if. z .i. raf. , .12....3 .. an. mm ‘ i , a... as. z“ 81:: (is. {Q .nwuttw . 1 . "my. fix . .3. «fig. tantrum. at» a... 1 d 1.. » _.,.u.w..m 1-. .70»... z. . uwfirflufi tn? .. 3:. . 7: ‘ . ‘mewfimmwmfi . ifivxfl . u 1. if? ”zany V - 9. L1,. . 351.4?! 31.9... 24...».5: ., z. (t {T . .t . {UPI} 9 he i 1 .0: . .. ‘ .51.... «3*-asfiupzhmfiét . I u w: .va .1. '3 . .LI 1.. If. .5 IE! ill. .3» b. . 4.... m ICI‘IIGAN STATE UNIVERSITY LIBRARIES Milli!\llllllllllllllllllHllflLlljlll 3 1293 02074 2 Liananv Michigan State University This is to certify that the thesis entitled The Effect of Ascorbic Acid and L-histidine Therapy on Acute Mammary Inflammation in Dairy Cattle. presented by Anantachai Chaiyotwittayahm has been accepted towards fulfillment of the requirements for Master of Sciences ‘ ' ’ ' i . ' degree in Large Annual Clinical Scrences / /’ ”W223? June 17". 1999 I)ate 0-7639 MSU i: an Aflirmativc Action/Equal Opportunity Institution PLACE IN RETURN Box to remove thi To AVOID FINES return on MAY BE RECALLED with earlier 5 checkout from your record. or before date due. due date if requested. DATE DUE DATE DUE DATE DUE woo armor-peso.“ THE EFFECT OF ASCORBIC ACID AND L-HISTIDINE THERAPY ON ACUTE MAMMARY INFLAMMATION IN DAIRY CATTLE By Anantachai Chaiyotwittayakun A THESIS Submitted to Michigan State University in partial fullfilment of the requirements for the degree of MASTER OF SCIENCES Department of Large Animal Clinical Sciences 1 999 ABSTRACT THE EFFECT OF ASCORBIC ACID AND L-HISTIDINE THERAPY ON ACUTE MAMMARY INFLAMMATION IN DAIRY CATTLE By Anantachai Chaiyotwittayakun Eight, non-pregnant Holstein cows with endotoxin-induced mastitis were selected to determine the effects of intravenous administration of L-histidine (L-Hi s) and ascorbic acid (AA) by conducting in the Latin square crossover design. Repeated measurement analysis (SAS) was used to compare cows with an individual treatment groups; control, AA only, L-His only, and AA plus L-His by testing rectal temperature, milk production, somatic cell count, milk IgGl, antioxidant activities, heart rate, respiratory rate, ruminal contraction rate and dry matter intake. AA treatments has a beneficial potential effect to increase recovery of milk production, and help to maintain DMI. However, both AA & L-His were not affected heart and respiratory rate. ACKNOWLEDGEMENTS Many people need to be mentioned and thanked for their help and support of my marvelous study life here during the last there years. First, I would like to thank the Civil Service of Commission Office, Bangkok, Thailand for a great support by giving me a scholarship. I also thank for all convenience from staffs in Office of Educational Affair (now closed), the Royal Tlmi Embassy, Washington, DC. I also thank staffs at Michigan State University dairy farm particularly Robert Krefi (Bob), a manager, who tried very hard to find available cows for my experiment. Thanks to all cows for your corporations. I would like to acknowledge my committee members, Dr. Ronald J. Erskine (Uncle Ron, my major advisor), Dr. Paul C. Bartlett, Dr. Phillip M. Sears, and Dr. Thomas H. Herdt for your great help and support in research experience and knowledge. A special thanks to the Erskines and the Bartletts for giving me opportunities to learn a lot about American cultures in many occasions and for making me feel comfortable while I have been thousands miles away from home. I also thank Dr. R J. Harmon, University of Kentucky, Lexington, Kentucky, and David A. Brigham, Animal Health Diagnostic Laboratory, Nutrition Section, MSU for taking care a ton of my serum samples. Dr. Larry Gudge, you are my good friend who took care of me when I first arrived this institution, as well as Dr. Ozlem Akpinar and other fi-iends both in USA and in Thailand. Chris Phipps, a laboratory technician, are thanked for being a good friend and laboratory assistant. You were the one who trained students to assist me both in mastitis iii laboratory and at MSU dairy farm. Those students include Brian Dawson, Brian Preston, Jason Valente, Terese Burns, Julie and Michelle. At last, I would like to thank my grandfather (Prasit Phakam), my parents (Pongpetch & Nurot), my Siblings, Ponganan & Anucha for your love and support. And thanks for always giving me a great inspiration to have a strong mind and to fight any obstacle in my life in every moments. Thanks to everybody for everything. iv TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... vii LIST OF FIGURES ........................................................................................................ xi KEY TO ABBREVIATIONS ....................................................................................... xvii INTRODUCTION ........................................................................................................... 1 REVIEW OF LITERATURES ........................................................................................ 3 Epidemiology of Clinical and Acute Clinical Mastitis ............................................ 3 Economic Impact .................................................................................................. 5 Pathogenesis ......................................................................................................... 5 Acute Phase Response ........................................................................................ 10 Therapy and Prevention. ..................................................................................... 11 Nutrition in Coliform Mastitis ............................................................................. 14 Antioxidants as Therapy ..................................................................................... 14 Ascorbic Acid & L-histidine ................................................................................ 15 MATERIALS AND METHODS ................................................................................... l9 Cows ................................................................................................................. 19 Endotoxin—induced Mastitis ................................................................................ 19 L-histidine & Ascorbic acid Solution Preparation. ............................................... 19 Experimental Design. .......................................................................................... 20 Milk Collection. .................................................................................................. 21 Blood Collection. ................................................................................................ 21 Ascorbic Acid Protocol ....................................................................................... 22 Antioxidant Activities (AOA) Protocol ............................................................... 23 Clinical Monitoring ............................................................................................. 24 Milking Procedure .............................................................................................. 24 Statistical Analysis .............................................................................................. 24 RESULTS ..................................................................................................................... 26 Rectal Temperature ............................................................................................ 26 Somatic Cell Count ............................................................................................. 26 Quarter Milk Production. .................................................................................... 31 Composite Milk Production. ............................................................................... 31 Milk I gG; ........................................................................................................... 31 Antioxidant Activities ........................................................................................ 41 Heart Rate ......................................................................................................... 41 Respiratory Rate ................................................................................................. 41 Ruminal Contraction Rate ................................................................................... 46 Dry Matter Intake ............................................................................................... 46 DISCUSSION ............................................................................................................... 50 APPENDICES ............................................................................................................... 55 APPENDIX A. .............................................................................................................. 56 APPENDIX B ................................................................................................................ 84 APPENDIX C ................................................................................................................ 86 APPENDIX D .............................. ' ................................................................................. 97 Electrochemical Detection/HPLC Analysis of Ascorbic Acid. ..................................... 97 Materials .................................................................................................. 97 Specificatiom ........................................................................................... 97 HPLC System ......................................................................................... 97 Mobile Phase ................................................................................................... 97 Approximate Settings ................................................................... 98 HPLC Mobile Phase Preparation. .............................................................. 98 Mobile Phase Solution. .................................................................. 98 HPLC Standard. ........................................................................... 99 Standard Buffer ............................................................................ 99 Standard Solution. ....................................................................... 100 Sample Preparation. .................................................................... 101 Tissue Buffer .................................................................. 101 Phsma Preparation. ......................................................... 101 Measurement of Ascorbic Acid and Uric Acid. ............................................. 101 Precipitation. ..................................................................................... 101 Analyzing with HPLC-EC ................................................................. 102 Calcuhtion of Results ....................................................................... 103 Troubleshooting ............................................................................................ 103 APPENDD( E ....................................................................................................................... 105 Phycoerythrin Fluorescence-Based Assay for reactive oxygen species ...................... 105 Reagents ............................................................................................................... 105 Experirmntal Procedure .......................................................................... 105 APPENDD( F ....................................................................................................................... 107 SAS & Output. .......................................................................................................... 107 REFFERENCES .................................................................................................................... 118 vi ‘ ‘ 0‘ ( all-\VLaC LIST OF TABLES Table 1 Mean Rectal Temperature by Hours Following LPS Challenge in Four Different Treatments; 00: LPS alone, CO: LPS + Ascorbic acid, OH: LPS + Histidine, and CH: LPS + Ascorbic acid + Histidine in dairy cattle. ........................... Table 2 Mean Rectal Temperature as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Diflerent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ....................................................................................................................... Table 3 Change of Mean Rectal Temperature as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ....................................................................................................................... Table 4 Mean Somatic Cell Count (SCC) by Hours Following LPS Challenge in Four Difl‘erent Treatments; OO: LPS alone, CO: LPS + Ascorbic acid, OH: LPS + Histidine, and CH: LPS + Ascorbic acid + Histidine in dairy cattle. ........................... Table 5 Mean Somatic Cell Count (SCC) as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ....................................................................................................................... Table 6 Mean Quarter Milk Production by Hours Following LPS Challenge in Four Different Treatments; OO: LPS alone, CO: LPS + Ascorbic acid, OH: LPS + Histidine, and CH: LPS + Ascorbic acid + Histidine in dairy cattle. ........................... Table 7 Mean Quarter Milk Production as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in F our Different Treatments; 00 (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ....................................................................................................................... Table 8 Mean Daily Quarter Milk Production as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Diflerent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ....................................................................................................................... vii 56 57 58 59 60 61 62 63 Table 9 Change of Mean Daily Quarter Milk Production as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Difi‘erent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. .......................................................................................................... Table 10 Mean Composite Milk Production by Hours Following LPS Challenge in Four Difi‘erent Treatments; OO: LPS alone, CO: LPS + Ascorbic acid, OH: LPS + Histidine, and CH: LPS + Ascorbic acid + Histidine in dairy cattle. ........................... Table 11 Mean Composite Milk Production as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Difl'erent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ....................................................................................................................... Table 12 Mean Daily Composite Milk Production as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ....................................................................................................................... Table 13 Change of Mean Change of Daily Composite Milk Production as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Diflerent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ................................................................................ Table 14 Mean Milk IgGr by Hours Following LPS Challenge in Four Diflemnt Treatments; OO: LPS alone, CO: LPS + Ascorbic acid, OH: LPS + Histidine, and CH: LPS + Ascorbic acid + Histidine in dairy cattle. ................................................. Table 15 Mean Milk IgG. as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ........................ Table 16 Mean Antioxidant Activities (AOA) as % Inhibition by Hours Following LPS Challenge in Four Different Treatments; OO: LPS alone, CO: LPS + Ascorbic acid, OH: LPS + Histidine, and CH: LPS + Ascorbic acid + Histidine in dairy cattle. Table 17 Mean Antioxidant Activities (AOA, % inhibition) as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours viii 65 66 67 68 69 70 71 Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. .......................................................................................................... Table 18 Mean Plasma Ascorbic Acid concentration by Hours Following LPS Challenge in CO (11 = 8): LPS + Ascorbic acid, and CH (n = 8): LPS + Ascorbic acid + Histidine in dairy cattle. ................................................................................................................ Table 19 Mean Heart Rate by Hours Following LPS Challenge in Four Different Treatments; OO: LPS alone, CO: LPS + Ascorbic acid, OH: LPS + Histidine, and CH: LPS + Ascorbic acid + Histidine in dairy cattle. ................................................. Table 20 Mean Heart Rate as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Different Treatments; 00 (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ........................ Table 21 Mean Respiratory Rate by Hours Following LPS Challenge in Four Different Treatments; OO: LPS alone, CO: LPS + Ascorbic acid, OH: LPS + Histidine, and CH: LPS + Ascorbic acid + Histidine in dairy cattle. ........................... Table 22 Mean Respiratory Rate (per minute) as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ....................................................................................................................... Table 23 Mean Ruminal Contraction Rate by Hours Following LPS Challenge in Four Diflerem Treatments; OO: LPS alone, CO: LPS + Ascorbic acid, OH: LPS + Histidine, and CH: LPS + Ascorbic acid + Histidine in dairy cattle. ........................... Table 24 Mean Rumiml Contraction Rate (per minute) as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Difl‘erent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. .......................................................................................................... Table 25 Mean Dry Matter Intake (lbs.) by Hours Following LPS Challenge in Four Different Treatments; OO: LPS alone, CO: LPS + Ascorbic acid, OH: LPS + Histidine, and CH: LPS + Ascorbic acid + Histidine in dairy cattle. ........................... Table 26 Mean Dry Matter Intake (lbs) as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy ix 72 73 74 75 76 77 78 79 80 cattle. ....................................................................................................................... 81 Table 27 Mean Dry Matter Intake (% Reduction) by Hours Following LPS Challenge in Four Difl‘erent Treatments; OO: LPS alone, CO: LPS + Ascorbic acid, OH: LPS + Histidine, and CH: LPS + Ascorbic acid + Histidine in dairy cattle. ........ 82 Table 28 Mean Dry Matter Intake (% Reduction) as Treatment Groups; AA: C0 + CH, H: OH + CH, Non-AA: 00 + OH, Non-H: 00 + CO, by Hours Following LPS Challenge in Four Difierent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + Histidine), and CH (LPS + Ascorbic acid + Histidine) in dairy cattle. ....................................................................................................................... 83 churn—rs u -v-n __. 53-. LIST OF FIGURES Figure 1 Mean Rectal Temperature as Treatment Groups; AA (n = 16): C0 + CH, H (n = 16): OH + CH, No AA (11 =16):OO + OH, No H (n =16):OO + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L-histidine) in dairy cattle. Mean rectal temperature AA cows is significantly lower than in non-AA cows from 24 to 48 hours after LPS challenge (P = 0.0393). However, there were no differences between H and non-H cows all over the experimental period. ....................... 27 Figure 2 Mean Rectal Temperature as Period Groups; Period 1, 2, 3, & 4 (n = 16 each), by Hours Following LPS Challenge in Four Difl‘erent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L- histidine) in dairy cattle. Mean rectal temperature in period 1 was significantly lower than inperiod2and3 fromOto4hr(P<0.04). Meanrectaltemperatureinperiod4wasalso significantly lower than in period 2 and 3 from 3 to 4 hr (P < 0.056). ............................. 28 Figure 3 Mean Somatic Cell Count (SCC) as Treatment Groups; AA (n = 16): C0 + CH, H (n = 16): OH + CH, No AA (n =16):OO + OH, No H (n =16):OO + CO, by Hours Following LPS Challenge in Four Difi‘erent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L-histidine) in dairy cattle. Mean SCC in AA cows is significantly lower than in non-AA cows at 24-36 hour post-LPS challenge (P = 0.0261). Moreover, from 6 to 24 hours after challenge. Mean SCC ianows is significantly lowerthaninnon-H cows fi'om6 to 24 hourafier LPS challenge (P = 0.0164). .......................................................................................... 29 Figure 4 Logarithmical Somatic Cell Count (SCC) as Treatment Groups; AA (11 = 16): CO+CH,H (n= 16): OH+CH,NoAA(n= 16): 00+ OH, No H (n= 16): OO+CO, by Hours Following LPS Challenge in Four Difl‘erent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L- histidine) in dairy cattle. ................................................................................................ 30 Figure 5 Mean Quarter Milk Production as Treatment Groups; AA (n = 16): C0 + CH, H (n=16):OH+CH,NoAA(n= l6):OO+OH,No H (n= 16): OO+CO,byHours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L—histidine), and CH (LPS + Ascorbic acid + L-histidine) in dairy cattle. There are no significant difl'erences between AA and non-AA cows throughout the trial. Mean quarter milk production in H cows tended to be higher than in non-H cows at 12 hours post LPS challenge (P < 0.090). .............................................. 32 Figure 6 Change of Mean Quarter Milk Production as Treatment Groups; AA (11 = 16): CO+CH,H(n=16):OH+CH,NoAA(n=16):OO+OH,No H (n=16):OO+CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L- histidine) in dairy cattle. There are no significant differences between AA and non-AA cows throughout the trial. Mean quarter milk production in H cows tended to be higher than in non-H cows at 12 hours post LPS challenge (P < 0.090). ................................... 33 Figure 7 Daily Mean Quarter Milk Production as Treatment Groups; AA (11 = 16): C0 + CH, H (n = 16): OH + CH, No AA (11 =16):OO + OH, No H (n =16):OO + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L—histidine) in dairy cattle. There are no significant differences between AA and non-AA cows throughout the trial. ...................................................................................................... 34 Figure 8 Change of Daily Mean Quarter Milk Production as Treatment Groups; AA (n = 16): CO+CH,H (n= 16): OH+CH,NoAA(n= l6): OO+OH,NoH (n= 16): 00+ CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L- histidine) in dairy cattle. There are no significant difl‘erences between AA and non-AA cows throughout the trial. ............................................................................................. 35 Figure 9 Mean Composite Milk Production as Treatment Groups; AA (11 = 16): C0 + CH, H (n= 16): OH+CH,No AA(n= l6): OO+OH, No H (n= 16):OO+CO,by Hours Following LPS Challenge in Four Difi‘erent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L-histidine) in dairy cattle. Mean composite milk production in AA cows is significantly higher than in non-AA cows from 48 to 96 hours post LPS challenge (P < 0.02). Whereas there are no significant differences between H and non-H cows throughout the trail. ......................... 36 Figure 10 Change of Mean Composite Milk Production as Treatment Groups; AA (n = 16): C0 + CH, H (n= 16): OH + CH, No AA (n =16):OO + OH, No H (n =16):OO + CO, by Hours Following LPS Challenge in Four Difl‘erent Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L- histidine) in dairy cattle. Mean composite milk production in AA cows is significantly higher than in non-AA cows fi'om 48 to 96 hours post LPS challenge (P < 0.02). Whereas there are no significant differences between H and non-H cows throughout the trail. ..... 37 Figure 11 Daily Mean Composite Milk Production as Treatment Groups; AA (n = 16): C0 + CH, H (n=16):OH + CH, No AA (n= 16): 00 + OH, No H (n =16):OO + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L- histidine) in dairy cattle. There are no significant differences between H and non-H cows throughout the trail. . .................................................................................................... 38 Figure 12 Change of Daily Mean Composite Milk Production as Treatment Groups; AA (n=16):CO+CH, H (n=16):OH+CH,No AA(n=16):OO+OH,No H (n=16): 00 + CO, by Hours Following LPS Challenge in Four Difi‘erent Treatments; OO (LPS xii alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L-histidine) in dairy cattle. There are no significant differences between H and non-H cows throughout the trail. ............................................................................................. 39 Figure 13 Mean Milk IgG. as Treatment Groups; AA (11 = 16): C0 + CH, H (n = 16): OH + CH, No AA (11 = 16): 00 + OH, No H (n = 16): 00 + CO, by Hours Following LPS Challenge in Four Different Treatments; 00 (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L-histidine) in dairy cattle. Mean milk IgGl in AA cows tended to be higher than in non-AA cows at 6 and 24 hours after LPS challenge (P < 0.10). Mean milk IgG. in H cows also tended to be lower than in non-H cows from 3 to 9 hours post LPS challenge (P = 0.0555). ................................ 40 Figure 14 Mean Antioxidant Activities (AOA, % inhibition) as Treatment Groups; AA (11 =16):CO + CH, H (n = 16): OH + CH, No AA (11 =16):OO + OH, No H (n =16):OO + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L- histidine) in dairy cattle. Mean AOA in H cows is significantly lower than in non-H cows from 6 to 12 hours post-LPS challenge (P < 0.04). Whereas there were no significant differences of mean AOA between AA and non-AA cows at any time. ........................... 42 Figure 15 Mean Plasma Ascorbic Acid concentration by Hours Following LPS Challenge in CO (11 = 8): LPS + Ascorbic acid, and CH (11 = 8): LPS + Ascorbic acid + L-histidine in dairy cattle. There is no significant difference between CO and CH afier ascorbic acid infusion. ....................................................................................................................... 43 Figure 16 Mean Heart Rate as Treatment Groups; AA (n = 16): C0 + CH, H (n = 16): OH + CH, No AA (n = 16): 00 + OH, No H (n = 16): 00 + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L-histidine) in dairy cattle. There were no significant differences between AA and non-AA treatments, as well as between H and non-H treatments. ........................................................................................ 44 Figure 17 Mean Respiratory Rate (per minute) as Treatment Groups; AA (n = 16): C0 + CH,H (n=16):OH+CH,No AA(n=l6):OO+OH,NoH (n=16):OO+CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L-histidine) in dairy cattle. No significant difference between AA and non-AA cows, also between H and non-H cows. ................................................................................................................. 45 Figure 18 Mean Ruminal Contraction Rate (per minute) as Treatment Groups; AA (11 = 16): C0 + CH, H (n = 16): OH + CH, No AA (n= 16): 00 + OH, No H (n =16):OO + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L- histidine) in dairy cattle. Mean ruminal contraction rate in AA cows tended to be higher than in non-AA cows from 3 to 6 hours post-LPS challenge (P < 0.10). No significant xiii difi'erence between H and non-H cows at any time. ........................................................ 47 Figure 19 Mean Dry Matter Intake (lbs) as Treatment Groups; AA (11 = 16): C0 + CH, H (n= 16): OH + CH, No AA (n= 16): 00 + OH, No H (n =16):OO + CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L-histidine) in dairy cattle. Mean DMI in AA had been statistically significant higher (P < 0.06) than in No AA since the beginning, but there was no significant difference between H and No H . ...................................................................................................................................... 48 Figure 20 Mean Dry Matter Intake (% Reduction) as Treatment Groups; AA (11 = 16): CO+CH,H (n=16):OH+CH,NoAA(n=16):OO+OH,NoH (n= l6):OO+CO, by Hours Following LPS Challenge in Four Different Treatments; OO (LPS alone), CO (LPS + Ascorbic acid), OH (LPS + L-histidine), and CH (LPS + Ascorbic acid + L- histidine) in dairy cattle. No significant difference between AA and No AA, also between H and non-H cows . ............................................................................................................................ 49 Figure 21 Mean Rectal Temperature by Hours Following LPS Challenge in Four Difl‘erent Treatments; 00 (n = 8): LPS alone, CO (11 = 8): LPS + Ascorbic acid, OH (11 = 8): LPS + L-histidine, and CH (11 = 8): LPS + Ascorbic acid + L-histidine in dairy cattle. Difference between CO and CH approaches statistical significance (P < 0.10) at 48 hours post- LPD challenge. Mean rectal temperature in CO tended to be higher than in CH from at challenge and 2 hour post LPS challenge (P < 0.10). Mean rectal temperature in OH is significantly higher than in all other treatments (P < 0.040) at 36 hours post LPS challenge. Mean rectal temperature in 00 is significantly higher than in CO and CH at 24 and 48 hours post challenge (P < 0.050). ....................................................................... 86 Figure 22 Mean Somatic Cell Count (SCC) by Hours Following LPS Challenge in Four Diflerent Treatments; 00 (n = 8): LPS alone, CO (n = 8): LPS + Ascorbic acid, OH (11 = 8): LPS + L-histidine, and CH (11 = 8): LPS + Ascorbic acid + L-histidine in dairy cattle. Mean SCC in 00 is significantly higher than in CO (P = 0.0222), in OH (P = 0.0255), and in CH (P = 0.0058). ................................................................................................ 87 Figure 23 Mean Quarter Milk Production by Hours Following LPS Challenge in Four Different Treatments; OO (11 = 8): LPS alone, CO (n = 8): LPS + Ascorbic acid, OH (n = 8): LPS + L-histidine, and CH (11 = 8): LPS + Ascorbic acid + L-histidine in dairy cattle. Differences among treatments were not significant throughout the experiment. ............. 88 Figure 24 Mean Composite Milk Production by Hours Following LPS Challenge in Four Different Treatments; 00 (n = 8): LPS alone, CO (11 = 8): LPS + Ascorbic acid, OH (n = 8): LPS + L-histidine, and CH (11 = 8): LPS + Ascorbic acid + L-histidine in dairy cattle. Mean composite milk production in CO is significantly higher than in OH fi'om 48 to 96 hours post-LPS challenge (P = 0.0172). Mean composite milk production in CH is significantly higher than in OH fi'om 48 to 96 hours post-LPS challenge (P = 0.0100). .................................................................................................................................. 89 xiv Figure 25 Mean Milk IgGl by Hours Following LPS Challenge in Four Different Treatments; 00 (n = 8): LPS alone, CO (n = 8): LPS + Ascorbic acid, OH (11 = 8): LPS + L-histidine, and CH (11 = 8): LPS + Ascorbic acid + L-histidine in dairy cattle. Mean milk IgGl in CO tended to be higher than in OH from4 to 36 hours post-LPS challenge (P < 0.08). Mean milk IgG1 in CO is significantly higher than CH from 4 to 12 hours after challenge (P < 0.05). ............................................................................................................ 90 Figure 26 Mean Antioxidant Activities (AOA) as % Inhibition by Hours Following LPS Challenge in Four Diflerent Treatments; 00 (n = 8): LPS alone, CO (n = 8): LPS + Ascorbic acid, OH (11 = 8): LPS + L-histidine, and CH (11 = 8): LPS + Ascorbic acid + L- histidine in dairy cattle. Mean AOA in CO is significantly higher than in OH and CH at 6 hours post-LPS challenge (P < 0.03). There were no significant differences of mean AOA in 00 and other groups throughout the trial. ................................................................. 91 Figure 27 Mean Heart Rate by Hours Following LPS Challenge in F our Difl‘erent Treatments; 00 (n = 8): LPS alone, CO (11 = 8): LPS + Ascorbic acid, OH (n = 8): LPS + L-histidine, and CH (n = 8): LPS + Ascorbic acid + L-histidine in dairy cattle. There were no significant differences among four treatments throughout the experimental period. ...................................................................................................................................... 92 Figure 28 Mean Respiratory Rate by Hours Following LPS Challenge in F our Different Treatments; 00 (n = 8): LPS alone, CO (n = 8): LPS + Ascorbic acid, OH (n = 8): LPS + L-histidine, and CH (n = 8): LPS + Ascorbic acid + L-histidine in dairy cattle. Mean respiratory rate in CO tended to be lower than in OH from 12 to 36 hours post-LPS challenge (P < 0.10). ..................................................................................................... 93 Figure 29 Mean Ruminal Contraction Rate by Hours Following LPS Challenge in Four Different Treatments; 00 (n = 8): LPS alone, CO (11 = 8): LPS + Ascorbic acid, OH (11 = 8): LPS + L-histidine, and CH (11 = 8): LPS + Ascorbic acid + L-histidine in dairy cattle. Mean ruminal contraction rate in CO is significantly higher than in OH fiom 4 to 6 hours post-LPS challenge (P = 0.0504). .................................................................................. 94 Figure 30 Mean Dry Matter Intake (lbs.) by Hours Following LPS Challenge in Four Difl‘erent Treatments; OO (11 = 8): LPS alone, CO (11 = 8): LPS + Ascorbic acid, OH (n = 8): LPS + L-histidine, and CH (n = 8): LPS + Ascorbic acid + L-histidine in dairy cattle. Difference between 00 and CH is statistically significant (P < 0.04) from D(-l) to D2. Difference between OH and CH is also statistically significant (P < 0.02) from D(-l) to D2. Difference between 00 and CO group approaches statistical significance (P < 0.10) from D3 to D5. ...................................................................................................................................... 95 Figure 31 Mean Dry Matter Intake (% Reduction) by Hours Following LPS Challenge in Four Different Treatments; 00 (n = 8): LPS alone, CO (n = 8): LPS + Ascorbic acid, OH (11 = 8): LPS + L-histidine, and CH (11 = 8): LPS + Ascorbic acid + L-histidine in dairy cattle. In D0 and D1, difference between 00 and OH approaches statistical significance (P < 0.10). .................................................................................................................... 96 XV LIST OF SYMBOLS & ABBREVIATIONS AA = ascorbic acid AAPH = 2,2'-azobis (2-arnidinopropane) hydrochloride AAUA=ascorbicacid&uricacid ADCC = antibody-dependent cell mediated cytotoxicity AOA = antioxidant activities B-PE = B-phycoerythrin BSA = bovine serum albumin cfu = colony-forming unit DHIA = Dairy Herd Improvement Association DNA = deoxyribonucleic acid DMI = dry matter intake EDTA = ethylenediaminotetraacetate ERS = electronic spin resonance FL = fluorescence GSH-Px = glutathione peroxidase H = L-histidine H202 = hydrogen peroxide HPLC = high performance (pressure) liquid chromatography HR = heart rate HSCC = high somatic cell count xvi IgG = immunoglobulin G IgG} = immunoglobulin 61 IL = interleukin 1M] = intramamrmry infection IU = international unit KLH = keyhole limpet hemocyanin L-His = L-histidine LSCC = low somatic cell count LPS = lipopolysaccharide LT = leukotriene LTB4 = leukotriene B4 MHC = major histocompatibility NSAIDs = non-steroidal anti-inflammatory drugs 'OH = hydroxyl radical PG = prostaglandin PGE; = prostaglandin E2 PGan = prostaglandin an PMN(s)= polymorphonuclear neutrophil(s) PSS = physiological saline solution RCR = ruminal contraction rate rpm = round/min RR = respiratory rate xvii SAS = Statistical Analysis System SCC = somatic cell count SEM = standard error of the means SRID = Single Radial Inununodifiirsion TMR = total mixed ration(s) TNF-a = tumor necrosis factor-a INTRODUCTION Mastitis, or mammary inflammation, is generally considered to be the most costly disease in dairy cattle throughout the world. Economic losses due to mastitis include decreased production, discarded milk, culling, mortality, labor, veterinary service, medication, and delayed genetic progress (Reneau, 1993). Based on the degree of inflammation, it may be classified as subclinical, subacute clinical, acute, peracute, chronic, and nonbacterial mastitis. Hogan et al. (1989a) gave four guidelines of clinical mastitis cases determined by retrospective reports of clinical signs and culture results of foremilk samples of environmental mastitis reported by Smith et al. (1985). First, a new case of clinical mastitis occurred when a 14-day period had elapsed between reports of clinical signs, regardless of the bacteriological status of the quarter. Second, a new case of clinical mastitis occurred when a different pathogen was isolated fiom a clinical quarter regardless of the number of days between isolation of dissimilar pathogens. Third, when one or more pairs of duplicate milk samples were cultured during a 14—day period and a pathogen was isolated from one or more pairs of samples, but the remainder of samples were bacteriological negative, the isolated pathogen was determined the cause of the clinical cases. Finally, a new case of clinical mastitis was not recorded if the same pathogen was isolated or if samples were bacteriological negative when less than 14 days had elapsed between reports of clinical signs. A proper mastitis control program, i.e. post-milking teat dipping, total dry cow therapy, culling, and proper maintenance of milking equipment (Bramley et al., 1984), cannot completely eliminate mastitis from a dairy herd, particularly mastitis caused by As ther systc dais deter; mam] ascorl environmental pathogens. Among clinical mastitis cases, coliform organisms are the most common cause of severe cases. The problem of coliform mastitis has not been effectively solved. However, nutrition, especially supplementation of antioxidant micronutrients, is an important part of coliform mastitis prevention because of its critical role in mammary resistance (Bowers, 1997; Erskine, 1993). However, therapeutic potential of antioxidants for the treatment of coliform mastitis has not been investigated. As antioxidants, L-histidine and ascorbic acid have been suggested as potential therapeutics to alleviate free radical-mediated damage in a variety of clinical models. My hypothesis is that therapy with histidine and/or ascorbic acid will reduce the systemic and local inflammatory response resulting from endotoxin-induced mastitis in dairy cattle. Therefore, this study was conducted with two primary objectives. (1) To determine the effect of parenteral histidine and/or ascorbic acid treatment on acute mammary inflammation. (2) To determine the effect of parenteral histidine and/or ascorbic acid on systemic variables resulting from acute mammary inflammation. REVIEW OF LITERATURE Epidemiology of Clinical and Acute Clinical Mastitis. Dairy herds that have controlled contagious mastitis can still have an unacceptable incidence of intramammary infection (IMI) and clinical cases caused by environmental pathogens (Hogan et aL, 1989a; Smith et al., 1985). Procedures, such as post-milking teat dipping, total dry cow therapy, culling, and proper maintenance of milking equipment are successful in reducing the reservoir of contagious pathogens (Bramley et al., 1984). However, they are not generally effective in the control of environmental pathogens (Smith et al., 1985) because, as opposed to contagious pathogens, infections do not generally occur during milking The average herd incidence of clinical mastitis in low-somatic-cell-count (LSCC) herds fi'om California, Ohio, and Pennsylvania was 45-50 cases/100 cow-years. And coliforms, lactose—fermenting, gram-negative bacilli of the family Enterobacteriaceae, were the predominant pathogen, isolated from 30 to 40% of the clinical cases (Erskine et al., 1988; Gonzales et aL, 1990; Hogan et aL, 1989a; Smith et al., 1985). In two additional studies fiom Ohio, 46.5% of microbiological cultures of milk samples fi'om clinical mastitis cases (824/1772) yielded coliform organisms And E. coli, Klebsiella sp., and Enterobacter sp. accounted for 14.6%, 2.6%, 2.8% of the clinical cases, respectively (Bartlett et al., 1992). It is suspected that when no bacteria are isolated on culture it fi'equently results fiom coliform infections that have been eliminated by the cow’s defenses (Bartlett et al., 1993). In the Pennsylvania study, the proportion of clinical mastitis cases attributable to coliform bacteria was significantly (P < 0.005) higher in low somatic cell count (LSCC, S 150,000 cells/ml) herds (43.5 i 3.5%, n = 12) than in high sormtic cell count (HSCC, 2 700,000 cells/ml) herds (8.0 :t 3.4%, n = 6) (Erskine et al., 1988). An increased incidence of coliform mastitis is also associated with the firstmonth of lactation, and warm humid weather (Erskine et al., 1988; Hogan et al., 1989a; Smith et al., 1985). Bedding materials are implicated as primary sources of environmental pathogens during inter-milking periods. The number and type of bacteria in bedding are related to microbial numbers on the teat end (Janzen et al., 1982; Natzke et al., 1976). Hogan et al. (1989b) reported that organic bedding materials, such as sawdust and chopped straw, had significantly higher moisture content and coliform bacteria concentrations (P < 0.05) than did sand and crushed limestone. Fundamentally, moisture, available nutrients, and proper temperature are the ecological factors for colonization and multiplication of bacteria. Thus, these factors are critically associated with significantly greater bacterial counts in organic as compared to inorganic bedding materials (Hogan et al., 1989b). The average coliform count in organic materials is significantly higher (P < 0.05) during summer (6.5 i 0.3 colony-forming unit (cfu) logm/g dry weight) than other seasons (5.7 :1: 0.4 cfir logm /g dry weight) (Hogan et al., 1989b). The greater coliform counts are probably related to a higher ambient temperature (Hogan et al, 1989b), and coincided with the highest rate of clinical mastitis during summer (Erskine et al., 1988; Hogan et al, 1989a). Additionally, increasing parity is associated with an increased rate of coliform mastitis (Smith et al., 1985). Hogan et al. (1989a) also found that coliforms accounted for 58.9% (56/95) of cl ‘4 r3. severe clinical cases, and 29.2% (56/ 192) of clinical coliform cases were classified as severe mastitis with abnormal milk, swelling of quarter, and systemic signs as summarized in a review by Eberhart et a1. (1979). Economic impact. Severe coliform mastitis causes a tremendous reduction in both milk quantity and quality (Dobbins, 1977; Kitchen, 1981; Schalm, 1977). Losses to dairy producers include decreased production, discarded milk, culling, mortality, labor, veterinary service, medication, and delayed genetic progress (Reneau, 1993). In 50 Ohio dairy herds with a total of 4068 cow-years, the costs per cow-year for clinical cases of mastitis caused by E. coli was $3.21 :1.- 0.12, which was higher than mastitis caused by other pathogens (Miller et al., 1993). Pathogenesis Polymorphonuclear neutrophil (PMN) phagocytosis is the most critical part of the cow’s mammary defense for bacterial clearance (Kehrli, 1994). Four important physiological functions of PMNs are involved in the inflammatory reaction, namely chemotaxis, diapedesis, phagocytosis, and intracellular killing (Burvenich et al., 1994). In response to inflammatory stimuli, PMNs are released from circulating and marginal storage pools, and adhere to blood vessel walls (Burvenich et al., 1994). Then, there is a rapid and massive influx of neutrophils fi'om peripheral blood into the alveolar lumen of the mammary gland, thus markedly increasing the somatic cell count in milk (Burvenich et al., 1994). Lin et a1. (1995) supported this concept and demonstrated in vitro the process of bovine neutrophil diapedesis across bovine mammary gland epithelial cells (MAC-T). (/ ofier Sever \‘egSe Wages, Light and transmission electron microscopy revealed the sequential neutrophil transmigration, accumulation of neutrophils on the surface of epithelial monolayer, projection of pseudopods into intercellular junctions and movement of neutrophils between adjacent epithelial cells, reapproximation of the lateral epithelial cell membranes, and reformation of the epical tight junctions after neutrophils crossed the bovine mammary gland epithelium (Lin et al., 1995). Following diapedesis, neutropenia or neutrophilia in blood initially occurs with a possible lefl shifl appearance. Neutropenia and depletion of bone marrow reserves of neutrophils follows (Jain et al., 1978). Replenishment of blood and bone marrow neutrophil pools from compensatory stimulation of granulopoiesis is often associated with subsidence of acute mastitis and recovery (Jain et al., 1978). The speed at which neutrophils are mobilized into the gland is a primary determinant of the severity of coliform mastitis cases during lactation (Hill, 1981). The emigration of leukocytes, particularly PMNs and monocytes, out of the blood vessels takes place independently of the increased vascular permeability of acute inflammation (Tizard, 1996). This process, known as an extravasation, depends on adhesive interactions activated by the local release of inflammatory mediators (Janeway et al., 1997; Tizard, 1996). Binding results when endothelial cells express adherence molecules. This expression is triggered by bacterial components (e.g. lipopolysaccharide, LPS) or inflammatory mediators, i.e. thrombin, histamine, tumor necrosis factor [TNF-a] and interleukin-1 (IL-1) (Janeway et al., 1997; Tizard, 1996). Adhesive glycoprotein molecules namely P-selectin (CD62P) and E-selectin (CD62E) mediate the first step of the process (Janeway et al., 1997). P-selectin, which is normally stored in granules (Weibel- Ti to ex- (la Car 199 the exp, infill: 19% Palade bodies) in endothelial cells, is translocated to the endothelial cell surfaces within a few minutes of exposure to Leukotriene-B4 (LTB4), C5a, or histamine (Janeway et al., 1997). E-selectin appears a few hours after exposure to LPS or TNF-a. These selectins can bind to carbohydrate side chains, sialyl-Lewis" moiety (s-Le") on neutrophil surface glycoproteins (Janeway et al., 1997). The adhesion in this step is weak and allows leukocytes to roll along the vascular endothelial surface (Janeway et al., 1997; Tizard, 1996). This adhesive interaction enhances the stronger interactions of the second step, which depends upon the leukocyte integrins, LFA-l (CD1 lazCD18), and the immunoglobulin-related molecule ICAM-l on endothelial surfaces (Janeway et al., 1997; Tizard, 1996). Platelet-activating factor (PAF) secreted by endothelial cells activates the rolling neutrophils (Janeway et aL, 1997; Tizard, 1996). Then, LFA—l is increasingly expressed on the neutrophil surface, which results in an increased affinity for ICAM-2 (Janeway et al., 1997; Tizard, 1996). IL-8 produced fi'om endothelial cells by the induction of IL-1 triggers a conformational change in LFA-l , which increases its adhesive capacity. Subsequently, neutrophils adhere firmly to the endothelium (Janeway et al., 1997; Tizard, 1996). IL-8 also acts as a chemotactic molecule to attract more PMNs to the area (Tizard, 1996). In the third step, CD31, an immunoglobulin-related molecule, is expressed on leukocytes and at the intercellular junction of endothelial cells. This enhances neutrophils to penetrate across the endothelium. The last step is under the influence of cytokines, thereafter, phagocytosis take places (Janeway et al., 1997; Tizard, 1996). In phagocytosis, PMNs primary granules fuse with the phagosomes to form phagolysosomes. Since this fusion may occur before the pathogen or LPS is completely ingested, the lysosomal contents may be released into the mammary tissues (Janeway et al., 1997; Tizard, 1996). After the ingestion of pathogens, phagocytes will increase their oxygen consumption 10 times as much as that of resting cells. This cellular oxidative mechanism of the PMN termed “the respiratory burst” (Chew, 1996; DeChatelet, 1978) generates potent oxidizing agents also called oxygen-derived radicals, molecule with an odd number of electrons (V anSteenhouse, 1987). They include singlet oxygen ('02), peroxides (11202), and fiee hydroxyl radicals (' OH). H202 is included because its potential for the rapid production of 'OH in the presence of an iron catalyst via the F enton reaction in equation [[1 (V anSteenhouse, 1987). (Haber-Weiss) 02' + H202 02 + 'OH + 'OH .............. (Equation 1) 02' + Fe” 02 + Fe2+ ......................... (Equation H) (Fenton) Fe2*+ H202 Fe3*+ 'OH + 'OH ............. (Equation 111) These agents destroy the invading microbes or their products (e.g., LPS). Concomitantly, they also provide the harmful activities associated with oxidative damage to host cell membrane, enzymes and nucleotides in DNA (Bendich, 1993; Chew, 1996; Machlin & Bendich, 1987; VanSteenhouse, 1987). PMNs isolated from mammary secretion are less efficient than PMNs isolated fi'om peripheral blood (Pappe et al., 1977). Their decreased phagocytic and bactericidal activities had been associated with many factors—decreased intracellular glycogen reserves, ingested milk fat globules and casein, (Pappe et al., 1977) inadequate level of opsonins, and cortisol levels (Fox et al., 1981). Endotoxin also called lipopolysaccharide is a virulent factor and a cell wall component of gram-negative bacteria (Raetz, 1993). It is released fi'om the gram—negative bacteria upon cell death and composed of three basic subunits; O-specific polysaccharide, Lipid A, and R-core (Raetz, 1993). Although endotoxin itself has no direct damaging efi‘ect on mammary epithelium (Frost, 1984), it can cause pathophysiological effects, which are predominantly dose—dependent (Giri, et al., 1984; Lohuis et al., 1988b). Generally, as dosage increases latency time decreases, the peak efi‘ect becomes more pronounced, and the duration of the effect protracted (Lohuis et aL, 1988b). It also induces host inflammatory mediators (Shuster et al., 1993). When mammary tissues are stimulated, phospholipases act on the phospholipids in cell wall to release fatty acids including the most important unsaturated long-chain fatty acid, arachidonic acid (Tizard, 1996). Two enzymes, including 5-lipoxygenase and cyclooxygenase result in arachidonic acid metabolites (Tizard, 1996). Under the influence of the former, arachidonic acid is converted to biologically active lipids called leukotrienes (LT). While under the influence of the latter, arachidonic acid yields prostaglandin (PG) series, i.e., PGA; (Thromboxane- A2), PGEz, PGFZa, PGIz (Prostacyclin) (Tizard, 1996). Other inflammatory mediators include cytokines, interleukin (IL-1, IL-6, IL-8) and tumor necrosis factor-a [TNF-a], and complements, such as C5a as chemotactic factors, and vasoactive factors such as histamine (Tizard, 1996). The mechanism results in local inflammation with five cardinal signs, i.e. redness, swelling, pain, heat, and disturbed rmmmary function within a few hours after intramamrnary infusion of endotoxin (Lohuis et al., 1988b; Tizard, 1996). However, a study in non-pregnant, lactating cows demonstrated that the intravenous administration of 100 ug of LPS did not induce clinical mastitis (Shuster et al., 1991c). The subsequent absorption of endotoxin-induced inflammatory endogenous mediators in the udder rather than absorption of endotoxin itself into the circulation (Lohuis et al., 1988) causes systemic signs, i.e., fever, acute phase reactants, metabolic changes, and vascular responses of the host (Lohuis et al., 1988b; Tizard, 1996). Endotoxin by either intracisternal or intravenous route causes pathophysiological effects on lactational performance by suppressing milk yield in affected quarters as well as unaffected ones (Shuster et al., 1991a, 1991b, 1991c). However, a more severe and prolonged suppression occurred in infirsed quarters compared to uninfused ones, which are consequently affected by systemic responses (Shuster et al., 1991a). Intramamrnary infirsion of endotoxin does not result in as markedly decreased rumen motility in contrast to the intravenous route (Lohuis et aL, 1988), or experimental and natural E. coli mastitis (V erheijden et al., 1983). However, clinicopathological changes including expanded plasma volume, hyponatremia, transient hyperchloremia and hypophosphatemia, hypocalcemia, and decreased serum activities of liver- and muscle- specific enzymes, have been well demonstrated (Tyler et al., 1994a). Acute Phase Response. The systemic events that result fi'om acute endotoxin-induced mastitis are collectively termed the acute phase response (Bishop et al., 1976). These include fever, increased serum cortisol, increased serum concentrations of proteins (fibrinogen, complement, haptoglobin, and ceruloplasmin), transient decreases of serum Fe and Zn, and mobilization of leukocytes. These events have been demonstrated in cows with acute mastitis (Conner et al., 1986; Erskine et al., 1989; Erskine et al., 1993; Jackson et al., 10 1990; Lohuis et al., 1990; Shuster et al., 1992). In cows experimentally administered E. coli endotoxin, the average serum cortisol peaked significantly (P < 0.05) higher (100 vs. 82 ng/ml) and sooner (2.5 vs. 4.5 hr posttreatment) in intravenous (n = 4) as compared to the intramammary (n = 12) treatment group (Jackson et al., 1990). In cows intracisternally challenged with 50 colony-forming units (cfu) of E. coli, mean serum concentrations of Zn and Fe decreased 21-24% and 28-3 5%, respectively (Erskine & Bartlett, 1993; Lohuis et al., 1988), and mean serum concentration of Cu decreased to 52% of prechallenge concentrations (Erskine & Bartlett, 1993). Three plasma proteins; haptoglobin, ceruloplasmin, and al-antitrypsin classified as acute phase reactants were higher in the cows with mastitis than non-affected cows (Conner et al., 1986; Tizard, 1996). In particular, the iron-binding protein haptoglobin is considered a major acute- plmse protein in ruminants (Alsemgeest et al., 1994; Tizard, 1996). A 52-fold increase in serum haptoglobin was detected by a high performance liquid chromatography (HPLC) method fiom serum samples taken fi'om eight cows with experimentally E. coli-induced mastitis. (Salonen et al., 1996). Politis et a1. (1991) found that various concentrations (0-30 ug/ml) of E. coli LPS did not affect the expression of major histocompatibility (MHC) class II molecules on the surfice of bovine mammy macrophages in vitro. In addition, LPS was unable to enhance the proliferation of antigen-specific T-cells (Politis et al., 1991). Therapy & Prevention. Anderson (1989) suggested the therapeutic management of acute coliform mastitis should be based on early, accurate detection and careful clinical assessment. Therapeutic ll principles identified for management of acute coliform mastitis include elimination of bacteria from the mammary gland, neutralizing the effects of endotoxin, and providing supportive therapy (Anderson, 1987). Antimicrobial agents, particularly extra-labeled drugs, have been promoted as the primary regimen for bacterial elimination. Antimicrobial agents alone, however, have minimal benefit in the treatment of clinical gram-negative mastitis (Erskine et al., 1992; Jones et al., 1990). Most cases spontaneously recover without antimicrobial therapy (Anderson, 1989). Additionally, milk discard costs to avoid drug residues in marketed milk are potentially the most costly consequence of antibiotic use (Erskine et aL, 1991, 1992). Thus, antimicrobial treatment should only be considered as an adjunct to other supportive care to alleviate the effects of shock (Erskine et al., 1991). This includes anti-inflammatory treatment (Anderson etal., 1986; DeGraves et al., 1993; Lohuis et al., 1988a, 1989), and administration of calcium, glucose, and hypertonic saline solution (Anderson, 1989; Constable et al., 1991; Cullor, 1993; Tyler et aL, 1994b). Anti-inflammatory treatment of coliform mastitis with either steroids (Lohuis et al., 1989); dexamethasone (Lohuis et al., 1988a) or non-steroidal anti-inflammatory drugs (NSAIDs) such as flunixin meglumine (Anderson et al., 1986), and Ibuprofen (DeGraves et al., 1993) enhances clinical outcomes of experimental coliform mastitis. Endotoxin-induced shock is complex involving cardiogenic, hypovolemic, neurogenic and other mechanisms (Constable et al., 1991; Smith, 1986). The technique of hypertonic saline infirsion has proved to be a useful adjunct in treatment ofthe outcome of those mechanisms (Erskine et al., 1994; Sargison et al., 1996; Tyler et al., 1994). Intravenous administration of 5ml/kg of hypertonic saline solution (7.2-7.5% NaCl) 12 increased plasma volume in cows with endotoxin-induced mastitis and endotoxin induced shock compared to cows that were administered isotonic NaCl solution (Erskine et al., 1994; Sargison et al., 1996; Tyler et al., 1994). Mechanisms of action of hypertonic saline may include redistribution of body water, which enhances circulatory blood volume and tissue perfusion, a vagal-mediated ionotropic effect on the heart, and altered peripheral vascular resistance or a combination of these factors (Sargison et al., 1996). Effective and economic coliform mastitis control programs rely on prevention rather than treatment (Erskine et al., 1993); therefore, milking hygiene, teat dipping, and environmental sanitation should be major objectives (Anderson, 1989). Additionally, vaccination programs, including E. coli 15 vaccine can be helpfirl. The E. coli J5 vaccine is a bacteria produced from a mutation of E. coli 01 11:B4 strain J5 (Rc mutant), which lacks the “0” antigen capsular portion of the cell wall (Cullor, 1991; Gonzalez et al, 1989). This mutant thus has the core antigen (LPS) portion of the cell wall exposed to possrhle irmnune recognition (Cullor, 1991; Gonzalez et al, 1989). Using the core antigen as an immuno gen reduces the requirement for antibody diversity. This is important because coliform mastitis infections are caused by numerous serotypes of gram-negative bacteria (Fang & Pyorala, 1996; Gonzalez et al., 1989; Tyler et al., 1990). The severity of clinical signs in experimental infections (Hogan et al., 1992b), and the incidence of clinical cases of coliform mastitis during the first three month of lactation have been decreased through vaccination (Gonzalez et al., 1989; Hogan et al., 1992a). Escherichia coli J5 vaccination should be profitable when incidence of coliform mastitis exceeds 1% (DeGraves et al., 1991). 13 Nutrition in Colifonn Mastitis. Nutrition, particularly supplementation of antioxidant rnicronutrients, plays a critical role in mammary resistance and phagocytic firnction (Bowers, 1997; Erskine, 1993). The role of antioxidant vitamins including vitamin A, vitamin E, ascorbic acid and B-carotene has been studied as well as minerals—Selenium (Se), Zinc (Zn), Copper (Cu), and Iron (Fe) (Chew, 1996; Erskine, 1993). In particular, studies have demonstrated the role of vitarrrin E and Se in host resistance to coliform mastitis. Grasso et a1. (1990) demonstrated that dietary Se supplementation in cows increased bovine PMN phagocytosis and killing, and decreased extracellular hydrogen peroxide (H202) production. Experimentally induced intrammmary E. coli (15-40 cfir) infections were significantly (P < 0.05) more severe, and of longer duration (114.4 3: 18.0 hr) in Se- deficient Holstein cows (162.0 i 12.0 hr, n = 10) than in a Selenium-supplemented group (Erskine et. al., 1989). Supplementation of Se and vitamin E during the dry period decreased (62%) the duration of clinical mastitis, while the incidence of clinical mastitis was reduced (37%) by vitamin E (740 IU/d) alone (Smith et al., 1984). Selenium and vitamin E are associated with lower milk SCC (Erskine et al., 1987). Selenium is required for glutathione-peroxidase (GSH-Px) activities (Bendich, 1993). The whole blood concentrations of Se and GSH-Px activity were significantly higher (P < 0.01) in low SCC dairy herds (n = 16, 0.133 i 0.01 ug/ml and 35.6 i 2.95 mU/mg obe) than in high SCC herds (n = 16, 0.074 3: 0.007 ug/ml and 20.2 :1: 2.38 mU/mg of Hb) (Erskine et al., 1987). Antioxidants as Therapy. Therapeutic modulation of the local inflammatory and systemic response of clinical 14 coliform mastitis is not fully understood. Fundamentally, cellular mechanisms of host defense should not be totally obstructed by therapy. The role of antioxidant vitamins and minerals as part of a therapeutic regimen has not been studied. Studies of single nutrients may be misleading because interactions are not considered; therefore, more research on the effects of multiple nutrients is needed (Jacob, 1995). Ascorbic Acid & L—histidine. Ascorbic acid Ascorbic acid is produced by the liver of many animals including cattle (Eicher- Pruiett, et al., 1992;1tze, 1984). Hence, the biosynthetic capacity for ascorbic acid in adult ruminants is suflicient to cover the ascorbic acid requirement (Itze, 1984). Nonetheless, ruminants can be prone to ascorbic deficiency due to an impaired synthesis, and a rapid destruction by the ruminal microflora via oral administration of ascorbic acid (Itze, 1984). Because of the irritation following intramuscular and subcutaneous injection, the best means of administration of ascorbic acid is by intravenous injection (Loscher et al., 1984). Ascorbic acid is a water-soluble cytosolic chain-breaking antioxidant (Machlin & Bendich, 1987). It quenches fiee radicals as well as singlet oxygen (Bodannes et al., 1979; Machlin & Bendich, 1987; Niki, 1991b) by providing hydrogen atoms to pair up with unpaired electrons on free radicals in the aqueous compartments such as blood plasrm and cell cytosol (Jacob, 1995). Dwenger et al. (1994) suggested that scavenging of reactive oxygen metabolites by ascorbic acid is responsible for the improvement of endotoxin-induced acute lung injury. In vitro, chemiluminescence response of following 15 zymosan exposure was significantly higher in PWS collected from sheep treated with endotoxin (0.5 rig/kg body weight, i.v.; E. coli endotoxin 055:B5) than in PMNs from sheep treated with endotoxin endotoxin and ascorbic acid group (0.5 rig/kg body weight, E. coli endotoxin 055:B5 & 1 g/kg body weight, iv. bolus injection followed by 0.2 g/kg per hr continuous infusion of ascorbic acid) (Dwenger et al., 1994). The mortality rate fi'om bacterial septicemia in channel catfish decreased with increased dietary ascorbic acid from 100% (0 mg/kg) to 15% (300mg/kg) and 0% (3000 mg/kg) (Li et al., 1985). In chickens, 330 mg of ascorbic acid/ kg of feed reduced mortality and pericarditis (46/60, 76%) afier challenging with Escherichia coli (01 :K1) in air sacs compared to unsupplemented controls (12/63, 19%) (Gross et al., 1988). In cattle, given 20 mg/kg body weight ascorbic acid subcutaneously, (n = 15) neutrophil oxidative metabolism and capability of neutrophils to mediate antibody-dependent cell rmdiated cytotoxicity (ADCC) were enhanced (P < 0.05) (Roth et al., 1985). Conversely, in young calves, ascorbic acid appeared to lmve beneficial as well as adverse effects (Eicher-Pruiett et al., 1992). Young calves (n = 10) supplemented orally with 10 g of ascorbic acid had reduced ocular and nasal discharge (P < 0.01), but lard more fluid feces and impaired neutrophil function (neutrophil-mediated phagocytosis and antibody- dependent cellular cytotoxicity) (Eicher-Pruiett et aL, 1992). Cummins and Brunner (1989) determined that ascorbic acid (1.75 g/d) decreased plasma IgG concentrations and plasma antibody titers to a specific antigen (keyhole limpet hemocyanin, KLH) in young calves (n = 6), but also decreased the incidence of scouring. Ascorbic acid can also synergistically interact with other antioxidants. Ascorbic 16 acid synergistically restores radical scavenging activity of vitamin E (Machlin & Bendich, 1987; Niki, 1991b), and protects cell membrane against peroxidation (Eicher-Pruiett et al., 1992; Niki, 1991b). Ascorbic acid interacts with the tocopheroxyl radical in order to regenerate tocopherol, the active form of vitamin E (Jacob, 1995; Machlin et al.,1987). In young calves, the adverse effect of ascorbic acid supplementation on neutrophil functions was negated by simultaneously feeding of 57 lU/kg of vitamin E in dry milk replacer (Eicher-Pruiett et al., 1992). L-histidine L-histidine, an essential amino acid (Chalupa & Sniffen, 1991; Peterson et al., 1998), has been classified as an antioxidant (Kawamoto et al., 1997; Peterson et al., 1998). Evidence supported L-His as an extremely efl‘ective scavenger of 'OH by decreasing electron spin resonance (ESR) signal intensity of 5,5-dimethyl- l -pyrroline-N- oxide (DMPO)-OH spin adduction in electron-spin—resonance spectroscopy (Nagy & Floyd, 1984). In mice, L-His reduced intestinal membrane permeability in a model experimental bacterial diarrhea (Peterson et al., 1998). The mean fluid-accumulation response in irrtraperitoneally L-His-treated mice (100 pl of 238 mM, n = 22) challenged with Salmonella typhimurium was 76:14 til/cm, which was significantly lower (47%, P = 0.0002) than that of the S. typhimurium-challenged control mice (143 i 10 ul/cm, n = 28) (Peterson et al., 1998). Kawamoto et a1. (1997) reported L—His protected against ischemic/reperfusion-induced injury in the cerebrum of the rat. Intravenous administration of 50 mg/kg and 100 mg/kg L-His delayed neuronal death and maintained the neuronal density of the forebrain in rat hippocampus (P < 0.01) (Kawamoto et al., 1997). Because 17 of a short half-life, L-His is rapidly metabolized and/or excreted (45 min in mice and 1.7 hr in human) (Peterson et al., 1998; Sitton et al., 1988). L-histidine serves as a precursor of histamine, which is synthesized locally by the enzyme histidine decarboxylase in mast cells (Babizhayev et al., 1994; Maslinski et al, 1993), but not in enterocytes (Guihot & Blachier, 1997). Maslinski et a1. (1993) found that histamine concentrations inbovine milk were higher (317 i 29 nmol/l, n= 6) thanthat in bovine plasma (4.83 i 0.82 nmolll, n = 5). Histamine concentration in bovine milk was higher (600 nmng) than in other mammals' milk (guinea pig, mouse, rat, and pig) (Maslinski et al, 1993). Histamine affects blood vessels, smooth muscle and exocrine glands (Tizard, 1996), and is believed to contract myoepithelial cells of alveoli and small ducts in rmmmary gland, that in turn stimulate milk secretion or milk ejection (Maslinski et al, 1993). Hence, L-His may indirectly elicit these responses through histamine induction during clinical mastitis. Although studies demonstrating the potential benefits of ascorbic acid and L-His in various laboratory animals are well recognized (Bushell et al., 1996; Cummins and Brunner, 1989; Eicher-Pruiett et al., 1992; Gross et al., 1988; Kawamoto et al., 1997; Li et al., 1985; Maslinski et a1, 1993; Peterson et al., 1998; Roth et al., 1985). Potential benefits in cows with coliform mastitis are unknown. As antioxidants, they may ameliorate clinical changes caused by shock, and perhaps shock caused by endotoxin- induced mastitis. l8 MATERIALS AND METHODS I. Cows We selected eight, non-pregnant Holstein cows with clinically normal milk and mammary glands, quarter somatic cell counts less than 500,000 cells/ml and negative bacterial cultures at 24 hr before endotoxin challenge. Cows were fed a total mixed ration (TMR) balanced for 90-lb milk production and housed in tie stalls. Data regarding age, milk production, day of lactation, and lactation number was recorded. A jugular catheter was aseptically inserted at 12 hr before endotoxin challenge and remained in each cow until the end of data collection. I]. Endotoxin-induced mastitis Endotoxin solution (20 ug/ml) was prepared by dissolving 100 pg of a commercial (Sigma) Escherichia coli 0111:B4 endotoxin in 5 ml of pyrogen-free physiological saline solution (PSS), which was then filtered by a 0.22-um low extractable filter unit (Sterile‘D D-GS, Millipore Industria E. Comerico Ltda.). The suspension was stored at 4 °C and vigorously shaken before infirsion. On Tuesday mornings, soon after milking, the entire 100-ug preparation was intracisternally infirsed into 1 quarter/cow via syringe and 1 1/3” disposable J-12 teat infusion cannula. Before infusion, the teat was aseptically prepared with alcohol The infused teats and quarters were immediately massaged for 15—20 seconds in order to distribute endotoxin. III. L-histidine & Ascorbic Acid Solution Preparation. L-histidine (ICN Biomedicals Inc.) sohrtion was prepared by dissolving 25 g of L- 19 His in 500 ml of pyrogen-free PSS and dissolving with a stir bar on a warm magnetic stirrer for approximately 35 min. The solution was then filtered with a 0.22-um low extractable filter unit (Sterileq’ D-GS, Millipore Industria E. Comerico Ltda.). Ascorbic acid solution was also prepared from ascorbic acid injectable solution (The Butler Company) by diluting 25 g of ascorbic acid into in 500 ml of pyrogen-free PSS. The solution was vortexed and then filtered with a 022-th low extractable filter unit (Sterileg D-GS, Millipore Industria E. Comerico Ltda.). Two 25-g doses of L-His and/or ascorbic acid were slowly administered by intravenous injection via the jugular catheter. In order to mimic a clinical case of coliform mastitis, the first dose was administered intravenously at 3 hr after endotoxin challenge to allow time for clinical signs to appear. Thereafter, the second dose of 25 g was administered at 5-hr post endotoxin challenge. IV. Experimental design The Latin square cross-over design (4 x 4 table) was used in dififerent orders. Each Holstein cow was randomly selected to complete each of the four treatments. The treatments included LPS challenge as control (00), LPS and ascorbic acid (CO), LPS & L-His (OH), and LPS, L-His & ascorbic acid (CH) (Table 29, Appendix B). The experiment was started by using the left-front (LF) quarter of the first cow. The other cows were then randomly assigned to treatment by selecting cow numbers by drawing fi'om a box and procwding in order down the table (Table 29, Appendix B). Each quarter was used one time for endotoxin-induced mastitis, thus all four quarters were used over the four different periods (each of four successive weeks). 20 V. Milk Collection Milk samples were collected at 12 hr before challenge, immediately before challenge, and 2, 3, 4, 6, 9, 12, 24, 36, 48, 60, 72, 96 hr and 1 week after challenge (Table 30, Appendix B). After aseptic preparation and discarding foremilk, milk samples were collected to determine somtic cell count, bacteriology, and immunoglobulin G, (IgG) concentration. All milk samples were stored in crushed ice immediately after collection. We collected one vial for somatic cell count preserved with a bronopol pellet. This was sent to the DHIA laboratory of Michigan. A second vial was collected for bacteriological culture on 5% sheep blood agar, and incubated for 24 hr at 37 °C. A third sample was collected into a vial with 0.05 ml of 1 M Benzamidine HCl as a protease inhibitor and then centrifuged at 1000 x g for 15 min to separate cells and fat. Whey was prepared for IgG measurement by modifying Guidry’s procedure (1980). The skim layer beneath the fat was transferred to a new vial, and 5 ul of glacial acetic acid added to precipitate casein. The solution was then centrifirged at 13,000 rpm (Biofirge pico) for 13 min. The supernatant was decanted into another clean cryovial, 5 ul of KOH was added, and frozen at -20 °C. Commercial IgG; Single Radial Inununodifiirsion (SRID) kits (VMRD, Inc.) were used to determine IgG] concentration. V1 Blood Collection Blood samples were collected at 12 hr before challenge, immediately before challenge, and 2, 3, 4, 6, 9, 12, 24, 36, 48, and 72 hr afier challenge (Table 30, Appendix B). Blood samples were obtained fi‘om a jugular catheter into heparinized vacutainers, and immediately placed in crushed ice. Sodium citrate was used at each blood sampling to 21 insure anticoagulation in the catheter. Blood samples were centrifuged at 3,000 x g, 4 °C for 15 min to separate plasma. Duplicate 200-ul and 750-u1 samples of plasma were pipetted into cryovials for ascorbic acid and antioxidant capacities, respectively. Nitrogen gas was added and samples were stored at -80 °C. Ascorbic acid analysis was performed at the Animal Health Diagnostic Laboratory, Nutrition Section, Michigan State University, Michigan. Antioxidant capacities were analyzed at the laboratory of Dr. R]. Harmon, University of Kentucky, Lexington, Kentucky. VII. Ascorbic Acid (AA) Protocol Plasma ascorbic acid was measured by HPLC using isocratic mobile plnse buffers and a reverse-phase, C18 column coupled with electrochemical detection and compared with a known ascorbic acid standard (AAUA-1010). The protocol is described in detail (Appendix D). Briefly, eachsarnple (200 quasmixedwithbuffer(400 ulofl mM90%rnethanolin water saturated with EDTA) to precipitate protein. They were vortexed, incubated on ice for 10min,andcentrifirgedat3000rpm,4°Cfor 15min. Thesupermtarrtswerethentransfen'ed toanothersetofplasticmicrotubesandplacedonice. Beforenmningthesamples,thecohnnn waspreparedbypassingthemobilephase solutionthroughtheentire systemandrinsingthe pump seal with different comentrations ofMeOH. Ten-microliter samples were injected into thepreparedHPLCcohunn, andquantifiedbysingle-poirrt cah'brationagainstaknown ascorbic acid standard (AAUA-lOlO). Peak height or area integration was considered as the respome factor. TthofascoflacidwascalcuhtedMonnticallyonaMMumspreadsheet. Thehuermlandemernalstandmdvahwwemusedmflwqmmificmionandddemmwdbyflm 22 specific peak of each sample and AAUA, respectively. Both inter- and intra-assay coefficient of variations which were 13.6% and 5.9%, respectively, were also included in the calculations. The content for each sample was divided by injection volume (10 p1), multiplied by adihrtion factor (3, the addition oftissue buffer), and finally reported ian concentrations ofascorbic acid. VIH. AntioxidantActivitia (AOA) Protocol The assay measured the antioxidant ability of plasma, which inhibited chemical damage to phycoerythrin induced by the oxidative agent (i.e., mM 2,2'-azobis (2- amidinopropane) hydrochloride, AAPH) and was detected by the rate of phycoerythrin fluorescence emission. It was previously performed (Glazer, 1988), and described in detail for this experiment (Appendix E). Briefly, as a control, the 4-ml final reaction mixtures contained 3.58 ml of 75 mM sodium phosphate buffer (pH 7.0), 0.02 ml of 1.7x10'6 M B- phycoerythrin (B-PE), and 0.4 ml of 40 mM 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH, an initiator of the oxidative reaction) at 37 °C. Into each sample tube, 0.2 m1 diluted plasma (1:320 dilution in the final volume) was added in the same mixtures in place of 0.2-ml buffer. The solution was excited at 525 nm, aml emission was read at 575 nm. The fluorescence was measured at 37 °C in a digital fluorometer Wdiately before and at 5-minute intervals for 40 min after the addition of AAPH. Sample AOA was calculated and reported as percentage inhibition values of the decay of fluorescence (FL) of the compound phycoerythrin. The percentage inhibition was calculated as: % = [(change FL control - change FL sample) / (change FL control)] x 100 The change in FL was that which occurred over the 40-minute incubation. The greater the 23 % inhibition, the greater the antioxidant capacity of the plasma. The control in the assay is the rate of decay of fluorescence with no antioxidant present. Hydroxyl radicals are generated by this reaction and cause the decay. The samples were measured in duplicate and the means are displayed on graphs. DC Clinical Monitoring Cows were clinically monitored at 12 hr before challenge, immediately before challenge, and 2, 3, 4, 6, 9, 12, 24, 36, and 48 hr after challenge (Table 30, Appendix B). Rectal temperature, ruminal contraction rate, heart rate, and respiratory rate were all concomitantly measured. Quarter and milk appearance were also observed compared with the appearance before LPS challenge. X Milking Procedure Cows were milked twice daily at approximately 10-12-hour intervals (A.M.-P.M.) by a quarter milking machine fiom Monday evening through Saturday morning. All quarters were post-dipped with a post-dipping solution soon after each milking. Dry matter intakes (DMTs) were also recorded each day. )0. Statistical Analysis A repeated measurement analysis (Statistical Analysis System, SAS” Institute 1989-1996), was used for comparisons among the four treatments (00, CO, OH, CH). Specific contrasts were used to determine the effects of ascorbic acid, histidine, non- ascorbic acid, and non-histidine group. Period (week 1, 2, 3, & 4), front or hind quarter, and cow were also included as independent variables. The test for sphericity on the GLM printout (Mauchly's criterion) applied to Orthoganol components was used to indicate if a 24 multivariate analysis was needed. With this statistical method, the following dependent variables were tested: milk production, rectal temperature, log SCC, milk IgG], AOA, Heart rate, respiratory rate, and ruminal contraction rate. A period variable was included to adjust for carryover effects of the previous endotoxin or treatments sufficiently long time was not allowed between the administration of different treatments to the same cow. Due to a sumll sample size (8 cows) with many repeated measure, repeated measures were grouped into 3-4 groups in order of the time. A copy of the SAS and output is shown in AppendixF. 25 RESULTS The data was analyzed to compare cows that were administered ascorbic acid (AA) with cows not treated with ascorbic acid, and to compare cows treated with L- histidine (H) with cows not treated with L-His. Comparisons among individual treatment groups (control, ascorbic acid only, L-His only, and ascorbic acid + L-His) were made, however in order to present a concise discussion on the critical hypothesis of this research, the comparisons among treatments are attached in Appendix C. I. Rectal Temperature Mean rectal temperature in AA treated cows was significantly lower than in non- AA cows from 24 to 48 hr afler LPS challenge (P = 0.0393, Figure 1). However, there was no difference between H and non-H cows over the experimental period (Figure 1). Mean rectal temperature in period 1 was significantly lower than in period 2 and 3 from 0 to 4 hr post LPS challenge (P < 0.04, Figure 2). Mean rectal temperature in period 4 was also significantly lower than in period 2 and 3 fiom 3 to 4 hr post LPS challenge (P < 0.056, Figure 2). The data is plotted in Figures 1 and 2. II. Somatic Cell Count Mean somatic cell count in non-AA cows was significantly higher (P = 0.0261, Table 4) than in AA cows at 24 and 36 hr post LPS challenge. Moreover, mean SCC in H cows was significantly lower than in non-H cows fi'om 6 to 24-hr after challenge (P = 0.0164). Log SCC looked consistent among treatments (Figure 3). 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Quarter Milk production There was no significant difference between AA and non-AA cows, and H and non-H cows throughout the trial. Mean quarter milk production tended to be higher in H cows as compared to non-H cows at 12 hour post LPS challenge (P = 0.0875, Table 7). The data is plotted in Figures 5 and 6. Daily quarter milk production and daily change are shown in Figures 7 and 8. Milk fiom non-challenged quarters remained normal both in appearance and bacteriologically negative throughout the period of study. IVE Composite Milk production The lowest amount of mean composite milk production (kg i SEM) was at 12 hr post-LPS challenge for all treatment groups (AA = 7.28 i 0.92, non-AA = 6.94 :l: 0.81, H = 6.36 :t 0.86, and non-H = 6.70 :t 0.89) (Table 11). The mean milk production following AA treatments was significantly higher from 48 to 96 hr post-LPS challenge as compared to the milk production in the non-AA treatments (P < 0.02, Table 11). 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IO .. 5.5- m 8+ 5.5 m. 5.5 .... 55 w 96 APPENDIX D ASCORBIC ACID PROTOCOL: ELECTROCHEMICAL DETECTION/HPLC ANALYSIS OF ASCORBIC ACID Animal Health Diagnostic Laboratory, Nutrition Section, Michigan State University Mater-ink: Ascorbic acid (Sigrm A—7506), dodecyln-imethylammonium bromide (Sigma D863 8), ethylenediaminetetraacetic acid disodium salt (EDTA, Baker), methanol (Baxter), o- phosphoric acid (85%, Fisher), sodium acetate (Baker 3470-01), Sodium phosplnte (monobasic anhydrous, and Sigma S-O751) were used without further purification Specifications 1) Ascorbic Acid (C6H806) MW: 176.12 2,3—endiol-L-gulonic acid-gamma-Iactone (Ascorbic acid) pK1= 4.17 pKz = 11.57 Both free acid and salt are colorless, crystalline, highly water-soluble; not stable at pH >10. Absorbance WM: 245 nmat acid medium; 265 at neutralmedium HPLC system: TheI-IPLC systemdevebpedtomasm'eascorbicacidusesisocraticnnbflephase bufl‘er delivering and reverse phase C18 column coupled with electrochemical detection. Mobile Phase:0.05 M sodium phosphate, 0.05 M sodium acetate, 97 300 mg/l dodecyltrimethylanmionimn bromide 40 M EDTA in 5% methanol in water (v/v), pH 4.8. Column: 3.9x 150mmNova-Pak C18 60A4 pm(Waters) andaguard-pak cartridge Holder with Nova-Pak C13 guard-pak precolumn insert (Waters) Detector: BSA Coulochem II Multi-electrode Detector, Model 5200 Analytical Cell: ESA Model 5010 Guard Cell: ESA Model 5020 Approximate Settings: Potential Current Guard cell: 350 mV Electrode l: -200 mV 2—400 1 to 5 uA depending on samples Electrode 2: 300 mV $300 1 to 5 uA depending on samples Dummy cell is not connected HPLC Mobile Phase Preparation Mobile Phase Solution: 0.05 M sodiumphosplnte (NaH2P04) 0.05 M sodiumacetate (CH3COONa) 300 mg/l dodecyltrimethylarmnonium Br 40 M EDTA (disodium salt) 5% (v/v) methanol in H20; pH: 4.8 For 2 Liter Solution: 98 .3 g; l iw 1) Add 1800mloffieshdouble distilled waterina2 L beaker. 2) Add and stir: 13.8 g NaHzPO4 x H20 (12.0 anhydrous) 8.2 g CH3COONa (anhydrous) 600 - 800 mg dodecyltrimethylanunonimn Br 3) Add29.8mgdisodiumEDTAsaltandstir(Donotuseahigherconcentration ofEDTA as it will increase background current) 4) WhenEDTAistotallydissolvedadd 100mlof100% methanol. u” 5) Transfer solution to a 2000 ml graduated cylinder and q.s. to 2000 ml with E. double distilled water. 6) Adjust pH to 4.8 with 85% o-phosphoric acid (about 3 ml) 7) Filter with 0.2 pm Nylon-66 filter (Rainin, vacuum filtration) into a 2 liter vacuum flask Leave the vacuum on for another 20 min for degassing. HPLC Standards Standard Bufl'er: 0.05 M sodiumphosphate (NaH2P04) 0.05 M sodiumacetate (CH3COONa) 0.1 mM EDTA (disodium salt) 5% (v/v) methanol in H20; pH: 4.8 For 500 ml solution: 1) 2) 3) Add400mldoubledistilledwaterto abeaker. Add and stir: 3.45 g NaH2P04 x H20 (3.0 anhydrous) 2.05 g CH3COONa Add 18.6 mg EDTA disodiumsalt and stir. 4) Add25mloflOO% methanol 5) q.s.to500mlina500mlgraduatedcylinderwithdoubledistilledwater. 6) Adjust pH to 4.8 with 85% o-phosphoric acid (about 0.75 ml) 7) Filter with 0.2 pm Nylon—66 filter (Rainin, vacuum filtration) and degas with vacuum for an additional 20 min. 8) Sealandstoreat4°C. Standard Solutions (keep tubes in ice): 1) 2) 3) 4) Stock solution A a) WeighoutSmgascorbicacidintofoilwrappedtesttube. b) Add 10 ml standard bufi‘er (always use newly-made cold standard buffer) This will leave a 500 pig/ml solution. Stock solution _B a) Take40ulofstocksolutionAandputintoaibilwrappedtesttube. b) Add 9.96 ml standard bufl‘er, and this will leave a 2 pg/ml (or 20 ng/10 pl) solution. Stock solution Q a) WeighomlmgmicacidandpmintoafoilwrappedSOmlvohuneu‘icflask. b) Add 50 ml standard bufi‘er (always use newly-made cold standard hifier) and mix well. This will leave a 20 ug/ml (or 200 ng/10 pl) solution. Note: the solubility of uric acid in water is very limited. Do not try to make a higher concentration. Stock solution 2 100 a) Takelmlofstocksohltiongandpmintoafoilwrappedtesttube. b) Add9mlstandardbufl‘er. Thiswillleavea2.0ug/ml(20ng/10ul)soh1tion. 5) 2 mix standard solution (AAUA-1010) Nfixequalvolumesofstocksohrtbnfland2(10ngascorbicacidand lOnguricacid per 10 p.1(AAUA-1010)) Place sample inmicro tubes. 6) AAUA-lOlO is stored at -80 °C and is good for at least a month Sample Preparation 5.1 Tune Butler: in 90%methamlinwatersatmatedwithEDTA(fimlconcenfiationisabom 1 mM) Add 37.2 mg EDTA in 100 ml of90% methanol/water solution, stir for 20 min and store in refrigerator. Phsma Preparation 1) Bbedmunnlsandcoflectbbodmvacuummbeswhichcomammparinmnahermfive is to collect blood into EDTA tubes, but heparin gives better results) 2) Centrifuge inatable-topmicrofi1geat3000rpmat4°C for 15min. 3) Label 1.5 ml plastic microcentrifiige tubes. 3) Transfer 200 u] of supenmtant (plasrm) to each microcentrifiige tube. 4) Store in ultra-low freezer (-80 °C) or analyze for ascorbic acid as described in the following sections. Measurement of Ascorbic Acid Precipitation: 1) Remove6-8 samplesfiomultra-lowfieezerandplacemarackinice-coldwaterto lOl 2) 3) 4) 5) thaw the plasma or continue from the previous section. Onevohnneofplasnnismixedwithtwo vohunes (400pfinthiscase) oftissuebuffer. Vortexandincubate onice for 10min. Centrifiigeat3000rpm,4°Cfor 15min. Transfer supernatant to another set of plastic microtubes and place them on ice (ascorbicacidarestable only foracoupleofhoursonice, sodo notprcparetoonnny tubes each time) Analyzing with HPLC-EC 1) 2) 3) 4) 5) Readythecohmmandanalyticalcellforthemobilephase by: a. Running in filtered 50% MeOH-water for 30 min at .5 mein. b. Rumiing in fresh filtered 5% MeOH/distilled water 30 min at 1.0 ml/min. c. Rtmningfi‘eshfilteredmobilephasetoeqmlibrateovemightat 1.0ml/min. Take wastetubefiomMeOHwaste containerandplaceitintothemobilephasecontainerto recycle. Dothisonlywhenyouaresmethemobilephasehaspassedthmughtheentire system. Daily, pfiortonmninganysarnplesrinsepumpsealwith 10%MeOH. It is important to mix samples prior to each injection. Inject 10 pl AAUAlOlO standard before starting injection ofsamples; runtwo or more standard injections. Inject lOnlpreparedsamplesonto HPLCcolurmi. IncludeCaninePlasmal if possible. Atthe end ofsample injections orthe endofthe day, inject 10 plAAUAlOlO standard 102 6) two times. Leave equilibrating overnight if you will use it again the next day. a. reverse potentials onthe electrodes for 10-15 min. while nmning mobile phase to waste. b. run fresh filtered 5%MeOH/distilled water at lml/min for 30 min. c. nm filtered 50% MeOH/water at .5 ml/min for 30 min. d. nm filtered 100% MeOH at .5 ml/min for 1 hour. e. rtm 50% MeOH for 30 min again and store in 50% MeOH/water. Calculation of results (calculated automatically on Millenium) 1) 2) 3) 4) PhcemmsmaspreadsleetlflteExcelMflkruumflhehnermlandextermlsthmd valuewereused inthequantificationanddeterminedbythe specificpeakofeach sample and AAUA, respectively to calculate the content ofascorbic acid in samples. Content ofascorbic acid or uric acid divided by injection volume (10 pl) gives concentrations of these acids in the injected sample. Include dilution factor (3x - due to addition of tissue buffer to precipitate protein) Inter-assay coefficient of var'mtion (%) was calculated by (standard deviation / mean of all samples) x 100. But intra-assay coefficient of variation (%) was based on any single sampleatanysingledayforabom24databasisandcakuhtedmthesamennnner. In this study, inter- and intra-assay coefiicient of variation are 13.6 % and 5.9 %, respectively. Report Imits in mg/dl. They will be converted into mM concentrations on the report. Troubleshooting 103 Due to electrode fouling, it is necessaryto electrically set the condition of the ebcuodespefiodkany.1hemocedmeismtomfinedmflrennnuals.thmobflephase numingtowastesetelectrodes to 1000 mV for 10-15 min. Thenreversethepotentialto -400 mV for 10-15 min. Firnlly, rmke anew HDVA afierreconditioning. 104 Wflfielfi '3 .‘Q r— APPENDIX E PHYCOERYTHRIN FLUORESCENCE-BASED ASSAY FOR REACTIVE OXYGEN SPECIES The assay for reactive oxygen species depends on the detection of chemical damage to phycoerythrin through the decrease in its fluorescence emission. The fluorescence of phycobiliproteins is highly sensitive to the conformation and chemical integrity of the protein and prosthetics. Under the appropriate conditions, in the presence of reactive oxygen species, therateoflossofphycoerytlninfluorescenceisanirxlexoffieeradicaldamage. Theefl‘ectof addedcompoundsontherate ofthis fluorescence lossisaneasureoftheirabilityto protect the protein (Glazer, 1990). Reagents Porphyridium cruenturn B-phycoerythrin (B-PE; Sigrm, St. Louis, MO), a very soluble protein (>10 mg/ml in the 75 mM sodium phosphate buffer, pH 7.0) used in this assay. This stock solutionscanbe stored in4 °C formonths. A40 nMstock solutionofthe water- soluble fiee radical initiator 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH; MW 267; Polysciences)wasprepared inthepH 7.0bufl'erimmediate1ybeforeusearxl stored onice. Contaminating metal ions were removed fi'om 75 mM sodium phosphate butler, pH 7.0, by passage through a lO—ml colunm ofChelex with 100 resin (50-100 dry mesh), sodium form (Sigma, St. Louis, MO). WM 105 The assay for measuring the AOA in plasma was previously performed (Glazer, 1988). The final reaction mixtures contained 0.85x10‘8MB-PE, 2 mMAAPH, and other additives in 75 mM phosphate bufl‘er, pH 7.0 (made fi'om 75 mMK2HP04 and 75 mM NaH2P04) at 37 °C inafinalvolume of4 ml, in 12x75 mmround borosilicate glasstubes. The mixture was added to the control tube in the following order: 3.58 ml of phosphate buffer, 0.02 ml of 1 .7x10‘MB—phycoerythrin, and 0.4 ml of40 mMAAPH. Into each sample tube, 0.2-ml diluted plasma was added in place of the same volume of buffer. All final dilution of plasma in all runs is 1:320. ThereactionwasinhiatedbyaddhrgO.4mlof40mMAAPH(fieshlypreparedmid stored on ice) to the other components in 3.60 ml at 37 °C. Compensation for the temperature drop due to this addition required approximately 2 min. The solution was excited at 525 nm using a #58 filter, and emission was read at 575 mnusing a #23A filter. The emission intensity wasadjustedto areadablerangeusingtheexcitationwirxiowsetat 10X. Fluorescencewas treasured at 37 °C in a Turner Model 112 Digital Fluorometer (Sequoia-Turner Corporation, Mountain View, CA) immediately before and at 5-min interval for 40 min after addition of AAPH. Sample antioxidant activity was converted to percentage inhibition using the formula given below: % Inhibition = [(Change FL Control - Change FL Sample) / (Change FL Control)] x 100 106 APPENDIX F SAS 8: OUTPUT SAS PROGRAM EDITOR ‘libname 'c:\ac'; em; Data one; set ac.commilk; *define treatments; lftx = 1 then AA=0; if tx = 1 then hist=0; if tx = 2 then AA=1; if tx = 2 then hist=0; if tx = 3 then AA=1; iftx = 3 then hist=l; if tx = 4 then AA=1; iftx = 4 then hist=1; *Define groups G3=(m12+h0+h12)/3; GZ=(h24+h36)/2; Gl=(h48+h60+h72+h84+h96)/5; proc 21111; class period tx cow half; model g1 g2 g3= tx cow half period; repeated group 3 (1 2 3)/summary printe; 107 contrast'AA'tx-l 1-1 1; contrast'Hist'tx-l -1 1 1; contrast'OO-OH‘tx-l 0 10; contrast'OO-OC'tx-l 100; contrast'OO-CI-l'tx-IOO 1; contrast 'CO-OH‘ txO 1-10; contrast'CO-CI-l'txo 1 0-1; contrast 'OH-CH'tXOO l -1; title 'AA & L-Histidine'; “mi SAS OUTPUT Class Levels PERIOD 4 TX 4 COW 8 HALF 2 AA & L-Histidine General Linear Models Procedure Class Level Information Values 1234 1234 2612 2813 2926 2937 2952 3049 3133 3268 12 Numberofobservatimsindataset=32 General Linear Models Procedure Dependent Variable: G1 Somce DF Sum of Squares Mean Square F Value Pr > F Model 14 445.92015016 31.85143930 24.54 0.0001 Error 17 22.06521784 1.29795399 Corrected Total 31 467.98536800 R-Square C.V. Root MSE G1 Mean 108 0.952851 Source DF TX 3 COW 7 HALF 1 PERIOD 3 Source DF TX 3 COW 7 HALF 1 PERIOD 3 Dependent Variable: 62 Source DF Model 14 Error 1 7 Corrected Total 31 R-Square 0.848026 Source DF TX 3 COW 7 HALF 1 PERIOD 3 Source DF TX 3 COW 7 HALF 1 10.22049 Type I SS 12.57249100 424.88535400 0.00017904 8.46212612 Type 111 SS 13.40584709 424.88535400 0.83765716 8.46212612 Sum of Squares 245.83894152 44.05674598 289.89568750 C.V. 18.65801 Tlvpe 1 SS 3.58623750 229.72368750 1.42224516 1 1.10677136 Type 111 SS 3.380191 10 229.72368750 0.68090402 1.13927784 11.14700000 Mean Square F Value Pr > F 4.19083033 3.23 0.0486 60.69790771 46.76 0.0001 0.00017904 0.00 0.9908 2.82070871 2.17 0.1287 MeanSquare FValue Pr>F 4.46861570 3.44 0.0403 60.69790771 46.76 0.0001 0.83765716 0.65 0.4329 2.82070871 2.17 0.1287 Mean Square F Value Pr > F 17.55992439 6.78 0.0002 2.59157329 Root MSE G2 Mean 1.60983642 8.62812500 MeanSquare FValue Pr>F 1.19541250 0.46 0.7130 32.81766964 12.66 0.0001 1.42224516 0.55 0.4689 3.70225712 1.43 0.2692 Mean Square F Value Pr > F 1 . 12673037 0.43 0.7309 32.81766964 12.66 0.0001 0.68090402 0.26 0.6148 109 PERIOD Dependent Variable: GB Source Model Error 3 DF 14 17 CorrectedTotal 31 Source TX COW HALF PERIOD Source COW HALF PERIOD R-Square 0.914005 DF 11.10677136 Sum of Squares 423 . 12637277 39.81041577 462.93678854 C.V. 15.30817 Type I SS 3.02667326 408.2901 1354 11.41746810 0.39211786 Type 111 SS 2.79063300 408.29011354 4.32716270 0.39211786 3.70225712 1.43 0.2692 MeanSquare FValue Pr>F 30.22331234 12.91 0.0001 2.34178916 Root MSE G3 Mean 1.53029055 9.99656250 Mean Square F Value Pr > F 1.00889109 0.43 0.7336 58.32715908 24.91 0.0001 11.41746810 4.88 0.0413 0.13070595 0.06 0.9821 Mean Square F Value Pr > F 0.93021 100 0.40 0.7567 58.32715908 24.91 0.0001 4.32716270 1.85 0.1918 0.13070595 0.06 0.9821 Repeated Measures Analysis of Variance Repeated Measures Level Information Dependent Variable G1 Level of GROUP 1 G2 G3 2 3 AA & L-Histidine General Linear Models Procedure Repeated Measures Analysis of Variance 110 Partial Correlation Coefficients fi'om the Error SS&CP Matrix / Prob > |r| DF = 17 61 GZ G3 01 1.000000 0.476577 0.463698 0.0001 0.0455 0.0526 02 0.476577 1.000000 0.535779 0.0455 0.0001 0.0219 G3 0.463698 0.535779 1.000000 0.0526 0.0219 0.0001 E = Error SS&CP Matrix GROUPN represents the contrast between the nth level of GROUP and the last GROUPJ GROUP.2 GROUPJ 34.38924576 18.48803187 GROUP.2 18.48803187 38.99054271 Partial Correlation Coefficients from the Error SS&CP Matrix of the Variables Defined by the Specified Transformation / Prob > M DF = 17 GROUP] GROUP.2 GROUPJ 1.000000 0.504893 0.0001 0.0326 GROUP.2 0.504893 1.000000 0.0326 0.0001 Test for Sphericity: Mauchly‘s Criterion = 0.742153 Chisquare Approximation = 4.7711976 with 2 df Prob > Chisquare = 0.0920 Applied to Orthogonal Components: Test for Sphericity: Mauchly's Criterion = 0.9947029 Chisquare Approximation = 0.0849795 with 2 df Prob > Chkquare = 0.9584 111 Manova Test Criteria and Exact F Statistics for the Hypothesis of no GROUP Effect H = Type III SS&CP Matrix for GROUP E = Error SS&CP Matrix S=l M=0 N=7 Statistic Value F Num DF Den DF Pr > F Wilks' Lambda 0.15197217 44.6412 2 16 0.0001 Pillai's Trace 0.84802783 44.6412 2 16 0.0001 Hotelling-Lawley Trace 5.58015209 44.6412 2 16 0.0001 Roy's Greatest Root 5.58015209 44.6412 2 16 0.0001 Manova Test Criteria and F Approximations for the Hypothesis of no GROUP‘TX Efl‘ect H = Type HI SS&CP Matrix for GROUP*TX E = Error SS&CP Matrix S=2 M=0 N=7 Statistic Value F Num DF Den DF Pr > F Wilks' Lambda 0.696591 19 1.0568 6 32 0.4084 Pillai's Trace 0.30915168 1.0361 6 34 0.4194 Hotelling-Lawley Trace 0.4273 1799 l .0683 6 30 0.403 1 Roy's Greatest Root 0.4070651 1 2.3067 3 17 0.1132 NOTE: F Statistic for Roy's Greatest Root is an upper bound. NOTE: F Statistic for Wilks' Lambda is exact. Manova Test Criteria and F Approximations for the Hypothesis of no GROUP‘COW Effect H = Type III SS&CP Matrix for GROUP‘COW E = Error SS&CP Matrix S=2 M=2 N=7 Statistic Value F Num DF Den DF Pr > F Wilks' Lambda 0.31042105 1.8168 14 32 0.0800 Pillai's Trace 0.81363088 1.6656 14 34 0.1 1 10 112 Hotelling-Lawley Trace 1.82180623 1.9519 14 30 0.0609 Roy's Greatest Root 1.56673825 3.8049 7 17 0.0115 NOTE: F Statistic for Roy’s Greatest Root is an upper bound. NOTE: F Statistic for Wilks' Lambda is exact. Manova Test Criteria and Exact F Statistics for the Hypothesis of no GROUP‘HALF Effect H = Type III SS&CP Matrix for GROUP‘HALF E = Error SS&CP Matrix S=1 M=0 N=7 Statistic Value F Num DF Den DF Pr > F Wilks' Lambda 0.79035271 2.1221 2 16 0.1523 Pillai's Trace 0.20964729 2.1221 2 16 0.1523 Hotelling-Lawley Trace 0.26525789 2.1221 2 16 0.1523 Roy's Greatest Root 0.26525789 2.1221 2 16 0.1523 Manova Test Criteria and F Approximations for the Hypothesis of no GROUP‘PERIOD Effect H = Type III SS&CP Matrix for GROUP'PERIOD E = Error SS&CP Matrix S=2 M=0 N=7 Statistic Value F Num DF Den DF Pr > F Wilks' lambda 0.71389075 0.9789 6 32 0.4555 Pillai's Trace 0.30577154 1.0227 6 34 0.4274 Hotelling-Lawley Trace 0.37323212 0.9331 6 30 0.4859 Roy's Greatest Root 0.27195727 1.5411 3 17 0.2402 NOTE: F Statistic for Roy's Greatest Root is an upper bound. NOTE: F Statistic for Wilks' Lambda is exact. General Linear Models Procedtu'e Repeated Measures Analysis of Variance 113 Tests of Hypotheses for Between Subjects Effects Type 111 SS Mean Square F Value Pr > F 12.25154126 4.08384709 1.00 0.4162 1028.38350418 146.91192917 36.02 0.0001 1.32018762 1.32018762 0.32 0.5768 13.08439260 4.36146420 1.07 0.3883 69.33787520 4.07869854 Univariate Tests of Hypotheses for Within Subject Efl‘ects Source DF TX 3 COW 7 HALF 1 PERIOD 3 Error 1 7 Source: GROUP or Type 111 ss 2 101.76916158 Source: GROUP*TX DF Type 111 SS 6 732512993 Source: GROUP*COW DF Type 111 SS 14 34.51565086 Source: GROUP’HALF DF Type 111 SS 2 4.52553626 Source: GROUP‘PERIOD Adj Pr > F MeanSquare FValue Pr>F G-G H-F 50.88458079 47.28 0.0001 0.0001 0.0001 Adj Pr > F MeanSquare FValue Pr>F G-G H-F 1.22085499 1.13 0.3638 0.3639 0.3638 Adj Pr > F MeanSquare FValue Pr>F G-G H-F 2.46540363 2.29 0.0243 0.0246 0.0243 Adj Pr > F MeanSquare FValue Pr>F G-G H-F 2.26276813 2.10 0.1378 0.1381 0.1378 Adj Pr>F 114 DF TypeIIISS MeanSquare FValue Pr>F G-G H-F 6 6.87662274 1.14610379 1.06 0.4025 0.4024 0.4025 Source: Error(GROUP) DF Type 111 SS Mean Square 34 36.59450439 1.07630895 Greenhouse-Geisser Epsilon = 0.9947 Huynh-Feldt Epsilon = 2.0540 Contrast Variable: GROUP.1 Analysis of Variance of Contrast Variables GROUP.N represents the contrast between the nth level of GROUP and the last Source DF Type 111 SS Mean Square F Value Pr > F MEAN 1 42.35220613 42.35220613 20.94 0.0003 TX 3 1 1.33778106 3.77926035 1.87 0.1733 COW 7 9.1793 1421 1.31133060 0.65 0.7112 HALF 1 8.97253772 8.97253772 4.44 0.0504 PERIOD 3 7.63272646 2.54424215 1 .26 0.3203 Error 17 34.38924576 2.02289681 Contrast Variable: GROUP.2 Som’ce DF Type 111 SS Mean Square F Value Pr > F MEAN 1 59.92387812 59.92387812 26.13 0.0001 TX 3 0.85442009 0.28480670 0. 12 0.9445 COW 7 43.60529687 6.22932812 2.72 0.0438 HALF l 1.57506076 1.57506076 0.69 0.4188 PERIOD 3 9.24101001 3.08033667 1.34 0.2936 115 Error 17 3 8.99054271 2.293 56 1 34 General Linear Models Procedure Dependent Variable: G1 Contrast DF Contrast SS Mean Square F Value Pr > F AA 1 10.39184007 10.39184007 8.01 0.0116 Hist 1 1.02102050 1.02102050 0.79 0.3875 OO-OH 1 3.21792600 3.21792600 2.48 0.1338 OO-OC 1 1.49940025 1 .49940025 1 .16 0.2975 OO-CH 1 2.53178904 2.53178904 1.95 0.1805 CO-OH 1 9.04547188 9.04547188 6.97 0.0172 CO-CH 1 0.14246730 0.14246730 0.1 1 0.7445 OH-CH 1 10.88542634 10.88542634 8.39 0.0100 Dependent Variable: 02 Contrast DF Contrast SS Mean Square F Value Pr > F AA 1 0.38623893 0.38623893 0.15 0.7042 Hist 1 2.96461250 2.96461250 1.14 0.2998 OO-OH 1 1.20990007 1.20990007 0.47 0.5036 OO-OC 1 0.30802500 0.30802500 0.12 0.7345 OO-CH 1 0.58290358 0.58290358 0.22 0.6413 CO-OH 1 2.72271323 2.72271323 1.05 0.3197 CO-CH 1 1.72552200 1.72552200 0.67 0.4258 OH—CH 1 0.10755360 0.10755360 0.04 0.8410 Dependent Variable: G3 Contrast DF Contrast SS Mean Square F Value Pr > F AA 1 0.60724713 0.60724713 0.26 0.6171 Hist 1 1.3764170] 1.3764170] 0.59 0.4538 OO-OH 1 0.04042907 0.04042907 0.02 0.8970 116 In.” .. an; tat-.321- OO-CH CO-OH CO-CH OH-CH 1.41015625 0.07099087 1.89917907 2.08362975 0.00405974 1.41015625 0.07099087 1.89917907 2.08362975 0.00405974 117 0.60 0.03 0.81 0.89 0.00 0.4484 0.8638 0.3804 0.3588 0.9673 REFERENCES Alsemgeest, S. P. M., H. C. Kalsbeek, Th. Wensing, J. P. Koeman, A. M. van Ederen, and E. Gruys. 1994. Concentrations of serum amyloid-A (SAA) and haptoglobin (HP) as parameters of inflammatory diseases in cattle. The Veterinary Quarterly. 16(1): 21-23. Anderson, K. L. 1987. Management of coliform mastitis in dairy cows. Agri-Practice. 17-21. Anderson, K. L. 1989. Therapy for acute coliform mastitis. The Compendium Continuing Education: Food Animal. 11(9):1125-1133. Anderson, K. L., H. Kindahl, A. Petroni, A. R. Smith, and B. K. Gustafsson. 1985. Arachidonic acid metabolites in milk of cows during acute coliform mastitis. Am. J. Vet. Res. 46(7):1573-1577. Anderson, K. L., A. R. Smith, R. D. Shanks, L. E. Davis, and B. K. Gustafsson. 1986. Efficacy of flunixin meglumine for the treatment of endotoxin-induced bovine mastitis. . Am. J. Vet. Res. 47(6):1366-1372. Anderson, R. 1985. The irnmunostirnulatory, anti-inflammatory, and anti-allergic properties of ascorbate. Advanced In Nutritional Research. 6: 19-45. Anderson, R., and P. T. Jones. 1982. Increase leukoattractant binding and reversible inhibition of neutrophfl motility mediated by the peroxidase/I120; lhalide system: effect of ascorbate, cysteine, dithiothreito], levamisole and thiamine. Clin. Exp. Immunol. 47:487. Babizhayev, M. A., M. C. Seguin, J. Gueyne, R P. Evstigneeva, E. A. Ageyeva, and G. A. Zheltukhina. 1994. L-Camosine (fl-alanyl-L-histidine) and carcinie (,B-alanyl histidine) act as natural antioxidants with hydroxyl-radical-scavenging and lipid peroxidases activities. Biochem. J. 304:509-516. de Quiroga, G. B., Lopez-Torres, M., Perez-Campo, R. and Rojas, C. 1991. Simultaneous deterrninationoftwo antioxidants, uricandascorbic acid, inanirnaltissueby higli-perfornnnce liquid cinematography. Am] Biochem. 199:81-85. Bartlett, P. C., and G. Y. Miller. 1993. Mastitis microbiology: What is considered normal? Agri-Practice. 14(6):]2-14. Bartlett, P. C., G. Y. Miller, S. E. Lance, and L. E. Heider. 1992. Clinical mastitis and intrannmrmry infections on Ohio dairy farms. Preventive Veterinary Medicine. 118 12:59-71. Becker, B. F., N. Reinholz, B. Lipert, et a1 1991. Role of uric acid as an endogenous radical scavenger and antioxidant. Chest. 100:176S-181S. Bendich, A. 1993. Physiological role of antioxidants in the immune system. J. Dairy Sci. 76:2789-2794. Bodannes R. S. and P. C. Chan. 1979. Ascorbic acid as a scavenger of singlet oxygen. FEBS Lett. 105:195-196. Bishop, J. G., F. L. Schanbacher, L.C. Ferguson, et a1. 1976. In vitro growth inhibition of mastitis-causing coliform bacteria by bovine apo-lactoferrin and reversal of inhibition by citrate and high concentrations of apo-lactoferrin. Infect. Immun. 14:91 1. 1".._fi 1N...' N». no: Bowers, T. L. 1997. Nutrition and immunity part 2: The role of selected micronutrients and clinical significance. Veterinary Clinical Nutrition. 4(3):96-101. Bramley, A., and F. Dodd. 1984. Review of the progress of dairy sciences: Mastitis control—progress and prospects. J. Dairy Sci. 51:481-512. Broadley, C., and R. L. Hoover. 1989. Ceruloplasmin reduces the adhesion and scavenges superoxide during the interaction of activated polymorphonuclear leukocytes with endothelial cells. Am. J. Pathol. 135:647-650. Burton, G. W., and K. U. Ingold. 1988. Mechanisrm of antioxidant action: preventive and chain-breaking antioxidants, in CRC Handbook of Free Radicals and Antioxidants in Biomedicine, Volume II, 29-43. Burvenich, C., M. J. Paape, A. W. Hill, A. J. Guidry, R. H. Miller, R. Heyneman, W. D. J. Kremer, and A. Brand. 1994. Role of the neutrophfl leukocyte in the local and systemic reactions during experimenatally induced E.coli mastitis in cows immediately after calving. The Veterinary Quarterly. 16(1):45-50. Bushell, A., L. Klenerman, H. Davies, 1. Grierson, and M.J. Jackson. 1996. Ischemic— reperfusion-induced muscular damage. Protective effect of corticosteroids and antioxidants in rabbits. ActaOrthop. Scand. 67(4):393-8(Abstr.). Cantoni, 0., P. Sestili, A. Guidarelli, P.U.Giacomoni, and F. Cattabeni. 1992. Effect of L-histidine on hydrogen peroxide-induced DNA damage and cytotoxicity in cultured mammalian cells. Molecular Pharmacology. 41:969-974. Guidarelli, A., P. Sesteli, A. Cossariza, C. Franceschi, F. Cattabeni, and O. Cantoni. 1995. Evidence for dissimilar mechanisms of enhancement of inorganic and 119 organic hydrogen peroxide cytotoxicity by L-histidine. The Journal of Pharmacology and Experimental therapeutics. 275: 1575-1582. Chalupa, W., C. J. Snifi‘en. 1991. Protein and amino acid nutrition of lactating dairy cow. In: Snifien C.J. and Herdt. T.H (eds.) Dairy nutrition management. Veterinary Clinics of North America: Food Animal Practice. 7(2):353-372. Chew, B. P. 1996. Importance of antioxidant vitamins in immunity and health animals. Animal Feed Science Technology. 59:103-114. Conner, J. G., and P. D. Eckersall. 1986. Acute phase response and mastitis in the cow. R. Vet. Sci. 41:126-128. Constable, P. D., L. M. Schmall, W. W. Muir, G. F. Hoffsis, E. R. Shertel. 1991. Hemodynamic response of endotoxemic calves to treatment with small-volume hypertonic saline solution. Am. J. Vet. Res. 52(7); 981-989. Cullor, J. S. 1991 . The Escherichia coli J5 vaccine: investigating a new tool to combat coliform mastitis. Veterinary Medicine. 86 (8):838-842. Cullor, J. S. 1993. The control, treatment, and prevention of the various types of bovine mastitis. Veterinary Medicine. 571-579. Cummins, K A. and C. J. Bnmner. 1989. Dietary ascorbic acid and immune response in dairy calves. J. Dairy Sci. 72:129-134. Dhariwal, K. R., Hartzell, W. O. and M. Levine. 1991. Ascorbic acid and dehydroascorbic acid measurements inhuman plasma and sertun. Am. J. Clin. Nutr. 54(4):712-716. DhariwaLK. R., Washko, P. W. andM. Levine. 1990. Determinationofdehydroascorbicacid using high-performance liquid chromatography with coulometric electrochemical detection. Anal Biochem. 189:18-23. Dobbins, C. N. 1977. Mastitis Losses. J. Am. Vet. Med. Assoc. 170:1129-1132. DeChatelet, L. R. 1978. Initiation of the respiratory burst in human polymorphonuclear neutrophils: A critical review. J. Reticuloendothelial Soc. 24:73-91 . DeGraves, F. J ., and K. L. Anderson. 1993. Ibuprofen treatment of endotoxin-induced mastitis in cows. Am. J. Vet. Res. 54(7):]128-1132. DeGraves, F. J., and J. F etrow. 1991. Partial budget analysis of vaccinating dairy cattle against coliform mastitis with an Escherichia coli J5 vaccine. J. Am. Vet. Med. Assoc. 199(4):451-455. 120 Doha, J. W. 1992. Ion-pair problems, LC-GC. 10:744-746. Dwenger, A., H. C. Pape, C. Bantel, G. Schweitzer, K. Krumm, M. Grotz, B. Lueken, M. Funck, and G. Regel. 1994. Ascorbic acid reduces the endotxin—induced lung injury in awake sheep. European Journal of Clinical Investigation. 24:229-235. Eberhart, R. J., R. P. Natzke, F. H. S. Newbould, B. Nonnecke, and P. Thompson. 1979. Coliform mastitis—a review. J. Dairy Sci. 62:1-22. Eicher-Pruiett, S. D., J. L. Morrill, F. Blecha, J. J. Higgins, N. V. Anderson, and P. G.Reddy. 1992. Neutrophfl and lymphocyte response to supplementation with vitamin C and E in young calves. J. Dairy Sci. 75:1635-1642. Erskine, R. J. 1993. Nutrition and Mastitis. In: Anderson K L (ed.) Update on bovine mastitis. Veterinary Clinics of North America: Food Animal Practice. 9(3):551-6. Erskine, R. J., and P. C. Bartlett. 1993. Serum concentrations of copper, iron, and zinc during Escherichia coli mastitis. J. Dairy Sci. 76:408-413. Erskine, R. J., R. J. Eberhart, L. J. Hutchison, and R. W. Scholz. 1987. Blood selenium concentrations and glutathione peroxidase activities in dairy herds with high and low somatic cell counts. J. Am. Vet. Med. Assoc. 190(11);l417-l421. Erskine, R J., R. J. Eberhart, P. J. Grasso, and R. W. Scholz. 1989. Induction of Escherichia coli mastitis in cows fed selenium-deficient or selenium-supplemented diets. Am. J. Vet. Res. 50:2093-2100. Erskine, R. J., R. J. Eberhart, L. J. Hutchison, S. B. Spencer, and M. A. Campbell. 1988. Incidence and types of clinical mastitis in dairy herds with high and low somatic cell counts. J. Am. Vet. Med. Assoc. 192(6):766-768. Erskine, R. J., J. W. Tyler, M. G. Riddell, and R C. Wilson. 1991. Theory, use, and realities of efficacy an food safety of antimicrobial treatment of acute coliform mastitis. J. Am. Vet. Med. Assoc. 198:980-984. Erskine, R. J., R. C. Wilson, M. G. Riddle, J. W. Tyler, H. J. Spears, and B. S. Davis. 1992. Intrarnamrnary administration of gentamicin as treatment for experimentally induced Escherichia coli mastitis in cows. Am. J. Vet. Res. 53(3):375-381. Erskine, R. J., J. H. Kirk, J. W. Tyler, and F. J. DeGraves. 1993. Advanced in the therapy for mastitis. In: Anderson K.L.(ed.), Veterinary Clinics of North America: Food Animal Practice. 9(3):499-517. Erskine, R. J., R C. Wilson, J. W. Tyler, K A. McClure, R. S. Nelson, and H. J. Spears. 1995. Cetiofur distribution in serum and milk from clinically normal cows and 121 cows with experiment Escherichia coli-induced mastitis. Am. J. Vet. Res. 56(4):481-485. Fang W. and S. Pyorala. 1996. Mastitis-causing Escherichai coli: serum sensitivity and susceptibility to selected antibacterials in milk. J Dairy Sci. 79:76-82. Fleiss, J. L. 1986. The crossover study. In: The design and analysis of clinical experiments. John Wiley & Sons, Inc. 263. Fox, L.K, C.W. Heald. 1981. Effect of cortisol on the bactericidal function of the bovine milk neutrophil in vitro. Am. J. Vet. Res. 42:1933-1936. Frost, A. J., B. E. Brooker, and A. W. Hill. 1984. The Effect of Escherichia coli endotoxin and culture filtrate on the lactating bovine mammary gland. Aust. Vet. J. 61 :77. Giri, S. N., Z. Chen, E. J. Carol], R Mueller, M. J. Schiedt, and L. Panico. 1984. Role of prostaglandins in pathogenesis of bovine mastitis induced by Escherichia coli endotoxin. Am. J. Vet. Res. 45(3):586-591. Glazer, A. N. 1988. Fluorescence-based assay for reactive oxygen species: a protective role for creatinine. FASEB. 2:2487-2491. Glazer, A. N. 1990. Phycoerythrin fluorescence-based assay for reactive oxygen species. Method in Enzymology. 186:161. Gonzalez, R. N., J. S. Cullor, D. E. Jasper, T. B. Farver, R. B. Bushnell, and M. N. Oilver. 1989. Prevention of clinical coliform mastitis in dairy cows by a mutant Escherichia coli vaccine. Can. J. Vet. Res. 53:301-305. Gonzalez, R. N. , D. E. Jasper, N. C. Kronlund, et a1. 1990. Clinical mastitis in California dairy herds participating in contagious rmstitis control program. J. Dairy Sci. 73:648-660. Grasso, P. J., K W. Scholz, R J. Erskine, and R. J. Eberhart. 1990. Phagocytosis, bactericidal activity, and oxidative metabolism of milk neutrophils from dairy cows fed selenium-supplemented and selenium-deficient diets. Am. J. Vet. Res. 5 1(2):269-274. Gross, W. B., D. Jones, and J. Cherry. 1988. Effect of ascorbic acid on the disease caused by Escherichia coli challenge infection. Avian Diseases. 32:407-409. Guidry, A. J., M. J. Pappe, and R. E. Pearson. 1980. Efl‘ect of udder inflammation on milk irmnunoglobulins and phagocytosis. Am J. Vet. Res. 41(5):751-753. 122 Guihot, G. and F. Blachier. 1997. Histidine and histamine metabolism in rat enterocytes. Mol. Cell Biochem. 175(1-2):143-8 (Abstr.). Halliwell,B.1987. Freeradiealsandmetalionsinhealthand disease. Proc.Nutri. Soc. 46:13. Hatch, L. L. and Sevanian, A. 1984. Measurement of mic acid, ascorbic acid and related metabolites in biological fluids. Anal Biochem. 138:324-328. Hill, A. W. 1979. The pathogenesis of experimental Escherichia coli mastitis in newly calved dairy cows. Res. Vet. Sci. 26:97-101. Hill, A. W. 1981. Factors influencing the outcome of Escherichia coli mastitis in the dairy cow. Res. Vet. Sci. 31:107-112. Hill, A. W., A. L. Shears, and K G. Hrhbitt. 1979. The pathogenesis of Escherichia coli mastitis in newly calved dairy cows. Res. Vet. Sci. 26:97-101. Hogan, J. S., K. L. Smith, K H. Hoblet, P. S. Schoenberger, D. A. Todhunter, W. D. Hueston, D. E. Pritchard, G. L. Bowman, L. E. Heider, B. L. Brockett, and H. R. Conrad. 1989a. Field survey of clinical mastitis in low somatic cell count herds. J. Dairy Sci. 72:1547-1556. Hogan, J. S., K L. Smith, K H. Hoblet, D. A. Todhunter, P. S. Schoenberger, W. D. Hueston, D. E. Pritchard, G. L. Bowman, L. E. Heider, B. L. Brockett, and H. R. Conrad. 1989b. Bacterial counts in bedding materials used on nine Ohio commercial dairies J. Dairy Sci. 72:250-258. Hogan, J.S., KL. Smith, D.A. Todhunter, and RS. Schoenberger. 1992a. Field trial to determine of an Escherichia coli J5 mastitis vaccine. J. Dairy Sci. 75:78-84. Hogan, J. S., W. P. Weiss, D. A. Todhunter, and K L. Smith. 1992b. Eflicacy of an Escherichia coli J5 mastitis vaccine in experimental challenge trial. J. Dairy Sci. 75:415-422. Itze, L. 1984. Ascorbic acid metabolism in ruminants. In: Wagger, 1., F .J. Tagwerker, and J. Moustgaard (eds) Ascorbic Acid in Domestic Animals. The royal danish agriculture society, Copenhagen, DK 120-130. Jackson, J. A., D. E. Shuster, W. J. Silvia, and R. J. Harmon. 1990. Physiological responses to intramammary or intravenous treatment with endotoxin in lactating dairy cows. J. Dairy Sci. 73:627-632. Janeway, C. A., P. Travers, S. Hunt, M. Walport. 1997. Host defense against infection. In: Immunobiology: The immune system of health and disease, Part V. 9: 1-9:52. 123 Janzen, J. J., J. R. Bishop, A. B. Bodine, C. A. Caldwell, and D. W. Johnson. 1982. Conposted dairy waste solids and crushed limestone as bedding in free stalls. J. Dairy Sci. 65:1025. Jain, N. C., O. W. Schahn, and J. Lasrnanis. 1978. Neutrophil kinetics in endotoxin- induced mastitis. Am. J. Vet. Res. 39(10):]662-1667. Jones, G. F., and G. E. Ward. 1990. Evaluation of systemic gentamicin for treatment of coliform mastitis in cows. J. Am. Vet. Med. Assoc. 197:731-735. Jacob, R. A. 1995. The Integrated Antioxidant System. Nutrition Research. 15(5):755- 766. Kawamoto, T., Y. Ikeda, and A. Teramoto. 1997. Protective effect of L-histidine (singlet oxygen scavenger) on transient forebrain ischemia in the rat. No-To-Shinkei. 49(7):612-8.(Abstr.). Kehrli, M. E., and D. E.Shuster. 1994. Factors fleeting milk somatic cells and their role in health of the bovine mammary gland. J. Dairy Sci. 77:619-627. Kitchen, B. J. 1981. Review of the progress of dairy science: Bovine mastitis: milk compositional changes and related diagnostic tests. Journal of Dairy Research. 48: 167-1 88. Li Y. and R. T. Lovell. 1985. Elevated levels of dietary ascorbic acid increase immune responses in Channel catfish. J. Nutri. 115:123-131. Lin, Y., L. Xia, J.D. Turner, X. Zhao. 1995. Morphological observation of neutrophil diapedesis across bovine mammary gland epithelium in vitro. Am. J. Vet. Res. 56(2):203-207. Lohuis, J. A. C. M., W. Van Leeuwen, J. H. M. Verheijden, A.S.J.P.A.M. Van Miert, and A. Brand. 1988a. Effect of dexamethasom on experiment Escherichia coil mastitis in the cow. J. Dairy Sci. 71:2782-2789. Lohuis, J. A. C. M., J. H. M. Verheijden, C. Bervenich, and A. S. J. P. A. M. Van Miert. 1988b. Pathophysiological effects of endotoxins in ruminants. 1. Changes in body temperature and reticulo-rumen motility, and the effect of repeated administration. The Veterinary Quarterly. 10(2):109-125. Lohuis, J. A. C. M., W. Van Leeuwen, J. H. M. Verheijden, A. Brand, and A. S. J. P. A. M. Van Miert. 1989. Effect of steroid anti-inflammatory drugs on Escherichia coil endotoxin-induced mastitis in the cow. J. Dairy Sci. 72:241-249. 124 Lohuis, J. A. C. M., Y. H. Schukken, J. H. M. Verheijden, A. Brand, and A. S. J. P. A. M. Van Miert. 1990. Effect of severity of systemic signs during the acute phase of experimentally induced Escherichia coli mastitis on milk production losses. J. Dairy Sci. 73:333-341. Loscher, W., G. Jaeschke, and H. Keller. 1984. Pharmacokinetics of ascorbic acid in horses. Equine Veterinay Journal 16:59-65. Lu, M. 1997. Phycoerythrin Fluorescence-Based Assay for reactive oxygen species. MS. Thesis. The Dairy Section, University of Kentucky, Lexington, Kentucky. Machlin, L.J., and A. Bendich. 1987. Free radical tissue damage: Protective role of antioxidant nutrients. FASEB. 1:441-446. Margolis, S.A., Paule, RC. and Ziegler, RG. 1990. Ascorbic and dehydroascorbic acid measmed in plasma preserved with dithiothreitol or metaphosphoric acid. Clin. Chem. 36:1750-1755. Margolis, S.A., Ziegler, R.G. and Helzlsouer, KJ. 1991. Ascorbic and dehydroascorbic acid measurement inhuman serum and plasma. Am. J. Clin. Nutr. 54:13158-18S. Maslinski C., D. Kierska, W. A. Fogel, A. Kinnunen, and P. Panula. 1993. Histamine: Its metabolism and localization in mammary gland. Comp. Biochem. Physiol. 105C(2):269-273. Miller, G. Y., P. C. Bartlett, S. E. Lance, J. Anderson, and L. E. Heider. 1993. Costs of clinical mastitis and mastitis prevention in dairy herds. J. Am. Vet. Med. Assoc. 202(8): 1230-1236. Morin, D. E., P. D. Constable, and G. C. McCoy. 1998. Use of clinical parameters for differentiation of gram-positive and gram-negative mastitis in dairy cows vaccinated against lipopolysaccharide core antigens. J. Dairy Sci. 212: 1423-143 1. Nagy, I. Z. S., and R. A. Floyd. 1984. Hydroxyl fiee radical reactions with amino acids and protein studies by electron spin resonance spectroscopy and spin-trapping. Biochim. Biophys. Acta. 790:238-250. Natzke, R. P., and B. J. LeClair. 1976. Coliform contaminated bedding and new infections. J. Dairy Sci. 59:2152. Niki, E. 1991a. Action of ascorbic acid as a scavenger of active and stable oxygen radicals. Am J. Clin. Nutr. 54:1119S-24S. Niki, E. 1991b. Vitamin C as an antioxidant. In: Sirnopoulos, A. P (ed ) Selected vitamins, minerals, and fimctional consequencesof maternal malnutrition. World 125 Rev. Nutr. Diet. Basel, Karger. 64:1-30. Pappe, M. J. and W. P. Wergin. 1977. The leukocytes as a defense mechanisms. J. Am. Vet. Med. Assoc. 170(10(2)):1214-1223. Peterson, J. W., I. Boldogh, V. L. Popov, S. S. Saini, and A. K Chopra. 1998. Anti- inflarmnatory and antisecretory potential of histidine in Salmonella-challenged mouse small intestine. Laboratory Investigation. 78(5):523-534. Politis, I., X. Zhao, B.W. McBride, and J.H. Burton. 1991. The effect of lipopolysaccharide on bovine marmnary macrOphage ftmction. Can J Vet Res. 55:220-223. Raetz, C. R. H. 1993. Bacterial endotoxin: extraordinary lipids that activate eukaryotic signal transduction. Journal of Bacteriology. 175:5745-53. Reneau, J. K 1993. Clinical mastitis records in production medicine programs. The compendium: Food animal. 15(3):497-503. Roth, J. A and M. L. Kaeberle. 1985. In vivo efl‘ect ascorbic acid on neutrophil function in healthy and dexarnethosone-treated cattle. Am. J. Vet. Res. 46(12):2434-2436. Sargison, N. and P. Scott. 1996. Supportive therapy of generalized endotoxemia in cattle using hypertonic saline. In Practice. 18(1):]8-19. Schalm, O. W. 1977. Pathologic changes in the milk and udder of cow with mastitis. J. Am. Vet. Med. Assoc. 170: 1137-1140. Sevanian, A., Davies K J. A and P. Hochstein. 1991. Serum mate as an antioxidant for ascorbic acid. Am. J. Clin. Nutr. 54:1129S-34S. Shuster, D. E., and R. J. Harmon. 1991. Lactating cows become partially refi'actory to fi'equent intramammary endotoxin infusions: recovery of milk yield despite a persistently high somatic cell count. Res. Vet. Sci. 51:272-277. Shuster, D. E., and R. J. Harmon. 1992. High cortisol concentration and mediation of the hypogalactia during endotoxin-induced mastitis. J. Dairy Sci. 75:739-746. Shuster, D.E., RJ. Harmon, J. A. Jackson, and R. W. Hemken. 19913. Suppression of milk production during endotoxin-induced mastitis. J. Dairy Sci. 74:3763-3774. Shuster, D. E., R. J. Harmon, J. A. Jackson, and R. W. Hemken. 1991b. Endotoxin rmstitis in cows milked four times daily. J. Dairy Sci. 74:1527-1538. Shuster, D. E., R. J. Harmon, J. A. Jackson, and R. W. Hemken. 1991c. Reduced 126 lactational performance following intravenous endotoxin administration to dairy cows. J. Dairy Sci. 74:3407-3411. Shuster, D. E., M. E. Kehrli Jr., and M. G. Stevens. 1993. Cytokine production during endotoxin-induced mastitis in lactating dairy cows. Am. J. Vet. Res. 54(1):80-85. Sitton N. G., J. S. Dixon, C. Astbury, R. J. Francis, H. A. Bird, and V. Wright. 1988. Kinetic investigations into the possible cause of low serum histidine in rheumatoid arthritis. Ann. Rheum. Dis. 47:48-52 (Abstr.). Smith, B. P. 1986. Understanding the roles of endotoxins in gram-negative septicemia. Vet. Med. 12:1148-1060. Smith, K L., J. H. Harrison, D. D. Hancock, D. A., Todhunter, and H. R. Conrad. 1984. Effect of vitamin E and selenium supplementation on incidence of clinical mastitis and duration of clinical symptoms. J. Dairy Sci. 67:1293. Smith, K.L., D.A. Todhunter, and P. Schoenberger. 1985. Environmental rmstitis: cause, prevalence, prevention. J. Dairy Sci. 68:1531-1553. Solonen, M, J. Hirvonen, S. Pyorala, S. Sankari, and M. Sandhohn 1996. Quantitative determination of bovine serum haptoglobin in experimentally induced Escherichai coli mastitis. Res. Vet. Sci. 60(1):88-91. Stowe, H. D. 1992. Project proposal to Ralston Pmina. Reneau, J. K 1993. Clinical mastitis records in production medicine program. The Compendium: Food Animal. 15(3):497-503. Tanaka M., N. Muto, E. Gohda, and I. Yamarnoto. 1994. Enhancement by ascorbic acid 2-glucosides or repeated additions of ascorbate of mitogen-induced IgM and IgG productions by human peripheral blood lymphocytes. Jpn J Pharmacol (K07). 66:451-6 (Abstr.). Tachon, P., A. Deflandre, and P. U. Giacomoni. 1994. ModuLation by L-histidine of H202-mediated damage of cellular and isolated DNA Carcinogenesis. 15(8):]621-1626. Tizard, I. R. 1996. Inflammation. In: Veterinary immunology, An introduction, fifth edition. W.B. Saunders Company, Philadelphia. 43-54. Tyler, J. W., J. S. Cullor, Spier, S. J. and B. P. Smith. 1990. Immunity targeting common core antigens of gram-mgative bacteria. Journal Internal Medicine. 4:17-25. Tyler, J. W., E. G. Welles, R. J. Erskine, Hui-Chu Lin, M. A. Williams, J. S. Spano, J. T. 127 Gaslin, and K A McClure. 1994a. Clinical and clinicopathalogical changes with endotoxin-induced mastitis treated with snmll volumes of isotonic or hypertonic sodium chloride administered intravenously. Am. J. Vet. Res. 55(2):278-287. Tyler, J. W., F. J. Degraves, R. J. Erskine, M. G. Riddle, Hui-Chu Lin, and J. H. Kirk. 1994b. Milk production in cows with endotoxin-induced mastitis treated with isotonic or hypertonic sodium chloride solution. J. Am. Vet. Med. Assoc. 204(12); 1949-1952. Van Steenhouse, J. L. 1987. Free radicals: Relation to tissue damage—a review. Vet Cli Pathol. 116:29-35. Verheijden, J. H. M., A. S. J. P. A. M. Van Miert, A. J. H. Schotrnan, and C. T. M. Van Duin. 1983. Pathophysiological aspects of E. coli mastitis in ruminants. Vet. Res. Commun. 7:229. Washko, P. W. Hartzell, W. O. and M. Levine. 1989. Ascorbic acid analysis using high- perforrmnce liquid chromatography with coulometric electrochemical detection. Anal Biochem. 181:276-282. W'mkler, J. K 1986. Mastitis. In Howard, 1. L. (ed): Current Veterinary Therapy 2: Food Animal Practice. Philadelphia, W.B. Saunders. 765-771. Zar, J. H. 1996. The latin square & repeat-measurement experimental designs. In: Biostatistic Analysis, 3rd edition. 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