diet consisted of soybean meal, ground corn grain, whole cottonseed, urea, minerals and vitamins (Table 1). To make treatment B diets, a portion of the ground corn and soybean meal from C was replaced with B (Table 1), thus reducing DCAD (Table 2). Urea was included in C and HCI diets to supply an equivalent amount of the NPN supplied by B. Ammonium sulfate, MgCl2-6HZO, and NH4C| were included in AS to supply an equivalent amount of NPN, S, Mg and Cl as supplied by B. Sodium hydroxide was added to AS to increase the Na concentration to be similar to that of B. In HCI diets, HCI, water and liquid sugar cane molasses were mixed with ground corn and soybean meal. Calcium carbonate replaced ground corn in appropriate amounts to increase the Ca concentration to an average of 2.1% of 64 DM in the high dietary Ca treatments across C and each anion source. Cows were placed in tie-stalls for 14 d before the beginning of the experiment and fed C (with low dietary Ca). They were fed individually, and from 7 d before through 21 d after switching to dietary treatments, orts were weighed once daily at 1100 h. Orts were dried at 60 °C for 48 h for use in calculation of DMI. Cows were fed once daily at 1200 h. Water intake (VIII) was measured daily by in-line flow meters (Omega, Stamford, CT). Forage samples were taken weekly, dried (60 °C), ground through a 5 mm screen and composited. Samples of grains and concentrates were taken weekly and composited. Individual composite samples were ground through a 2 mm screen and sent to the Northeast DHIA Laboratory (Ithaca, NY) for analyses. Urine samples were collected into plastic cups by manually stimulating the area around the vulva at 0900 h. Urine samples were taken daily from d 2 before switching to dietary treatments until d 7 after feeding treatments, and on d 14 and 21 after the switch. Urine pH was measured immediately after collection using a calibrated hand-held pH tester (Hach Company, Loveland, CO). Statistical Analysis A repeated measures model was used to analyze the data based on the PROC MIXED procedure of SAS (1996). Means of DMI and WI for each cow from 7 d before switching to dietary treatments were included in the model as a covariate. Mean urine pH for each cow from d 2 before switching to dietary 65 treatments were used in the model as a covariate. The general model consisted of treatment, day , treatment by day interaction, covariate and residual (test term), allowing for a compound symmetry structure on the correlation between residuals within cows. Treatment, day and the interaction of treatment by day were tested by orthogonal contrasts. Orthogonal mean contrasts were: treatment C vs. B, AS, HCI; B vs. AS, HCI; AS vs. HCI; the interaction of contrasts involving anion source by dietary Ca concentration; low dietary Ca vs. high dietary Ca; the linear, quadratic, cubic and quartic effects of day; and the interaction of anion source by day and Ca concentration by day. A multivariate, repeated-measures model (PROC MIXED; SAS, 1996) was performed on DMI and water intake, DMI and urine pH, and WI and urine pH to calculate residual correlations between each of these characters. Least-squares treatment means are presented and statistical significance between treatment means was declared at P < 0.05. RESULTS AND DISCUSSION Diets averaged 15.0% CF (Table 2). Dietary treatments had similar ADF and NDF concentrations. Diets averaged 0.49% for low. dietary Ca and 2.10% Ca for high dietary Ca. Concentrations of K and Na were similar across diets. Pooled across dietary Ca concentrations, inclusion of anion sources increased analyzed concentrations of Cl from 0.58% in C to 0.85% in B, 0.93% in AS, and 1.52% in HCI treatments. Also, concentrations of S increased from 0.14% in C 66 and HCI treatments, to 0.30% in B and 0.35% in AS treatments. Increasing amounts of anions in dietary treatments reduced DCAD from +16.9 (C) to -4.4 (B), -8.7 (AS) and -10.1 meq/100 g of DM (HCI), pooled across dietary Ca concentrations. Pooled across dietary Ca concentrations, mean DMI and standard deviation for the 7 days before switching to dietary treatments were 20.4 and 3.1, 17.9 and 2.8, 17.4 and 2.1, 17.4 and 3.4 kgld, for cows fed C, B, AS and HCI, respectively. Over the 21-d period after dietary treatments were introduced, cows fed C had greater DMI (7.5% higher) compared with cows fed B, AS and HCI, pooled across dietary Ca concentrations and day (Table 3). Also, cows fed AS had greater DMI than cows fed HCI. In previous studies, lower DMI prepartum was observed when cows were fed diets containing supplemental anions with DCAD of -15 meq (Moore et al., 1997), -8 meq (Goff and Horst, 1997a) and -7 meq/100 g of DM (Joyce et al., 1997; Schoenbaum et al., 1994) compared with no anion supplementation. In the current experiment, the lowest DMI was observed for cows fed HCI which had the numerically lowest DCAD (~10.1 meq/100 g of DM). Lower DMI observed in cows fed HCI may be due to a greater systemic acidifying effect of HCI treatment compared with AS and B, because DCAD were not statistically different among treatments with supplemental anions (P < 0.15). There was an anion source by day interaction on DMI (P < 0.01; Figure 1). Cows .fed HCI had lower DMI than cows fed AS at the beginning and the end of the 21-d period, however from d 11 through 16 DMI 67 were similar. Pooled across anion sources, mean DMI and standard deviation for the 7 days before switching to dietary treatments were 18.0 and 3.5, and 18.6 and 2.7 kgld, for cows fed 0.5% and 2.1% Ca, respectively. Pooled across anion sources and day, cows fed low dietary Ca had greater DMI (4.8%) than cows fed high dietary Ca ( P < 0.03; Table 4). Contrary to this, Goff and Horst (1997a) reported increased DMI (3.2%) in cows fed 1.5% Ca compared with cows fed 0.5% Ca. Differences in responses between the two studies may be due to different dietary Ca concentrations or to palatability of the Ca sources used. Goff and Horst (1997a) fed 1.5% dietary Ca and the sources of Ca were CaCOa, CaSO., CaCl2 and CaHPO4. In the current study, there was a dietary Ca concentration by day interaction ( P < 0.04; Figure 2). Cows fed high dietary Ca generally had lower DMI during the 21-d experiment compared with cows fed low dietary Ca; however, DMI were similar on d 9, 17 and 18. Pooled across dietary Ca concentrations, mean WI and standard deviation for the 7 days before switching to dietary treatments were 50.2 and 9.4, 42.8 and 9.2, 41.8 and 8.8, 41.5 and 11.9 Ud, for cows fed C, B, AS and HCI, respectively. Pooled across Ca concentration and day, there was no effect of anion sources on WI (Table 3). Over time cows fed AS had a somewhat different WI pattern compared with cows fed HCI ( P < 0.02; Figure 3) based on a significant interaction of AS vs. HCI contrast with day. Comparing WI of cows fed HCI and AS, WI ranking between the groups switched back and forth during 68 the first 9 d of the experiment. From d 10 through 21, WI was similar or higher for cows fed HCI compared with those fed AS. Differences in WI patterns across time may be due to the differences observed in DMI pattern across time when cows were fed these dietary treatments. Residuals of DMI with WI were correlated positively (r = 0.37; P < 0.01). This indicates that unaccounted for noise in both characters is jointly associated. Pooled across anion sources, mean WI and standard deviation for the 7 days before switching to dietary treatments were 42.7 and 12.3, and 45.6 and 8.4 Ud, for cows fed 0.5% and 2.1% Ca, respectively. There was no effect of Ca concentration on WI across time; however, there appeared to be a tendency of cows fed low Ca to have higher WI on many days of the experiment (Figure 4). Pooled across anion sources and day, cows fed low dietary Ca had greater WI than cows fed high dietary Ca (Table 4). One of the major factors that influences WI is DMI (Murphy, 1992). Greater WI of cows fed low compared with high dietary Ca, may be due to the greater DMI when fed low dietary Ca. Pooled across dietary Ca concentrations, mean urine pH and standard deviation for the 2 days before switching to dietary treatments were 7.9 and 0.12, 7.9 and 0.11, 7.9 and 0.11, 7.8 and 0.11 Ud, for cows fed C, B, AS and HCI, respectively. Pooled across Ca concentrations and day, cows fed C had average urine pH of 8.1, whereas cows fed anion sources had urine pH of 6.1 ( P < 0.01; Table 3). Reduced urine pH was reported when anion sources were fed to prepartum cows (Goff and Horst, 1997a; Moore et al., 1997; Rodriguez et 69 al., 1996) and to nonlactating nonpregnant cows (Goff et al., 1997; Giesy et al., 1997). Reduced un'ne pH of cows is due to the contribution of Cl and S from the anion sources (Horst et al., 1997). In the current experiment, cows fed anion sources responded differently across time compared with cows fed C. Cows fed B, AS and HCI had lower urine pH (about 6.0) within 2 d after initiation of feeding the dietary treatments, whereas urine pH of cows fed C remained around pH 8.0 or above ( P < 0.01; Figure 5). Also, cows fed B responded differently across time compared with cows fed AS and HCI ( P < 0.01). The urine pH of cows fed B increased nearly 1 pH unit from d 7 to 21, whereas urine pH of cows fed AS and HCI remained around pH 6.0. Mean urine pH of some cows tended to increase across time suggesting some metabolic adaptation. However, the increase in mean urine pH observed for cows fed B from d 7 through 21 may be due to a greater decrease in DMI of the TMR by cows fed B compared with cows fed AS and HCI during the same experimental period. Dry matter intake of cows fed B decreased 3.0 kgld from d 7 through 21; however, cows fed AS and HCI decreased 2.2 and 0.7 kgld, respectively, during the same period of time. Variation in urine pH among cows may be explained partially by variation in DMI and the daily intake of anions because the residuals of DMI and urine pH were correlated negatively (r = -0.14; P < 0.01). Pooled across anion sources and day, there was no effect of dietary Ca on mean urine pH (Table 4). However, there was a Ca treatment by day 70 interaction on mean urine pH ( P < 0.01; Figure 6). Urine pH of cows was similar on d 0 and 1. From d 2 through 7 and at d 14, cows fed 0.49% Ca had higher mean urine pH than cows fed 2.1% Ca, but on d 21 pH was lower for cows fed 2.1% Ca. Un'ne pH was highly correlated with DCAD intake (meqldlkg of BVV) from d 2 through 21 of the experimental period (r = 0.90; P < 0.01; Figure 7). Cows fed negative DCAD treatments had lower mean urine pH compared with cows fed positive DCAD, pooled across Ca concentrations. However, cows fed B had mean urine pH ranging from 7.3 to 5.2 when consuming -1 meq/dlkg of BW. Cows fed AS and HCI had less variation in mean urine pH during the same experimental period. CONCLUSIONS Feeding different anion sources and high dietary Ca (2.1%) reduced DMI in nonlactating nonpregnant cows compared with C and 0.5% dietary Ca. Practically, feeding 2.1% dietary Ca may be too high when using anion sources due to the potential for DMI reduction. Introduction of anion sources to cows previously fed cationic diets with low dietary Ca changed temporal patterns of DMI depending on the anion source used. Cows fed HCI had the lowest DMI and urine pH, indicating a stronger acidifying effect. Hydrochloric acid can be used as anion source to change acid-base status in dairy cows. Changes in urine pH should be expected to occur within 2 d after cows are introduced to 71 anion sources if the DCAD is -7.7 meq/100 g of DM. 72 Table 1. Ingredient composition of dietary treatments with different anion sources and Ca concentrations (% of dietary DM). Treatments‘ Ingredients CL CH BL BH ASL ASH HCIL l-ICIH Alfalfa, silage 19.7 19.7 19.7 19.7 19.7 19.7 19.7 19.7 Com, silage 39.4 39.4 39.4 39.4 39.4 39.4 39.4 39.4 Cottonseed, whole 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 Com, ground 27.1 23.6 23.9 20.3 26.2 22.6 25.7 22.2 Soybean, meal 3.5 3.5 . . . . . . 3.5 3.5 3.5 3.5 Urea 0.65 0.65 . . . . . . . . . . . . 0.65 0.65 Mineral-vitamin mix2 0.56 0.58 0.56 0.56 0.64 0.64 0.56 0.58 CaCOa . . . 3.67 . . . 3.63 . . . 3.68 . . . 3.65 M90 0.26 0.13 0.23 0.16 0.07 . . . 0.26 0.13 NaCl 0.47 0.47 . . . . . . . . . . . . 0.47 0.47 NH4PO4 H2 0.35 0.34 0.20 0.22 0.37 0.39 0.35 0.34 Bio-Chlorm" . . . . . . 8.00 8.00 MgCl2-6HZO . . . . . . . . . . . . 0.49 0.49 NH4CI . . . . . . . . . . . . 0.54 0.53 (NH,)2SO4 . . . . . . . . . . . . 0.80 0.79 NaOH . . . . . . . . . . . . 0.30 0.30 HCI‘ 0.82 0.82 Liquid molasses . . . . . . . . . . . . . . . . . . 0.51 0.51 ‘ Dietary treatments: C = control, no anion source added; B = Bio-Chlor'”; AS = anionic salts; HCI = hydrochloric acid; L = low dietary Ca; H = high dietary Ca. 2 Composition: 3670 ppm Zn, 3675 ppm Mn, 974 ppm Cu, 29.4 ppm I, 10.6 ppm Co, 29.3 ppm Se, 87690 IU/kg vitamin A, 26140 lU/kg vitamin D, 297 lU/kg vitamin E. 3 Bio-ChlorT", Biovance, Omaha, NE. ‘ Muratic acid, 30% Cl. 73 la (7 (I) "f‘ 9V Table 2. Analyzed chemical composition of dietary treatments with different anion sources and Ca concentrations (% of DM)‘. Treatments2 CL CH BL BH ASL ASH HCIL HCIH GP 15.17 14.75 15.14 14.86 15.23 14.83 15.12 14.73 ADF 23.5 23.5 23.8 23.7 23.7 23.6 24.2 23.8 NDF 34.0 33.3 34.5 34.1 33.4 33.3 33.4 33.2 Ca 0.50 2.19 0.48 2.13 0.48 2.04 0.50 2.05 P 0.40 0.38 0.40 0.38 0.38 0.36 0.44 0.40 I Mg 0.37 0.29 0.39 0.31 0.41 0.33 0.40 0.31 E‘ K 1.20 1.18 1.20 1.17 1.19 1.18 1.22 1.21 Na 0.28 0.24 0.17 0.17 0.21 0.20 0.27 0.21 CI 0.63 0.53 0.87 0.82 1.00 0.85 1.57 1.47 S 0.14 0.13 0.30 0.29 0.36 0.34 0.14 0.14 DCAD3 +16.4 +17.3 -4.7 -4.0 -10.9 -6.4 -10.2 -10.0 ‘ Feed samples were taken weekly, dried (60 °C), ground through a 2 mm screen and composited. 2 Dietary treatments: C = control, no anion source added; B = Bio-Chlor”; AS = anionic salts; HCI = hydrochloric acid; L = low dietary Ca; H = high dietary Ca. 3 Dietary cation-anion difference = meq [(Na+K) - (CI+S)]I100 g of dietary DM. 74 Ill 3‘“: Table 3. Least square means of dry matter and water intake, and urine pH of cows fed different anion sources and Ca concentrations, pooled across Ca concentrations and day of experiment. Treatmenta‘ Contrasts ( P < ) C vs. B, B vs. Measurement C B AS HCI SEM AS, HCI AS, HCI AS vs. HCI DMI, kgld 17.4 16.3 16.6 15.4 0.3 0.01 N82 0.02 DMI, % BW 2.5 2.1 2.0 2.0 0.01 0.01 NS NS WI3, Ud 39.1 37.5 37.6 38.8 1.32 NS NS NS Urine pH 8.1 6.2 6.1 6.0 0.06 0.01 NS NS ‘ Dietary treatments: C = control, no anion source added; B = Bio-Chlor‘“; AS = anionic salts; HCI = hydrochloric acid. 2 NS = nonsignificant( P < 0.05). 3 Water intake. 75 III Table 4. Least square means of dry matter and water intake, and urine pH of cows fed different anion sources and Ca concentrations, pooled across anion sources and day of experiment. 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P h r L p I L r p m d D I. a. e D and e a. . nadiQO d .66 an. E e 01 .Hi "4.. Edei‘. B1 44 i 0 EH. 4. £‘ .0 nu<<00 Cl C D 4 CI El 0 a! n4 . 6 4 .4 .66 544 D n .. n. -3 O O 09890 O n. 9.0 00 .90 .o :9 00 0.90 00 0900009090906 0‘ 00 O O O . 3.. Hd «Ir-In 83 CHAPTER 5 ANION SOURCES AND DIETARY CALCIUM CONCENTRATION REDUCED DRY MATTER INTAKE AND CHANGED ACID-BASE STATUS OF NONLACTATING NONPREGNANT HOLSTEIN COWS ABSTRACT Objective was to determine if the addition of dietary anions from different anion sources and varying dietary Ca content affected feed intake, acid-base status, and macromineral metabolism and utilization. Thirty-one nonlactating, nonpregnant Holstein cows were assigned in a randomized incomplete block design with three periods of 21 d each. Totally mixed diets were 20% alfalfa silage, 40% corn silage, and concentrates (dry basis). Dietary treatments were: Control (no anion source added), dietary cation-anion difference (DCAD; meq [(Na+K)-(CI+S)]l100 g of DM) = +15.5 meq; Bio-Chlorm; an Anionic Salts mixture designed to match the anionic and cationic contributions of Bio-Chlorm; and HCI. DCAD of treatments with supplemental anions averaged -9.7 meq/100 g of dietary DM. Factored with Control and treatments were dietary Ca concentrations of 0.48 or 2.0% (dry basis) with supplemental CaCO3 (4 x 2 factorial arrangement of treatments). Pooled across dietary Ca concentrations, 34 cows fed Control had 3.6% greater DMI than cows fed anion sources, but there was an anion source by Ca concentration interaction on DMI. Cows fed anionic salts mixture had similar DMI when fed 0.48% or 2.0% Ca, however, cows fed HCI had lower DMI when fed 2.0% compared with 0.48% Ca. Pooled across anion sources, cows fed 0.48% Ca had 4.3% greater DMI than cows fed 2.0% Ca. Cows fed anion sources had lower urine pH (6.0) compared with cows fed Control (8.1). Cows fed anion sources had higher blood plasma Cl and lower HCOa‘ concentrations than cows fed Control indicating a mild metabolic acidosis. Pooled across anion sources, cows fed 2.0% Ca had lower plasma 1,25(OH)2 vitamin 03 concentrations, but greater apparent absorption and retention of Ca and P than cows fed 0.48% Ca. Pooled across dietary Ca concentrations, cows fed greater concentrations of Cl excreted more CI in urine but also retained more CI in the body than cows fed Control. Supplemental anions from all sources were acidogenic. Greater absorption of Ca when cows were fed high dietary Ca suggests an alternate mechanism to active transport for Ca absorption. Abbreviation key: AA = apparent absorption; AS = anionic salts; B = Bio- ChlorT"; C = control; DCAD = dietary cation-anion difference; iCa = ionized Ca; IV = intravenous; PTH = parathyroid hormone; NAE = net acid excretion; SID = strong ion difference; WI = water intake; 1,25(OH),D3 = 125-dihydroxyvitamin 03. 85 INTRODUCTION A variety of tissues and physiologic processes require Ca: for normal bone formation, nerve transmission, muscle contraction, and blood clotting (Horst, 1986; Horst et al., 1994). Milk fever, a hypocalcemic disorder, is associated with parturition and the initiation of lactation in dairy cows. It is caused by the rapid and large removal from the blood of Ca to be secreted into colostrum and milk during the periparturient period (Horst, 1986). Almost all multiparous cows experience some degree of hypocalcemia in the first few days after calving (Horst et al., 1994). The dietary cation-anion difference (DCAD) is calculated as: meq [(Na + K) - (Cl + S)]I100 g of dietary DM. Several research studies showed that manipulation of DCAD during the prepartum period reduced the incidence of hypocalcemia (Block, 1984; Goff et al., 1989; Goff et al., 1991; Oetzel et al., 1988; Wang, 1990) and other postpartum disorders (Joyce et al., 1997; Lema et al., 1992; Wang, 1990). The DCAD has a direct impact on blood acid-base physiology. Diets with low or negative DCAD increase blood H+ concentration and decrease HCO3' concentration and pH (Block, 1994). Feeding low or negative DCAD changes systemic acid-base status, can increase resorption of Ca from bone (Block, 1984; Leclerc and Block, 1989), increase urinary Ca excretion (Gaynor et al., 1989; Wang and Beede, 1992a), and possibly increase Ca absorption from the gastrointestinal tract (Goff et al., 1991, Freeden et al., 1988a) 86 Ender et al. (1971) fed cows DCAD of -2 meq/100 g of dietary DM with 0.34% or 1.27% dietary Ca. Cows fed high dietary Ca had a more positive Ca balance up to 6 d before parturition and after parturition compared with cows fed 0.34% dietary Ca. Also, incidence of subclinical hypocalcemia (plasma iCa < 4 mgldl, Curtis et al., 1983) was 58% for cows fed +19 meq/100 g of dietary DM and 42% for cows fed -8 meq with 0.60% dietary Ca (Oetzel et al., 1988). However, when cows were fed 1.17% dietary Ca the incidence of subclinical hypocalcemia was reduced from 75% with positive DCAD to 17% with negative DCAD. More recently, Goff and Horst (1997a) used intravenous (IV) Ca treatment per milk fever case as an indicator of Ca status. Cows fed 1.5% dietary Ca required fewer lV Ca treatments (1 .4/case ) compared with cows fed 0.5% dietary Ca (2.1/case). Therefore, it seems that it may be beneficial to feed higher dietary Ca concentrations prepartum than current NRC (1989) recommendations, especially when cows are fed low or negative DCAD. However, varying the DCAD fed to cows before calving can reduce DMI (Joyce et al., 1997; Schoenbaum et al., 1994; Moore et al., 1997) and energy balance (Moore et al., 1997). Also, Oetzel and Barmore (1993) reported different depressions of DMI of concentrate mixtures when cows'were fed different anion sources. The advantages of lowering DCAD in prepartum diets to control or reduce hypocalcemia may be offset by the depression in DMI sometimes observed in cows fed anionic diets. It is important to find sources of anions that effectively alter acid-base status and have minimal effects on DMI. Therefore, 87 the objective of this study was to determine if the addition of dietary anions from different anion sources and dietary Ca from CaCO3 affected feed intake, acid- base status, and macromineral metabolism and utilization using nonlactating nonpregnant Holstein dairy cows as a model. MATERIALS AND METHODS Design, Cows and Diets Thirty-one nonlactating nonpregnant Holstein cows that had completed at least one lactation were assigned to a randomized incomplete block reversal design with three 21—d periods. Basal diet was formulated to have approximately 1.6 Mcal NELIkg of dietary DM and 15% CP and the same amounts of forage, consisting of corn silage and alfalfa silage in a ratio of 2:1 (DM basis, Table 1). The basal diet was 60:40 forage-to-concentrate. Basal concentrate consisted of ground corn grain, whole cottonseed, minerals and vitamins. Dietary treatments were Control (C, no anion source added); Bio-ChlorTM (B, Biovance, Omaha, NE); anionic salts (AS, a mixture of MgCl2-6H20, NH4CI, (NH4)ZSO, and NaOH designed to match the anionic and cationic profile of B); and hydrochloric acid (HCI) with or without addition of CaCO3 to give total dietary Ca concentrations 0.48% or 2.0% (dry basis, Table 2). Concentrate mixes for each dietary treatment for the entire experiment were mixed and put into plastic lined-bags before starting the experiment. For treatment B diets, a portion of the ground corn and soybean meal was replaced 88 of Fe C1: by B thus reducing DCAD (T able 2). Urea was included in C and HCI diets to supply the equivalent amount of NPN supplied by B. Ammonium sulfate, MgCl2-6H20, and NH4CI were included in the AS to supply an equivalent amount of CI, S, NPN, and Mg as supplied by B. Sodium hydroxide was added to the AS concentrate mix to achieve a Na concentration similar to that of B. In HCI diets, HCI, water and liquid sugarcane molasses were mixed with ground corn and soybean meal. Calcium carbonate replaced ground corn in appropriate amounts to increase the Ca concentration to an average of 2.0% of DM for the high dietary Ca treatments across C and each treatment with an anion source. Feeding, Sampling and Analytical Methods Feed and Orts. Cows were placed in tie-stalls for 14 d before the beginning of the experiment and fed C (with low dietary Ca). Cows were fed once daily at 1200 h. Orts were weighed once daily at 1100 h from d 5 through 21 of each of three periods. Orts were dried at 60 °C for 48 h for use in calculation of DMI. Free water intake (WI) was measured daily by in-line flow meters (Omega, Stamford, CT) from d 5 through 21. Forage samples were taken weekly, dried (60 °C), ground through a 5 mm screen in a Thomas-Nley mill (Arthur Thomas Company, Philadelphia, PA) and composited within each period. Samples of grains and concentrates were taken weekly and composited within period. Individual composite samples for each period were ground through a 2 mm screen. Ground feed samples were sent to the Northeast DHIA 89 Laboratory (Ithaca, NY) for CP, NDF, ADF, and mineral element analyses. Cows were weighed on d 21 of each period. Blood. Blood samples (20 ml) were taken from the tail vein using Li- heparin coated glass tubes at 0900 h (prior to daily feeding) on d 20 and 21 of each period and immediately placed on ice. Samples were centrifuged immediately after sampling at 2800 x g for 20 min to harvest plasma. A plasma aliquot was analyzed within 2 h of centrifuging with a Stat Profile 4 blood gas and mineral element analyzer (Nova Biomedical, Walthman, MA) to determine pH, p002, and concentrations of H003, ionized Ca (iCa), Na, K, and CI. The remainder of the sample was divided into equal parts and frozen at -20 °C in 5- ml plastic tubes. A frozen plasma sample was sent overnight on dry ice to the USDA National Animal Disease Center (Ames, IA) for parathyroid hormone (PTH) and 125-dihydroxyvitamin D3 [1,25(OH),D,] analyses. Plasma PTH was determined using an inmunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA) previously validated for use with bovine plasma (Goff et al., 1989). Plasma 1,25(OH)2D3 was analyzed using a radioreceptor assay (Reinhardt et al., 1984). Plasma was deproteinized with 4 ml of 12.5% trichloroacetic acid and analyzed for Ca and Mg by flame atomic absorption spectrophotometry (Smith- Heiftje 4000, Therrno Jarrel Ash Corporation, Franklin, MA), and for P by colorimetric assay (Gomori, 1942). 90 Urine. Urine samples were collected into plastic cups by manually stimulating the area around the vulva at 0900 h (about 3 h before feeding) on d 7, 14 and 21 of each period. Urine pH was measured immediately using a calibrated hand-held pH tester (Hach Company, Loveland, CO). Urine pH data were analyzed as the average of d 7, 14 and 21 of each period and as H” concentrations. On d 16 of each period after feeding, a subset of 16 cows were fitted with urethral catheters for total collection of urine (d 17 through 21). Each dietary treatment was represented twice in each period, so that there was a total of 6 cow-periods! dietary'treatment for the entire experiment. A 22 french (75 cc ribbed balloon) Bardex" Foley urethral catheter (C.R. Bard, lnc., Covington, GA) was inserted into the bladder and connected to a 25 L plastic collection container using Tygon" R-3603 tubing (Norton, Akron, OH) for total urine collection. Catheter balloons were inflated using 50 ml of sterile saline solution. Ten ml of a 37% formaldehyde solution were added to each collection container as a preservative before connecting the tubing. Urine excretion was measured (by weight) twice daily at 0800 and 2000 h from d 17 through 21. After mixing 12-h collection in each container, a 100-ml sample was filtered through eight layers of cheese cloth, placed in plastic cups, and frozen at -20°C. A composite sample (by weight) for the 5-d collection period was analyzed for Ca, Mg, K, and Na by flame atomic absorption spectrophotometry and for P colorimetrically. Urine samples were sent to the Northeast DHIA Laboratory (Ithaca, NY) for analysis of 91 Cl using an automatic potentiometric titration with silver nitrate using a Brinkman Metrohm 716 Trtrino titration unit (Brinkman Instruments Inc, Westbury, NY) with a silver electrode (Cantliffe et al., 1970). Plasma and urine samples were analyzed for creatinine colorimetrically (Procedure 555, Sigma Diagnostics, St.Louis, M0) at 500 nm. Concentrations of plasma and urine creatinine and urine volume were used to determine glomerular filtration rate and fractional excretion of different mineral elements as described by Delaquis and Block (1995b). Titratable acidity and ammonium concentrations were determined by titration of 5-ml urine samples (Chan, 1972). Net acid excretion (NAE) in urine was determined by summing titratable acidity and ammonium concentration (Chan, 1972). Urine bicarbonate concentrations were determined also by titration (Lin and Chan, 1973). Feces. Chromic oxide was used as an inert digesta marker to estimate total fecal excretion. Lock-ring gelatin capsules (T orpac Inc., Fairfield, NJ) each filled with 7.5 g of Cr203 were given orally by balling gun from d 7 through 21 of each period at 0900 and 2100 h. Approximately 400 g of feces were collected every 15 h for 5 consecutive d starting at 0900 h on d 17 of each period. All fecal samples were dried (60°C), ground through 5 mm and 2 mm screens and composited within each period for each cow. Approximately 1 g of fecal sample was wet-ashed (AOAC, 1990) in a mixture of 10 M perchloric acid (3 ml) and 14 M nitric acid (20 ml) using a PC-500 Corning hot plate (Corning, NY). Digested samples were diluted with deionized distilled water and analyzed for Cr, Ca, Mg, 92 Na, and K by flame atomic absorption spectrophotometry and for P colorimetrically. Fecal samples were sent to the Northeast DHIA Laboratory (Ithaca, NY) for CI analysis. Mineral element balances (gld) were calculated using the mineral intake minus mineral excretion in feces and urine. Dry matter fecal excretion was calculated dividing daily Cr intake by Cr concentration in feces. Mineral fecal excretion was calculated multiplying fecal DM excretion by mineral element concentration in feces. Mineral balances were calculated from the subset of 16 cows with total urine collection (6 cow-period observations/dietary treatment). Statistical Analysis Data were analyzed by method of least squares ANOVA using the general linear model procedures of SAS (1996). The model consisted of treatment, period, cow, and residual error. Main effects of treatments were tested by orthogonal contrasts: treatment C vs. B, AS, HCI; treatment B vs. AS, HCI; treatment AS vs. HCI; low dietary Ca vs. high dietary Ca; and, the interaction of the contrasts of anion sources by Ca concentration. Correlation coefficients were analyzed using PROC CORR (SAS, 1996). A multivariate repeated measures model (PROC MIXED; SAS, 1996) was used to analyze the correlation between residuals effects on different characters. This indicates the relative degree of association of the joint influence of unknown causal variables and environmental noise on these characters. Least-squares treatment means 93 are presented. Statistical significance was declared at P < 0.05. RESULTS AND DISCUSSION Diet Composition Diets averaged 15.2% CF (Table 2). Dietary treatments had similar ADF and NDF concentrations. Diets averaged 0.48% and 2.0% for low and high dietary Ca, respectively. Concentrations of P and K were similar among dietary treatments. Concentrations of Mg in low Ca diets were somewhat higher which may be due to the inclusion of MgO. Dietary treatments were formulated to have concentrations of Na similar to those in B; however, analyzed Na concentrations were somewhat higher in C and HCI treatments. Pooled across dietary Ca concentrations, inclusion of anion sources increased analyzed concentrations of Cl from 0.57% in C to 0.88% in B, 0.97% in AS, and 1.47% in HCI treatments. Sulfur concentrations were higher in B and AS treatments because of the anions sources used in formulation. Increasing amounts of anions in dietary treatments reduced DCAD from +155 meq (C) to -7.3 meq (B), -11.2 meq (AS) and -10.6 meq/100 g of DM (HCI), pooled across dietary Ca concentrations. For the other dietary treatments, no attempt was made in formulation to equalize the higher CI in the HCI treatment. Dry Matter and Water Intake Cows fed anion sources had 3.5% lower DMI than cows fed C, pooled 94 across dietary Ca concentrations ( P < 0.04; Table 3). Cows fed AS and HCI had 4.5% lower DMI than cows fed B ( P < 0.01). However, there was an anion source by dietary Ca concentration interaction on DMI ( P < 0.02). Cows fed AS with 0.48 or 2.0% Ca had similar DMI, however, cows fed HCI had lower DMI when fed 2.0% Ca compared with 0.48% Ca. Moore et al. (1997) found reduced DMI when late pregnant nonlactating cows were fed a diet containing anionic salts with a DCAD of -15 meq/100 g of dietary DM. In other studies where the DCAD was similar to that of the current study reduced DMI was observed (Goff and Horst, 1997a; Joyce et al., 1997; Schoenbaum et al., 1994). Lower DMI of cows fed anion sources may be due to the induced metabolic acidosis observed when cows are fed diets high in anions (Goff and Horst, 1997a). In the current study, lowest DMI was observed for cows fed HCI and 2.0% dietary Ca (13.9 kgld), the treatment that caused the lowest urine pH (5.7; Table, 4). Pooled across anion sources, DMI of cows fed 2.0% Ca was reduced by 4.3% compared with cows fed 0.48% Ca. Contrary to this, Goff and Horst (1997a) reported a 3.2% increase in DMI in late pregnant cows fed 1.5% compared with 0.5% Ca. Differences in responses between the current experiment and that of Goff and Horst (1997a) may be attributable to different dietary Ca concentrations or to the palatability of the different Ca sources used (CaCO3, CaSO., CaCI, and CaHPO,; in the study of Goff and Horst, 1997a). Cows fed 2.0% Ca had lower free WI and total WI compared with cows fed 0.48% Ca, pooled across anion sources (Table 3). Lower total WI in cows 95 fed 2.0% Ca was due to the combination of lower free WI and lower feed WI. Major factors affecting water intake are DMI, milk production, temperature, humidity and Na intake (Murphy, 1992). Residuals for DMI and WI were correlated highly (r = 0.77; P < 0.01). Lower total WI in cows fed 2.0 compared with 0.48% Ca may be partially due to the lower DMI. Water apparently absorbed (total water intake minus total water excreted in feces, Ud) was lower for cows fed 0.48% compared with 2.0% Ca, pooled across anion sources. Lower water apparently absorbed in cows fed high dietary Ca may be due to the lower DMI, because water absorbed as a percentage of total daily water consumption did not differ among cows fed different dietary treatments. Urine Measurements Pooled across dietary Ca concentrations, cows fed anion sources increased urine volume by approximately 1 L compared with cows fed C (Table 4), however, cows fed AS had similar urine volume to cows fed C. Also, cows fed HCI had greater urine output than cows fed AS. Contrary to this, lowering DCAD decreased urine volume of dry cows (Delaquis and Block, 1995a) and of lactating cows in early and mid-lactation, but not in late lactation (Delaquis and Block, 1995b). Even though there were no differences in total WI among cows fed anion sources in our experiment, increased urine volume may be explained partially by the increased apparent absorption (AA) and urinary excretion of Na 96 as percent of Na intake compared with cows fed C (Table 11). Finco (1989) reported increased Na ingestion caused water retention and expansion of the extracellular space. Increased hemodilution reduces the peritubular capillary oncotic pressure in the kidney, resulting in less water and Na reabsorption. Cows fed C had higher urine pH than cows fed B, AS and HCI, pooled across dietary Ca concentrations (Table 4). Cows fed B had higher urine pH than cows fed AS and HCI. The DCAD of B was somewhat less negative than that of AS and HCI, -7.2, -11.2 and -10.6 meq/100 g of dietary DM, respectively. There was an anion source by dietary Ca concentration interaction ( P < 0.01) in urine pH. Urine pH of cows fed AS increased from 5.9 to 6.1, whereas that of cows fed HCl decreased from 6.0 to 5.7 when fed 0.48% Ca and 2.0% Ca, respectively. Reduced urine pH was observed in cows fed anionic salts (Goff and Horst, 1997a; Moore et al., 1997; Rodriguez et al., 1996; Wang and Beede, 1992a), HCI (Goff et al., 1997; Goff and Horst, 1997c), and in goats fed HCI (Freeden et al., 1988a). The lowest average urine pH and DMI were observed in cows fed HCI. DCAD of total intake (meq/d per kg of BW) were computed for each cow- period by multiplying daily DMI (kg) times meq [(Na + K) - (Cl + S)]l100 g of dietary DM. Pooled across dietary Ca concentrations, urine pH and DCAD intake (meq/d per kg of BW) were correlated highly (r = 0.93; P < 0.01); however, residuals between these two variables were not correlated ( P > 0.22). Cows fed AS and HCI had greater DCAD intake (meq/d per kg of BW) and less 97 variation in urine pH (SD = 0.28 and 0.23, respectively) compared with cows fed B (SD = 0.43; Figure 1). Cows fed C had less variation in urine pH than cows fed anion sources as absolute meq of DCAD intake varied (SD = 0.10; Figure 1). Also, lower DMI and urine pH in cows fed HCI may indicate a greater acidifying effect of HCI than other anion sources (Goff and Horst, 1997c; Goff et al., 1997). Strong ion difference (SID) is defined as meq [Na + K - CI]IL, (Stewart, 1983). Trtratable acidity (meq/L) is measured by titration of urine samples with NaOH to pH 7.4 multiplied by the normality of NaOH. The NAE is defined as the sum of titratable acidity plus ammonium concentration in urine (Chan, 1972). The NAE measures the effect of renal excretion on plasma SID (Lunn and McGuirk, 1990). Pooled across dietary Ca concentrations, cows fed anion sources had greater titratable acidity, NAE, and lower bicarbonate concentrations in urine than cows fed C. The major mechanism by which the kidney can affect plasma H’ concentration is by adjusting plasma SID by differential removal of Na, K and Cl from plasma to urine (Stewart, 1983). Greater excretion of H+ (lower urine pH) and lower of bicarbonate concentration in urine observed in this study resulted in more excretion of Cl (Table 13) and more reabsorption of K (Table 12) in urine of cows fed anion sources compared with cows fed C. Urine pH and NAE were correlated negatively (r = -0.81; P < 0.01), but residuals were not correlated ( P > 0.35). Urine glomerular filtration rate was estimated multiplying urine creatinine concentration times urine volume divided by plasma creatinine concentration, 98 and was not affected by dietary treatments (Table 5). Fractional excretion of mineral elements was calculated by multiplying urine mineral element concentration by urine volume per day, and dividing by glomerular filtration rate times plasma mineral element concentration. Pooled across dietary Ca concentrations, fractional excretions of Ca and P increased in cows fed anion sources compared with cows fed C. Also, cows fed NH4CI and (NH,)2SO, had greater fractional excretion of Ca compared with cows fed control diets (Wang and Beede, 1992a). Delaquis and Block (1995a) found no differences in fractional excretion of mineral elements in cows fed different DCAD. Lack of difference in fractional excretion of Ca in the Delaquis and Block (1995a) study may be due to the small difference in DCAD between dietary treatments (+48 vs. +33 meq/100 g of dietary DM). Pooled across dietary Ca concentrations, cows fed AS had lower fractional excretions of Na compared with cows fed HCI. Differences were mainly due to the higher fractional excretions of Na in cows fed HCI and 0.48% Ca. Pooled across dietary Ca concentrations, cows fed C had lower fractional excretions of Cl than cows fed anion sources. Also, cows fed B had lower fractional excretions of Cl compared with cows fed AS and HCI. Cows fed HCI had the greatest fractional excretions of Cl. Differences among dietary treatments in fractional excretions of Cl are mainly due to differences in the concentrations and amounts of Cl fed. 99 Plasma Measurements Cows fed C had higher plasma pH and bicarbonate concentrations than cows fed anion sources, pooled across dietary Ca concentrations (Table 6). Similarly, lower blood pH (Goff and Horst, 1997a; Wang and Beede, 1992a) and bicarbonate concentrations (Goff and Horst, 1997a) were observed in cows fed negative compared with positive DCAD. There was an anion source by dietary Ca interaction on plasma pH, pCOz, bicarbonate and SID concentrations ( P < 0.06). Cows fed C and 2.0% Ca had lower plasma pH, and greater plasma pCOz, bicarbonate, and SID concentrations compared with cows fed C and 0.48% Ca; whereas, cows fed anion sources had similar plasma pH, pCOz, bicarbonate and SID concentrations irrespective of dietary Ca treatment. Greater plasma p002, and bicarbonate and SID concentrations observed with cows fed C and 2.0% Ca, may be due to a greater base load when feeding CaCOa. Stewart (1983) indicated that the entirety of acid-base balance can be understood quantitatively using the independent variables pCOz, SID and total protein, and their regulation by lungs, kidneys, gut, and liver. Because most membranes separating body fluids are not permeable to proteins, changes in SID and p002 will determine changes in blood pH and bicarbonate concentrations. Our data suggest that a decrease in plasma SID resulted in a lower plasma pH in cows fed anion sources compared with cows fed C. Greater concentrations of plasma bicarbonate in cows fed C and 2.0% Ca are the result of increased SID; however, plasma pH was lower and can not be explained by 100 this theory. Cows fed anion sources had slightly higher plasma Cl concentrations compared with cows fed C, pooled across dietary Ca concentrations. Also, Block (1984) and Freeden et al. (1988a) observed increased plasma Cl concentrations when negative DCAD were fed. Increased plasma Cl concentrations may be due to the high dietary Cl concentrations when cows were fed B, AS and HCI. However, based on results of cows fed all dietary treatments, plasma Cl concentrations appear to be highly regulated. There was an anion source by dietary Ca interaction in total plasma Ca ( P < 0.04; Table 7). Cows fed C had lower total plasma Ca when fed 2.0% Ca compared with cows fed 0.48% Ca, but cows fed B, AS and HCI had more similar plasma Ca concentrations when fed 0.48 or 2.0% Ca. An interaction was detected also in plasma iCa concentrations (P < 0.01), but in an opposite manner. Cows fed C had greater plasma iCa concentrations when fed 2.0% compared with 0.48% Ca, whereas similar plasma iCa concentrations were observed in cows fed B, AS and HCI and 2.0 or 0.48% Ca. Pooled across Ca concentrations, cows fed C had lower plasma iCa concentrations than cows fed B, AS and HCI. This effect is due mainly to the response to HCI; cows fed HCI had higher plasma iCa concentrations than cows fed AS ( P < 0.01). Similarly, Wang and Beads (1992a) reported greater concentrations of blood iCa, but no differences in total blood Ca in cows fed anionic salts. Changes in plasma iCa were most likely due to changes in plasma pH, lower plasma pH was associated 101 with higher plasma iCa. Plasma pH and iCa were negatively correlated ( r = - 0.67; P < 0.01) as were their residuals (r = -0.53; P < 0.01). Although plasma concentrations of PTH were not affected strongly overall by dietary treatments, greater concentrations of plasma 1,25(OH)2D3 were observed in cows fed anion sources compared with cows fed C, pooled across dietary Ca concentrations (Table 7). Similarly, renal production of 1,25(OH)203 increased in Jersey cows fed negative DCAD prepartum (Goff et al., 1991). It is theorized that the increase in plasma 1,25(OH)203 concentration is due to increased tissue responsiveness to PTH in cows fed anion sources (Horst et al., 1997). Cows fed 2.0% Ca had lower concentrations of plasma 1,25(OH)203 compared with cows fed 0.48% Ca. Although PTH concentrations were not affected by dietary Ca, lower plasma 1,25(OH)2D3 concentrations in cows fed 2.0% Ca may be explained partially by the increased apparent absorption of Ca for cows receiving higher dietary Ca (Table 8). Hypercalcemia has a direct effect by inhibiting kidney 1o<-hydroxylase (Horst, 1986). Pooled across dietary Ca concentrations, plasma Mg concentrations were greater for cows fed B, AS and HCI than for cows fed C. Similarly, greater plasma Mg concentrations were observed in cows fed anionic diets compared with cows fed cationic diets (Gaynor et al, 1989), but sources of Mg were different between dietary treatments. Dietary Ca concentration also tended to affect the concentrations of plasma Mg. Pooled across anion sources, cows fed 0.48% Ca had greater plasma Mg concentrations than cows fed 2.0% Ca (P < 102 0.06). Similarly, Goff and Horst (1997a) reported greater Mg concentrations in plasma of cows fed 0.5% Ca compared with cows fed 1.5% Ca; however, cows fed 0.5% Ca had greater dietary concentrations of Mg. Mineral Balances: Ca, Mg and P Table 8 summarizes results of measurements used in calculation of apparent Ca balance for each dietary treatment. As expected cows fed 2.0% Ca had greater Ca intakes than cows fed 0.48% Ca, pooled across anion sources. Fecal concentrations of Ca also were greater for cows fed 2.0% compared with 0.48% Ca. However, cows fed B, AS and HCI tended to have lower fecal Ca concentrations than cows fed C, pooled across dietary Ca concentrations. More importantly, there was an anion source by dietary Ca interaction in fecal Ca excretion (P < 0.06). Cows fed C and 2.0% Ca excreted 227 g/d more Ca than cows fed C and 0.48% Ca, but cows fed anion sources excreted just 176 g/d more Ca when fed 2.0% Ca compared with 0.48% Ca. Pooled across anion sources, fecal Ca excretion (gld) was greater in cows fed 2.0% compared with cows fed 0.48% Ca. Apparent absorption of Ca (g/d and as percent of Ca intake) were greater for cows fed AS and HCI than for cows fed B, pooled across dietary Ca concentrations. The greatest AA of Ca was observed in cows fed HCI. No differences in AA of Ca were observed in cows fed different DCAD (Delaquis and Block, 1995a); however DCAD in that study were +48 and +33 meq/100 g of dietary DM. No difference in Ca absorption was observed in sheep 103 fed different DCAD (T akagi and Block, 1991a; Takagi and Block, 19910), but it increased during a Ca challenge (T akagi and Block, 1991c). Similarly, nonlactating nonpregnant goats had 33% Ca absorption when fed a cationic diet compared with 43% when fed a HCI diet (Freeden et al., 1988b), but during a Ca challenge, goats fed HCI maintained Ca absorption at 45% whereas those fed a cationic diet reduced Ca absorption to 18%. Also, increased Ca absorption was observed in goats fed anionic diets during lactation, but not during pregnancy (Freeden et al., 1988a). Calcium can be absorbed from the gastrointestinal tract by a transcellular or a paracellular mechanism (Bronner, 1992). The primary regulator of transcellular intestinal Ca transport is 1,25(OH)203 (Fullmer, 1992). Increased plasma concentrations of 1,25(OH)2D3 may result in stimulation of the active transport mechanism of Ca in the gastrointestinal tract (Goff et al., 1986). Takagi and Block (1991a) observed increased Ca absorption in sheep fed positive DCAD and injected with vitamin D. In the current study, increased Ca absorption observed in cows fed 2.0% Ca compared with cows fed 0.48% Ca could not be explained by increased concentrations of plasma 1,25(OH)203, in fact plasma 1,25(OH)203 concentrations were lower in cows fed 2.0% Ca. Braithwaite (1979) reported that, as Ca intake reached 100 mg of Ca/d per kg of BW in mature sheep, active absorption of Ca decrease to a negligible level. Also, he found a highly significant linear relationship (r = 0.94) between the rate of Ca absorption and Ca intake when sheep had Ca intakes between 100 and 400 mg of Cald per 104 kg of BW. In our study, cows fed low dietary Ca consumed an average of 100 mg of Cald per kg of BW, whereas cows fed high dietary Ca consumed an average of 389 mg of Cald per kg of BW, pooled across anion sources (data not shown). Paracellular transport of Ca could be an alternate mechanism to active transport used for cows fed anion sources and 2.0% dietary Ca resulting in an increase in AA of Ca. Paracellular movement of Ca is necessarily down a chemical gradient because the concentration of Ca in the intestinal lumen is higher than at the serosal pole of the intestinal cell (Bronner, 1992). Pooled across anion sources, cows fed 2.0% Ca had greater AA of Ca (gld and as percent of Ca intake) than cows fed 0.48% Ca. Also, sheep fed 0.81% Ca had greater Ca absorption than sheep fed 0.47% Ca (T akagi and Block, 1991a). The intracellular transport of Ca is likely not responsible for the increased AA of Ca in cows fed 2.0% Ca because concentrations of plasma 1,25(OH)203 were lower in cows fed 2.0% Ca compared with cows fed 0.48% Ca. Hypercalciuria was observed in cows fed anion sources in several studies (Gaynor et al., 1989; Wang and Beede, 1992a), in sheep (T akagi and Block, 1991a) and in goats (Freeden et al., 1988a). In metabolic acidosis, hypercalciuria maintains a high Ca flux through the Ca pool (Freeden et al., 1988b). There was an anion source by dietary Ca concentration interaction in urine excretion of Ca ( P <0.07). Cows fed C increased urinary excretions of Ca from 0.4 g/d when fed 0.48% Ca to 1.2 g/d when fed 2.0% Ca; however, when 105 cows were fed anion sources urinary excretions of Ca averaged about 5.2 g/d when fed 0.48% or 2.0% Ca. Urinary Ca concentrations and excretions were greater in cows fed anion sources compared with cows fed C, pooled across dietary Ca concentrations (data not shown). Also, cows fed AS had greater urine Ca concentrations and excretions than cows fed HCI. Increased flux of Ca through the Ca pool may be responsible partially for the increased urine Ca excretion observed in cows fed anion sources. Metabolic acidosis may be responsible partially for the increased Ca excretion in urine because NAE and urine Ca excretion were correlated positively (r = 0.64; P < 0.01). There was an anion source by dietary Ca interaction on urine Ca excretion as percent of Ca intake (P < 0.01). Cows fed C excreted about 1% of Ca intake in urine when fed 0.48% or 2.0% Ca, but cows fed anion sources excreted an average of 6.9% when fed 0.48% Ca compared with 1.7% when fed 2.0% Ca. Also, pooled across anion sources, cows fed 0.48% Ca excreted greater Ca in urine as percent of intake than cows fed 2.0% Ca. Differences in urine excretion of Ca as percent of intake between low and high dietary Ca are mainly due to the wide range in dietary Ca concentration. Cows fed HCI excreted more Ca in urine as percent of apparently absorbed Ca compared with cows fed AS, pooled across dietary Ca concentrations. This may be due to better acidifying effect of HCI compared with AS because NAE and urinary Ca excretion were correlated positively. Apparent balance of Ca (gld and as percent of Ca intake) was greater for 106 cows fed AS and HCI compared with cows fed B. Also pooled across anion sources, cows fed 2.0% Ca had greater apparent Ca balance (gld and as percent of Ca intake) than cows fed 0.48% Ca. Braithwaite (1979) reported increased body retention of Ca in mature sheep as Ca intake and absorption increased from 100 to 400 mg of Ca/d per kg of BW. The increased Ca retention reflected an increased skeletal retention which was due to a decrease in the rate of bone resorption relative to the rate of bone accretion. An alternative possible explanation for at least part of the increase in AA and balance of Ca for cows fed high vs. low dietary Ca might be increased residence time of dietary Ca in the digestive tract. This would result in some residual dietary Ca being considered absorbed, although it truly was not. This would cause an over-estimation of AA and balance of Ca for cows fed high dietary Ca. However, this does not negate the fact that pooled across dietary Ca concentrations, AA and balance of Ca were greater for cows fed AS and HCI vs. B, and HCI vs. AS ( P < 0.08). Pooled across anion sources, P intakes were greater for cows fed 0.48% Ca compared with cows fed 2.0% Ca (Table 9). Differences in P intakes may be partially due to lower concentrations of P in AS diets and to the lower DMI of cows fed HCI and 2.0% Ca. Pooled across anion sources, fecal concentration and excretion of 7P were lower for cows fed 2.0% Ca compared with cows fed 0.48% Ca. Due to lower excretion of P, greater AA of P was observed in cows fed 2.0% Ca compared with cows fed 0.48% Ca. Similarly, greater AA of P was observed in sheep fed 0.85% Ca compared with sheep fed 0.47% dietary Ca 107 (T akagi and Block, 1991a). Also, increasing Ca absorption by injecting sheep with 1-«-hydroxycholecalciferol increased absorption and retention of P (Braithwaite, 1980). Braithwaite (1976) reported skeletal reserves of Ca and P are diminished during lactation, but cows are able to replenish reserves during late lactation and the dry period. Although most of the P absorbed is incorporated into bones and teeth during the growth period, some can be incorporated into bone in older animals (NRC, 1980). Greater AA of Ca in cows fed 2.0% Ca may have created a more efficient absorption of dietary P compared with cows fed 0.48% Ca. The rate of excretion of endogenous fecal P is related directly to the rate of absorption and the intake of P, but it is inversely related to the rate of Ca absorption (Braithwaite, 1976). Also, Braithwaite (1979) reported P is retained in direct relation to the rate of Ca retention. Cows used in this study were taken from the lactation herd and dried-off 30 d before starting the experiment. Although the cows were nonlactating and nonpregnant, greater absorption of Ca and P observed in cows fed 2.0% Ca, may indicate some mineral deposition into bone. Pooled across dietary Ca concentrations, urinary excretion of P increased in cows fed anion sources compared with cows fed C. Urine P excretion as percent of P intake was lower for cows fed C compared with cows fed anion sources, pooled across dietary Ca concentrations. Contrary to this, urine P excretion as percent of P intake was not affected in goats fed anionic diets during pregnancy and lactation (Freeden et al., 1988a). Metabolic acidosis leads 108 to increased excretion of the major urinary buffer HPO; (Lunn and McGuirk, 1990). Differences in urinary excretion of P when cows were fed anion sources were very small and did not have major effect on the apparent balance of P. However pooled across anion sources, cows fed 2.0% Ca apparently retained 21.5 g/d or 36% of intake of P compared with 9.8 gld and 15% of intake of P for cows fed 0.48% Ca. Table 10 shows the measurements for apparent balance of Mg for each dietary treatment. Pooled across anion sources, intakes and fecal excretions of Mg were lower for cows fed 2.0% Ca compared with cows fed 0.48% Ca, likely due to the lower concentration of Mg of these diets and lower DMI for cows fed these treatments. There were two interactions of anion source by dietary Ca on daily urine Mg excretion. Cows fed C and 2.0% Ca had lower urinary Mg excretion than cows fed C and 0.48% Ca; however, urinary Mg excretion was more similar when cows were fed anion sources with 0.48% or 2.0% Ca ( P < 0.02). Also, cows fed B had lower urine Mg excretion when fed 0.48% Ca compared with 2.0% Ca; however, cows fed AS and HCI had greater urine Mg excretion when fed 0.48% Ca compared with 2.0% Ca ( P < 0.01). Cows fed C had greater urine excretion of Mg as percent of Mg intake when fed 0.48% Ca compared with 2.0% Ca, but cows fed anion sources had greater urine excretion of Mg as percent of Mg intake when fed 2.0% Ca. Pooled across anion sources, cows fed 2.0% Ca had greater urine excretion of Mg as percent of Mg intake compared with cows fed 0.48% Ca. Greater urine excretion of Mg as percent of 109 Mg intake in cows fed high dietary Ca may be possibly due to the lower intake of Mg in cows fed these diets because urine concentrations and excretion of Mg were not affected by dietary Ca concentration. Pooled across anion sources, apparent Mg balance (gld) was greater in cows fed 0.48% Ca compared with cows fed 2.0% Ca. Greater apparent Mg balance for cows fed 0.48% Ca may be due to greater Mg intake compared with cows fed 2.0% Ca. Also, high dietary Ca may have affected Mg absorption from the digestive tract (Chicco et al., 1973; Martens and Rayssiguier, 1980) Mineral Balances: Na, K and CI Pooled across dietary Ca concentrations, intakes of Na were greater in cows fed C compared with cows fed anion sources (Table 11), due mainly to the greater Na concentrations in C diets. Also, cows fed B had lower Na intakes compared with cows fed AS and HCI because dietary Na concentrations were lower in B. There was an anion source by dietary Ca concentration interaction on intake of Na ( P < 0.04). Cows fed AS had similar intakes of Na with 0.48% or 2.0% Ca; however, cows fed HCI had lower Na intake when fed 2.0 compared with 0.48% Ca. . There was an anion source by dietary Ca concentration interaction on fecal concentrations and excretion of Na ( P < 0.01). Cows fed C had lower fecal concentration and excretion of Na when fed 2.0% compared with 0.48% Ca, but cows fed anion sources had more similar concentrations and excretions of Na 110 regardless of the dietary Ca concentration. Pooled across dietary Ca concentrations, fecal concentrations and excretions of Na were greater in cows fed C compared with cows fed anion sources, which likely was due to the greater Na intake. There were two anion source by dietary Ca concentration interactions on AA ( P < 0.01) and balances of Na ( P < 0.04). Cows fed C and 2.0% Ca had greater AA and balance of Na than cows fed C and 0.48% Ca, whereas cows fed anion sources had more similar AA and balances of Na when fed 0.48 or 2.0% Ca. Lower fecal excretions of Na resulted in greater AA and balances of Na in cows fed C and 2.0% Ca compared with cows fed C and 0.48% Ca. Contrary to this, lowering DCAD reduced Na digestibility in sheep fed 0.83% Ca, but had no effect in sheep fed 0.47% Ca (T akagi and Block, 1991a). Also, cows fed B had greater AA and balance of Na (gld) when fed 2.0% compared with 0.48% Ca; however, cows fed AS and HCI had lower AA and balance when fed 2.0% compared with 0.48% Ca (P < 0.04). Although cows fed anion sources excreted less Na in feces compared with cows fed C, apparent balance of Na (gld) was not different due to greater urinary excretion and lower intake of Na when cows were fed anion sources. However, apparent balance of Na as percent of Na intake was greater for cows fed anion sources compared with cows fed C, mainly due to the lower Na balance as percent of intake for the low Ca treatment in C. Greater AA and urinary excretion of Na by cows fed anion sources compared with cows fed C, may be one of the mechanisms used to try to adjust acid-base 111 balance in cows fed anion sources. There were not major differences in apparent balances of K due to anion sources or dietary Ca concentration (Table 12). Pooled across anion sources, cows fed 2.0% Ca had lower intake and fecal excretion of K compared with cows fed 0.48% Ca. Lower intake and fecal excretion of K in cows fed 2.0% Ca, may be due to the lower DMI observed for cows fed high dietary Ca because dietary K concentrations were similar among dietary treatments. Pooled across dietary Ca concentrations, cows fed C had greater urine K concentrations than cows fed anion sources. However, total K excretions in urine were not different probably due to the greater urine volume of cows fed anion sources. Similarly, lowering DCAD had no effect on K balance in sheep fed two dietary Ca concentrations (T akagi and Block, 1991a) or in urinary K excretion in Jersey cows (Gaynor et aL,1989) Intakes of Cl were lower in cows fed C compared with cows fed anion sources, pooled across dietary Ca concentrations (Table 13); dietary treatments B, AS and HCI contained higher concentrations of Cl. Pooled across dietary Ca concentrations, cows fed AS had lower Cl intake compared with cows fed HCI because Cl concentrations were higher in HCI. Also pooled across anion sources, intake of CI was lower for cows fed 2.0% Ca compared with cows fed 0.48% Ca. Because fecal excretion of Cl was not affected by treatments, differences in AA of (gld) resembled the differences observed in CI intakes. Apparent absorption of Cl as percent of Cl intake was very high ( >94%) in all 112 dietary treatments. Similarly, high digestibility of Cl was observed in sheep fed different DCAD with high or low dietary Ca (T akagi and Block, 1991a). In the current study, cows fed anion sources had greater urine Cl concentration and excretion than cows fed C, pooled across dietary Ca concentrations. Cows fed AS and HCI had greater urine Cl concentration and excretion than cows fed B. Also, cows fed HCI had greater urine Cl excretion than cows fed AS. Although cows fed anion sources had greater urinary excretion of Cl, the apparent balance was greater for cows fed anion sources compared with cows fed C ( P < 0.07). Similarly, Takagi and Block (1991a) reported greater AA, urinary excretion and apparent retention of CI in sheep fed lower DCAD when dietary Cl was increased. Cows fed greater concentrations of Cl absorbed more Cl, excreted more CI in urine, but also retained more Cl within the body, affecting acid-base status of cows fed anion sources. The amount of chloride retained especially in cows fed HCI seemed to be quite high. Cows excrete chloride in urine, feces, skin secretions and milk (Coppock, 1986). In this study we measured excretion of CI in urine and feces, but not in milk because cows were nonlactating. It is difficult to believe that skin excretions accounted for a great amount of the Cl retained by cows fed the HCI treatments. However, when plasma Cl concentrations are high, the Cl ion can diffuse from plasma to the erythrocyte (chloride shift) exchanged by HCOa’ (Reece, 1993). 113 CONCLUSIONS Feeding anion sources to nonlactating nonpregnant cows reduced DMI, but HCI resulted in the greatest DMI reduction which may be due to its greater acidifying effect. Feeding 2.0% Ca reduced DMI possibly due to the unpalatability of the Ca source used, CaCOa. Anion supplementation changed acid-base status and reduced urine pH. Even though dietary treatments had similar DCAD, the greater the concentration of CI in the diet, the lower the urine pH. This indicates a better acidifying effect or a better absorption of the Cl compared with S. Also, cows fed higher dietary concentrations of Cl had greater retention of CI in the body. Diets with relatively more Cl anion can have a greater-impact on cow's acid-base status. Dietary treatments had no effect on PTH; however, anion supplementation increased concentrations of 1,25(OH)203 in plasma. Greater plasma 1,25(OH)203 may indicate greater tissue responsiveness to PTH in anion supplemented cows. Although feeding 2.0% Ca reduced plasma 1,25(OH)2D3, greater absorption and retention of Ca in cows fed 2.0% Ca may indicate paracellular transport of Ca from the digestive tract. Hypercalciuria was evident in cows fed anion sources. Greater excretions of Ca in urine may increase the concentration gradient between gastrointestinal tract and the bloodstream, increasing the flux of Ca through the system. Cows fed high dietary Ca had greater AA and balance of Ca and P than cows fed low dietary Ca. Greater retention of Ca in cows fed high dietary Ca may have created a more efficient use of dietary P compared with cows fed low dietary Ca. 114 Therefore, we conclude from this study that Cl-based anion sources have greater acidifying effect than S-based and can be used to control hypocalcemia in periparturient dairy cows. Feeding high dietary Ca increased AA and retention of Ca in nonlactating nonpregnant cows; however, effects of high dietary Ca fed with negative DCAD need to be researched in periparturient cows. 115 Table 1. Dietary ingredients (% of dietary DM) of treatments with different anion sources and Ca concentrations. Dietary Treatments‘ Ingredients CL CI-I BL BH ASL ASH HCIL HCIH Alfalfa silage 19.7 19.7 19.7 19.7 19.7 19.7 19.7 19.7 Com silage 39.4 39.4 39.4 39.4 39.4 39.4 39.4 39.4 Cottonseed, whole 8.0 8.0 8.0 8.0 8.0 8.0 8.0 8.0 Corn, ground 27.1 23.6 23.9 20.4 26.2 22.5 25.7 22.2 Soybean, meal 3.50 3.50 . . . . . . 3.50 3.50 3.50 3.50 Urea 0.65 0.65 . . . . . . . . . . . . 0.65 0.65 Mineral-vitamin mix2 0.56 0.58 0.56 0.56 0.64 0.64 0.56 0.58 Caco3 . . . 3.67 . . . 3.63 .. . 3.68 . . . 3.65 M90 0.26 0.13 0.23 0.16 0.07 . . . 0.26 0.13 NaCl 0.47 0.47 . . . . . . . . . . . . 0.47 0.47 NH,.PO,.H2 0.35 0.34 0.20 0.22 0.37 0.39 0.35 0.34 Bio-ChlorT’“ . . . . . . 8.00 8.00 MgCIZ-BHZO . . . . . . . . . . . . 0.49 0.49 NH4CI . . . . . . . . . . . . 0.54 0.53 (NH4)2SO4 . . . . . . . . . . . . 0.80 0.79 NaOH . . . . . . . . . . . . 0.30 0.30 HCI“ 0.82 0.82 Liquid molasses . . . . . . . . . . . . . . . . . . 0.51 0.51 ‘ Dietary treatments: C = control, no anion source added; B = Bio-ChlorT"; AS = anionic salts; HCI = hydrochloric acid; L = low dietary Ca; H = high dietary Ca. 2 Composition: 3670 ppm Zn, 3675 ppm Mn, 974 ppm Cu, 29.4 ppm I, 10.6 ppm Co, 29.3 ppm Se, 87690 lU/kg vitamin A, 26140 lUlkg vitamin D, 297 lUlkg vitamin E. 3 Bio-ChlorTM (Biovance, Omaha, NE); composition: 51% CP, 4.6% ADF, 18.2% NDF, 015% Ca, 0.82% P, 0.36% Mg, 1.25% K, 1.95% Na, 2.12% S, 7.95% CI. ‘ Muratic acid, 30% Cl. 116 Table 2. Analyzed chemical composition (% of dietary DM) of treatments with different anion sources and Ca concentrations‘. 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L . RI.'IL .IILII . m u 66 _ C ‘ r md .5... . .. fi‘ 5 4 <6 1 a 6 5 a ...6 < m. W 6 45.54 .64 4 6 a ,- 5.5 n n. d T N H D - ms 6 . I 6 6 6 6 6 5 666 6m 6 «66 6 6 6 - m6 129 CHAPTER 6 HIGH (1.98%) VERSUS LOW (0.48%) CALCIUM IN PREPARTUM DIETS WITH HYDROCHLORIC ACID AFFECTS ACID-BASE STATUS AND CALCIUM HOMEOSTASIS ABSTRACT Objective was to determine if increasing dietary Ca concentration in a prepartum diet with negative dietary cation-anion difference (DCAD; meq [(Na+K)-(CI+S)]I1OO g of dietary DM) improved periparturient Ca status and mineral metabolism of multiparous Holstein cows. Twenty-two nonlactating, pregnant multiparous Holstein cows were assigned randomly to two dietary treatments 28 d before expected day of calving. Before parturition, totally mixed diets were 18% alfalfa silage, 42% corn silage and concentrates (dry basis). Dietary treatments had 0.48% or 1.98% Ca (supplemental CaCOa). The DCAD was( -11.2 meq/100 g of dietary DM) from the inclusion of extruded heat-treated soybean meal treated with hydrochloric acid. After parturition all cows were fed a totally mixed diet containing 21% alfalfa silage, 21% corn silage and concentrates, with 0.98% Ca. Plasma and urine samples were taken daily from 10 d before through 10 d after parturition. Dry matter intake was measured from 130 d 21 prepartum through d 21 postpartum and was not affected by prepartum dietary treatments; however, there was a 30% decrease in DMI from wk 3 prepartum through wk 1 prepartum across both treatments. Cows fed 0.48% Ca had lower plasma pH and bicarbonate concentrations before parturition than cows fed 1.98% Ca prepartum. Cows fed 0.48% Ca had greater plasma Cl concentrations than cows fed 1.98% Ca prepartum. For both treatments, urine pH was lower the week before parturition (about 6.1) compared with the week after parturition (about 8.0). Incidence of clinical hypocalcemia tended to be greater in cows fed 1.98% Ca (36%; P < 0.12) compared with cows fed 0.48% Ca prepartum (9%). Incidence of subclinical hypocalcemia (iCa < 4.0 mgldl) was greater for cows fed 1.98% Ca (64%; P < 0.03) compared with cows fed 0.48% Ca prepartum (18%). Cows fed 1.98% Ca had greater total plasma Ca from d - 10 through d -1, but lower plasma Ca on the day of parturition compared with cows fed 0.48% Ca. Cows had similar plasma ionized Ca concentrations from d -10 through d -1 prepartum regardless of dietary Ca concentration; however, on the day of and the day after parturition, cows fed 1.98% Ca had lower plasma ionized Ca concentrations than cows fed 0.48% Ca prepartum. On d D cows fed 0.48% Ca averaged 4.44 mgldl of plasma iCa compared with 3.68 mgldl for cows fed 1.98% Ca prepartum. Also, cows fed 1.98% Ca prepartum had greater concentrations of parathyroid hormone and 125-dihydroxyvitamin 03 the day of calving and for 2 d after parturition. Peripartum plasma osteocalcin and urine deoxipiridinoline (indicators of bone formation and resorption, respectively) were 131 not different due to prepartum dietary treatments, but each tended to be consistently lower over the entire sampling period for cows fed 1.98% Ga prepartum. Also, cows fed 0.48% Ca had greater plasma concentrations of hydroxyproline from d -10 through d 0 compared with cows fed 1.98% Ca prepartum. Feeding 1.98% Ca prepartum with negative DCAD prepartum increased alkalinity of the blood before parturition and affected Ca metabolism reducing bone Ca mobilization during the periparturient period. (Key words: cation-anion difference, hypocalcemia, calcium metabolism, dairy cows) Abbreviation key: AA = apparent absorption; DCAD = dietary cation-anion difference; iCa/Ca = the ratio of ionized Ca to total plasma Ca; iCa = ionized Ca; IV = intravenous; PTH = parathyroid hormone; SID = strong ion difference; 1,26(OH),D3 = 125-dihydroxyvitamin 03. INTRODUCTION Periparturient hypocalcemia is caused by the rapid and large removal of Ca from the blood into colostrum and milk. Its incidence is approximately 6% for all cows, but incidence in multiparous cows was 13.5% in Michigan (Dyk, 1995). Subclinical hypocalcemia [blood ionized Ca (iCa) < 4 mgldl] can reduce smooth muscle function creating postpartum digestive and reproductive problems (Curtis et al., 1983). Reducing the dietary cation-anion difference (DCAD: meq [(Na + 132 K)- (Cl + S)]I100 9 dietary of DM) prepartum causes mild metabolic acidosis (Moore et al., 1997; Rodriguez et al., 1996; Wang, 1990), increases Ca mobilization from bone (Block, 1984, Freeden et al., 1988a) and possibly enhances absorption of Ca from the digestive tract (Freeden et al., 1988b, Goff et al., 1991a). Several studies showed improved Ca status and reduced incidence of milk fever and other metabolic disorders when cows were fed negative DCAD compared with positive DCAD prepartum (Block, 1984; Goff et al., 1991a; Oetzel et al., 1988; Wang, 1990). Cows fed -2 meq/100 g of dietary DM and 1.27% dietary Ca had a more positive Ca balance up to 6 d before parturition and for 8 d after parturition compared with cows fed -2 meq/100 g of dietary DM and 0.34% dietary Ca (Ender et al., 1971). Also, incidence of subclinical hypocalcemia was 58% for cows fed +19 meq/100 g of dietary DM and 42% for cows fed -8 meq/100 g of dietary DM, each with 0.60% dietary Ca (Oetzel et al., 1988). However, when cows were fed 1.17% dietary Ca the incidence of subclinical hypocalcemia was reduced from 75% with positive to 17% with negative DCAD. Recently, Goff and Horst (1997a) studied the interaction of dietary Ca and K in prepartum diets on peripartum Ca status of Jersey cows. Cows fed diets with 1.1% K (negative DCAD) had 10% incidence of milk fever; however, increasing dietary K to 2.1 and 3.1% (both positive DCAD) increased milk fever incidence to 50 and 48%, respectively. Using intravenous (IV) Ca treatment per milk fever case as an indicator of Ca status, cows fed 1.5% dietary Ca required 133 fewer IV Ca treatments (1 .4lcase) compared with cows fed 0.5% dietary Ca (2.1/case). These results indicate that high dietary K concentrations in prepartum diets reduce the cow's ability to maintain Ca homeostasis and that dietary Ca concentration may influence peripartum Ca status. Several experiments were conducted to study varying DCAD and dietary Ca, but in each study, the two factors were confounded (Moore et al., 1997; Rodriguez et al., 1996; Wang, 1990), or negative DCAD was fed with high dietary Ca (Goff et al., 1991a), or negative DCAD was fed with low dietary Ca (Block, 1984). Results from some of these studies suggest that it may be beneficial to feed higher dietary Ca prepartum than the current recommendation (NRC, 1989), especially with low or negative DCAD. Therefore, the objective of this study was to determine if increasing dietary Ca concentration using CaCO3 in prepartum diets with negative DCAD improves periparturient Ca status and utilization in multiparous Holstein dairy cows. MATERIALS AND METHODS Cows and Diets Twenty-two nonlactating pregnant multiparous Holstein cows were assigned randomly to one of two dietary treatments 28 d before expected day of calving. Treatments were low (0.48%) or high (1 .98%) dietary Ca in the prepartum diet. Diets had the same amount of corn and alfalfa silage with a forage-to-concentrate ratio of 60:40. The low Ca diet had extruded heat-treated 134 soybean meal, extruded heat-treated soybean meal treated with HCI, ground corn grain, oat hulls, soybean oil, minerals, and vitamins (I' able 1). In the high Ca diet, oat hulls were replaced by CaCOs to increase dietary Ca concentration (T able 2). Calcium carbonate was used as the source of supplemental Ca because this is the most common source used in the dairy industry. Prepartum diets had 1.65 Mcal NEL/kg and 15.9% CP. dry basis. Cows were kept in tie-stalls beginning 28 d before expected day of calving, and fed dietary treatments. When signs of parturition were evident, cows were moved to maternity pens and fed treatment diets until parturition occurred (typically just for a few hours). After parturition, all cows were fed the same postpartum diet formulated to have 1.72 Mcal NELIkg and 19.3% CF (Table 2). Postpartum diet had a 47:53 forage-to-concentrate ratio and consisted of alfalfa silage, alfalfa hay, corn silage, soybean meal, whole cottonseed, corn distillers grains, protein pellets, minerals, and vitamins (Table 1). Sampling and Analytical Methods Feed and Orts. Cows were fed individually once per day at 0900 h and orts were weighed daily from d 21 before through d 21 after parturition. Cows were fed to have at least 10% orts daily (as-fed basis). Forage samples were taken weekly and dried (60°C). Concentrate samples were taken weekly. All feed samples were ground through a 2 mm screen in a Thomas-Wiley mill 135 (Arthur Thomas Company, Philadelphia, PA) and composited every 2 wk. Ground feed samples were sent to the Northeast DHIA Laboratory (Ithaca, NY) for CP. NDF, ADF, and mineral element analyses. Blood. Blood samples (20 ml) were taken daily from the tail vein using Li- heparin coated glass tubes at 0800 h from d 14 before expected day of calving through d 10 after calving. Samples were centrifuged at 2800 x g for 20 min to harvest plasma. A plasma aliquot was analyzed within 30 min after sampling in a Stat Profile 4 blood gas and mineral element analyzer (Nova Biomedical, Walthman, MA) to determine pH, p002, and concentrations of H005, iCa, Na, K, and CI. The remainder of the sample was divided into equal parts and frozen at - 20°C in 5 ml plastic tubes. Plasma pH data were analyzed as H” concentration, and data from 10 d before through d 10 after parturition were used in statistical analyses. A frozen plasma sample was sent overnight in dry ice to the USDA National Animal Disease Center (Ames, IA) for parathyroid hormone (PTH), 1,25- dihydroxyvitamin 03 [1,25(OH),D,], and hydroxyproline analyses. Plasma PTH was determined using an inmunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA) previously validated for use with bovine plasma (Goff et al., 1989). Plasma 1,25(OH)2D3 was analyzed using a radioreceptor assay (Reindhart et al., 1984). Plasma hydroxyproline was determined colorimetrically (Daved and Struck, 1971). Plasma osteocalcin was determined using a competitive immunoassay 136 (Novocalcin; Metra Biosystems, Inc., Mountain View, CA). Plasma was deproteinized with 3 ml of 15% tricholoroacetic acid and analyzed for Ca and Mg by flame atomic absorption spectrophotometry (Smith-Heiftje 4000, Therrno Jarrel Ash Corporation, Franklin, MA), and P was by colorimetric assay (Gomori, 1942). Un'ne. Urine samples were collected daily into plastic cups by manually stimulating the area around the vulva at 0700 h from d 14 before expected day of parturition until parturition. Urine pH was measured immediately using a calibrated hand-held pH meter (Hach Company, Loveland, Co). Urine pH data were analyzed as H“ concentration. Urine ketone concentrations were measured from parturition to 9 d postpartum using Ketostix (Bayer, Elkhart, IN). On the morning of d 1 after calving, a 22 french (75 cc ribbed balloon) Bardex‘D Foley urethral catheter (C.R. Bard, lnc., Covington, GA) was inserted into the bladder and connected to a 100 L plastic collection container using Tygon“ R-3603 tubing (Norton, Akron, OH) for total urine collection. Catheter balloons were inflated using 50 ml of sterile saline solution. Ten ml of a 37% formaldehyde solution were added to each collection container as a preservative before connecting the tubing. Urine excretion was measured (by weight) daily at 0700 h from d 1 through d 9 after parturition. Urine specific gravity was measured using a A3OOCL clinical refractometer (Atago Co., LTD, Tokyo, Japan). After mixing 24-h collections, a 100-ml sample was placed daily in plastic cups and frozen at - 20°C. Urine samples were analyzed for Ca, Mg, Na and K by flame atomic 137 absorption spectrophotometry, and for P colorimetrically. Urine samples were sent to the Northeast DHIA Laboratory (Ithaca, NY) for analysis of Cl using an automatic potentiometric titration with silver nitrate using a Brinkman Metrohm 716 titrino titration unit (Brinkman Instruments Inc., Westbury, NY) with silver electrode (Cantliffe et al., 1970). Urine deoxypyridinoline crosslinks were determined using a competitive enzyme immunoassay (Pyrilinks-D; Metra Biosystems, Inc., Mountain \fiew, CA). Plasma and urine samples were analyzed for creatinine colorimetrically (Procedure 555, Sigma Diagnostics, St.Louis, M0) at 500 nm. Concentrations of plasma and urine creatinine were used to determine fractional excretion of different mineral elements as described by Lunn and McGuirk (1990). Feces. Cows were trained to wear fecal bags with harnesses (Wyoming Tent & Awning, Cheyenne, WY) from d -28 through d -21. Fecal bags and harness were placed on each cow from d 1 through d 9 after parturition. Fecal bags were emptied into a plastic storage container twice daily at 0700 and 1800 h. Feces were mixed well and approximately 900 g of feces were subsampled and frozen at -20°C. All fecal samples were dried (60°C) and ground through 5 and 2 mm screens. Fecal samples were sent to the Northeast DHIA Laboratory (Ithaca, NY) for analysis of Ca, P and Mg by inductively coupled atomic emission spectrometry (Sirois et al., 1994). Internal standards were interspersed and sent with each set of experimental samples. Milk. Cows were disconnected from the urine collection container and the 138 tubing was clamped off before each milking. Cows walked to the milking parlor. Milking was in a double-7 herringbone milking parlor twice daily at 0700 and 1700 h. Milk yield was measured using a Perfection 3000 Boumatic weigh meter (Boumatic, Madison, WI) and milk samples were taken using a proportional sampler at each milking. Milk samples from each milking were mixed well and approximately 100 ml was transferred into plastic cups and frozen at -20°C. Milk (5 ml) from both milkings was composited by volume according to milk yield at each milking and placed in a 100-ml volumetric Hach flask (Hach Company, Loveland, CO) and wet-ashed with 10 ml of nitric acid using a PC-520 Corning hot plate (Corning, NY) at 320°C. Red fumes appeared and samples were evaporated to dryness. Dry samples were reconstituted with 4 ml of nitric acid and approximately 2 ml of hydrogen peroxide were added slowly dropwise until dryness. This procedure was repeated as needed and samples were considered digested when all red fumes disappeared. Digested samples were diluted with 4 ml of nitric acid and deionized distilled water. Samples were analyzed for Ca and Mg by flame atomic absorption spectrophotometry and for P colorimetrically. Mineral Balances. Postpartum mineral element balances were calculated as mineral intake (gld) minus mineral excretion in feces and urine, and secretion into milk for d 1 through d 9 postpartum. Intake, excretion and secretion data from the same day were used to compute daily balance. Amounts of mineral elements used during treatment of hypocalcemic cows were added to the mineral balance calculation. 139 Hypocalcemia Categories Cows were considered clinically hypocalcemic when recumbent or when their plasma iCa concentration was < 3.0 mgldl. When clinically hypocalcemic cows were treated with IV administration of 10.7 g of Ca as Ca gluconate (CMPK-solution; Vedco Inc., St. Joseph, MO), oral administration of 39 g of Ca as CaCl2 (CMPK—oral; Vedco Inc., St. Joseph, MO), and intramuscular administration of 0.52 g of P as 4-dimethylamino-2-methy-phenyl phosphinic acid (PhosphAid; Vedco Inc., St. Joseph, MO). They were considered to be subclinically hypocalcemic when plasma iCa was below 4 mgldl at any time during the periparturient period (Curtis et al., 1983). Statistical Analysis Data were analyzed by multivariate repeated measures model using PROC MIXED (SAS, 1996). The model consisted of treatment, day, treatment by day interaction, and residual error. Data from d -21 through d 21 were used in analysis of treatment and day effects on DMI. Data from d 0 through d 21 were used in analysis of treatment and day effects on milk yield. Data from d -10 through d 10 were used in analysis of treatment and day effects on plasma and urine acid-base, and plasma and urine mineral element concentrations. Data from d 1 through d 9 were used in analysis of treatment and day effects on mineral element balances. Data from all cows were included in all data summaries and statistical analyses for all variables. Correlations between 140 variables were analyzed using PROC CORR (SAS, 1996). Categorical variables such as incidence of milk fever, abomasum displacement, retained placenta, and mastitis were analyzed using contingency table analysis. Least-squares treatment means are presented. Statistical significance was declared at P < 0.05, unless otherwise indicated. RESULTS AND DISCUSSION Diet Composition Prepartum diets averaged 15.9% CF and postpartum diet was 19.3% CF (I' able 2). Dietary treatments had similar ADF and NDF concentrations prepartum and postpartum. Diets averaged 0.48% for low and 1.98% for high dietary Ca prepartum, and 0.98% Ca postpartum. Concentrations of dietary P, Mg, K, Na, CI and S were similar for prepartum diets. Postpartum dietary concentrations of P, Mg, Na and K were greater than prepartum, but Cl concentrations were greater in the prepartum diets due to the inclusion of extruded heat-treated soybean meal treated with HCI. Sulfur concentrations were similar between prepartum diets. Increasing the amount of CI in the prepartum dietary treatments lowered DCAD to about -1'1 meq/100 g of dietary DM, whereas in the postpartum diet DCAD was + 31 meq/100 g of dietary DM. DMI, Milk Yield and Metabolic Disorder Incidence 141 Figure 1 shows peripartum DMl from d 21 before through d 21 after parturition. Dry matter intake over this time interval was not affected by dietary treatments. There was a day effect on DMI (P < 0.01). Cows decreased DMI from 14.7 kgld (mean from d -21 through d -15) to 10.5 kgld (mean from d -7 through -1) before parturition. Similar, reductions in DMI of prepartum cows was reported previously in the literature (Bertics et al., 1992; Goff and Horst, 1997a). After parturition, DMI increased at a rate of 2.9%/d from d 0 through d 21 pooled across treatments. When data were analyzed separately before and after calving, there was a treatment by day interaction (P < 0.01) on DMI before calving. Cows fed 0.48% Ca had slightly higher DMI from d 18 through d 14, and lower DMI from d 9 through d 3 before parturition; however, they had greater DMI the day before and the day of parturition compared with cows fed 1.98% Ca prepartum. Goff and Horst (1997a) reported a 3.2% greater DMI in cows fed 1.5% Ca compared with cows fed 0.5% Ca during the 10 d prior to calving, but dietary Ca had no effect on DMI the first 2 weeks after calving. There were no effects of prepartum dietary treatments on postpartum DMI intake. Milk yields from d 0 through d 21 were not affected by prepartum dietary treatments (Figure 2). There was a day effect as expected in early lactation with milk yield increasing over this period of time (P < 0.01). Pooled across prepartum dietary treatments, daily mean milk yield on d 0 was 21.6 kg increasing to 45.4 kg on d 21. Incidence of clinical hypocalcemia of cows fed the prepartum dietary 142 treatments was not different (P < 0.12; Table 3), but was numerically lower for cows fed 0.48% Ca, 9% (1/11 cows) compared with 36% (4/11 cows) for cows fed 1.98% Ca prepartum. However, incidence of subclinical hypocalcemia (iCa < 4.0 mgldl) was greater for cows fed 1.98% Ca (64%, 7/11 cows; P < 0.03) compared with cows fed 0.48% Ca prepartum (18%; 2/11 cows). Several experiments reported lower incidence of clinical and subclinical hypocalcemia by lowering DCAD (Block, 1984; Moore et al., 1997; Rodriguez et al., 1996; Wang, 1990). However, in these studies low or high dietary Ca was used, or dietary Ca was increased at the same time DCAD was reduced. Very few studies have tested the interaction of DCAD and dietary Ca concentration. Ender et al. (1971) found no difference in the incidence of clinical hypocalcemia between cows fed 0.34% or 1.27% Ca with negative DCAD prepartum; however, the incidence was 56% in cows fed positive DCAD. Similarly, Oetzel et al. (1988) found no difference in the incidence of clinical hypocalcemia in cows fed 0.6 and 1.17% Ca with negative DCAD prepartum; however, incidence of subclinical hypocalcemia was lower in cows fed negative DCAD and high compared with low dietary Ca. Recently, Goff and Horst (1997a) studied the effect of varying Ca and K in prepartum diets on the incidence of clinical hypocalcemia. In their study, cows fed positive DCAD had greater incidence of clinical hypocalcemia than cows fed negative DCAD, pooled across dietary Ca concentrations. However, there was no incidence of clinical hypocalcemia in cows fed low dietary K (negative DCAD) with 0.5% Ca, but the incidence was 20% when cows were 143 fed negative DCAD with 1.5% Ca. Postpartum urine ketone concentrations were not affected by prepartum dietary treatment, but increased across time (P < 0.01; data not shown). Pooled across prepartum dietary treatments, urine ketone concentrations gradually increased from 0 mgldl at parturition about 20 mgldl on d 10 postpartum. Incidence of abomasum displacement, retained placenta, and mastitis were not affected by prepartum dietary treatment (Table 3). However, overall incidence were 14% (3/22 cows), 5% (1/22 cows) and 9% (2122 cows) for abomasum displacement, retained placenta, and mastitis, respectively. Plasma Acid-Base Status There was a treatment by day interaction (P < 0.01) on peripartum plasma pH (Figure 3). Cows fed 0.48% Ca had lower plasma pH from d 6 prepartum through parturition, but greater plasma pH on d 1 after parturition, and then lower plasma pH from d 3 through 10 compared with cows fed 1.98% Ca prepartum. Greater plasma pH on d 1 for cows fed 0.48% Ca prepartum may be due to the greater DMI of these cows on the day of parturition. Pooled across prepartum dietary treatments, cows had lower plasma pH prior to parturition compared with after parturition. This was due to the lower DCAD fed prepartum compared with postpartum. Feeding diets with negative DCAD reduced plasma pH in nonlactating pregnant Jersey cows before parturition (Goff and Horst, 1997a), and nonlactating nonpregnant Holstein cows (Wang and Beede, 1992a). 144 Pooled across prepartum dietary treatments, there was a day effect (P < 0.01) on plasma pCO2 (Figure 4). Plasma pCO, concentrations were lower from d 10 through d 1 before parturition, and increased during the first 10 d after parturition. When the prepartum data were analyzed separately, cows fed 0.48% Ca had lower plasma pCO, before parturition compared with cows fed 1.98% Ca prepartum, pooled across days. Greater plasma pCO2 prepartum in cows fed 1.98% Ca may be due to some alkalinizing effect of the supplemental Ca source (CaCOa). There was a treatment by day interaction (P < 0.03) in plasma HCOa' concentrations (Figure 5). Cows fed 0.48% Ca had lower plasma HCO; concentrations prior to parturition compared with cows fed 1.98% Ca prepartum; however, concentrations were similar after parturition. Pooled across prepartum dietary treatments, cows had lower plasma HCOa' concentrations prior to compared with after parturition, mainly due to the low DCAD fed prepartum. Similarly, cows fed negative DCAD had lower plasma HCOa' concentrations (Goff and Horst, 1997a; Rodriguez et al., 1997) compared with cows fed positive DCAD. Plasma Na, K, and Cl Prepartum dietary treatment had no effect on peripartum plasma Na and K concentrations (Figures 6 and 7), however there was a day effect ( P < 0.01). Pooled across prepartum dietary treatments, plasma Na and K concentrations 145 were greater prepartum than postpartum. Greater plasma Na and K concentrations were observed in cows fed negative DCAD prepartum and positive DCAD postpartum (Block, 1984). Contrary to this, Goff and Horst (1997a) reported no effect of DCAD, calving or dietary Ca on plasma concentrations of Na and K of Jersey cows fed negative DCAD prepartum. In the current study, greater plasma Na and K concentrations prepartum may be due to the negative DCAD fed prepartum compared with positive postpartum. Reducing excretion of Na and K may be one of the mechanisms used by the cow to maintain electroneutrality when large amount of anions (Cl') are fed. Pooled across dietary treatments prepartum, plasma Cl concentrations were about 390 mgldl prepartum; however, concentrations decreased to 344 mgldl by d 10 postpartum (Figure 8). Greater plasma Cl concentrations are due to the greater dietary CI fed prepartum (0.95%) compared with postpartum (0.36%). Also, greater plasma Cl concentrations prepartum than postpartum were observed in cows fed negative DCAD prepartum and positive DCAD postpartum (Block, 1984). Strong ion difference (SID) is defined as meq [Na + K - CI]IL (Stewart, 1983). The major mechanism by which the kidney can affect plasma H+ concentration is by adjusting plasma SID by differential removal of Na, K and Cl from plasma to urine. There was a treatment by day interaction in plasma SID (P < 0.01). Cows fed 0.48% Ca had lower plasma SID prior to parturition compared with cows fed 1.98% Ca prepartum, but after parturition SID was similar (Figure 146 9). Pooled across prepartum dietary treatments, greater plasma Cl (Figure 8) and lower plasma SID (Figure 9) prepartum resulted in lower plasma pH prepartum when cows were fed negative DCAD. However, when cows were fed positive DCAD postpartum, greater plasma pH and SID were observed. Lower plasma pH, HCOa' and SID, and greater plasma Cl concentrations in cows fed 0.48% Ca prior to parturition, may indicate some alkalinizing effect of supplemental CaCO3 in cows fed 1.98% Ca prepartum. Large amounts of Ca and Mg (strong cations) might be able to alkalinize the blood if provided in the diet in the carbonate form (Goff and Horst, 1997a). Plasma Ca, P, and Mg Peripartum (d -10 through d 10) total plasma Ca concentrations were not affected by prepartum dietary treatments (Figure 10). However, when pre- and postpartum data were analyzed separately, cows fed 1.98% Ca had greater total plasma Ca concentrations before parturition compared with cows fed 0.48% Ca, pooled across days. There was a treatment by day interaction on peripartum plasma iCa (P < 0.01; Figure 11). Cows maintained similar plasma iCa concentrations before and after parturition; however, cows fed 0.48% Ca had greater plasma iCa concentrations the day of and the day after parturition compared with cows fed 1.98% Ca prepartum. On d 0 cows fed 0.48% Ca averaged 4.44 mgldl of plasma iCa compared with 3.68 mgldl for cows fed 1.98% Ca prepartum. On average, cows fed 1.98% Ca prepartum were 147 considered subclinical hypocalcemic the day of parturition (subclinical hypocalcemia plasma iCa < 4 mgldl, Curtis et al., 1983). Pooled across prepartum dietary treatments, plasma total Ca and iCa concentrations decreased about 15% the day of parturition compared with concentrations the week before parturition. Similarly, lower plasma Ca concentrations were reported the day of parturition (Block, 1984; Gaynor et al., 1989; Goff and Horst, 1997a; Joyce et al., 1997). Figure 12 shows the ratio of plasma iCa to total plasma Ca (iCalCa) during the peripartum period. There was a treatment by day interaction (P < 0.05). Cows fed 1.98% Ca had lower iCa/Ca from 10 d prior to parturition to 1 d after parturition, but similar ratio from d 3 through d 10 after parturition. Pooled across prepartum dietary treatments, iCa/Ca was greater prepartum (0.50) than postpartum (0.47). Similarly, Ballantine and Herbein (1991) reported iCa/Ca of 0.49, 0.51 and 0.50, 2 wk before, on the day of and 2 wk after parturition, respectively. Joyce et al. (1997) reported greater iCa/Ca in cows fed negative DCAD compared with cows fed positive DCAD prepartum. In the current study, greater iCa/Ca of cows fed 0.48% Ca prepartum, and prior to compared with after parturition may be due to greater dissociation of protein-bound Ca during acidosis (Wang and Beede, 1992a) or to greater contribution of bone resorption relative to dietary Ca in meeting the Ca requirements (Ballantine and Herbein, 1 991 ). Peripartum plasma P concentrations were not affected by prepartum 148 dietary treatment but varied across time (P < 0.01; Figure 13). Pooled across prepartum dietary treatments, plasma P concentrations were lower the day before, the day of, and the day after parturition compared with P concentrations the week before and after parturition. Also, Block (1984) and Goff and Horst (1997a) observed lower plasma P concentrations around time of parturition. Colostrum has higher concentrations of minerals compared with milk (Jennes, 1985). Pool across prepartum treatments, lower plasma Ca and P m concentrations the day of parturition may be partially due to the withdraw of ,4» minerals elements from plasma to the formation of colostrum. Lower plasma Ca and P concentrations in cows fed 1.98% Ca prepartum the day of parturition may be due to lower bone mobilization (Figure 17 and 19); which will be presented subsequently. Measurements of Calcium Metabolism Plasma PTH concentrations were not affected by dietary treatment during the peripartum period (P > 0.11), however, the day of and the day after parturition cows fed 1.98% Ca had greater plasma PTH concentrations compared with cows fed 0.48% Ca prepartum (Figure 15). Higher plasma PTH concentrations in cows fed 1.98% Ca around calving may be due to the lower plasma Ca during this period of time, because PTH secretion is regulated by the plasma Ca concentration (Horst et al., 1994). Also, plasma PTH and iCa were negatively correlated (r= -0.61; P < 0.01) from d -10 to d 0 of the experimental 149 period (Table 5). Pooled across prepartum dietary treatments, plasma PTH concentrations increased six-fold the day of parturition compared with concentrations 10 d before parturition. Also, greater plasma PTH concentrations were reported in cows at parturition (Goff and Horst, 1997a; Goff et al., 1991a). There was tendency for a treatment by day interaction ( P < 0.07) in peripartum plasma 1,25(OH)203 concentrations (Figure 16). Cows fed 0.48% Ca had greater plasma 1,25(OH)2D3 concentrations before parturition, but lower concentrations the day of and the first 2 d after parturition compared with cows fed 1.98% Ca prepartum. Pooled across prepartum dietary treatments, plasma 1,25(OH)2D3 concentrations increased two-fold the day after parturition compared with concentrations before parturition. A similar plasma 1,25(OH)2D3 peak was observed in Jersey cows after parturition (Goff and Horst, 1997a; Goff et al., 1991a). Greater concentrations of plasma PTH and 1,25(OH).,,D3 during the time of parturition are due to the withdraw of Ca for the formation of colostrum. Both plasma PTH and 1,25(OH)2D3 are produced in response to hypocalcemia and aid to increase the entry of Ca into the Ca pool (Horst et al., 1997). Plasma 1,25(OH)2D3 was negatively correlated with plasma iCa (r= -0.69; P < 0.01) and positively correlated with plasma PTH (r= 0.54; P < 0.01) from d -10 to d 0 of the experimental period (Table 5) . Cows fed 1.98% Ca prepartum had the greatest plasma PTH and the lowest plasma Ca concentrations the day of and the day after parturition, may be because plasma PTH concentrations are regulated 150 “w— mainly by plasma Ca concentrations (Horst et al., 1994). Parathyroid hormone stimulates renal 1oc-hydroxylase and production of 1,25(OH)203 (Engstrom et al., 1987). Plasma PTH and 1,25(OH)2D3 act synergistically increasing renal reabsorption of Ca and stimulating osteoclastic bone resorption activity (Horst et al., 1997). Also, plasma 1,25(OH)2D3 stimulates active absorption of Ca from the digestive tract (Horst et al., 1994). Goff et al. (1991 b) reported that production of 1,25(OH)2D3 and osteoclastic bone resorption were temporarily refractory to PTH stimulation in cows fed cationic compared with anionic diets. It is theorized that the increase in plasma 1,25(OH)2D3 concentration is due to increased tissue responsiveness to PTH in cows fed anion sources (Horst et al., 1997). In our study, cows fed 1.98% Ca prepartum had greater plasma PTH and 1,25(OH)203 concentrations after parturition than cows fed 0.48% Ca prepartum. This may be due to the lower plasma Ca concentration at time of parturition in cows fed 1.98% compared with 0.48% Ca prepartum. Cows fed 1.98% Ca prepartum increased plasma 1,25(OH)203 (d 0 through d 2), and somehow high dietary Ca prepartum affected peripartum Ca metabolism, increasing the incidence of clinical and subclinical hypocalcemia. Greater incidence of hypocalcemia in cows fed 1.98% Ca prepartum was due to lower bone mobilization before and on the day of parturition as indicated by peripartum concentrations of plasma hydroxyproline (Figure 17). Osteocalcin is a protein produced by osteoblasts which is thought to be involved in bone formation (Wasserrnan et al., 1993; Delmas, 1995). 151 Osteocalcin is incorporated into the extracellular matrix of bone, but a fraction is released into circulation and can be measured by radioimmunoassay (Lian et al., 1988). Thus, osteocalcin in plasma was measured as an indicator of deposition of Ca into bone. Peripartum plasma osteocalcin concentration was not affected by prepartum dietary treatments (Figure 18). However, cows fed 1.98% Ca had numerically lower plasma osteocalcin concentrations during the peripartum period compared with cows fed 0.48% Ca prepartum. Pooled across prepartum dietary treatments, plasma osteocalcin averaged 18 nglml at d 10 before parturition, decreased to 8 nglml at parturition, and increased to about 15 nglml after parturition. Similarly, Naito et al. (1990) reported lower plasma osteocalcin concentrations in Holstein cows around parturition compared with concentrations before or after parturition. Lower plasma osteocalcin concentrations near parturition likely indicates lower rate of bone formation (Naito el at., 1990). Plasma osteocalcin was negatively correlated with plasma PTH (r= -0.30; P < 0.01) and plasma 1,25(OH)1,D3 (r= -0.36; P < 0.01) and positively correlated with plasma iCa (F 0.34; P < 0.01) from d -10 to cl 0 of the experimental period (Table 5). This indicated cows under Ca stress (periparturient period) reduced the amount of Ca stored into bone. 1 The major organic constituent of bone matrix is type I collagen (Wassennan et al., 1993). Type I collagen is crosslinked by specific molecules such as pyridinium, pyridinoline, and deoxypyridinoline (Delmas 1995; Robins et al., 1994). Deoxypryridoline is released into circulation during the bone 152 resorption process, is excreted intact in the urine, and can be measured as a bone resorption marker (Robins et al., 1994). Urinary excretion of deoxypyridinoline is increased at the time of menopause (Uebelhart et al., 1991) and in osteoporotic humans (Robins et al., 1994). To our knowledge there are no data available characterizing urine deoxypyridinoline in ruminants. There was a tendency for urine deoxypyridinoline concentrations to be greater during the peripartum period in cows fed 0.48% Ca prepartum (P < 0.07; Figure 19). Pooled across prepartum dietary treatments, urine deoxypyridinoline concentrations were low (3.7 nM/mM of creatinine) before parturition, spiked at parturition (5.5 nM/mM of creatinine), and after a 2-d decline increased to about 10 nM/mM of creatinine at d 10 after parturition. Greater urine deoxypyridinoline concentrations on the day of and after parturition indicated greater bone mobilization after the initiation of lactation. Cows fed 1.98% Ca had greater total plasma Ca prepartum compared with cows fed 0.48% Ca prepartum (Figure 10). The primary regulator of transcellular intestinal Ca transport is 1,25(OH)203 (Fullmer, 1992). Increased active transport of Ca from the gastrointestinal tract is likely not responsible for the greater total plasma Ca concentrations before parturition in cows fed 1.98% Ca, because they had lower plasma 1,25(OH)2D3 concentrations before parturition compared with cows fed 0.48% Ca prepartum. However, paracellular transport of Ca from the gastrointestinal tract could be an alternate mechanism to active transport. Paracellular movement of Ca occurs down a concentration 153 gradient because the concentration of Ca in the intestinal lumen is higher than at the serosal pole of the intestinal cell (Bronner, 1992). It appears that before parturition, cows fed 1.98% Ca had greater absorption of Ca because plasma total Ca and urine Ca concentrations were greater compared with cows fed 0.48% Ca prepartum. ln metabolic acidosis, hypercalciuria eliminates the absorbed Ca, therefore maintaining a high flux of Ca through the Ca pool (Freeden et al., 1988b). It is likely that cows fed 1.98% Ca absorbed more Ca passively, but also excreted more Ca in the urine compared with cows fed 0.48% Ca prepartum (Figure 27). Recent evidence suggest that diets with low DCAD fed before parturition caused mild metabolic acidosis and increased responsiveness of bone and kidney to PTH (Gaynor et al., 1989; Goff et al., 1991a). In our study, cows were in mild metabolic acidosis as evidenced by the lower plasma and urine pH before parturition. However, around parturition cows fed 1.98% Ca prepartum had greater concentrations of plasma PTH and 1,25(OH)2D3 compared with cows fed 0.48% Ca prepartum. Greater plasma PTH and 1,25(OH)2D3 concentrations around parturition may be in response to the lower total plasma Ca and iCa when cows were fed 1.98% Ca prepartum because plasma PTH concentrations increase with the onset of hypocalcemia (Horst et al., 1994). Cows fed 1.98% Ca decreased bone mobilization as evidenced by the lower plasma hydroxyproline, and osteocalcin, and urine deoxypyridinoline concentrations compared with cows fed 0.48% Ca prepartum. In nonlactating 154 pregnant cows, Ramberg et al. (1984) reported that as dietary Ca intake increased the fraction of Ca absorbed from the gastrointestinal tract and the fraction removed from bone decreased prepartum. However, total entry of Ca from the gastrointestinal tract to the Ca pool increased, whereas total entry from bone decreased. At parturition in cows fed high dietary Ca, entry of Ca to the Ca pool was completely supplied by absorption of Ca, whereas mobilization of Ca from bone was virtually zero. One day after the onset of lactation, total entry of Ca from the gastrointestinal tract to the Ca pool increased, however, entry of Ca from bone did not increase until d 7 after parturition. Also, Kichura et al., (1982) reported lower plasma Ca, greater peak of plasma 1,25(OH)ZD3, and lower plasma hydroxyproline in cows fed 86 g/d vs. 10 9 Id of dietary Ca prepartum. In our study, it appears that cows fed 1.98% Ca were unable to mobilized bone Ca stores to the same degree as cows fed 0.48% Ca prepartum, as evidenced by the lower plasma iCa and hydroxyproline concentrations, and the greater incidence of clinical and subclinical hypocalcemia at the time of parturition. According to the literature (Ender et al., 1971; Oetzel et al., 1988; Rodriguez et al., 1996; Wang, 1990), it seems that feeding higher dietary Ca with negative DCAD improved Ca status around parturition. However, the high Ca concentration in our study (1 .98%) and that of Goff and Horst (1997a; 1.5%), may be too high, and negatively affected Ca homeostasis around parturition. 155 Urine Measurements Figure 20 shows peripartum urine pH responses of cows fed each dietary treatment across time. Lower excretion of H” (slightly higher urine pH) by prepartum cows fed 1.98% Ca may be due to some alkalinizing effect of CaCOa, because these cows also had higher plasma pH and H00; concentrations, and lower plasma Cl concentrations and SID prepartum compared with cows fed 0.48% Ca prepartum. After parturition urine pH values were similar among cows fed low or high dietary Ca prepartum. Pooled across prepartum dietary treatments, urine pH prepartum averaged 6.0 when cows were fed negative DCAD, but increased to 8.0 by d 3 through d 10 postpartum when cows were fed positive DCAD. Reduced urine pH was observed in cows fed anionic salts (Goff and Horst, 1997a; Moore et al., 1997; Rodriguez et al., 1996; Wang and Beede, 1992a) or HCI (Goff et al., 1997; Goff and Horst, 1997c, Rodriguez et al., 1997) and in goats fed HCI (Freeden et al., 1988a). There was a treatment by day interaction in peripartum urine K concentration (P < 0.04; Figure 23). Cows fed 0.48% Ca had lower urine K concentrations from d 10 before to d 1 after parturition compared with cows fed 1.98% Ca prepartum, however concentrations were similar after d 2 postpartum. This may be due to the lower urine Cl concentration in cows fed 0.48% Ca compared with 1.98% Ca prepartum. The kidneys balances Cl (anion) excretion against Na (cation) excretion to regulate plasma SID in humans (Stewart 1981). However, in cows where dietary K concentrations are greater than Na 156 concentrations, K may be the cation of preference to maintain cow's electroneutrality. There was no effect of prepartum dietary Ca on fractional excretion of K during the peripartum period; however, when analyzed separately cows fed 1.98% Ca had greater fractional excretion of K prepartum than cows fed 0.48% Ca (P < 0.03; Figure 24). Pooled across prepartum dietary treatments, fractional excretion of K decreased before and increased after the day of parturition. This may be due to the greater K concentration and intake of K from the postpartum diet (1 .5%) compared with prepartum (1 .25%). Urine Cl concentrations were lower in cows fed 0.48% Ca from 10 d before through 3 d after parturition compared with cows fed 1.98% Ca prepartum; however, urine Cl concentrations were similar from d 4 through d 10 after parturition (P < 0.03; Figure 25). Pooled across prepartum dietary treatments, urine Cl concentrations were greater prepartum than postpartum. Greater excretion of H“ in urine resulted in greater fractional excretion of Cl and lower fractional excretion of Na and K prepartum when cows were fed negative DCAD. After calving when cows were fed positive DCAD, fractional excretion of Na and K increased and fractional excretion of Cl decreased, increasing plasma SID and lowering plasma H” concentration (higher pH). ‘ Cows fed 0.48% Ca had lower urine Ca concentrations from d 10 before to d 2 after parturition compared with cows fed 1.98% Ca prepartum. However, urine Ca concentrations were similar from d 3 through d 10 after parturition (P < 0.01; Figure 27). Greater urine Ca concentrations in cows fed 1.98% Ca 157 prepartum may be due to greater Ca absorption, and higher total plasma Ca concentration, because urinary Ca excretion is related directly to plasma Ca concentration and is a controlling mechanism to prevent hypercalcemia when Ca entry into plasma is excessive (Ramberg et al., 1984). Pooled across prepartum dietary treatments, urine Ca concentrations declined from about 700 ppm at d 10 before parturition to about 30 ppm at d 2 after parturition, and remained low until d 10 after parturition. Similarly, greater urine Ca concentrations (Joyce et al., 1997) and fractional excretion of Ca were reported when cows were fed negative DCAD (Wang and Beede, 1992a). Peripartum fractional excretion of Ca (Figure 28) was not affected by prepartum dietary treatment. However, fractional excretion of Ca declined from 11% at d 10 before, to 4% the day of parturition and to less than 1% at d 2 after parturition. Plasma Ca concentration is controlled by a coordinated effort of PTH and 1,25(OH)2D3 (Horst et al., 1997). A decrease in plasma Ca causes PTH secretion, and within minutes renal reabsorption of Ca from the glomerular filtrate increases (Horst et al., 1997). Declining fractional excretion of Ca as parturition approaches may be due to the increased PTH concentrations observed around the time of parturition when ca demand is increased (Figure 15). I Pooled across prepartum dietary treatments, urine P concentrations and fractional excretion were greater postpartum than prepartum (Figure 29 and 30). Greater postpartum urine P concentrations may be due to the greater dietary P (0.62%) compared with the prepartum diets (0.41%), or partially due to the 158 intramuscular injections of P in cows with clinical hypocalcemia. There was treatment by day interaction on urine Mg concentration (P < 0.01; Figure 31). Cows fed 1.98% Ca had greater urine Mg concentrations from d 10 before to d 2 after parturition compared with cows fed 0.48% Ca prepartum. However, urine Mg concentrations were somewhat higher in cows fed 0.48% Ca prepartum from d 5 through d 10 after parturition. There was treatment by day interaction on urine fractional excretion of Mg (P < 0.02; Figure 32). Fractional excretion of Mg was similar from d 10 before through d 4 after parturition. However, cows fed 1.98% Ca had lower fractional excretion of Mg from d 5 to d 10 after parturition compared with cows fed 0.48% Ca prepartum. Mineral Balances: Ca, P and Mg Pooled across prepartum dietary treatments, Ca intake increased from 136 gld on d 1 to 151 gld on d 10 postpartum mainly due to increasing DMI during this period of time (Figure 33; P < 0.01). There was a treatment by day interaction on fecal Ca excretion (P < 0.01; Figure 34). Cows fed 1.98% Ca had greater fecal excretion of Ca from d 1 through d 7 postpartum compared with cows fed 0.48% Ca prepartum, but excretion was similar on d 8 and 9 postpartum. Greater fecal excretion of Ca for cows fed 1.98% Ca prepartum is very likely due to the greater intake of dietary Ca cows prepartum and the residual canyover of Ca consumed prepartum on postpartum Ca excretion. There was a treatment by day interaction in apparent absorption (AA) of Ca 159 postpartum (P < 0.01; Figure 35). Cows fed 0.48% Ca prepartum maintained an apparent absorption of Ca around 65 gld during the experimental period; whereas, cows fed 1.98% Ca prepartum had negative AA from d 2 through d 5 postpartum. This response in mainly due to the greater fecal Ca excretion observed in cows fed high dietary Ca prepartum and the likely residual effects of the unabsorbed Ca in the digestive tract. Postpartum secretion of Ca in milk and urine were not affected by prepartum dietary treatment (Figure 36 and 37). Pooled across prepartum dietary treatments, milk Ca secretion increased from 41.7 gld on d 1 to 53.7 gld on d 9 postpartum, mainly due to the increased milk production during this period of time. Balance of Ca was calculated as mineral intake (gld) plus oral Ca treatment minus mineral excretion in feces and urine, secretion into milk, plus lV Ca administration for d 1 through d 9 postpartum. There was a treatment by day interaction on apparent balance of Ca (P < 0.01; Figure 38). Average Ca balance for cows fed 0.48% Ca was 14.6 gld after parturition; however, cows fed 1.98% Ca prepartum were in negative balance throughout the experimental period. Negative balance of Ca for cows fed 1.98% Ca prepartum was due mainly to greater fecal excretion of Ca. The most plausible explanation for the negative AA and balance of Ca in cows fed high vs. low dietary Ca prepartum was the high amount of dietary Ca remaining in the digestive tract from the prepartum diet even after parturition. On average, cows fed fed 1.98% Ca 160 consumed 230.8 g Ca/d during the last 10 d before calving compared with 51.6 g Cald for cows fed 0.48% Ca. This likely resulted in considerable residual dietary Ca being excreted postpartum, although it was ingested prepartum. This caused an underestimation of AA and balance of Ca postpartum for cows fed high dietary Ca. Pooled across prepartum dietary treatments, intake of P increased from about 83 gld on d 1 to 95 gld on d 9 postpartum, mainly due to the increased DMI during the same period (P < 0.01; Figure 39). Pooled across prepartum dietary treatments, fecal excretion of P increased from 29 gld on d 1 to 47 gld on d 9 postpartum (P < 0.01; Figure 40). Greater fecal excretion of P was due mainly to the increased intake of P because rate of fecal excretion of P is related directly to the intake of P (Braithwaite, 1976). The AA of P, secretion of P into milk and excretion in urine, and apparent balance of P were no affected by prepartum dietary treatments or day (Figure 41, 42, 43, and 44). However, pooled across prepartum dietary treatments P secreted into milk (38 gld), excreted in urine (0.25 gld) and apparently retained (8.8 gld) represented 81, 0.5 and 18.7% of the P apparently absorbed. Contrary to this, ewes fed to ARC (1980) Ca and P recommendations were in negative P balance during early lactation (Braithwaite, 1983b). In our study, positive P balance during early lactation may be due to the high dietary P (0.62%) of the early lactation diet. Pooled across prepartum dietary treatments, intake of Mg increased from 69.4 to 79.8 gld and Mg fecal excretion increased from 29.6 to 50.7 gld from d 1 161 through d 9 postpartum (P < 0.01; Figure 45 and 46). Secretion of Mg in milk averaged 4.4 gld and accounted for 16% of the Mg apparently absorbed from d 1 through d 9 postpartum (Figure 48). Urine Mg excretion increased from 3.1 gld on d 1 to 4.9 g/d on d 9 postpartum (Figure 49). Greater excretion of Mg in urine may be due to progressively greater intake of Mg from d 1 through d 9 postpartum Mg because excretion of Mg in urine reflects availability of Mg in the diet (Littledike and Goff, 1987). Ewes fed plentiful Ca and P or to ARC (1980) recommendations (restricted) experienced negative Ca (Braithwaite, 1983a) and P (Braithwaite, 1983b) balances during early lactation. In our study, cows were in negative Ca balance the first 9 d after parturition. However, cows fed negative DCAD and high dietary Ca prepartum had a more positive Ca balance postpartum compared with cows fed positive DCAD or negative DCAD with low dietary Ca prepartum (Ender et al., 1971). Also, Braithwaite (1976) reported dairy cows in early lactation were in negative Ca balance due to high demand for Ca in milk. In our study, P and Mg balances regardless of dietary Ca prepartum, and Ca balance of cows fed 0.48% Ca prepartum were positive. Differences in Ca balance in the current study, and those from the literature likely are due to the increased residual Ca from the prepartum diet in cows fed high dietary Ca. Also, differences in the P and Mg balances may be due to the greater dietary concentrations of P and Mg, 0.62 and 0.52% postpartum compared with 0.41 and 0.35% prepartum, respectively. 162 ‘ . CONCLUSIONS Higher plasma pH, H00; and lower Cl concentrations in cows fed 1.98% Ca prepartum indicated a slight alkalinizing effect of supplemental CaCO3 on prepartum acid-base status. However, both dietary treatments successfully placed cows in mild metabolic acidosis as shown by the low urine pH prepartum (6.1) compared with postpartum (8.0). Greater incidence of subclinical hypocalcemia and lower plasma iCa the day of parturition showed that high dietary Ca prepartum affected peripartum Ca metabolism. Greater total plasma Ca concentrations and increased urine Ca excretion indicated a greater flux of Ca through the Ca pool in cows fed high dietary Ca, which may be due to greater passive absorption of Ca. Due to the greater concentrations of plasma parathyroid hormone and 1,25(OH)203 the first 2 d after parturition in cows fed high dietary Ca, it seems that the slight alkalinizing effect of supplemental CaCO3 had no effect on plasma 1,25(OH)2D3 production. Lower plasma hydroxyproline and numerically lower plasma osteocalcin and urine deoxypyridinoline when cows were fed high dietary Ca prepartum, indicated slower bone mobilization around the time of parturition, likely due to the greater flux of Ca through the Ca pool. Greater fecal excretion of Ca for cows fed 1.98% Ca prepartum is very likely due to the greater intake of dietary Ca by cows prepartum and to the residual carryover of Ca consumed prepartum on postpartum Ca excretion. Feeding 1.98% Ca prepartum with negative DCAD prepartum was too high and 163 negatively affected Ca homeostasis during the periparturient period. Additional research on the potential interaction of DCAD and dietary Ca prepartum with lower dietary Ca concentrations may prove worthwhile. 164 Table 1. Dietary Mrsdients (% of dietary DM) of prepartum and postpartum dietary treatments. Prepartum fludlsnts 0.48% Ca 1.98% Ca Postpartum Alfalfa silage 17.6 17.6 21.0 Corn silage 42.2 42.2 21.5 Extruded heat-treated SBM1 4.4 4.4 Extruded heat-treated SBM-HCI2 12.3 12.3 Ground corn 8.1 8.1 Mineral-vitamin mix“ 11.0 11.0 Soybean oil 0.7 0.7 Oat hulls 3.7 0.0 Ca carbonate 0.0 3.7 Protein pellets‘ . . . . . . 5.4 Soybean meal . . . . . . 10.3 Cottonseed, whole . . . . . . 7.1 Corn distiller grains . . . . . . 9.2 Alfalfa hay . . . . . . 4.3 Mineral-vitamin mix5 . . . . . . 21.1 ‘ Extruded heat-treated soybean meal; composition: 45.1% CF, 11.1% ADF, 20.0% NDF, 0.36% Ca, 0.64% P, 0.29% Mg, 2.16% K, 0.01% Na, 0.04% CI, 0.33% S. 2 Extruded heat-treated soybean meal, with HCI; composition: 43.5% CP, 10.0% ADF, 15.6% NDF, 0.4% Ca, 0.64% P, 0.30% Mg, 2.15% K, 0.01% Na, 6.31% CI, 0.32% S. 3 Composition: 11.7% CP. 4.4% ADF, 38.7% NDF, 0.14% Ca, 1.2% P, 1.2% Mg, 0.4% K, 0.07% Na, 0.1% CI, 1.3% S, 1.8 ppm Co, 146 ppm Cu, 4.5 ppm l, 382 ppm Mn, 2.9 ppm Se, 398 ppm Zn, 179000 lUlkg vitamin A, 26700 lU/kg vitamin D, 912 lUlkg vitamin E. Ground corn as carner. ‘ Composition: 28.8% CP. 9. 0% ADF, 19 0% NDF, 3.1% Ca, 1. 06% P, 0.55% Mg, 1 2.2% K,0.68% Na, 0.,96%Cl 0..39%S 5 Composition. 10. 7% OF, 5.0% ADF, 11.8% NDF, 1.5% Ca, 1.15% P, 1.25% Mg, 0.44% K, 1.31% Na, 0.24% CI, 0.14% 8,760 ppm Fe, 110 ppm Zn, 230 ppm Mn, 40 ppm Cu, 15400 lUlkg vitamin A, 3500 lUlkg vitamin D, 260 lUlkg vitamin E. Ground corn as carrier. 165 Table 2. Analyzed chemical composition (% of dietary DM) of the prepartum and postpartum dietary treatments‘. Item Prepartum 0.48% Ca 1.98% Ca Postpartum CF 16.0 15.8 19.3 ADF 21.6 20.6 21.4 NDF 33.5 31.7 32.0 Ca 0.48 1.98 0.98 P 0.41 0.41 0.62 Mg 0.34 0.35 0.52 K 1.26 1.24 1.53 Na 0.03 0.02 0.34 CI 0.95 0.95 0.36 S 0.28 0.28 0.21 DCAD’ -11.1 -11.2 +30.8 ‘ Feed samples were taken weekly, dried (60 °C), ground through a 2 mm screen and composited every 2 wk. 2 Dietary cation-anion difference = meq [(Na+K) - (CI+S)]I100 g of dietary DM. 166 Table 3. Incidence of metabolic diseases by cows fed different dietary Ca concentrations with - 11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + 811100 9 of dietary DM) prepartum, and +308 meq/100 g of dietary DM with 0.98% dietary Ca postpartum. Treatments Variable 0.48% Ga 1.98% Ga Total P < Clinical hypocalcemia 1I11 4/11 5/22 0.12 Subclinlcal hypocalcemia1 2/1 1 711 1 9l22 0.03 Abomasum displacement 2/1 1 1I1 1 3/22 NS2 Retained placenta OI1 1 1/1 1 1/22 NS Mastltls 1I11 1I11 2/22 NS ‘ Plasma iCa concentration < 4 mgldl. 2P>0.13 167 Table 4. Mineral element balances pooled across days (1 to 9 postpartum) by cows fed different dietary Ca concentrations with -11.2 meq/100 g of dietary DM (DCAD: meq [Na + K] - [Cl + S]/100 g of dietary DM) prepartum, and +30.8 meq/100 g of dietary DM with 0.98% dietary Ca Postpartum Ca Balance P Balance Mg Balance Items I % Ca 0.48% 1.98% 0.48% 1.98% 0.48% 1.98% Intake, gld 138.7 139.3 87.7 85.7 73.0 71.5 Fecal excretion, g/d 75.7 157.8“ 41.1 41.1 47.0 46.0 AA‘, gld 66.8 -16.0** 48.8 48.1 27.9 28.4 Urinary excretion, gld 0.36 0.85‘ 0.22 0.27 4.7 3.9" Secretion in milk, gld 50.5 49.2 39.4 38.2 4.5 4.3 Apparent retention, gld 16.0 -66.9“ 9.1 9.5 18.6 20.0 ‘ AA = Apparent absorption. " Pairs of treatments means differed (P < 0.01). ' Pairs of treatments means differed (P < 0.05). 168 l_ Table 5. 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Subclinical and clinical hypocalcemia can reduce smooth muscle function, which can cause digestive tract stasis leading to abomasum displacement and (or) reduced feed intake, and poor reproductive performance, including reduced uterine motility, increased retained placenta, and slow uterine involution. Manipulation of DCAD in prepartal diets was successful in reducing the incidence of hypocalcemia in several studies (Block, 1984; Goff et al., 1989; Goff et al., 1991a; Oetzel et al., 1988; Wang, 1990; Wang and Beede, 1992) and other postpartum disorders (Joyce et al., 1997; Lema et al., 1992; Wang, 1990). However, reducing the DCAD by anion supplementation in the field has not always improved Ca status and reduced peripartum problems; possibly due to unknown DCAD, unpalatability inherent with some anion sources and (or), 220 improper ration mixing. Having supplemental anion sources as a separate mix with a carrier allows alteration of the anion inclusion rate through time. Also, urine pH may be a useful measure of the cow's acid-base status when feeding anion sources, helping to access whether the ration is having the desired physiologic effects or not. Therefore, the objective of the Experiment 1 in Chapter 3, was to characterize the temporal pattern of changes in urine pH and blood variables in relation to time of feeding of non-lactating pregnant multiparous Holstein cows fed varying amounts of an anion-Ca supplement. The objective of the Experiment 2 in Chapter 3, was to evaluate acid-base and Ca status, and peripartum health of Holstein cows fed one of three different dietary treatments varying in DCAD and Ca content. In Experiment 1, urine pH averaged 8.1 during the first 4 d when cows were fed +17 meq/100 g of DM. Urine pH decreased from 8.1 to 5.8, and plasma iCa increased linearly for cows fed the anion-Ca supplement. Changes in urine pH and blood plasma measurements related to acid-base status occurred within 2 d after reducing DCAD. In Experiment 2, lowering DCAD while increasing dietary Ca concentration, decreased urine pH and reduced postpartum subclinical hypocalcemia. The overall incidence of milk fever was low (3.4%). The low incidence of milk fever observed in this study likely was due partially to the relatively large proportion (47%) of first parity cows in the experiment. Dietary treatments had no effect on milk fever incidence; however, 221 cows fed anion-Ca supplement had improved plasma iCa status compared with cows fed control. The incidence of displaced abomasum was greater in cows fed anion-Ca supplement compared with those fed control. More than 60% (5l8) of cases of abomasal displacement occurred in first parity cows which did not have subclinical hypocalcemia. I conclude from this study that urine pH can be used as a tool to monitor changes in acid-base status of cows fed supplemental anions before calving. The anion-Ca supplement inclusion rate can be adjusted to vary the DCAD to achieve a targeted urine pH through time, thus reducing periparturient hypocalcemia. It was not beneficial to feed anion-Ca supplement to first parity cows before calving. Varying the DCAD fed to cows before calving can reduce DMI (Joyce et al., 1997; Schoenbaum et al., 1994; Moore et al., 1997). The advantages of lowering DCAD in prepartum diets to control hypocalcemia may be offset by the depression in DMI sometimes observed in cows fed anionic diets. Therefore, it is important to find sources of anions that effectively alter acid-base status and have minimal effects on DMl. Therefore, the objective of Experiment 3 reported in Chapters 4 and 5 was to determine if the addition of different anion sources (Bio-Chlorm, an anionic salts mixture designed to match the anionic and cationic profile of Bio-Chlorm, and HCI) and different dietary Ca concentration from CaCO3 affected feed intake, acid-base status, and macromineral metabolism and utilization of nonlactating nonpregnant Holstein dairy cows. Feeding anion sources to cows previously fed 222 cationic diets with low dietary Ca changed temporal patterns of DMl depending on the anion source used. All anion sources were acidogenic, however, cows fed HCI had the lowest DMI and urine pH, indicating a stronger acidifying effect. I conclude from this study that Bio-Chlorm, anionic salts, and HCI can be used as anion sources to change acid-base status in dairy cows. Changes in urine pH occured within 2 d after cows were fed the anion sources. Feeding anion sources to nonlactating nonpregnant cows reduced DMI, but HCI resulted in the greatest DMI reduction compared with Bio-ChlorTM and anionic salts, which may be due to its stronger acidifying effect. Feeding 2.0% Ca reduced DMI possibly due to the unpalatability of the supplemental Ca source used, CaCOa. Anion supplementation changed acid-base status and reduced urine pH. Even though dietary treatments had similar DCAD (-9.7 meq/100 g of dietary DM), the greater the concentration of CI in the diet the lower the urine pH. This indicates a better acidifying effect or a better absorption of the Cl compared with 8. Also, cows fed higher dietary concentrations of Cl had greater retention of CI in the body. Dietary treatments had no effect on PTH; however, anion supplementation increased concentrations of 1,25(OH)203 in plasma. Greater plasma 1,25(OH)203 may indicate greater tissue responsiveness to PTH in anion supplemented cows. Although feeding 2.0% Ca reduced plasma 1,25(OH)203, greater absorption and retention of Ca in cows fed 2.0% Ca may indicate paracellular transport of Ca from the digestive tract. Hypercalciuria was evident in cows fed anion sources. Greater excretion of Ca in urine may increase the 223 concentration gradient between the gastrointestinal tract and the blood stream, increasing the flux of Ca through the system. Cows fed high dietary Ca had greater AA and balance of Ca and P than cows fed low dietary Ca. I conclude from this study that diets with relatively more Cl anion were better acidifiers and had greater impact on cow's acid-base status. High dietary Ca increased the amount of Ca absorbed and retained in the body, affecting the Ca regulating honnones. Several experiments were conducted to study varying DCAD and dietary Ca, but in each study, the two factors were confounded (Moore et al., 1997; Wang, 1990), or negative DCAD was fed with high dietary Ca (Goff et al., 1991a), or negative DCAD was fed with low dietary Ca (Block, 1984). Results from some of these studies suggest that it may be beneficial to feed higher dietary Ca prepartum than current recommendation (NRC, 1989), especially with low or negative DCAD. However, available studies are inconclusive. Therefore, the objective of Experiment 4 reported in Chapter 6 was to determine if increasing dietary Ca concentration using CaCO3 in prepartum diets with negative DCAD (-11.2 meq/100 g of dietary DM) resulted in improved Ca status and utilization by periparturient multiparous Holstein dairy cows. Both low and high dietary Ca treatments successfully placed cows in mild metabolic acidosis as shown by the low urine pH prepartum (6.1) compared with postpartum (8.0); however, CaCO3 did increase urine and blood pH slightly for cows fed high dietary Ca. Greater incidence of clinical and subclinical 224 hypocalcemia, and lower plasma iCa the day of parturition showed that high dietary Ca prepartum affected peripartum Ca metabolism. Greater total plasma Ca concentrations and increased urine Ca excretion indicated a greater flux of Ca through the Ca pool in cows fed high dietary Ca, which may be due to greater passive absorption of Ca. Due to the greater concentrations of plasma PTH and 1,25(OH)203 the first 2 d after parturition in cows fed high dietary Ca, it seems that the slight alkalinizing effect of supplemental CaCOa had little effect on plasma 1,25(OH)2D3 production. Lower plasma hydroxyproline, and numerically lower plasma osteocalcin and urine deoxypyridinoline when cows were fed high dietary Ca prepartum, indicated lower bone mobilization around the time of parturition. I conclude from this study that cows fed 1.98% Ca and negative DCAD prepartum had a greater flux of Ca through the Ca pool, decreasing bone mobilization before parturition, and may be active transport of Ca from the digestive tract. In conclusion, urine pH can be used as a tool to monitor acid-base changes in response to changing DCAD before calving. Anion supplement inclusion rate can be adjusted to control DCAD and to achieve a targeted urine pH through time. Hydrochloric acid had greater acidifying effect than Bio-ChlorTM and anionic salts, and can be used to reduce hypocalcemia in periparturient dairy cows. Feeding 2.0% dietary Ca increased AA and retention of Ca in nonlactating nonpregnant cows, but cows fed high vs. low dietary Ca might had increased residence time of dietary Ca in the digestive tract causing over- 225 eslir will hor die as he estimation of AA and retention of Ca. In periparturient cows, feeding 1.98% Ca with negative DCAD prepartum was too high and negatively affected Ca homeostasis during the periparturient period. Additional research is needed on the potential interaction of DCAD and dietary Ca prepartum with lower dietary Ca concentrations. In the practical aspect, factoring different dietary Ca concentrations with negative DCAD will help to determine which is the optimum Ca concentration for prepartum diets. Also, the optimal time of feeding negative DCAD needs to be determined. lf acid-base changes occur within 2 d as indicated in the current research, we must determine if the length of time of feeding supplemental anions can be shortened. If urine pH will be the recommended tool for measuring acid-base status, more work is needed to determine the optimum urine pH range and the optimum DCAD prepartum to achieve physiological changes. Practical studies with new and more economical sources of anions and Ca always will be worthwhile. In the basic aspect, it has been hypothesized that metabolic alkalosis changes 1,25(OH)2 D3 receptor structure or function. Further research is needed to determine the role of acid-base status on the production of 1,25(OH)2 D3 and other Ca-regulating hormones. Past research with sheep found absorption of Ca from the rumen (Holler et al., 1988b). Further study is needed to know if and how much Ca can be absorbed from the rumen of dairy cows. Also, better characterization of the relative contributions of the two potential routes of Ca transport from the gut, paracellular or transcellular, could be very worthwhile, to 226 improve our understanding of optimal dietary Ca supplementation to periparturient dairy cows. 227 APPENDIX A TABLES OF STATISTICAL ANALYSIS FOR CHAPTER 3 TABLE A-t. Least squares analysis of variance for dry matter intake (DMl,kgId), urine pH, urine [H’], plasma pH. plasma lH‘l. Exper'rnent1 (days 1 through 0). Source df Dflf‘ Urine pl-l Urine [H1 Plasma Plasma pH ["1 Treatment2 2 30.1 10.57“ 1762.63 0.0241” 0.0060“ Cow (Treatment) 3 23.3 1.18 2159.73 0.0006 0.0001 Day 7 24.4 12.92" 221490" 0.0035 0.0009 Treatment x Day“ 14 10.6 2.59“ 604.10 0.0020 0.0004 Cow (Treatment) x Day 21 9.0 0.60 763.51 0.0032 0.0007 Hour“ 3 0.12‘ 43.67 0.0132“ 0.0032“ Day it Hour' 21 0.04 14.81 0.0066“ 0.0019 Treatment x Hour‘ 6 006+ 70.01“ 0.0028 0.0008 Treatment x Day x Hour 42 0.03 30.76 0.0019 0.0004 CONTRASTS T1 vs T2,T3” 1 20.37‘ 3120.48 0.00004 0.0000 T2 vs T32 1 0.78 405.17 0.0482“ 0.0120“ Day (Linear, L)’ 1 68.68” 9397.73” 0.00531 0.00179 Day (Quadratic, Q)’ 1 6.23“ 2696.59 0.00451 0.00143 Day (Cubic,C)3 1 0.08 44.17 0.00013 0.00017 Day (Quartic, Qr)‘| 1 11.61 " 1502.16 0.00058 0.00008 T1 vs T2,T3 x L3 1 23.75“ 4594.70‘ 0.00526 0.00149 T1 vs T2,T3 x Q’ 1 3.53‘ 1340.95 0.00001 0.00001 T1 vs T2,T3 x C’ 1 1.28 23.57 0.00951 0.00210 T1 vs T2,T3 x Or3 1 3.22' 724.08 0.00156 0.00020 T2 vs T3 x L3 1 1.49 545.69 0.00019 0.00002 T2 vs T3 at Q’ 1 0.03 105.16 0.00081 0.00025 T2 vs T3 x C’ 1 0.60 1.63 0.00093 0.00014 T2 vs T3 it Or“ 1 0.03 1.54 0.00014 0.00009 ”P<0.01; 'P<0.05; +P<0.10 ‘ Degrees of freedom for DMI were: 2 for Treatment, 3 for Cow (Treatment). 7 for Day, 14 for Treatment x Day and 47 for Total. ' 2 Tested using Cow (Treatment) as a test term. ’Tested using Cow (Treatment) at Day as atestterrn. ‘ Tested using Treatment x Day x Hour as a test term. 5 T1 = Treatment 1, DCAD I + 17 meq/1009 of DM and 0.61% Ca. T2 8 Treatment 2, DCAD = -11 meq/1009 of DM and 1.01% Ca. T3 a Treatment 3, DCAD = -26 meq/1009 of DM and 1.66% Ca. 228 TABLE A-2. Least squares analysis of variance for plasma ionized Calcium (iCa, mgldl), Sodium (mgldl), Potassium (mgldl) and Chloride (mgldl), Experiment 1 (days 1 through 8). Source df ICa Na K CI Treatment' 2 0.042 33.42 7.476 5281 .4 Cow (Treatment) 3 0.150 123.85 8.269 6717.1 Day 7 0.075' 100.99“ 4.360‘ 6617.1” Treatment x Day"' 14 0.025 37.91 1.752 1831.7 Cow (Treatment) x Day 21 0.030 25.66 1.550 1731.8 Hour“ 3 0.031 245.33” 6.0314 927.2 Day 1! Hour' 21 0.029 20.66‘ 0.922 931.7 Treatment x Hour“ 6 0.044 19.03 1.564 363.0 Treatment x Day It Hour 42 0.019 11.11 0.930 785.6 CONTRASTS T1 vs T2,T3” 1 0.0712 59.89 1.621 4353.7 T2 vs T31 1 0.0138 6.95 13.32 6209.1 Day (Linear, L)2 1 0.0015 157.12‘ 0.076 36145.3” Day (Quadratic, Q)2 1 0.0519 24.61 15.61 1" 56.4 Day (Cubic.C)’ 1 0.238' 1 13.19' 7.1 14' 2400.4 Day (Quartic, Qr)’ 1 0.0620 254.01' 3.176 4203.2 T1 vs T2,T3 x L2 1 0.1099 180.30' 7.933‘ 629.7 T1 vs T2,T3 x Q2 1 0.0180 29.80 1.889 78.2 T1 vs T2,T3 x C2 1 0.0576 75.40 2.285 1150.2 T1 vs T2,T3 x Gr2 1 0.0050 2.55 1.685 1416.0 T2 vs T3 x L2 1 0.0000 42.02 2.712 12393.9' T2 vs T3 x Q2 1 0.0087 11.61 0.097 726.3 T2 vs T3 x C2 1 0.0040 81.89 3.363 5106.6 T2 vs T3 x Qr’ 1 0.0035 9.53 1.046 1914.4 fi< 0.01; ' P < 0.05; + P < 0.10 ‘ Tested using Cow (Treatment) as a test term. 2 Tested using Cow (Treatment) x Day as a test term. ’ Tested using Treatment x Day x Hour as a test term. ‘ T1 = Treatment 1, DCAD = + 17 meq/1009 of DM and 0.61% Ca. 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N «2...... 8.» +8....” 9.3 8.3 33 .8. .3 .83 ooo.o m8... . .888 r... .... .8 .20 b.» .u x ..z 8. «58... 2.8... ... 85% .N .coEteaxm 8.2.8.8.. ... ...5. 2.958.. :0 2.8 new ......oEE :05. 22.3.8... .380... .06. 8:22.... ..o. 9.2.... .83.... 8.5.8 ......9... 22.88.. ......9... ......8m ......8. .8.. 8.0.8 858. «as... ...... 8.33 .... 8.2.... .o. 88...... .o «.38.. 8.28 .33 ...... 22.. 231 TABLE A-5. Chi-aquam values from Chi-squat. analysis for the caboodcal variable: milk favor (MF). displaced abomasum (DA). retained placonta (RP). metritis (MT) and dead calf (DC) Experiment 2. Com df ”F DA RP HT DC Tm 1 vs 2,3' 1 0.951 7.162” 0.209 0.051 0.161 TM 2 vs 3 1 0.030 0.714 0.007 0.669 0.513 Parity 1 vs 2.3+: 1 0.000 0.826 1.004 0.502 3317+ PIM 2 vs 3+ 1 7.481“ 0.334 0.546 0.018 1.713 “P<0.01;'P<0.05;+P<0.10 ‘T1=Treatment1.DCAD-+11meql1009 ofDM and 0.63% Ca. T2=Treatmen12. DCAD=-11 meq/1009 ofDMand 0.95% Ca. T'3-TroaUnont3.DCAD=-26mql1009010Mand1.17%Ca. ’Pafity1scawsca1ving 1attimo.Parity2=cowscalvin92ndtimeand Parity3+=cowswith30rmorecalvings. 232 APPENDIX B TABLES OF STATISTICAL ANALYSIS FOR CHAPTER 4 Er'_-_'1' ~ TABLE 8-1. Test of fixed effects. F values for dry matter intake (DMI). water intake 0M) and urine plflUpH). Source dt DMI WI UpH Covariate‘ 1 169.48” 83.54” 0.15 Treatment 7 3.44' 1.71 6.09“ Day 20 8.48“ 11.31“ 14.15“ Treatment x Day 140 1.32' 1.58“ 1.72“ CONTRASTS’ C vs 8". AS,HCI 1 9.71“ 0.71 37.53“ B" vs AS,HCI 1 0.54 0.23 2.18 AS vs HCl 1 6.30' 0.57 0.99 Low Ca vs High Ca 1 5.59' 5.46' 0.21 C vs 8"", AS,HCI x Ca 1 1.51 1.88 0.14 8"" vs AS.HC| x Ca 1 0.01 0.01 1.53 AS vs HCI x Ca 1 0.15 2.75 0.42 Day (Linear. L) 1 16.01“ 4.70‘ 0.00 Day (Quadratic. Q) 1 1.36 0.30 70.97" Day (Cubic.C) 1 14.46“ 24.78“ 44.37" Day (Quartic. Or) 1 1 .59 22.28“ 1 .13 C vs 87", AS,HCI x L 1 0.77 0.13 0.00 C vs 8““, AS.HCI x Q 1 2.06 280+ 23.80“ C vs 8'“. AS,HCI x C 1 1.07 2.93+ 15.11“ C vs 8"". AS.HCl x Or 1 0.47 0.35 0.37 8““ vs AS.HC| x L 1 0.21 0.01 0.43 8““ vs AS.HCI x Q 1 0.04 0.72 3.97‘ 8"" vs AS.HCI x C 1 1.77 1.29 6.82“ B" vs AS.HC| x Or 1 0.22 0.63 0.30 AS vs HCI x L 1 14.6“ 593" 322+ AS vs HCI x Q 1 0.03 2.18 0.54 A8 vs HCI x C 1 4.78' 9.14“ , 280+ AS vs HCI x Qr 1 2.58 0.08 0.35 Low Ca vs High Ca x L 1 1.51 0.01 1.43 Low Ca vs High Ca x Q 1 0.35 1.53 5.49' Low CavsHigh CaxC 1 1.13 0.11 7.59” Low Ca vs HEB Ca x Or 1 4.37‘ 0.21 0.70 " P<0.01; ‘P <0.05; + P<0.10 ' Covariate for DMI and WI = means from d 7 before switching diets to d O; Covariate for urine pH = means from d 2 before switching diets to d O; 2 C =- control; B" =- Biochlor‘“; AS 8 Anionic Salts; HCI = Hydrochloric acid. Low Ca = 0.5% Ca; High Ca = 2.1% Ca 233 APPENDIX C TABLES OF STATISTICAL ANALYSIS FOR CHAPTER 5 \I‘Ilt :IIWYI. {1 .8 .....3 u 8 8.... .8 .8... u 8 5.. .28 22.8.2... u .o: 5.8 0.5.5.. u 2. 8.288 u an .228 n o . ...... v n. + .8... 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