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Use of Avian Antibodies against Lipopolysaccharide to Improve Performance in Early Lactation Dairy Cows

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Title:
Use of Avian Antibodies against Lipopolysaccharide to Improve Performance in Early Lactation Dairy Cows
Creator:
Ibarbia, Lucas
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
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Language:
english
Physical Description:
1 online resource (120 p.)

Thesis/Dissertation Information

Degree:
Master's ( M.S.)
Degree Grantor:
University of Florida
Degree Disciplines:
Veterinary Medical Sciences
Veterinary Medicine
Committee Chair:
DONOVAN,GERALD A
Committee Co-Chair:
GALVAO,KLIBS NEBLAN ALVES
Committee Members:
DILORENZO,NICOLAS
MAUNSELL,FIONA P
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Acidosis ( jstor )
Antibodies ( jstor )
Cattle ( jstor )
Dairy cattle ( jstor )
Diseases ( jstor )
Lactation ( jstor )
Milk ( jstor )
Milk production ( jstor )
Milk yield ( jstor )
Rumen ( jstor )
Veterinary Medicine -- Dissertations, Academic -- UF
antibodies -- dairy -- lipopolysaccharide -- polyclonal -- transition
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Veterinary Medical Sciences thesis, M.S.

Notes

Abstract:
Subacute ruminal acidosis (SARA) has been associated with decreased milk production. During SARA, increased free ruminal lipopolysaccharides (LPS) and its translocation to blood have been measured. Acute phase response is stimulated by LPS, and cytokines reduce DMI and milk production. An avian-derived anti-LPS polyclonal antibody preparation (PAP-LPS) was developed to use as a feed additive. Our objective was to evaluate the effectiveness of PAP-LPS in decreasing the intensity of the inflammatory response and increasing milk yield in early lactation dairy cows. Primiparous (Pr; n = 174) and multiparous (Mu, n = 226) Holstein cows from one herd were randomly allocated to one of three treatments: 1) PAP-LPS (n = 55 Pr and n = 70 Mu), 2) PLC (n = 66 Pr and 77 Mu), and 3) CTL (n = 53 Pr and 79 Mu). Cows on PAP-LPS received a dose of 3 g/d of PAP-LPS via oral drench in a water suspension, while PLC received the same dose of inactivated PAP-LPS. Control cows did not receive any treatment. Treatments were administered from d 0 (calving) until 14 DIM. Milk production and components were recorded from d 0 to d 98 and weekly averages, Energy Corrected Milk (ECM) and components were used in a repeated measures analysis using the MIXED Procedure of SAS and a first-order autoregressive covariate structure. Feeding PAP-LPS increased ECM (p = .0023) during the first 98 DIM. Total milk production also increase (p = 0.04) when compared with CTL (3881 and 3769 kg, respectively), while only tended to increase it (p = 0.07) when compared to PLC (3785 kg). A week x treatment interaction was observed (p = 0.097) for weekly average milk production, and was greater (p < 0.05) for PAP-LPS vs. Control on weeks 3, 4, and 10. On week 6, cows fed PAP-LPS had greater (P < 0.05) milk production than both, PLC and CTL. No difference in haptoglobin, ceruloplasmin or health disorders was observed between groups. In conclusion, feeding PAP-LPS increased milk production in early lactation dairy cows. Mechanism of action is unknown, but increased dry matter intake was proposed. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (M.S.)--University of Florida, 2014.
Local:
Adviser: DONOVAN,GERALD A.
Local:
Co-adviser: GALVAO,KLIBS NEBLAN ALVES.
Statement of Responsibility:
by Lucas Ibarbia.

Record Information

Source Institution:
UFRGP
Rights Management:
Copyright Ibarbia, Lucas. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Classification:
LD1780 2014 ( lcc )

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U SE OF AVIAN ANTIBODIES AGAINST LIPOPO LYSACCHARIDE TO IMPROVE PERFORMANCE IN EARLY LACTATION DAIRY COWS By LUCAS IBARBIA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR TH E DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014

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© 2014 Lucas Ibarbia

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To my grandma, Bela Also to all those who encouraged me to perus e a higher comprehensive level

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4 ACK NOWLEDGMENTS I would like to thank Dr. Art hur G. Donovan , chairman of the supervisory committee and Dr Nicolas DiLorenzo , member of the committee , who provided me guidance, motivation, support and trust but also were actively involved in the design of the experiment, analysis and interpretation of results and elaboration of the manuscript . My appreciation is extended to the committee members Dr. Fiona Maunsell and Dr Klibs Galvao ; who provide valuable insight in the analysis and interpretation of results . . Also, I want to thanks to John Arthlington and his lab technicians , who provide help in laboratory analysis during this thesis . Ver y special thanks to Dr . Risco and Dr. Rae for the ir continuous support and clinical training , and for allowing me to pursue this master degree simultaneously with my residency program . I would like also to express my gratitude to Drs. Gaby Maier, Dr Joao Bittar, inter and resident in our service and Delores Foreman, Laura Newman and Doddy Dee, Office manager, and technicians , for their help during the conduction of this study. I woul d like to particularly thank North Florida Holsteins in B ell, FL , they all provided an incredible support during the conduction of the farm work. I would like to extend my appreciation to Mr . Don B ennink for the unconditional support to b uild knowledge on dairy science and because he allowed to use his cows and facilities . I also want to thanks to all the people from the ir crew, the hard workers of dairy industry. Particularly I want to extend my th ankfulness to Federico Cunha who leads the whole group in my absence at the farm or at the lab. I must also thank to all the students from Animal Science and other colleagues without whom it would ha ve been impossible to implement the project: Kyle Craig, Jackie Shen (10), Christina Chang (10), Kelly Mills and Kelly Hill, Rose Worobec, Yoanna Martin, Lia Missena, Kevin Sanches, Sydney Hayter, Rachel Shedden, Alyssa

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5 Faltin , Hannah Poole, Rachel Lucia , Stephanie Osborn, Jessica Zimermann, Rodolfo Daetz and St eve that participated very intensively of the routine of this project. I would like also to thanks Camas Inc., for providing with the product. Final and special thanks to my family : parents and bros, and my friends from Argentina and in Gainesville that su pport ed me during this stage of my career.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURES ................................ ................................ ................................ ......................... 9 LIST OF ABBREVIATIONS ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................................ ... 12 CHAPTER 1 LITERATURE REVIEW ................................ ................................ ................................ ....... 14 The Transition Cow ................................ ................................ ................................ ................ 14 Subacute Ruminal Acidosis ................................ ................................ ................................ .... 17 Definition and Cause ................................ ................................ ................................ ....... 17 Prevalence ................................ ................................ ................................ ........................ 19 Consequences of SARA ................................ ................................ ................................ .. 19 Decreased or erratic feed intake ................................ ................................ ............... 20 Reduced milk yield ................................ ................................ ................................ ... 21 Altered milk components ................................ ................................ ......................... 22 Diarrhea and gastrointestinal damage ................................ ................................ ...... 22 Abscessation ................................ ................................ ................................ ............. 23 Laminitis ................................ ................................ ................................ ................... 23 SARA During the Early Postpartum Period ................................ ................................ .... 24 Prevention of SARA ................................ ................................ ................................ ........ 25 Primiparous vs. Multiparous ................................ ................................ ........................... 27 Economics ................................ ................................ ................................ ....................... 28 Lipopolysaccharide ................................ ................................ ................................ ................. 28 Lipopolysaccharide Structure ................................ ................................ .......................... 29 LPS An tigenicity and Mechanism of Action ................................ ................................ ... 30 LPS and Subacute Rumen Acidosis ................................ ................................ ................ 31 LPS Translocation to Circulation ................................ ................................ .................... 32 LPS, Vaccines and Oral Challenges ................................ ................................ ................ 34 Acute Phase Proteins ................................ ................................ ................................ .............. 36 IgY PAP ................................ ................................ ................................ ................................ . 41 IgY Structure ................................ ................................ ................................ ................... 41 Stability of IgY ................................ ................................ ................................ ................ 42 Mechanism of Action ................................ ................................ ................................ ...... 43 Experiments ................................ ................................ ................................ ..................... 43 2 MILK PRODUCTION AND MILK COMPONENTS ................................ .......................... 46

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7 Introduction ................................ ................................ ................................ ............................. 46 Materials and Methods ................................ ................................ ................................ ........... 50 Farm Description ................................ ................................ ................................ ............. 50 Cows, Housing and Feeding Management ................................ ................................ ...... 50 Exclusion Criteria ................................ ................................ ................................ ............ 52 Experimental Design and Treatments ................................ ................................ ............. 52 Sample size ................................ ................................ ................................ ............... 52 Treatments ................................ ................................ ................................ ................ 52 Preparation, delivery and timing of treatments ................................ ........................ 53 Randomization ................................ ................................ ................................ ......... 53 Data Collected ................................ ................................ ................................ ................. 54 Variables ................................ ................................ ................................ ................... 54 Calving and transition related disease definition ................................ ..................... 55 Statistical Analysis ................................ ................................ ................................ .......... 56 Results ................................ ................................ ................................ ................................ ..... 57 Descriptive Statistics ................................ ................................ ................................ ....... 57 Milk Production ................................ ................................ ................................ ............... 57 Milk Components ................................ ................................ ................................ ............ 58 Discussion ................................ ................................ ................................ ............................... 59 Conclusion ................................ ................................ ................................ .............................. 64 3 ACUTE PHASE RESPONSE AND HEALTH DISORDERS ................................ .............. 74 Introduction ................................ ................................ ................................ ............................. 74 Materials and Methods ................................ ................................ ................................ ........... 77 Experimental Design and Treatments ................................ ................................ ............. 78 Blood sample collection ................................ ................................ ........................... 78 Ceruloplasmin assay ................................ ................................ ................................ . 78 Haptoglobin assay ................................ ................................ ................................ .... 80 Variables ................................ ................................ ................................ .......................... 83 Statistical Analysis ................................ ................................ ................................ .......... 83 Results ................................ ................................ ................................ ................................ ..... 87 Descriptive Statistics ................................ ................................ ................................ ....... 87 Acute Phase Proteins ................................ ................................ ................................ ....... 87 Health Issues ................................ ................................ ................................ .................... 88 Discussion ................................ ................................ ................................ ............................... 88 Conclusion ................................ ................................ ................................ .............................. 93 4 GENERAL DISCUSION AND CONCLUSION ................................ ................................ ... 99 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ....... 119

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8 LIST OF TABLES Table page 2 1 Diet composition and ingredients of pr epartum and postpartum diets. ............................. 65 2 2 Vaccination protocol of prepartum heifers and cows and postpartum cows ..................... 66 2 3 Descriptive s tatistics for PAP LPS, Placebo and Control groups. ................................ ..... 67 3 1 Preparation of Controls for Cp assay ................................ ................................ ................. 85 3 2 Pearson Correlation Coeffi cient for APP and milk production ................................ ......... 97 3 3 Incidence of health disorders and cull cow in the first 98 days of lactation ...................... 98

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9 LIST OF FIGURES Figure page 2 1 Last squares means of weekly average milk yield through the first 98 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) and those not treated with dry egg yolk (CTL) ................................ ...... 68 2 2 Last squares means of total milk yield through the first 98 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS), inactivated PAP L PS (PLC) and those not treated with dry egg yolk (CTL) ................................ ................................ ......... 69 2 3 Last squares means of ECM through the first 98 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) and those n ot treated with dry egg yolk (CTL) ................................ ................................ .................. 70 2 4 Last squares means of Milk Protein percentage of primiparous and multiparous cows through the first 98 days in lactation in cows treated with avian an ti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) and those not treated with dry egg yolk (CTL) ................................ ................................ ................................ ................................ . 71 2 5 Last square means of Fat Yield (kg) through the first 98 days in lactation in cows t reated with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) and those not treated with dry egg yolk (CTL) ................................ ................................ ......... 72 2 6 Last squares means of Protein yield of primiparous and multiparo us cows shown through the first 98 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) and those not treated with dry egg yolk (CTL) ................................ ................................ ................................ ................................ . 73 3 1 Di agram of samples disposition in the rack of Cp assay ................................ ................... 85 3 2 Racks set up and labeling of tubes for Hp assay ................................ ................................ 86 3 3 Flat bottom 9 6 well plate for Hp Assay, same distribution as racks ................................ .. 86 3 4 Last squares means of plasma haptoglobin (Hp) concentration curves from 1 14 days in lactation in cows treated with avian anti LP S antibodies (PAP LPS), inactivated PAP LPS (PLC) and those not treated with dry egg yolk (CTL) .................... 95 3 5 Last squares means of plasma ceruloplasmin (Cp) concentration curves from 1 14 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) and those not treated with dry egg yolk (CTL) .................... 96

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10 LIST OF ABBREVIATIONS APP Acute Phase Proteins APR Acute Phase Response CNS Ce ntral Nervous System CTL Control Cp Ceruloplasmin DIM Days in milk DMI Dry matter intake DM Dry matter ECM Energy corrected milk Hp Haptoglobin Ig Immunoglobulin IL Interleukin LPS Lipopolysaccharide LTA Lipoteichoic acid MF % Milk Fat percentage MFD Milk fat depression MP % Milk Protein percentage NDF Neutral Detergent Fiber NEB Negative energy balance NEFA Non esterified fatty acids NFC Non fiber carbohydrates OR Odds ratio PAP Polyclonal antibody preparation peNDF Physically effective Neutral Detergent Fiber

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11 PLC Placebo SARA Sub acute rumen acidosis SCFA Short chain fatty acids S OP Standard operation procedure TLR Toll Like Receptor TNF Tumor Necrosis Factor VFA Volatile fatty acid

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12 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Masters of Science U SE OF AVIAN ANTIBODIES AGAINST LIPOPOLYSACCHARIDE TO IMPROVE PERFORMANCE IN EARLY LACTATION DAIRY COWS By Lucas Ibarbia August 2014 Chair: Arthur G. Donovan Major: Veterinary Medical Sciences Subacute ruminal acidosis (SARA) has been associated with decreased milk productio n. During SARA, increased free ruminal lipopolysaccharides (LPS) and its translocation to blood have been measured . Acute p hase response is stimulated by LPS, and cytokines reduce DMI and milk production. An avian derived anti LPS polyclonal antibody prep aration (PAP LPS) was developed to use as a feed additive. Our objective was to evaluate the effectiveness of PAP LPS in decreasing the intensity of the inflammatory resp onse and increasing milk yield i n early lactation dairy cows. Primiparous (Pr; n = 17 4) and multiparous (Mu, n = 226) Holstein cows from one herd were rando ml y allocated to one of three treatments: 1) PAP LPS (n = 55 Pr and n = 70 Mu), 2) PLC (n = 66 Pr and 77 Mu), and 3) C TL (n = 53 Pr and 79 Mu). Cows on PAP LPS received a dose of 3 g/d of PAP LPS via oral drench in a water suspension, while PLC received the same dose of inactivated PAP LPS. Control cows did not receive any trea tment. T reatments were administered from d 0 (calving) until 14 DIM. Milk production and components were recorde d from d 0 to d 98 and weekly averages , Energy Corrected Milk (ECM) and components were used in a repeated measures analysis using the MIXED Procedure of SAS and a first order autoregressive covariate structure. Feeding PAP LPS increased ECM ( p = .0023) du ring the first 98 DIM . Total milk production also increase ( p = 0.04) when compared with C TL (3881 and

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13 3769 kg, respectively), while only tended to increase it ( p = 0.07) when compared to PLC (3785 kg). A week x treatment interaction was observed ( p = 0.09 7 ) for weekly average milk production, and was greater ( p < 0.05) for PAP LPS vs. Control on weeks 3, 4, and 10. On week 6, cows fed PAP LPS had greater ( P < 0.05) milk production than both, PLC and CTL . No difference i n haptoglobin, ceruloplasmin or healt h disorders was observed between groups. In conclusion, feeding PAP LPS increased milk production in early lactation dairy cows. Mechanism of action is unknown, but increased dry matter intake was proposed .

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14 CHAPTER 1 LITERATURE REVIEW The Transition Cow Lactogenesis starts weeks before parturition when colostrum production starts (Bell, 1995) approximately 6 weeks, 3 weeks before to 3 weeks a fter parturition. During this period, intricate endocrine and metabolic processes adjust the metabolism to promote milk synthesis. The requirements of the postpartum cow are increased drastically after parturition to maintain lactation (Bell, 1995) , at the same time that the body needs to readjust to conceive again several months later. After parturition, milk production is rapidly increased, followed by increases in dry matter intake (DMI) (Bell, 1995; Ingvartsen and Andersen, 2000) . This was demo nstrated after administration of a n exogenous source of somatotropin , when cows enhance d milk yield and subsequently increased voluntary DMI (Ingvartsen and Andersen, 2000) . This review states that regulation is there One objective for the dairyman is to produce as much milk per cow per lactation as possible and Hutjens (2008) proposed that 225 to 250 more pounds of milk could be expected during lactation , per pound of milk yield increased at its peak of lactation . Since the energy consumed in the diet is not enough to cover milk output, body fat is mobiliz ed in order to fill this gap (Butler et al., 2006) . Nevertheless, because milk production is being increased, if DMI is not increased at a similar rate, the interna cover the energy needs. As a consequence of fat mobilization , high blood concentration of non esterified fatty acids (NEFA) could then depress DMI through the hepatic oxidation pathway (Allen e t al., 2009) . Another pathway that affects DMI and might be active during early

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15 postpartum is the cytokine pathway (Finck et al., 1998) . Researchers have demonstrated in mice models that when intraperitoneal injection of LPS results in decreased DMI , due to the action of increased , it stimulates the release of leptin producing the latter depression of appetite through inhibition of appetite center in the brain. Understanding and managing the transiti on period is extremely important in dairy production, as it is the most challenging period in the productive life of the dairy cow. The concept of multifactorial cause s needs to be kept in mind to understand and prevent production diseases during early lac tation and decrease the risk of culling (Andersen, 2003) . Other metabolic, immunologic and nutritional factors around parturition, excludin g the already mentioned negative energy balance, affect health and performan ce of transition animals (Mulligan and Doherty, 2008) . Hypocalcemia, ketosis, both with clinical or subclinical presentation, metritis, mastitis, and gastrointestinal tract alterations are the most common diseases f ound throughout early postpartum period (Benzaquen et al., 2007) . Some postpartum diseases have the ability to activate inflammatory processes. Metritis, the inflammation of all layers of the uterus (Lewis, 1997) , has been shown to b e closely associated to calcemium status (Martinez et al., 2012) and hypocalcemic cows are more severely affected than normocalcem ic cows. Gram negative bac teria , such as Escherichia coli, have been identified as a cause of metritis (Galvao et al., 2009) . Lipopolysacaride (LPS) is a component of the wall of gram negative bacteria , and it has been shown to increase the c oncentration of acute phase proteins ( haptoglobin ) . Some research had observed haptoglobin concentrations throughout the transition period and reported increased concentrations in healthy and metritic cows (Huzzey et al., 2009; Galvao et al., 2010) . The incidence of metritis varies depending on several fac tors, such as parity, season and management practices, and it has been reported to be

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16 as high as 40 % (Benzaquen et al., 2007; Galvao et al., 2010) . Mastitis is inflammation of the mammary gland (Bradley, 2002) ; E. coli has also been isolated in clinical cases (Blum et al., 2014) . Several proinflammatory cytokines, including IL phase proteins such as haptoglobin, serum amyloid A, and LPS binding protein have been detected in cows suffering clinical mastitis (Bannerman, 2009; Wenz et al., 2010; Larsen et al., 2010) . Mastitis incidence varies widely and several factors had been mention to affect this rate (Barkema et al., 19 99) . Ketosis is a metabolic condition that high producing cows normally undergo, although it can be a pathologic situation when it reaches a certain threshold (Oetzel, 2007; McArt et al., 2 011) . Ketosis incidence varies widely between herds , ranging from approximately 12 to 56 % (Duffield et al., 1998; Oetzel, 2007; McArt et al., 2011) . Oetzel (2007) p roposed 10 % as an alarm level for a herd incidence rate. Hepatic lipidosis or fatty liver is n ot a rare finding i n ketotic cows , with over conditioned cows being at higher risk (Rukkwamsuk et al., 1999) . Ketosis i s highly correlated with nutritional problems, and the incidence of abomasal displacements is often increased in herd s suffering ketosis (Oetzel, 2007) . The dairy industry is undergo ing a dramatic transformatio n with fewer co ws producing more milk in larger herds. Standardized milk yields have to be accomplished by the cows to remain in their herds , and in the periparturient dairy cows several diseases affect DMI and consequently milk yield. Great effort is done to prevent diseases and improve health status of this category of animals. Understanding and properly managing the transition period is one of the necessary pillars to build sustainable dairy herds and all this elements together are part of the reasons th at motivates the concretion of this trial.

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17 Suba cute Ruminal Acidosis Definition and Cause Rumen acidosis is cause d by abnormally low ruminal pH. Depending on the pathophysiology it can be defined as acute rumen acidosis, happening mainly under feedlot si tuations, and sub acute rumen or ruminal acidosis (SARA), found more commonly in dairy cattle. These two scenarios are a function of the type and amount of feed provided. Acute rumen acidosis is the result of a rapid drop in pH due to a rise in lactic acid concentration in the presence of diets high in rapidly fermentable carbohydrates and low in effective fiber, whereas SARA is caused by high dry matter intake and consequent high pro ductionof volatile fatty acids and the accumulation in the rumen which produce s the drop in pH (Goad et al., 1998; Plaizier et al., 2008) . Sub a cute Rumen Acidosis can be defined as the pathological decrease of the ruminal pH for several hours on consecutiv e days. Three elements are mentioned in the literature to depict SARA: pH, duration or time within the day that pH is below a threshold and an increase in free ruminal LPS concentration has a lso been proposed to be added to the definition (Gozho et al., 2005) . At present, there is no agreement on a pH threshold . Kleen et al. (2003) define SARA as rumen pH below 5.5 for some hours after concentrate feeding. Another group proposed the same threshold to diagnose cows with S ARA (Garrett et al., 1999) . Krause and Oetzel (2005) define the cutoff value as 5.6. Ruminal pH values of 5.8 have also been used as threshold for SARA (Beauchemin et al., 200 3) , but others consider pH in this range (5.8 to 5.6) as high risk for the presence of SARA (Stone, 1999) . With respect to duration of low rumen pH, some researchers proposed that at least 3 hours is needed (Gozho et al., 2005) but others suggest longer periods (5.4 h/day) (Zebeli et al., 2008) in order to diagnose SARA. This is based on the observation that fiber digestibility is

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18 decreased and levels of acute phase proteins are increased when this threshold is exceeded. Gozho et al. (2005a) propose to redefine SARA , suggesting that free ruminal LPS concentration should be considered. Diagnosis of SARA is not an easy task because it is often a s ubclinical disease (Mulligan and Doherty, 2008) . Obtaining a representative rumen fluid sample can be problematic. Different methods to measure ruminal pH are used , and each has advantages and disadvantages with res pect to obtaining the true ruminal pH value. The site where pH is measured also has an influence over this result. There are currently four methods to measure ruminal pH (Duffield et al., 2004) : cannulation and direct ruminal fluid sampling, indwelling pH meter, ruminocentesis (percutaneous needle aspiration), and oro ruminal probe, but only the last two are used under field conditions. The main disadvantage of oro ruminal probe is saliva contamination which could lead to erroneously high pH results and misdiagnosis (Nocek, 1997) . For this reason it is recommended to discard the first sample obtained and measure pH in a second sample . Duffield et al. (2004) evaluated and compared t he performance of ruminocentesis and oro ruminal (Geishauser) probe with the objective of identifying the most accurate method to measure ruminal pH , using samples obtained through a rumen cannula as the gold standard. They reported that ruminocentesis was a better field test to rule out rather than to confirm SARA when pH is marginal (5.8) but could be used to confirm the disease when pH is lower due to a higher specificity at low pH (5.5). A second sample taken by the oro ruminal probe method correlates b etter with ruminocentesis than the first one. Duffield et al. (2004) suggest s a cutoff value of 5.8 by this method to diagnose SARA. Rumen pH varies during the day (Keunen et al., 2002) ; therefore, sampling timing i s important in SARA diagnosis . It has been suggested to sample rumen pH 5 to 8 hours after

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19 feeding of total mixed rations (Stone, 1999) . A nother observation is that frequen t feeding of TMR maintained a stable but lower ruminal pH , compared to twice daily ( 2x ) feeding . Mean pH for cows f ed multiple times was lower (5.78 vs 6.02) than cows fe d 2 times . This was observed when pH from a n individual cow f ed 2X or 6X were compared (Oetzel and Nordlund, 1998) . Higher availability of organic matter in the rumen due to increase d DMI (21.3 vs 17.4 kg DM/day when 2X) leads to a higher VFA production and drop in pH. Prevalence Surveys indicate that SARA is prevalent in 19% of early lactatio n and 26% of the mid lactation cows in Wisconsin (Garrett et al., 1997) . Anothe r survey generated in the same re gion reported a prevalence of 20.1% in early and mid lactation cows (Oetzel and Nordlund, 1999) . Dairy cows under grazing systems are not exempt from SARA. It has been reported that in Australian and Irish farms, 10 to 15% of cows undergo this condition (Bramley et al., 2005; O'Grady et al., 2008) . In a publication by an Italian group, SARA was detected in three herds out of ten, with more than 33% of the cows with rumen pH below 5.5 (Morgante et al., 2007) . Additionally, f ive of these farms showed pH between 5.8 and 5.5, increasing the proportion of affected farms to 80%. A study of 18 Dutch dairy herds showed an overall prevalence of 13.8% ranging from 0 to 38% on a farm basis (Kleen et al ., 2009) . Consequences of SARA Consequences of SARA mentioned in the literature include feed intake depression or erratic DMI behavior, reduced fiber digestibility, reduced milk yield, altered milk components, gastrointestinal damage, abscesses and caud al vena cava syndrome, and laminitis (Krause and Oetzel, 2005; Gressley, 2014) . Production wise, SARA has also be en associated with high culling rate s and turnover rates because of unspecific ca uses, poor performance and poor body condition , although some disagree on this last concept (Kleen et al., 2003) .

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20 Decreased or erratic f eed i ntake S ubacute rumen acidosis occurs in bouts , with ruminal pH recovering to baseline value, without intervention, within several hours or days (Dohme et al., 2008) . This publication proposes that cows with SARA may alter their intake behavior in order to avoid future SARA eriment challenged cows to three acidosis eve nts in three consecutive 14 day periods. Highly variable feed consumption was seen, compared to their baseline days. There was a trend for lower DMI in the recovery phase of the first period, higher DMI in the s ame phase of the second period and lower consumption for the recovery period of the third event (Dohme et al., 2008) . Ruminal pH profile decreased with each subsequent acidosis challenge period and duration time (time pH below 5.8) was augmented after every challenge. Reduced fiber digesti on is one of the reasons why decreased DM I may occur during SARA. F ibrolytic rumen bacteria are pH sensitive and ruminal pH below 6 affects their survival (due to increased maintenanc e energy requirements reducing the absolute number of bacteria) (Shi and Weimer, 1992) . As fiber digestion slows, physical fill effect will limit feed intake , down regulating it by increasing the retention time of food in the rumen (Allen, 2000) . Another possible reason for lower DMI during SARA is due to the accumulation of propionate. Non fiber carbohydrate (NFC) fermentation leads to high propionate concentrations in the rumen. Propi onic acid has been shown to act as a short term regulator of DMI (Allen, 2000) . It has been proposed that when absorbed, propionate acts on the hepatocyte enhancing the energetic status of the cell , which will alter th e firing rate o f the vagus nerve, and finally inducing satiety at the central nervous system level. Another hypothesis for reduced DMI during SARA associates the drop in DMI with the generalized inflammation seen during endotoxic events, and with reduced r umen motility associated with increas ed concentration of LPS. When non lactating dairy cows were exposed

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21 every 3 weeks to different doses of intravenous LPS concentrations, a decrease in ruminal contractions was reported (Jacobsen et al., 2005) . The duration of rumen hypomotility increased with increasing dosage, lasting on average, 5, 24 and 36 hours for 10, 100 and 1000 ng LPS/kg challenged. Using the same experiment but in a different publication (Jacobsen et al., 2004) , acute phase proteins (APP) were analyzed and a rapid response was reported for haptoglobin and serum amyloid A (inflammatory markers). Gozho et al. (2005) induced SARA in steers through the usage of differe nt proportions of concentrates and forages, and was able to measure an erratic DM consumption during the induction period with high intakes some days followed by low intake on the following days. These finding were also correlated with increases in APP dur ing the induction period, particularly haptoglobin and serum amyloid A. Reduced milk y ield Reduced milk yield seen during S ARA or during recovery periods after induction of SARA may be the result of decrease d DMI. Krause and Oetzel (2005) induced SARA in lactating dairy cows and, as a consequence of decrease DMI, milk production was decrease d by 3.5 kg per day (35.2 vs 31.5 ± 2 kg) during the challenge and the recovery period. Another publication reported an approximated decrease of 6 pounds (2.7 kg) per d ay in a commercial dairy farm suffering from SARA (Stone, 1999) . In another study, SARA was induced in mid lactation dairy cows and compared with their controls. Average DMI and milk production were not different when diet, day or diet by day effect was analyzed (Gozho et al., 2007) . Zebeli and Ametaj (2009) reported a decrease in fat corrected milk (FCM) as well as milk energy efficiency (MEE), calculated as grams of milk fat divid ed by kg of DMI, when increasing amounts of barley was added to the diet of lactating dairy cows. Another group reported a linear decrease in

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22 milk yield from 35.9 to 32.7 kg per day when SARA was induce by replacing chopped alfalfa with pelleted alfalfa in mid lactation dairy cows (Khafipour et al., 2009a) . Altered milk c omponents SARA has been associated with a decrease in milk fat content. In general, diets high in energy content due to high amounts of rumen fe rmentable carbohydrates lead to decreased ruminal pH due to high VFA concentration in the rumen and also to milk fat depression (MFD). In an experiment w h ere increasing proportions of barley grain w ere fed in order to induce SARA, a linear decrease in milk fat content and milk fat yield was reported (Zebeli and Ametaj, 2009) . Khafipour et al . , (2009a) also reported a linear decrease in milk fat percentage and fat yield when dairy cows where challenged by gradually replac ing the physical chara cteristic of alfalfa (chopped vs pelleted) over 5 week period . In a clinical investigation (Stone, 1999) observed an increase in milk fat and protein levels of 0.3% and 0.1%, respectively, after d iets were adjusted to correc t a SARA scenario diagnosed in a herd. Diarrhea and gastrointestinal d amage Dia rrhea has been associated with SARA , although it is more likely to be found in acute acidosis. The main reason for this diarrhea is the increased amo unt of highly fermentable carbohydrates reaching the hindgut without being fermented in the rumen. Carbohydrates that escape the rumen are osmotically active, moving water from the tissues to the lumen of the gut, giving the aqueous consistency to feces. T hese nutrients are also an ene rgy source for hindgut bacteria and the hindgut act s as a small rumen causing secondary fermentation . VFA and CO 2 are produced being the first one absorbed, and the latter contributing to the foamy aspect of feces . It i s not u ncommon to find signs of intestinal inflammation, such as clear mucus or fibrin, mixed with feces in cows with SARA (Gressley, 2014) .

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23 Abscess ation During SARA, ruminitis can occur as a result of low ruminal pH ca used by accumulation of VFA, increased osmolarity, and also increased free LPS and biogenic amines (Gressley, 2014) . Emmanuel et al . , (2007) demonstrated in vitro that LPS and pH act synergistically to disrupt the rumen epithelial barrier. In healthy rumen epithelial cells, when butyrate is absorbed, (OH) butyrate, the nontoxic metabolite, and then moved to the portal circulation. It is also known that butyric acid causes inhibition of mitosis and stimulates cellular proliferation and differentiation (Thibault et al., 2010) . If butyrate accumulates in the rumen epithelial cells it leads to cellular differentiation, resulting in hyperkeratosis (thickening o f the stratum corneum) and or paraketarosis (retention of nuclei). Microscopical observations of rumen epithelium of non lactating dairy cows in which SARA was induced were done and rumen papillae lesions, sloughing of stratum corneum and presence of bact eria in the stratum granulosum and spinosum were reported (Steele et al., 2011) . They also reported an increase in rumen butyrate concentration of 330% during the first wee k of SARA induction. pH depression after ever y repeated SARA event gets deeper, and this may be explained by the hyper and paraketatosis suffered by the ruminal epithelium (Dohme et al., 2008) . If the rumen epithelium is breached, portal circulation is exposed and this c an lead to bacterial infection or endotexemia (Gressley, 2014) . Infections may be confined to the rumen epithelium leading to microabscesses, or they can impact other organs, primarily the liver (Nagaraja and Chengappa, 1998) . If bacteria bypassed the liver, other organs such as kidney s, lungs, brain, or joint s may be affected. Laminitis Laminitis is the aseptic inflammation of the dermal layers inside the foot and it is also known as pododermatitis aseptica diffusa (Nocek, 1997) . This pathology has been cited as a

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24 consequence of SARA and it has been proposed that SARA should be suspected in herds with a high prevalence of an imals with claw lesions. Stone ( 1999 ) reported a clinical investigat ion where this was the case. T he visible lesion of laminitis usually occurs sometime after the insulting period (Kleen et al., 2003) . The mechanism o f SARA induced laminitis is not well understood. The same mechanisms as previously mentioned for liver abscesses may apply, but other substances such as vasoactive amines and histamine have also been associated with this pathology. Nocek (1997) proposed th at inadequate physically e ffective neutral detergent fiber (peNDF) or excessive non fiber carbohydrates ( NFC ) in the diet, as well as feeding mismanagement, may produce SARA or acute acidosis leading to an increase in VFA and lactic acid accumulation. As a consequence of SARA and increased rumen permeability, LPS could be absorbed and these may stimulate the release of vasoactive substances, such as histamine, which will further decreased blood perfusion and leads to anoxia of the lamina. As a final result, the growing horn tissue will be debilitated and poorly resistant , providing weak support to the claw , ultimately resulting in claw lesions . SARA D uring the Early P ostpartum P eriod As SARA is caused by accumulation of short chain fatty acids ( SCFA ) in t he rumen, and since absorption capacity is the main buffer system in the ruminant (Allen, 1997) , it has been proposed that size and density of rumen papilla are a critical aspect in the patho physio logy of this disease. It has also been mentioned that fresh cows are at high risk of this disease due to a limited absorption capacity caused by a reduction in both physical characteristics of rumen papilla that develop during the dry period (Reynolds et al., 2004) . Penner et al. (2007) reported a higher SARA incidence after parturition in primiparous cows feed high concentrate diets pre partum and post partum than in control cows consuming low concentrate rations pre partum and high ene rgy post partum diets . In the same publication it is mentioned that SARA cases (pH 5.5

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25 for more than 3hs) occurred even though DMI was lower than grain induced SARA studies (Krause and Oetzel, 2005; G ozho et al., 2005) . Since cows during transition receive different diets, poorly adapted rumen microflora might lead to drastic drops in ruminal pH and increased risk of SARA. Off feed situations due to transition cow diseases , group changes and social reorganization and/or other management related issues could affect voluntary DMI and rumen microflora. In order to increase DMI and reach high levels of milk production, a rapid introduction to hi gh concentrate rations might be used as a strategy but this also will negatively affect SARA risk (Dirksen et al., 1985) . Prevention of SARA Two main areas should be considered in order to prevent SARA. The first is the factors affecting the physical and chemical properties of the diet, and the second includes the man agement factors that facilitate or alter DM consumption of cows. From the ration formulation standpoint, two major factors may affect SARA ; i nadequate dietary fiber (NDF) and / or insufficient peNDF will cause inadeq uate ruminal buffering cap acity and secondly, excessive consumption of rapidly fermentable carbohydrates. As previously mentioned, organic acid accumulation in the rumen leads to decreased rumen pH. It is known that ruminal absorption of VFA and salivary p roduction of buffers accounts for approximately 80 % of the buffering capacity of the rumen (Allen, 1997) . In order to keep these mechanisms active, rumination and rumen motility should be maintained. A minimum amount of NDF (28 35%) as the proportion of dietary DM has to be supplied in order to maintain a healthy rumen. Forage NDF (16 23%) is required to stimulate rumination and cud chewing. Rations that are limiting in fiber content predispose dairy cows to disturbanc es such as SARA (Santos, 2011) .

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26 Another important concept affecting cud chewing and rumination is physically effective fiber (peNDF), which is related to the particle size of the fiber (Mertens, 1997) . Low peNDF could lead to SARA , but also excessive intake of rapidly fermentable carbohydrates (NFC) needs ruminal acidosis (Nagaraja and Titgemeyer, 2007) . Inclusion of high amounts of NFC has been show n to be an effective strategy to induc e SARA experimentally (Gozho et al., 2005; Krause and Oetzel, 2005; Gozho et al., 2007; Zebeli and Ametaj, 2009) . In the case study by Stone (1999) the main die tary problem of the herd was an excessive amount of high moisture ear corn in the diet. Not only is the absolute amount of NFC in the diet important, but the degree of feed processing needs to be considered when formulating diets to avoid SARA events (Huntington, 1997) . With regard to management factors that affect DM cons umption of dairy cows, Morgante et al. (2007) proposed that feeding management is more important than the factors previously mentioned related to ration characteristics. Some of these factors concerns make up of feeds. For example, proper length of forage will decrease sorting against long particles in feed by cows, thus decreasing the risk of SARA by consumption of physically inconsistent diets (Leonardi and Armentano, 2003) . Mixing time also affects proper mixing and particle size of the ration delivered. Good feedbunk management practices are critical for SARA prevention. Incidence of SARA may be increased when cows eat fewer and larger meals more quickly (slug feeding) . Three main factors have been mention ed as a c ause of slug feeding: limited feed access , time restricted feeding, and an inconsistent feeding schedule (Krause and Oetzel, 2006) . In free stall housing, limited bunk space due to overcrowding, infrequent TMR push up and bunk

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27 competition are important factors associated with inconsistent feed intake (Krause and Oetzel, 2006) . Managing transition between diets is desirable in order to allow adequate adaptation of ruminal env ironment to highly fermentable diets. The effect of s ocial dominance and overcrowding are important considerations when young animals are mixed with older experienced cows. In order to keep an optimal capacity of pens, segregation of animals by parity is usually recommended. Overcrowding issues will impact feed bunk space availability. A minimum space of 45 cm per animal is recommended to avoid sorting in a group of cows (Krause and Oetzel, 2006) . Another consequen ce of overcrowding during warm weather is heat stress control. Thermal stress can also affect SARA incidence since increased temperature has a negative effect on DMI, consequently producing inconsistent consumption of the ration. Feed additives hve be en proposed to he lp in control of SARA (Krause and Oetzel, 2006) , although they do not address the cause of acidosis. Added buffers, such as sodium bicarbonate, are more likely to be beneficial when marginal amount s of effective fiber are provided by the diet (Krause and Oetzel, 2006) . All these factors must be considered in order to prevent SARA and to minimize inconsistence DMI to mai ntain high production in a dairy herd. Primiparous vs . Multiparous Primiparous cows have lower DM intakes than older cows (by about 10%), and because of this it might be thought that the risk of SARA in this group would be lower. However, Krause and Oetzel (2006) noted that primiparous cows m ay actually have a higher risk of SARA than multiparous cows. Prevalence of SARA in primiparous cows was higher (29% vs 19%) and they also appeared to be at risk for SA RA earlier in lactation than older cows. From observational data they proposed that prim iparous cows may need time to learn to regulate their feed intake

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28 when consuming a high energy diet, and when multiparous cows are present in the same group, primiparous cows may be challenged to get access to feedbunks for small and frequent meals. Econo mics It is not easy to accurately formulate a cost of SARA since so many factors affect the incidence and severity of the disease in a herd. Reduced milk yield, altered milk components and laminitis are some of the factors affecting this equation (Plaizier et al., 2008) . Furthermore, high culling rate, turnover because of unspecific causes and poor performance are also mentioned (Kleen et al., 2003) . Stone (1999) estimated a cost of 400 US$ to 475 US$ per cow per year due to SARA in a case study reported in a 500 cow herd in New York state. This was based on a decrease in milk production of 2.7 kg/d and also a decrease in milk fat and protein content in milk. In conclusion, SARA is a costly disease for the dairy producer . The diagnosis of SARA is not always easy since so many factors affect the outcomes. A long list of managerial strategies should be considered to prevent SARA but not many are availab le to prevent it s consequences. O n the other hand, little information is available concerning SARA early lactation dairy cows. This disease and it s silent consequence s are part of the motivation for implementing this study . Lipopolysaccharide The r umen microbial environment is composed of different species of protozoa, bacteria, fungi, methanogens, and viruses. Bacteria represent 50 to 80% of the microbial mass, about 10 11 CFUs per mL of rumen fluid. Lipopolisaccharides (LPS) are a major component of the cell wall structure of Gram n egative bacteria. Also known as endotoxin, LPS are heat stable amphiphilic molecules. They are composed of lipid A, the toxic component, which is the lipophilic region and is covalently linked to a poly or oligosaccharide portion, the hydrophilic region (Alexander and Rietschel,

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29 2001) . Gram negative bacteria have an extremely asymmetric outer membrane, with the external leaflet surface composed of about 75% of lipid A portion of th e LPS, and the internal half mad e u p by phos pholipids and lipoproteins (Alexander and Rietschel, 2001) . The LPS layer of the external leaflet act s as an effective barrier against the external environment . Lipopoly saccharide S tructure As previous ly mentioned, LPS is composed of a polysaccharide or oligosaccharide portion covalently linked to the second portion, the lipophilic component, a phospholipid. In the polysaccharide portion, two regions are described: the O specific chain and the Core regi on . The O specific chain consists of up to 50 repeating oligosaccharide units in most Gram negative bacteria. This chain is also characterized by considerable structural variability and constitutes the chemical basis for the serological classification of b acteria (Alexander and Rietschel, 2001) . This region is vital for the bacteria to survive in its host because it protects the bacterium from u ptake by phagocytes and attack f rom serum complement. The second, the cor e oligosaccharide region, has a predomina n t ly monosaccharide composition in an outer core region and an inner core, linked to the lipid A. The outer core region is more structurally variable and consists mainly of different hexoses. The inner region is les s variable in polysaccharide structure. `2 keto 3 deoxyoctulosonic acid, and L or D glycero D manno heptose often carrying additional anionic substituents predominates the chemical structure (Alexander and Rietschel, 2001) . Lastly, the lipophilic component of LPS represents the immunostimulatory or endotoxic component . Based on culture of Salmonella m innesota and E. coli , two different morphologies of colonies can be identified: smooth (S) or rough (R). The first one is the wild type but the second one, R type, has a genetic mutation which produces LPS lacking the O specific chain. This observation supports the fact that the endotoxic principle is in the lipophilic component since both types present endotoxic effect (Galanos et al., 1972) . The architecture of this portion is

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30 based on a backbone composed of linked disaccharide units (D glucosamine or D 2,3 diamino 2,3 dideoxy glucose, homo or heterodimeric convination) phospho rylated in positions 1 cylation pattern of the lipid A dictates its immunustimulatory effects in the eukaryotic o rganism and this had been elucidated by comparative studies of LPS and free lipid A. Reduced or no stimulation of the immune response has been observed in LPS structures lacking pho sphate residues or acyl chains (Ale xander and Rietschel, 2001) . LPS Antigenicity and Mechanism of A ction Endotoxemia is cause by systemic exposure to lipopoly saccharides. Several diseases in cattle such as puerperal metritis, toxic mastitis, neonatal coliform septicemia, and salmonellosis show common clinical signs produce d by the action of LPS and the resulting effect upon the immune response. Although considerable inter animal variation has been reported, these diseases are characterized by hyperthermia, anorexia, depression, ruminal stas is, shivering, tachyca rdia, and tachypnea. Some of the s e signs are influenced by the infective dose; ruminal hypomotility increase s with dose and peak rectal temperature decrease s with increasing dose (Jacobsen et al., 2005) . The primary immunoreactive c omponent of the LPS molecule is the lipid A region, due to the specific and sensitive recognition of this membrane lipid structure by numerous components of innate immunity, such as LPS binding protein (Hoffmann et al., 1999) . LPS has the ability to activate various receptors, such as Toll like receptor 4 (TLR4) an mammalian phagocytes. These cells secrete immune modulators such as interleukin 1 (IL 1), IL 6, and IL 12, whic h induce and enhance the mounting of an immune response (Medzhitov and Janeway, 2000) . Induction of an excessive and pathologically imbalanced immune response is, in mammalian species, the product of these immunosti mulatory effect s of LPS (Alexander and Rietschel, 2001) .

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31 LPS and Subacute Rumen A cidosis The rumen microbial population is composed mainly of Gram positive and Gram negative bacteria. Cell free rumen fluid from hay or grain fed cattle is a source of endotoxins (Nagaraja et al., 1978b) . Early studies show that at rumen pH of about 6, the bacterial population is mainly Gram negative. When animals were challenged by feeding alfa lfa or grains in order to induce rumen acidosis, the bacterial population shifted to a Gram positive predominance (Nagaraja et al., 1978a) . T his shift was based on an increase in the absolute amo unt of G ram positive bacteria, rather than a decrease in the Gram negative population. They also show ed that as pH decreased , r umen LPS concentration increased . The increase in free LPS is not only due to lysis of Gram negative bacteria but also because of the growth of this group. The systemic role of free rumen LPS in subacute rumen acidosis remains uncertain because when LPS are abso rbed , concentration is not always detected in peripheral blood due to hepatic clearance (Andersen et a l., 1994) . A number of experiments have been conducted in order to have a better understanding of SARA and the presence of LPS in rumen fluids and i n peripheral blood. One research group observed a four fold increase in rumen endotoxin con centration when SARA was induced in four mid lactation dairy cows using a restrict ed food access protocol on high grain diets. This was not translated into detectable levels of LPS in peripheral blood, although proteins of the acute phase response were increased (Gozho et al., 2007) . Another publication showed that feeding high proportions (30 and 45% DM basis) of barley grain to mid lactation dairy cows was associated with lower rumen pH, and increased LPS concentration in rumen flui d (Emmanuel et al., 2008) . Although LPS in peripheral blood was not measured , there was an increase in LPS binding Protein ( LBP ) , an acute phase protein synthesized by hepatocytes in response to specific cytokines, and which binds to endotoxins present in circulation (Tobias et al., 1999) .

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32 Gozho et al. (2005) induced SARA in three rumen fistulated steers by feeding different proportions of wheat barley pellets and chopped alf alfa hay. Significant differences in rumen LPS concentration were reported during the fo ur days of the induction period and the highest measurement was seen when SARA was detected. The duration of low rumen pH needed to achieve a subacute rumen acidosis si tuation was only reached during the last two days of the induction period. The highest concentration of LPS occurred when the duration of pH below 5.6 was the greatest (Gozho et al., 2005) . In another experiment, the sa me group reported a curvilinear increased in ruminal LPS concentration when the amount of concentrate in the diet increased (Gozho et al., 2006) . In this case, SARA was induced after stepwise adaptation to five diets c omposed of different forage to concentrate proportions. Induction of SARA by feeding with alfalfa pellets has also been attempted . Eight lactat ing dairy cows were initially f ed 50% concentrates and 50% chopped alfalfa. During a period of 5 weeks, the phys ical form of alfalfa was replace gradually (8% per week) by pelleted alfalfa in order to induce SARA. This process linearly decreased daily average rumen pH. From week 2 of induction, rumen pH was lower than 5.6 for more than 180 minutes per day. Free rume n LPS concentration was increased from week 0 to week 5 of the experiment showing a significant increase i n week 2 of the induction throughout the rest of the experimentation period. In this trial, peripheral blood LPS concentration was measured but it rem ained below detectable levels of 0.05 EU/ mL at all times; blood inflammatory markers were also not increased throughout the experimental period (Khafipour et al., 2009a) . LPS Translocation to C irculation Several res earch groups have investigated the association between SARA and the increase in free LPS concentration in the rumen, but not all found increased APP or free LPS in peripheral blood. When accumulation of organic acids in the rumen occurs and the capacity of

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33 the rumen to remove them is overwhelmed, there is a decrease in rumen pH and this may lead to the damage of rumen epithelium and consequent absorption of bacteria, animes and LPS that can cause inflammation or infection (Plaizier et al., 2008) . The increase in rumen LPS concentration and the reduction of the barrier function of th e mucosa due to SARA may lead to translocation of LPS from the rumen. Some of the c onsequences of SARA appear to be mediated through local ized or systemic inflammation following injury to the gastrointestinal epithelium (Gressley, 2014) . Rumen epithelium serves as a selective barrier. It is composed of four different stra tum: corneum, granulosum, sp in osum and basale, from lumen to center of papillae. In a healthy rumen, this barrier is kept functional due to highly keratinized squamous cells on the surface of the epithelium and by the presence of tight junction proteins in the stratum granulosum (mai nly) and in some cells of the stratum spinosum (Graham and Simmons, 2005) . This permeability is constantly challenged and it has been reported that low pH and the presence of LPS can disrupt it (Emmanuel et al., 2007) . When there is a breach of th e rumen epitelium , absorption of LPS may be observed. This is enhanced by local inflammation produced by mucosal associated lymphoid tissue (MALT) found on the surface of the gastrointe stinal tract. These microstructures also alter cytokine production; enhancing inflammation and further increasing permeability, allowing increase d entry of bacteria and LPS into the rumen papillae which enhance the inflammatory response (Kurashima et al., 2013) . Because events that occur in the rumen during SARA are mirrored in the large intestine and the intestinal epithelium is composed by a single layer of cells, absorption of LPS and bacteria at th is level has al so been proposed (Khafipour et al., 2009a) .

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34 LPS , Vaccines and Oral C hallenges Cows produce anti LPS antibodies after immunization with lysate of whole Gram negative bacteria . Lots of research effort has been made t o generate anti LPS vaccines but as far as I was able to find, no research of the effect of active immunization on SARA has been undertaken . The most extensive ly studied vaccine is the one formulated with a mutant strain of E coli O111:B4 (termed J5) which contain core LPS, membrane proteins and lipid A. These antigens are highly conserved among gram negative bacteria. Cross reactive anti Gram negative antibodies can be produce d after exposure to these vaccines (Tyler e t al., 1990) . These antigens elicit resistance against a wide variety of Gram negative bacteria in vaccinated cows (Erskine, 2012) . Early trials in commercial dairy farms reported a decreased incidence of clinical coliform mastitis, when cows were naturally exposed after vaccination with a lysate of J5 E coli (Gonzalez et al., 1989) . Another trial revealed that J5 immunization decreased the severity of the clinical signs alt hough it did not decrease the prevalence of the disease (Hogan et al., 1992) . However, p oor amnestic IgG1 and IgG2 responses were seen with these Gram negative bacterins . I ncreased number of doses during lactation res ults in an increased immune response and longer protection (Erskine, 2012) . In a recent publication wit h an experimental vaccine, researchers were not able to demonstrate a decrease in the severity of an intr a mamma ry infection when cows w ere, experimentally challenged with an E.coli wild type strain with the same R1 (rough) core that the experimental vaccine contained (Brade et al., 2013) , despite an immune response after challen ge . Oronasal administration of LPS has also been studied with the aim to elicit an improvement in production performance in periparturient dairy cows (Iqbal et al., 2013) . Only a trend for lower somatic cell count in favor of the treatment group was found throughout the first month after parturition. They reported that oronasal administration of LPS enhance d IgA salivary

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35 secretion (unpublished data), and suggested that this enhanced humoral immunity , lower ed pro in flammatory mediators and prevent ed subsequent translocation of LPS (Iqbal et al., 2013) . O ral administration of LPS had been studi ed (Ametaj et al., 2012) . They administer ed two doses a week at increasing concentration of LPS, starting 2 weeks before and completing it on day 7 after parturition. R esults showed in the treatment group an increased IgM anti LPS plasma concentration, starting on day 7 before parturition until day 14 after calving, and a decreased IgG anti LPS concentration. Differences were found 3 days before parturition until completion of the observation period on day 28 after parturition . Although the significance of the findings is not clear, Ametaj et al . , (2 012) proposed that the control group mounted an IgG anti LPS immune response after the ru minal environment was challenged with the postpartum diet, contrary to the treatment group that had a prepared mucosal immunity. In a similar experiment , cows were exp osed during the last 3 consecutive pre partum weeks to increased concentration of LPS and lipoteichoic acid ( LTA ) (Iqbal et al., 2014) . Increased concentration of salivary IgA was reported in the treated group while the plasma concentration of anti LPS IgA, IgG and IgM were decreased compared to the control group. They propose that secretory IgA protected the entrance of bacterial toxins without involving or stimulating the systemic primary humoral response (Iqbal et al., 2014) . LPS is a common factor present in several of the diseases suffered by dairy cows. LPS can cause systemic inflammatory processes. LPS are also increased during SARA processes and although inflammatory response h as been measured, littl e is known on its impact on DMI i n cattle. Elucidating possible alternatives to avoid the effect of ruminal LPS on dairy cows is another of the a spects that motivated this study .

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36 Acute Phase Proteins When the animal body is challenge d by an antigen, the innate immune system mounts a quick response in order to get rid of it. There is not only a local inflammatory response, but also a generalized response to the infection (Tizard, 2004) . The produ ction and secretion of a mixture of cytokines by phagocytic cells such as interleukin 1 (IL 1), i nterleukin 6 (IL 6) and Tumor Necro sis Factor including the brain , are affected by the se chemicals. Two mechanisms have been proposed for appetite suppression during an i nflammatory response (Tizard, 2004) . The first one is a neurogenic signal involving receptors found on sensory neurons in the vagus nerve. IL 1 stimulates these receptors causing central suppression of the appetite center (Tizard, 2004) . The second route is through a humoral pathway involving circulating cytokines. This mechanism is activated dir ectly by diffusion of cytokines to brain target areas or by release of cytokines by the brain macrophages. As a result, animal appetite is altered. Another organ affected by cytokines, especially by IL 6, is the liver , which is responsible for the product ion and secretion of acute phase response proteins (APP). During the inflammatory process, catabolism of proteins in peripheral tissue (muscles) is up regulated in order to provide amino acids for APP synthesis (Cecil iani et al., 2012) . Production is stimulated shortly after the injury or initial inflammation (a few hours) and usually decreases within 24 to 48 hours (Tizard, 2004; Kindt et al., 2007) . Differ ent mammalian species produce different APP responses. In the bovine, APP can be categorized in order of importance as major: Haptoglobin (Hp), Serum amyloid A (SAA), and Mammary Associated Serum Amyloid A3 (MSAA3), followed by the moderate proteins: Lipop oly 1 Acid

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37 1 anti proteinase (Ceciliani et al., 2012) . In this chapter we will focus on SAA, LBP, Hp, and Ceruloplasmin as the main proteins of interest, because these four a re the most commonly observed AP P in studies of subacute rumen acidosis. Serum amyloid A is the most important APP in cattle. It acts as a chemoattactant for neutrophils, monocytes and T cells, and because it is immunosuppressive, it has been proposed to regulate the immune response (Tizard, 2004) . Its name evolved due to its involvement in reactiv e amyloidosis. At least 3 different functions have been explored; chemotactic modulating activity is the first one , although in cattle it has not yet been demonstrated (Ceciliani, et al., 2012) . C holesterol binding activity is also an important SAA function . SAA acts as an apolipoprotein (replacing Apoprotein A1) in the high density lipoprotein , and has a role of exportation of cholesterol from the inflammatory site. The third described role is to act as an innate opsonin. It has been reported that SAA has a ntibacterial effects and although in humans its role is mainly against G ram negative bacteria , in the bovine, the spectrum is probably wider (against G and G+) (Molenaar et al., 2009) . LPS Binding Protein, one of the key members of the innate immune response against bacteria (Ceciliani et al., 2012) , is also important in cattle and rises rapidly after infection (Tizard, 2004) . It is ide ntified as a 50 KDa polypeptide, but after posttranslational processing it is found in the bloodstream as a 60 65 KDa glycoprotein (Khemlani et al., 1994) . Its main role is to modulate the innate immune response. I t has a LPS binding domain involved in binding and presenting the particle to macrophages, monocytes and granulocytes (Lamping et al., 1996) . Although during the acute phase response, high concentrations of LBP have a n anti inflammatory effect (by allowing clearance of LPS possibly through serum lipoproteins), low

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38 concentrations act as pro inflammatory mediators (Lamping et al., 1998) . LBP may also act as an opsonin by binding to bacteria and promoting their phagocytosis (Wright et al., 1989) . Haptoglobin (Hp) is also a major APP in ruminants. It is composed of two 20 KDa and two 35 (Morimatsu et al., 1991) . Four biological functions have been described for Hp. It binds hemoglobin and prevents oxidative damage s (Lim et al., 1998) . It also has an anti inflammatory role interacting and regulating monocytes and macrophages (Ceciliani et al., 2012) . Down regulation of neutrophil effects ha s also been cited (Saeed et al., 2007) . Hp has bacteriostatic effect s , inhibiting bacterial proliferation by binding iron molecules and reducing iron availability for invading bacteria such as E. coli (Tizard, 2004) . Angiogenesis enhancement and chaperone activity is also mentioned as important function s of the protein (Ceciliani et al., 2012) . 2 globulin with a molecul ar weight of 225.1 kDa when glycosylated and 106.1 kDA when it is not (Boivin et al., 2001) . It plays important physiological functions in copper storage and transport. In vertebrates, up to 95% of copper is stored in this structure. This glycoprotein exhibits multiple functions such as ferroxidase and amine oxidase activity, copper mobilization and antioxidant properties. Ceruloplasmin (Cp) is also involved in angiogenesis (Boivin et al., 2001) and is also responsible for limiting Fe availability for bacterial growth. There have been several experiments confirming that ceruloplasmin is an indicator of infection (Chassag ne et al., 1998; Sheldon et al., 2001) or stress (Arthington et al., 2008; Arthington et al., 2013) in cattle, although its usage is less common than other APP (Murata et al., 2004) . Numerous studies have attempted to characterize the acute phase response (APR) during sub acute ruminal acidosis (SARA) events. In one study, SARA was induced in eight lactating dairy cows by gradually decreasing the propo rtion of alfalfa hay and replacing it with alfalfa

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39 pellets (Khafipour et al., 2009a) . Although the free L PS concentration in rumen fluid was increased, LPS was not detected in peripheral blood. They hypothesized that th is could have been in part because they observed a decrease in free LPS concentration 6h post feeding. The same researchers induced SARA by feeding a high grain diet and Hp, SAA and LBP were measured (Khafipour et al., 2009b) . They observed a linear decrease during the time of the experiment from 56 to 12 µg/ mL for Hp, 23.1 to 6.9 µg/ mL for SAA and 7.2 to 2.6 µg/ mL for LBP being significant for Hp and SAA, but not for LBP. They also reported a significant decrease in milk yield, milk fat % and milk fat yield. In another study, when SARA was grain induced, an acute inflammatory response was detected (Gozho et al., 2007) . In this case four mid lactation cows were separated into tw o groups and in one of them SARA was induced by applying a TMR restriction period as well as replacing 25% of the total mixed ration (TMR) with the same amount of ground grain pellets; TMR composition change d from 44% to 68% concentrates. By doing this, th ey were able to decrease average rumen pH, and the duration that ruminal pH was below 5.6 increased from 187 to 309 min/d. During the 5 days of induction, the average free ruminal LPS concentration was significantly greater in SARA cows , although the LPS c once ntration of peripheral blood did not increase over th e detection limit of the test during any day of the induction. Nonetheless, serum amyloid A concentration showed a significant increase in the treat ed group. H igh Hp concentrations were also reported and, although no differences were found between groups, the author concluded that since healthy animals have negligible concentrations of Hp, these findings suggest ed active inflammatory processes in both groups. In another study aiming to measure ruminal free LPS concentration and acute phase response , eight mid lactation cows were fed increasing proportions of barley grain (Emmanuel et

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40 al., 2008) . Ruminal pH decreased with increased inclusion of barley grain in th e diet. Free ruminal LPS concentration increased with higher inclusion of concentrate and also increased SAA and LBP were reported for the higher barley grain inclusion groups (30 and 45%). Interestingly, concentration of both SAA and LBP decreased with th e experimental days. In this experiment Hp concentrations were also measured but no differences were found. Gozho et al. (2006) induced SARA in three Jersey steers by a weekly increase of wheat barley pellets in their diets. Free LPS ruminal concentration increased with concentrate enhancement until 61% of DM ( p <0.0001), but it was even higher ( p <0.05) when SARA was present with 76 % inclusion. The amount of LPS increased curvilinearly when the amount of concentrate in the diet increased. SAA numerically increased when concentrates were increased , but significantly increased ( p < 0.0001) when SARA was induced. Blood Hp concentration was higher ( p <0.01) in steers on 61% concentrate and SARA induction did not affect its concentration. In a previous study, t he same researchers induced SARA in three steers by offering different proportions of wheat barley pellets and chopped alfalfa hay for 5 days after a transition diet (Gozho et al., 2005) . No diet effect was reported for average rumen pH, time, and area below the curve with pH below 5.6, but SARA was effectively induced on day 4 and 5 of the induction. Nonetheless, a significant increase ( p <0.05) was reported for free LPS ruminal concentration on day 2 after induction co mpared with the transition diet (hay only). No difference between diets was reported for Hp, but significant incre ases were reported for days 3 and 5 during induction. SAA demonstrate d a different behavior, showing a significant increase from day 2 until d ay 5 of induction, similar to the slope of free ruminal LPS concentration. In conclusion, increased APP have been observed during early lactation or during stressful events in cattle. Because the influence of other diseases within the first 14 days after

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41 p arturition may mask measurements of APP responses to gastrointestinal damage, many of the available information is built on experiments involv ing cows after peak lactation or in mid lactation. Therefore, there is little information connecting SARA and its consequences and APR in early lactation. Enhancement of anim al health and lack of research i n this area is part of what encourage d us to find answers. IgY PAP During the last decades , public health concerns and perceptions regarding the usage of antibioti cs and hormones has increased (Simmons, 2011) . The use of compounds from natural microbial fermentation and plant extracts to specific antibodies has been postulated to improve animal productivity. Further developme nt is needed to obtain efficacious natural feed additives. Polyclonal antibody preparations (IgY) derived from the avian industry have been proposed as a possibility for prophylactic and therapeutic treatment of diseases in several species. IgY production is in alignment with the 3R principles of animal welfare: Reduction, Refinement and Replacement of laboratory animals (Schade and Hlinak, 1996) since this immune molecule can be obtained from eggs, and animals do not n eed to be bled in order to obtain the antibodies from their serum. Chickens can lay eggs almost every day, and because the yolk of an immunized hen's egg contains a high concentration of IgY, chickens are becoming popular as a source of customized antibodi es for research. IgY S tructure Three immunoglobulin isotypes has been described in the chicken: IgA, IgM, and IgY. The latter is a type found in birds, reptiles, amphibians and lungfish (Warr et al., 1995) . It is found in the egg yolk of laying chickens and is the main antibody isotype present in the yolk (Gassmann et al., 1990; Vega et al., 2011) . Of the immunoglobulins arising during the immune response, only I gY is found in chicken eggs. Thus, in pure preparations from chicken eggs, there

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42 is no contamination with Immunoglobulin A (IgA) or Immunoglobulin M (IgM). The structure of IgY is very similar to that of IgG. It has two heavy and two light chains (H2L2, is the configuration of the low molecular weight Ig) and a molecular weight of 180 kDa. Th e heavy chain is composed of four constant domains (Cv1 Cv4) in addition to the variable domain (Mine and Kovacs Nolan, 2002) . Both IgG and IgY are the major immunoglobulins providing defense against infectious agents in their respective species and appear in blood at high concentrations following synthesis of a higher molecular weight antibody (IgM). IgY posseses two antigen binding sites which have strong binding capacity (Warr et al ., 1995) . The steric flexibility of the IgY molecule is less than that of IgG. Chicken IgY does not activate mammalian complement systems, does not react with mammalian rheumatoid factors, and neither protein A or protein G bind to IgY. Stability of IgY The stability of IgY with regard to pH, heat t reatment and proteolytic enzyme exposure has been studied (Shimizu et al., 1992; Hatta et al., 1993) . Activity was decreased by incubating for 7h at pH 3 .5 and almost completely disappeared at pH 3. Hatta (1993) also studie d the activity at different pH and reported that there was a decrease of 40 and 35% efficacy when exposed to pH 4 and 3, respectively. Under alkaline conditions, activity was diminished when the preparations were exposed to pH of 12 or higher. In one publication, the stability of the molecule under heat conditions was also analyzed (Shimizu et al., 1992) . The activity of IgY antibodies was decreased w hen exposed to 70 °C or more for 15 min, although others report an activity of 50% at that temperature (Shimizu et al., 1992) . A review article states that IgY was fairly resistant to trypsin and chymotrypsin action, b ut sensitive to pepsin exposure (Chalghoumi et al., 2009) . These enzymes degrade antibodies

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43 into three different fragments and two of them retain some of their neutralizing activity (Akita, 1993). Mechanism of A c tion The major mode of action of IgY is its binding to certain specific components on the bacterial surface such as LPS , flagell a or fimbria (Chalghoumi et al., 2009) . This binding may impair the biological functio ns of those components, essential for the pathogenesis of the particular disease (Chalghoumi et al., 2009) . For example, Peralta et al. (1994) was able to prevent adhesion of Salmonella Enteritidis to enterocytes i n a mouse model after oral administration of egg yolk antibodie s specific for a fimbriae type. The mechanism of action of orally administered IgY preparations for pathogen reduction is still unknown. In this case, they proposed that IgY reduces bacterial v irulence by interfering with the adh esive properties of the fimbria, rather than a direct bactericidal or bacteriostatic effect (Peralta et al., 1994) . By definition, passive immunity is the transfer of preformed an tibodies with a given specificity (Kindt et al., 2007) from one individual to another (Chalghoumi et al., 2009) . Passive immunity can occur naturally or artificially, in the f irst case when maternal antibodies are transferred to the fetus t h rough placental circulation or in the second case as a product of systemic, intravenous or oral rout es (Chalghoumi et al., 2009) . Passive immunity c onfers immediate protection but is of short duration . A potential disadvantage of exogenous systemic or intravenous administration of antibodies is the ability of mounting an immune reaction (systemic an aphylaxis) against antibodies f r om other species caus ed by mast cell degranulation (Kindt et al., 2007) . Experiments Hens have developed the ability to produce large amounts of very effective antibodies upon experimental immunization, encouraging researchers to experim ent with the use of IgY for

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44 passive immunity in various forms: whole egg powder, whole yolk powder, water soluble fraction powder or purified IgY material (Chalghoumi et.al.,2009). In a review article by Mine and Kovacs Nolan (2002), a long list of articl es exploring the potential application of orally administered IgY are cited. Pathogens such as Rotavirus, Coronavirus, Escherichia coli , Salmonella , Yersinia , Staphyloccus aureus and Pseudomonas aeruginosa were challenged with diverse results. Induced bovi ne neonatal d iarrhea caused by rotavirus was successfully prevented u sing anti bovine rotavirus IgY (Kuroki et al., 1994) . In another trial , diarrhea was induced by experimental exposure of calves to coronavirus and th e control groups , which received no protection , experience severe diarrhea and all died . In groups feed egg yolk or colostrum, although not all lived , those receiving higher amount of antibodies gained weight throughout the experimental period (Ikemori et al., 1997) . Diarrhea consequences caused by Escherichia coli in piglets and calves was also prevented by prophylactic administration of IgY (Ikemori et al ., 1992; Marquardt et al., 1999) . In another trial, when calves were experimentally exposed to Salmonella sp. , the control group died within about a week of exposure. Although calves treated with low titer IgY experience d a high mortality (60 70 %), the h igh titer group had 100 % survival rate and suffered only diarrhea and fever. N ot much research has been published involving the usage of IgY on adult ruminant s , although some encouraging experiments can be mentioned . Several p olyclonal antibody preparatio ns ( PAP) against different bacteria have been studied. In one experiment, PAP against Fusobacterium necrophorum (PAP Fn) and Streptococcus bovis (PAP Sb) were fed to feedlot steers ; reduced ruminal S. bovis populations was reported, although they did not a ffect ruminal pH (DiLorenzo et al., 2006) . In a second series of experiments using the same PAPs, performance, carcass characteristics, and ruminal fermentation variables were evaluated . As a

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45 result, the PAP Sb grou p had enhanced feed efficiency when steers were fed high grain diets. They concluded that improved animal performance may be explained by ruminal microbial regulation and manipulation of ruminal fermentation metabolism. Severity of liver abscesses also dec reased when PAP Fn or both PAPs together were fed to steers (DiLorenzo et al., 2008) . In another experiment, cross bred heifers were fed multivalen t PAPs against several bacteria commonly found during acidosis, and they concluded that PAP may be effective in reducing the incidences of acidosis during transition to high grain diets from high forage diets (Blanch et al., 2009) . A few more publications evaluating varous aspects of t he cattle industry such as ruminal fermentation patterns (Marino et al., 2011) , feedlot performance, blood gas profile, and ruminitis (Pacheco et al., 2012) have been produced , but no research has been published regarding milking dairy cows. LPS IgY and the lack of research using early lactation dairy cows stimulated us to explore its effect on health and performance. An additional motivating factor in our research was the proven increased concentration of free ruminal LPS shown in cows undergoing SARA.

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46 CHAPTER 2 MI LK PRODUCTION AND MILK COMPONENTS Introduction In the last 30 years, US dairy production has dramatically changed. Individual milk yield of dairy cows had been almost doubled while the numbers of cows has continue d to decreas e , from about 11 million cows i n 1982 producing 5,600 kg /lct to roughly 9 million in 2013 yielding 10,000 kg/lct (USDA, National Agricultural Statistics Service, 2014) . With this select ion pressure there is very littl e room for mistakes since the genetics of the replacements are even better tha n those of t heir mothers in the majority of cases. Understanding and managing the transition period (the 3 weeks before and after parturition) is extremely important in dairy pro duction, as it is the most challenging period in the productive life of the dairy cow. In order to maximize milk production per cow , a high peak milk is needed since after this, the lactation curve will be defined and conditioned by the constant decrease in the number of active epithelial cells in the mammary gland. It has been stated that 200 to 225 more pou nd of milk should be produced for the entire lactation per every 1 pound increased at peak production (Hutjens, 1996; Hutjens, 2008) . Also, the concept of multifactorial diseases needs to be kept in mind in order to understand and prevent production diseases during early lactation and decrease the risk of culling (Andersen, 2003) . There are several factors around parturition, such as immunosuppression, calc emia status, fatty liver and energy balance, and subacute rumen acidosis (SARA) that affect health and performance of transition animals (Mulligan and Doherty, 2008) . Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria, which are present in the rumen. LPS is also known as endotoxin and when there is a rapid increase of b acterial mass or lysis of bacteria, LPS is released (Wells and Russell, 1996;

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47 Andersen, 2003) . Studies have demonstrated that decreased rumen pH during SARA leads to increased counts of free LPS in ruminal fluid. (Gozho et al., 2005; Gozho et al., 2007; Khafipour et al., 2009a) . These endotoxins may be absorbed or not, in both cases inducing increased concentration of i nflammatory markers. One factor associated with the risk of detecting LPS in peripheral blood is liver health. Hepatic lipidosis, one of the transition diseases, has been associated with a decreased capacity for endotoxin clearance by the liver (Andersen et al., 1996) . SARA is a pathological condition during which ruminal pH is decreased below 5.6 for at least 3 hours (K leen et al., 2003; Krause and Oetzel, 2005; Gozho et al., 2005; Plaizier et al., 2008) . The main dietary factor leading to SARA are an increase in the intake of rapidly fermentable carbohydrat es or the lack of long particle of forage in the diet , leading to ruminal accumulation of high concentrations of volatile fatty acids (Nagaraja and Titgemeyer, 2007) . The threshold of 3 hours is based on the observations that until that point fiber digestibility or acute phase response elements are not affected (Gozho et al., 2005) . Various prevalence s of SARA are cited in the literature , ranging from 10 to more than 30% (Garrett et al., 1997; Oetzel and Nordlund, 1999; Bramley et al., 2005; Morgante et al., 2007; O'Grady et al., 2008) depending on management and feeding systems. SARA is not an easy disease to diagnose , and its consequences c an be costly (Stone, 1999) . Consequences of SARA cited in the literature include depress ed or erratic DMI (Krause and Oetzel, 2005; Gressle y, 2014) , reduced fiber digestibility, reduced milk yield (Khafipour et al., 2009a; Khafipour et al., 2009b; Gressley, 2014) , altered milk components, gastrointestinal d amage, abscesses and laminitis (Gressley, 2014) . Several publications have reported increased concentrations of free ruminal lipopolysaccharides when SARA was present (Gozho et al., 2005; Gozho et al., 2007; Khafipour et al., 2009a; Khafipour et al., 2009b) .

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48 Feed intake depression is one of the consequences of SARA (Plaizier et al., 2008) and several theories have been proposed to explain this problem. Endotoxins have an effect on DMI. They stimulate the release of cytokines and the latter stimulate the increase of blood leptin concentration which is thought to inhibit a neu ropeptide at the central level. This inhibition blocks the appetite center (Finck et al., 1998) . Endotoxins also down regulate rumen motility (Jacobsen et al., 2005) which deepens with increased concentration o f LPS , ranging from 5 h to 36 h of stasis depending on the injected dose. Others have reported erratic DMI when SARA was induced , along with decrease d milk yield (Krause and Oetzel, 2005) . Although some studies have reported decreased yield during SARA (Stone, 1999; Krause and Oetzel, 2005; Khafipour et al., 2009b) others found no difference between cows with o r without SARA (Khafipour et al., 2009a) . Zebeli and Ametaj (2009) reported decreased fat corrected milk when SARA was induced. It has been proposed that early postpartum cows may be more susceptible to SARA. Their rumen epithelium must adapt to higher energy diets at the same time that ruminal microflora has to transition ; together this changed may overwhelm the absorption capacity of the rumen epithelium (Mulligan and Doherty , 2008) . Recent publications challenged thi s theory stating that epithelial changes during this period might be minor (Penner et al., 2007) . They stated that the previous theory might have applied years ago , but with current di ets in the transition periods, papillar absorption is no longer a limiting factor. T hey observed that SARA was increased in primiparous cows after parturition, when prepartum diets where formulated with higher energy. A sustained effort has bee n conducted to attenuate ru minally derived endotoxemic events in the transition period. The use of feed additives or antibiotics has been proposed (Krause and

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49 Oetzel, 2006) . During the last decades concern about pu blic health, animal welfare and polarized perception regarding the usage of antibiotics and hormones has increased (Simmons, 2011) . Va rious compound s , from natural microbial or plant extracts to specific antibodies , have been postulated as alternative to improve animal productivity. Polyclonal antibod y preparations (PAP) derived from immunized hens have been proposed for prophylactic and therapeutic treatment of different diseases in several species (Peralta et al., 1994; Ikemori et al., 1997; Mine and Kovacs Nolan, 2002; Zhen et al., 2011; Vega et al., 2011) . Avian IgY obtained from the yolk of hens immunized with E. coli 0111 lysate are available and show a high binding activity to LPS (Zhen et al., 2011) . Producing PAP or purified IgY from hens is technically easy and allows for production of large quantities co mpared to IgG obtained from serum of immunized mammals (Chalghoumi et al., 2009) . PAP against various bacteria has shown some efficacy in cattle (DiLorenzo et al., 2006; DiLorenzo et al., 2008; Blanch et al., 2009) . DiLorenzo and Gy lean f ound positive results when 48 beef steers in the final fattening process were feed 3 5 g of egg yolk daily containing PAP anti LPS ; DMI was improve d by about 10% and increased final body weight was reported (personal communication with the researcher). With these elements in mind, we hypothesized that feeding a polyclonal antibody preparation against lipopolysaccharides during the early postpartum p eriod w ould decrease the amount of free LPS in rumen and consequently diminish consequences of SARA. This effect should enhance overall health, and consequently improve milk yield in transition cows. The objective of the study is to determine the effects o f feeding 3 g/d of avian polyclonal antibody against LPS on milk yield and milk components of Holstein dairy cows in a commercial setting.

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50 Materials and Methods All procedures were approved by the Institutional Animal Care and Use Committee of the Univers ity of Florida (IACUC UFL # 201307790). Farm Description The study was done on a commercial dairy farm in north central Florida, milking approximately 4,800 Holstein cows thrice daily with a rolling herd average of approximately 11,500 kg/cow. Cows had ad libitum fresh water available, and TMR offered at least twice a day was formulated to meet or exceed the requirements of a second lactation Holstein dairy cow with the following characteristics: 680 kg of body weight producing 52 kg of milk/day with 3.5% fat and 3.0% of protein at 60 DIM. Cows were housed in tunnel ventilated freestall barns with primiparous and multiparous cows being housed separately after approximately 21 days in milk. Freestalls were cleaned thrice daily and bedded twice a week with sa nd. All barns were equipped with sprinklers over the feed area , which were programmed to engage when environmental temperature was > 2 4 ° C . PCDart ® software (Dairy Records Management Systems, Raleigh, NC) was used to record production, health and reproducti ve events. Allflex ® RFID ear tags (Allflex ® USA, Dallas, TX) were used as electronic devices for identification of animals and Smart Dairy ® Management Systems (BouMatic , USA, Madison, WI ) software was used for automatic measure ment of daily milk yield. Cows were vaccinated and treated according to standard operating procedures (SOP) for the farm. Cows , Housing and Feeding Management The farm where the experiment was conducted is located in central Florida. Cows are kept in different environments depending on lactation and gestational status. Briefly, at dry off,

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51 60 days prior to parturition, cows are moved to dry lots and feed is delivered daily in a concrete feed bunk. Once a week, 18 24 days before expected parturition, cows and heifers are moved to a free stall barn where they are housed until parturition . An all in all out system is use for intensive monitoring of calving. When cows show second stage of labor, they are moved to a sand packed bedding pen for delivery of the newborn. After parturition, cows are moved to a colostrum group, in a freestall barn where they spend a maximum of 16 hours. They are subsequently moved to the fresh cow pen in a different freestall barn until completion of the transition period. After 17 21 days in milk they are then mo ved to groups depending on milk yield, parity (1 st vs 2+) and genetic merit until the end of lactation. All cows and heifers receive d the same prepartum diet (Table 2 1) and the same post partum diets. Feed in the prepartum pens was delivered three times d aily, with the objective of keeping the feed bunk with food 24 hours a day. Feed delivery to fresh groups was done three times a day at 0700, 1500 and 2300 hours. Health protocols followed during the trial period were those currently used at the farm, and diagnosis and treatments were done by farm personnel. Routine health checks were performed on days 4, 6 and 10 postpartum . I f a cow was visually identified as being sick, or if the computerized measuring system noticed a deviation in milk yield, the cow w as automatically identified and sorted for posterior health check. The vaccination protocol used at the farm was the one currently in use, recommended by th e h erd veterinarian (Table 2 2 ).

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52 Exclusion C riteria All cows primiparous and multiparous were eligi ble to be enrolled except for cows having shorter gestation than predicted by PCDart ® , cows with calving difficulty of 5 (cesarean or fetotomy) or clinically lame cows ( locomotion score 4 and 5 were not selected ) . Experimental D esign and T reatments Sample s ize Sample size was calculated using Win Episcope 2.0 statistical software (EPIDECON University of Edinburgh). It was based on an expected difference of 1kg in milk yield, our main outcome of interest and based on previous reported SARA prevalence of 2 0% (Garrett et al., 1997) . Over the number obtained (120 cows per treatment), a 10% attrition was considered (voluntary culling in the first 90DIM) totaling 132 cows per treatment. Treatments Cows were rando mL y assign ed to one of 3 treatment groups: PAP LPS, received 20 cc orally of a solution containing 3 grams of active polyclonal antibody preparation (PAP) anti LPS; Placebo (PLC), received 20 cc orally of a solution containing 3 grams of inactivated PAP anti LPS; an d a Control (CTL) group that did not receive any treatment. The study was performed during the summer months of 2013: enrollment started on May 10 and ended on June 25. All cows were kept in the same pen for the treatment period and received the same ratio n. After the completion of the 3 wk fresh period, cows were moved following farm management criteria to high production groups. The treatment period, from day 0 14 of lactation, was based on two reasons: the highest incidence of transition diseases occu r within this period, and some dairy farms keep their cows in the fresh group for only 2 weeks. Treatment dosage was, as suggested by the producer (Camas

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53 Inc., LeCenter, MN) 3 grams of anti LPS polyclonal antibody preparation (PAP LPS). This amount was bas ed on previous trials with PAP products w h ere significant differences i n performance were observed in steers using lower amounts (DiLorenzo et al., 2006; DiLorenzo et al., 2008) . Preparation , d el ivery and timing of t reatments Three hundred grams of PAP, consisting of dried egg yolk from immunized hens were initially mixed with water to produce a cream like consistency. After that, more water was added to a final volume of 2000 cc. The treatmen t was m ix ed using a kitchen mixer to ensure the complete homogenization of the solution. After being mixed, 2000 cc bottles were kept at 4 ° C. Drenching guns equipped with an oral hook were used to deliver treatments; different models were used but all del ivered a standardized one shot 20 cc dose (3 grams of dry egg yolk) . Dosing o f healthy cows was done by research assistants after animals were restrained on a management rail . Treatments of cows in the maternity or hospital area were given by trained farm personnel after restraining in individual chutes. The time of day that treatments were administered varied depending on the day of treatment and health status. The initial dose was administered in the maternity facilit y within an hour of parturition. S ub sequent doses were administered to healthy cows between 1500 and 1600 after the fresh cows were milked, and in the hospital area between 1400 and 1500, after the rval between doses was 10 hours and 25 hours the maximum. Randomization Four hundred Holstein cows were used in a randomized design. To attain even enrollment within parity, two enrollment lists were prepared: first lactat

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54 Trained f calving, animals fitting the selection criteria were group assigned and processed, following a color coded list. Treatments (PAP L PS and PLC) were color coded by a third party . Color treatment code was maintained in a closed envelope until statistical analysis of data was performed to ensure blind ing . Color coding also facilitate d identification of cows and helped ensure correct admi nistration of treatments. Colored zip ties corresponding to treatment assignment were placed in each ear tag. On the last day of treatment (day 14), zip ties were removed. The control group was not identified with any color. Data Collected Production data were collected during the first 98 days of lactation (14 weeks). Milk yield w as collected daily, and monthly via DHIA test day data that included days in milk at test, milk production and milk component information (milk fat % and milk protein %). Da ta were obtain ed from PCDart ® which compiles information recorded daily by Smart Dairy ® , and information collected once a month by DHIA during milk test da y . For posterior analysis, daily individual milk production was weekly averaged. Because milk product ion on day 1 after partutition was so va riable, this day was excluded from analysis . Variables The analyzed dichotomous variables were: parity, induction of parturition, calving related problems (twins, stillbirth), retained fetal membranes, down cow synd rome, metritis, ketosis, mastitis, digestive issues (DA, scours, indig estion, constipation), lameness and cull. C ontinuous variables were : daily milk production (kg) and DHIA test data collected monthly ( milk fat percentage, milk protein percentage, milk f at yield (kg), milk protein yield (kg) ). C ategorical data was lactation number.

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55 Energy c orrected milk was calculated as ECM = milk corrected to 3.5% fat and 3.2 protein us ing the following equation: ECM kg = (12.82 x kg fat) + (7.13 x kg protein) + (0.323 x kg milk). (Gozho et al., 2007) Fat and protein yields were calculated as: Fat Yield = (milk yield (lb) reported by DHIA * 0.454)* fat percentage. Protein Yield = (milk yield (lb) reported by DHIA * 0.454)* protein p ercentage. Fat:Protein Ratio = Milk Fat Percentage / Milk Protein Percentage (both percentages reported by DHIA) Calving and transition related d isease d efinition The following are definitions to be used throughout this manuscript: A BORTION . D eliver y of a dead fetus of gestation length shorter than 260 days. S TILLBIRTH . Dead born fetus of gestation length longer than 259 days. C ALVING I NDUCTION . T he pharmacological induction of parturition, done twice a week on animals that excee d their expected calving date by 2 days through administration of 25 mg of PGF2 and 20 mg of dexamethasone. D YSTICIA . Defined as an abnormal parturition process. A ny c ow requiring assistance at c alving was considered a dystocia. Cows with calving ease score 5 (cesarean sectio n or fetotomy) were excluded from the study . C ALVING E ASE S CORE . 1 = calving without assistance; 2 = light force with use of obstetric chains; 3 = moderate force with use of obstetric chains; 4 = extreme force to deliver the calf; 5 = cesarean section or fetotomy. D OWN C OW S YNDROME . I nability to remain standing; could have different causes, hypocalcemia, trauma, etc. If any of these cows start a treatment, they were removed from the trial and replace d . R ETAINED F ETAL M EMBRANES . F etal membran es retained for after at least 2 4 hrs after parturition.

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56 M ETRITIS . Upon rectal palpation , a red brownish, fetid fluid vaginal discharge within 2 weeks postpartum. K ETOSIS . K etones body detected with urine test. R IGHT OR L EFT D ISPLACEMENT OF A BOMASUM . diagnosed by simultaneous auscultation and percussion on the right or left side between the 9 th to 13 th intercostal spaces. I NDIGESTION . W atery feces with indigested feed particles, mainly fiber from forage source. S COURS . Watery feces of unknown o rigin without measurable body temperature higher than normal. C ONSTIPATION . T he absence of feces at rectal palpation. M ASTITIS . A bnormal characteristics of the milk. Pain, swelling, warmth, and redness of the quarter involved could also be present . Onl y the first case was considered in health incidence analysis. L AMENESS S CORE . A scale from 1 being normal to 5 being clinically lame with almost complete weight transfer off the affected limb was used. This was judged by the farm hoof trimmer. C ULLING . A cow that leave s the herd, dead or alive during the observational period from day 15, until day 98 after parturition. Statistical A nalysis For baseline comparisons of dichotomous dependent variables the GENMOD procedure of SAS (SAS Inst. Inc., Cary, NC ) was used. The GENMOD procedure is used to fit generalized linear models such as logistic models for binary data. The dichotomous dependant variables in this study were calvprob, dystocia, milk fever, and retained fetal membranes. The MIXED procedure o f SAS (SAS Inst. Inc., Cary, NC) was used in order to make a statistical inference about the effectiveness of the PAP LPS treatment. The MIXED procedure fits a variety of mixed linear models to the data allowing for modeling not only the mean but also the data variances and covariances (SAS Institute, ) . A first order autoregressive covariate structure was used for all models and dependent variables in the analyses. Models were built up

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57 using stepwise regression. This procedure was performed by adding the explanatory variables with p > 0.05 according to Wald statistics criterion. Differences of p < 0.1 were considered significant when interaction was analyzed and p < 0.05 was considered significant for each of the individual explanatory variables. Results Descriptive Statistics Descriptive statistics for the different groups can be seen in Table 2 3 . Four hundred cows were enrolled, 174 primiparous and 226 multiparous, 398 completed the 14 day treatment period, and two died within the first 14 days postpartum. Final enrollment per group was 125 PAP LPS (55 primiparous and 70 multiparous), 143 PLC (66 primiparous and 77 multiparous) and 132 CTL (53 primiparous and 79 multiparous). Out of multiparous cows, 86 were sec ond lactation, 56 third and 84 fourth or higher. Fifty nine cows had their parturition induced and 113 of the 400 had at least one of the defined calving problems. No statistical differences were observed between parity, lactation number or calving problem s between the three groups. Milk P roduction Daily milk production was averaged weekly and this was compared. Cows peaked on average at week 6; overall production observed at this point was 43.7 ± 0.31 kg (45.67 kg multiparous and 34.56 kg primiparous). Cows receiving PAP LPS did not differ from the other groups when treatments were compared. A treatment by week interaction was observed ( p = 0.097). Significant differences ( p < 0.05) were observed on weeks 3, 4, 6, and 10 between PAP LPS and CTL, and the only significant difference between PAP LPS and PLC was observed at week 6 (P < 0.05). Lactation curves and di fferences are shown in Figure 2 1.

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58 For the total milk production analysis, only cows completing the first 100 days of lactation were considered ( n = 375 ) . Total milk production for PAP LPS, PLC, and CTL was 3881 ± 38.2, 3769 ± 38.6, and 3785 ± 37 kg respectively . Feeding PAP LPS increased (P = 0.04) total milk production by 112 kg in the first 98 DIM when compared to CTL, while only tended to incre ase it (P = 0.07) when compared to PLC (96 kg difference). These data are pr esented graphically in Figure 2 2. When energy corrected milk analysis was run, a treatment effect was observed ( p = .0023). Estimates of daily milk yield were 41.28 ± 0.3, 40. 43 ± 0.28 and 39.78 ± 0.3 kg for PAP LPS, PLC and CTL, respectively (Figure 2 3). Significant differences between groups were found between PAP LPS and the other two treatments ( p < 0.05), and no difference was found between PLC and CTL ( p = 0.1). Milk C om ponents Milk components (milk fat + milk protein) from monthly individual milk weight report s by DHIA service was summarized by week in milk. No differences between treatments were observed when milk fat percentage was evaluated ( p > 0.1). Only the effect of week was significant ( p < 0.0001). Protein percentage was affected by treatment ( p = 0.02) with 2.74 ± 0.01, 2.77 ± 0.01 and 2.77 ± 0.01 % for PAP LPS, PLC and CTL , respectively , throughout the first 98 days of production. PLC and CTL were both signific antly higher than PAP LPS ( p < 0.05). An effect of parity was seen; milk protein percentage was higher in primiparous than multiparous cows . A t reatment by pa rity interaction was observed for protein percentage ( p = 0.0091). Analyzing the data from pari ty standpoint (Figure 2.4), for multiparous cows there was a difference between PAP LPS (2.68 ± 0.01%) and PLC (2.74 ± 0.01, p = 0.001) and also between

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59 PLC and CTL (2.69 ± 0.01%, p = 0.01) with PLC being higher than the other two; there was no difference be tween PAP LPS and CTL. When primiparous cows were analyzed, only a difference between PAP LPS (2.80 ± 0.01%) and CTL (2.85 ± 0.01%, p = 0.01) was observed. Regarding fat yield, PAP LPS, PLC and CTL produce d on average 1.51 ± 0.01, 1.48 ± 0.01 and 1.45 ± 0 .01 kg of fat/day in the first 98 days of lactation. No treatment by parity or treatment by test week interaction was found. Fat yield was different between treatment groups ( p = 0.03), with PAP LPS showing a higher yield than CTL ( p = 0.008), while no dif ference was observed between PAP LPS and PLC or between PLC and CTL (Figure 2.5). There was also a parity effect (P=0.0001), with multiparous cows producing 1.69 ± 0.01 kg and primiparous cows 1.26 ± 0.01 kg of milk fat per day. When protein yield was anal yzed, a treatment by parity interaction was found ( p = 0.01). Results are illustrated in Figure 2.6. Within multiparous cows, PAP LPS, PLC, and CTL produce an average 1.28 ± 0.0 1 , 1.29 ± 0.0 1 and 1.24 ± 0.0 1 kg of protein per day during the 98 days observe d. While CTL was significantly lower than PAP LPS (P=0.002) and PLC (P= 0.0004), the latter two were not different. Different results were observed in primiparous cows between treatment groups. PAP LPS, PLC, and CTL produce in average 1.00 ± 0.0 1 , 0.98 ± 0 .0 1 and 0.99 ± 0.0 1 kg of protein per day during the 98 observed days. PAP LPS tend to produce more than PLC ( p = 0.09) but it was not different than CTL. CTL did not differ from PLC. Discussion As far as we know this is the first time that PAP LPS has bee n used i n early lactation dairy cows with the objective of enhancing milk yield. The randomized clinical trial herein reported was designed to evaluate the effect of oral daily administration for 14 days of an anti LPS polyclonal antibody preparation produ ced from eggs from immunized hens, on health, milk

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60 production and milk components. The hypothesis was that health and production effects would likely be t he result of prevention or lesse ning the effect s of SARA . We were not able to measure a significa nt difference in milk production measured on a weekly average between groups although PAP LPS, the treated group, did show increased milk yield during weeks 3, 4, 6 and 10. A hypothesis for the increase d milk production is that PAP LPS had a protective eff ect on rumen mucosa and because of this, there was reduced translocation of LPS to portal blood and consequently reduced release of cytokine s . Cytokines are attributable to reduction in DMI, thus, milk production. Research has shown increased concentration decrease d DMI either through increased leptin concentration or by a direct effect of the cytokine on the CNS (F inck et al., 1998; Ingvartsen and Andersen, 2000) . A previous trial using PAP LPS in 28 steers (data not published, DiLorenzo & Galyean) found that the treatment group ate, on average, 600 g more than placebo treated animals and this resulted in an increa se in final body weight after completion of the observational period. A second hypothesis could be that the product enhanced milk production by enhancing health status during transition ; no difference in milk production was seen during the first 2 weeks (t his is very difficult to see) but ruminal environment may have been better prepared to support further production as seen on weeks 3, 4 and 6. It has been mentioned that milk production leads DMI during lactation (Ingvartsen and Andersen, 2000) and when DMI is a limiting factor , then milk yield is suppressed. The difference in milk yield between PAP LPS and placebo was only seen in week 6. In previous trials w h ere placebos were used, the product used was dried eg g from non immunized hens, while in this study, we used PAP have any quality control test to assess that the inactivation process was 100% successful. There is

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61 some evidence that inactivation of IgY, th e main antibody in the egg, can vary depending on time and pH, since both parameters are needed for a complete inactivation of the preparation (Shimizu et al., 1992) . They observed decreased activity of the antibody w hen incubated at pH 3.5 or lower for 7 h. Total milk production over the first 98 days of lactation was increased significantly (112 kg) in favor of PAP LPS compared with CTL and tended to be higher (96 kg) compared with PLC. As proposed above, t his increa se in milk production could be the result of improved rumen health during the transition period, thus avoiding ruminitis and improving gastrointestinal health status. In an experiment where grain induced SARA was studi ed (Khafipour et al., 2009b) , decreased DMI and consequently a trend for decreased milk yield (31.6 ±1.7 control vs 28.3 ±1 .7 SARA) were observed. Two possible hypophagic causes were cited: endotoxemia was one and elevated concentration of propionic acid in rumen, related to hepatic oxidation theory and consequent suppression of appetite by decreasing stimulus on the vagus nerve was the second one. In a different experimental setting, Khafipour et al. (2009) induced SARA by gradually increasing % of pell eted alfalfa in the diet and reported a linear increase in DMI and a linear decrease in milk yield and fat %. Although they reported no translocation of LPS or increase in acute phase proteins, they explained that the smaller particle size could decrease r etention time and digestibility, and also low pH could inhibit rumen cellulolytic activity and reduced fiber digestibility. Another study of induced SARA showed a decreased milk yield from 35.2 to 31.7 kg that remained during the recovery period (Krause and Oetzel, 2005) . They also reported in a case study, reported an increase of 2.7 kg of milk after adjusting the diets i n a farm suffering SARA. Gozho et al. (2007) found no difference in milk yield or DMI between SARA induced

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62 and control cows; however, based on ruminal pH, the control group was also suffering from SARA. Emmanuel et al. (2008) reported increased milk produc tion when DMI was increased by substituting barley silage with rolled barley. In this publication they reported increased translocation of endotoxins from rumen fluid into the blood stream. In our study, ECM analysis showed an increase in milk yield in fa vor of PAP LPS. The difference between groups was 1.5 and 0.85 kg when compared with CTL and PLC, respectively. Gozho et al. (2007) reported a numerical decrease in ECM when SARA was induced, although the control group was also undergoing SARA. This findin g supports our previous observation in not measured in this experiment because it was performed in a commercial dairy herd. All the experiments that induced SARA by restriction protocols (Krause and Oetzel, 2005; Gozho et al., 2007) are difficult to compare with ours because their methodology of restricting access to feed for a period of time will necessari ly drive a decrease in milk yield. No treatment effect on milk fat percentage (MF%) was observed i n this trial . Stone (1999) suggested that MF% is not a confident indicator of ruminal pH and even less so in fresh cows because they are mobilizing body fat, and thus increasing MF%. In his case study, he reported decreased MF% when the whole herd was suffering SARA. Milk components were also reported and when diets were modified, MF% was increased from approx. 3.4 to 3.7 while milk protein percentage (MP%) was increased from 2.8 to 2.9. Average MF% for the first 98 day of lactation was 3.6 with the offered diet in our study. Milk protein percentage was statistically different between treatments although the biological significance of this finding is questionab le (2.77 ± 0.01, 2.77 ± 0.01 and 2.74 ± 0.01 for PLC, CTL and PAP LPS respectively). Several factors related to the dietary characteristics

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63 such as NE L intake, forage to concentrate ratio, amount of rumen fermentable carbohydrates, and dietary CP level amo ng others, affect protein % of the diet. Stone (1999) presented similar MP% values (2.8 to 2.9%) after dietary adjustments in a problematic herd. Khafipour et al. (2009b) reported MP% values of 3.42 and 3.29 for SARA and control groups, respectively, when grain induced SARA was studied. A trend for decreased MF% was observed in SARA compared with controls cows (2.93 and 3.30%, respectively). A significant effect of parity was seen on MP%. It has been reported that casein content of milk decrease as parity i ncrease (Ng Kwai Hang et al., 1984; DePeters and Cant, 1992) and the decrease in total protein is not highly affected due to an increase in the non casein N of milk. It would be hard to su pport a theory associating PAP LPS an increase MP% in animals suffering SARA, but in general, when this happens (Khafipour et al., 2009a; Khafipour et al., 2009b) , a decrease in MF% also occurs, and that is not the case in our trial. A significant difference in fat yield was observed between PAP LPS and CTL although it was of minimum amount (60 g/d). This difference could be explained mainly by the numerical difference in milk yield between both groups. Krause and Oetzel (2005) reported a numerical depression in fat yield when SARA was induced and it became significantly low during the recovery period. The y reported a change in milk yield. Khafipour et al. (2009) reported a linear decrease of 11 % in milk fat yield when gradually decreased chopped alfalfa to induce SARA while the difference observed in our experiment was of 4%. These findings support our th eory that PAP LPS acts locally by inducing an increase, or preventing a decrease, in DMI when SARA is present and finally enhancing milk production.

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64 Protein yield analysis found a significant interaction between treatment and parity. The observed d ifferenc es between treatments within parity categories seem biologically meaningless. The effect of parity on protein yield is correlated with higher milk production of the older group during the first third of lactation. There was a significant difference betwee n parity groups (280 g), and this is strictly related to milk yield. Conclusion In conclusion, analysis of daily milk production showed higher milk yield in cows treated with PAP LPS for in certain weeks , all of which were after completion of the treat ment period. Considering the total milk production of the first 98 days in lactation, there was also a difference in favor of the treatment group. This observation was reinforced and supported when milk was corrected for fat and protein content . Milk compo nents analysis did not help to clarify the advantage of using this tool , although fat yield was increase d in treated cows . Although DMI was not recorded in this experiment , previous research showed an increase in DMI in cattle fed this product , encouraging further investigation to explain our theory.

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65 Table 2 1 . Diet composition and ingredients of prepartum and postpartum diets . Ingredients (lb of DM) Prepartum Postpartum Springers Fresh High Production Fine Ground Corn 1.71 6.85 11.95 Soybean Meal 0.00 0.58 0.58 South Pivot Corn Silage 6 5.25 6.50 6.00 New Corn Silage 5 7 13 5.25 6.50 6.00 Citrus Pulp 0.00 3.00 4.35 Whole Cottonseed 0.00 3.00 3.64 Molasses Cane 0.54 1.36 1.98 Lacto Whey 0.00 1 .10 1.50 Wheat Straw 3.50 1.25 0.86 Bermuda Grass Hay 6 11 13 4.58 2.72 4.86 CSC Brewers Jacksonville 3.96 4.00 5.94 Amino Plus 0.00 2.39 2.77 Soybean Hulls GA 0.00 3.50 5.70 Lactation Mineral Blend 6 0.00 3.75 3.87 Bermu da Grass Silage 6 20 0.00 1.50 2.00 Springer Mineral Blend 5 2.62 0.00 0.00 Bermuda Grass green chop 4.59 0.00 0.00 Totals 32.00 48.00 62.00 Dry Matter % 39.61 46.08 46.53 Crude Protein % 16.67 18.06 18.07 RUP %Prot 31.65 36.34 37.74 RDP %Prot 68.35 63.66 62.26 RDP %DM % 11.39 11.5 11.25 SOL % Prot 43.61 35.86 32.95 SOL % DM % 7.27 6.48 5.95 NPN % 0.87 1.9 1.52 NEl CNCPS Mcal/lb 0.65 0.77 0.78 Fat % 3.56 5.16 5.0 1 ADF % 25.98 21.71 21.05 NDF % 48.39 35.03 34.51

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66 Table 2 2. Vaccination protocol of prepartum heifers and cows and postpartum cows Status Product Dose Manufacturer Fresh cows 14DIM J5 5 cc SQ Pharmacia® 56 DIM Bovi shield gol d + L5 HB 2 cc SQ or IM Pfizer® every 110 day in milk J5 5 cc SQ Pharmacia® 90 days pregnant Leptoferm 5 2 cc SQ Pfizer® Bovi shield gold + L5 HB 2 cc SQ or IM Pfizer® Dry off J5 5 cc SQ Pharmacia® Ultravac 7 5 cc SQ Pfizer® Scourguard 4KC 2 cc IM Pfizer® Cows entering springers group J5 5 cc SQ Pharmacia® Heifers entering springers group J5 5 cc SQ Pharmacia® Ultravac 7 5 cc SQ Pfizer® Scourguard 4KC 2 cc IM Pfizer®

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67 Table 2 3 . Descriptive statistics for PAP LPS, Placebo and Control groups. Treatment Groups Variable Level All Cows PAP LPS 1 PLC 2 CTL 3 P value N # (%) # (%) # (%) Parity 0.60 Primiparous 174 55 (44%) 66 (46%) 53 (40%) Multiparous 226 70 (56%) 77 (54%) 79 (60%) Totals 400 125 (100%) 143 (100%) 132 (100%) Lactation Number 0.92 1 174 55 (44%) 66 (46.1%) 53 (40.2%) 2 86 27 (21.6%) 31 (21.7%) 28 (21.2%) 3 5 6 19 (15.2%) 18 (12.6%) 19 (14.4%) 84 24 (19.2%) 28 (19.6%) 32 (24.2%) Totals 400 Induced Yes 59 17 (13.6%) 22 (15.4%) 20 (15.2%) 0.9 No 341 108 (86.4%) 121 (84.6%) 112 (84.8%) Calving Problems Yes 113 37 (30%) 42 (29.4%) 34 (25.8%) 0.74 No 287 88 (70%) 101 (70.6%) 98 (74.2%) 1 PAP LPS re c e i ved activated polyclonal antibodies preparation anti LPS from day 1 14 of lactation. 2 PLC received inactivated PAP LPS from day 1 to 14 of lactation. 3 CTL did not receive any treatment.

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68 Figure 2 1. Last squares means of weekly average milk yield through the first 98 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) and those n ot treated with dry egg yolk (CTL). PAP LPS was significantly different on weeks 3, 4 and 10 to CTL (P <0.05), and to both on week 6 (P <0.05).

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69 Figure 2 2. Last squares means of total milk yield through the first 98 days in lactation in cows treat ed with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) and those not treated with dry egg yolk (CTL). PAP LPS was significantly higher than CTL and tend to differ with PLC. ab b a

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70 Figure 2 3. Last squares means of ECM through the first 98 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) and those not treated with dry egg yolk (CTL) . ECM (kg) calculated as: ECM = milk corrected to 3.5% fat and 3.2 protein using the following equation: ECM, kg = (12.82 x kg fat) + (7.13 x kg protein) + (0.323 x kg milk). (Gozho et al., 2007) . PAP LPS was significantly higher than PLC and CTL. No difference between PLC and CTL was observed. a b b

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71 Figure 2 4. Last squares means of Milk Protein percentage of primiparous and multiparous cows through the first 98 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) an d those not treated with dry egg yolk (CTL). I nteraction between treatment and parity was observed . While in primiparous PAP LPS was different that CTL, in multiparous, PLC was significantly higher than the other two. a ab b a b a

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72 F igure 2 5. Last square means of Fat Yield (kg) through the first 98 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) and those not treated with dry egg yolk (CTL). PAP LPS was significantly different tha n CTL but was not different than PLC.

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73 Figure 2 6. Last squares means of Protein yield of primiparous and multiparous cows shown through the first 98 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS) , inactivated PAP LPS (PLC) and those not treated with dry egg yolk (CTL). While no diference between treatments was observed within Primiparous cows, in multiparous, CTL was significantly lower than the other two.

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74 CHAPTER 3 ACUTE P HA SE RESPONSE AND HEAL TH DISORDERS Introduction Understanding and managing the transition period requirements of a cow is extremely important in dairy production, since this period dictates fu t u r e performance of the animal. Multifactorial diseases affect this period and they ne ed to be understood and prevented to decrease the risk of culling and also to maximize milk production (Andersen, 2003) . There are immunological, metabolic and nutritional factors challenging the healt h of early lac tation dairy cows and it is well known that risk of diseases is increased during early postpartum (Mulligan and Doherty, 2008) . Within the metabolic diseases affecting the postpartum period, hypocalcemia and ketosi s, both presenting clinical and subclinical forms, are two of the most important. It has been stated in 2002 that incidence of hypocalcemia in US dairy herds averaged 5.2%. Although more recent data suggest decreased incidence of the clinical form, subclin ical hypocalcemia still remains an important problem to the dairy industry (Martinez et al., 2012) . Negative energy balance and lipomobilization are common features of high yielding dairy cows and ketosis is a conseq uence of this equilibrium (Oetzel, 2007; McArt et al., 2011) . Reported ketosis incidence varies widely and Oetzel (2007) proposed 10 % as an alarm level on herd incidence rate. Hepat ic lipid osis is not a r ar e finding i n ketotic cows and over conditioned cows are more likely to have hepatic lipidosis (Rukkwamsuk et al., 1999) . Metritis is the inflammation of all layers of the uterus (Lewis, 1997) and happens within the first 2 weeks after parturition. Presence of LPS during metritis and differential resp onse in acute phase proteins have been documented (Benzaquen et al., 2007; Galvao et al., 2009; Huzzey et al., 2009; Galvao et al., 2010) . The incidence of metritis varies between farms and parity but

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75 reported values ranges from 20 to 40 % (Benzaquen et al., 2007; Galvao et al., 2010) . Mastitis, another disease affecting postpartum cows, is the inflammation of the mammary gland (Bradley, 2002) . Escherichia col i , a Gram negative bacteria, is mentioned as one of the most frequent causes of mastitis (Blum et al., 2014) . Several proinflammatory cytok ines, including IL 6, amyloid A, and LPS binding protein had been detected in cows suffering clinical mastitis (Bannerman, 2009; Wenz et al., 2010; Larsen et al., 2010) . Subacute rumen a cidosis (SARA) is define as the pathological decrease of ruminal pH for prolonged periods of time each day, e.g. pH <5.6 for >3h/day (Plaizier et al., 2008) . SARA has been reported to be prevalent in 19% of early lactation cows in the USA (Mulligan and Doherty, 2008) . Although recently challenged by Penner et al . ( 2007), it has been proposed that early lactation cows are susceptible to this disease due to low rumen absorption capacity, poorly adapted rumen microflora and introduction to high concentrate rations in order to increase energy intake and milk production (Mulligan and Doherty, 2008) . Several experiment s have induced SARA using different grain source or decreasing length of forage source and all of them were able to detect increased concentration of free LPS in rumin al fluid (Gozho et al., 2005; Gozho et al., 2007; Emmanuel et al., 2008; Khafipour et al., 2009a) . It is well know that LPS cause s a n inflammatory respons e when it i s exposed to tissues. No t all the mentioned experiments look ed for presence of LPS i n peripheral blood but some were able to detect it (Khafipour et al., 2009b) although other s (Khafipour et al., 2009a) . Liver health status has been associated with the capacity for LPS clearance and ability the detect endotoxins on peripheral circulation (Andersen et al., 1996) . Besides that, all studies monitored different acute phase proteins in order to explain inflammatory processes.

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76 Haptoglobin (Hp), serum amyloid A, LPS binding protein, C reactive protein and ceruloplasmin (Cp) had been mentioned as inflammatory markers in different situations (Gozho et al., 2005; Arthington et al., 2005; Gozho et al., 2007; Emman uel et al., 2008; Khafipour et al., 2009a; Khafipour et al., 2009b; Arthington et al., 2013) . Some show ed increased serum Hp concentration when animals were SARA challenged (Goz ho et al., 2005; Gozho et al., 2006; Khafipour et al., 2009b) while others sh owed no difference (Gozho et al., 2007; Emmanuel et al., 2008) or even a linear decrease (Khafipour et al., 2009a) . S ome literature had observed Cp concentrations as m arker of inflammatory processes; besides that, references of this APP on SARA seems to be lacking (Chassagne et al., 1998; Sheldon et al., 2001; Arthington et al., 2013) . Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and which is released when these cells die (Andersen, 2003) . A rapid growth of these bacteria also leads to an increase in free LPS in the rumen (Wells and Russell, 1996) . Acidity of the rumen results in reduction of the number of cellulolytic b acter ia and a shift of the bacterial population from Gram negative to Gram positive (Nagaraja et al., 1978a) . A c onstant effort has been conducted to attenuate rumminally derived endotoxemic events in the transition peri od. Mainly, adjusted managerial practices are suggested for prevention of SARA in commercial herds but the use of feed additives or antibiotics ha ve also been promoted . Several reports show increasing public concern about human health and its relation to u sage of antibiotics and hormones in the cattle industry (Simmons, 2011) . The spectrum f rom natural microbial or plants extracts to specific antibodies has been postulated to improve animal productivity . P olyclonal a ntibod y preparations (PAP) derived from immunized hens have been proposed a s a viable alternative for prophylactic and therapeutic treatment of

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77 various diseases in several species (Peralta et al., 1994; Ikemori et al., 1997; Mine and Kovacs Nolan, 2002; Vega et al., 2011) . Avian IgY, a compound of PAP , obtained from the yolk of hens immunized with killed E. coli 0111 are available and show a high binding activi ty to LPS (Zhen et al., 2011) . Producing PAP or purified IgY is technically easy and allows for production of large quantities compared to IgG obtained from serum of immunized mammals (Chalghoumi et al., 2009) . Specific IgY has been reported to be beneficial in different trials (DiLorenzo et al., 2006; DiLorenzo et al., 2008; Blanch et al., 2009) . In a trial done using 48 beef steers in the final fattening process, daily dosage of 3 5 g of egg yolk containing polyclonal antibodies preparations (PAP) anti LPS, improved DMI by about 10% (personal communicat ion with the researcher). In this experime nt we hypothesized that feeding a fixed amount of anti LPS PAP during the first 14 days after parturition would decrease the amount of free LPS in rumen and consequently diminish consequences of SARA. This effect should enhance overall health, and conseque ntly improve milk yield in transitio n cows. The objective of the work presented in this chapter is to determine the effect of daily feeding of 3 g/ d of a polyclonal antibody preparation against LPS during the first 14 days post partum on hea lth performance and specific acute phase p rotein s of Holstein dairy cows in a commercial setting . Materials and Methods All procedures were approved by Institutional Animal Care and Use Committee from the University of Florida (IACUC UFL # 201307790). refer to Material and Methods i n Chapter 2.

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78 Experimental D esign and T reatments Blood sample c ollection Eighty one study cows had blood samples collected from the coccygeal vein on days 1, 6 and 14 after parturition. Samples on day one were taken approximately at 0600 or 1400 hours and samples f rom days 6 and 14 were taken between 1500 and 1600 hours, after the second milking of the day. Samples were collected in K2 EDTA 10 mL evacuated tubes (Vacutainer, Becton Dickinson, Fanklin Lakes, NJ) for plasma collection. Blood samples were maintained on ice and kept in refrigerator at 4 ° C until plasma was harvested by centrifuging tubes at 1,800 x g for 10 minutes. Within 12 hours of blood collection, pl asma was transferred to two polypropy lene vial s and stored at 80 ° C until assyed for ceruloplasmine and haptoglobin (Hp). All samples were collected by trained research assistants to ensure proper handling and collection of samples and more important, animal welfare. All samples were analyzed, but res ults from cows with any diagnosed health disorder were removed f r om the analysis in order to prevent bias in the observed results. Out of 81 cows sampled, 29, 27, and 25 were PAP LPS, PLC and CTL, respectively. Before statistical analysis, 5, 10, and 8 cow s from PAP LPS, PLC and CTL respectively, were removed leaving 24, 17, and 17 cows per group. Ceruloplasmin a ssay Ceruloplasmin assay was run as per (Demetriou et al., 1974) . The chemicals and reagents used were so dium acetate, 0.1 M acetate (0.57 mL glacial acetate/100 mL water), p phenylenediamine (PPD), sodium azide, and distilled water. Supplies needed for this assay were 16x75 mL disposable glass culture tubes (Fisherbrand®), reading cuvettes (Fisherbrand dispo sable plastic Cat. No. 14 955 127), 2 different pipette tips: 1 200 (yellow), and 101

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79 1000 (blue), and two adaptable syringes (Eppendorf®), one of 1.25 mL (1 =25 ) and the other one of 5.0 mL (1 = 100 ). Solutions . Sodium azide solution was prepared previous to the beginning of the assay by mixing 0.1 g Na azi de to 100 mL buffer. The solution was stored in the refrigerator and warmed to room temperature before usage. A p phenylenediamine (PPD) solution was prepared in a foil wrapped flask, just before it was needed and placed into a water bath for 5 minutes at 37 ° C to be used. For each three racks run, or a total of 180 tubes, 0.250 g of PPD crystals were mixed in 75 mL of the buffer solution previously mentioned and pH was stabilized to 6.0 by addition of 1 M NaOH. Lastly, 25 mL of buffer solution was added t o yield a final volume of 100 mL . Procedure . Tubes, racks and assay sheets were labeled before 60 glass disposable culture tubes (Fisherbrand®) of 75 mL were set up per rack. Three racks were analyzed per run. All samples were processed in triplicate with the first being the blank sample and the remaining two, the replicates. At the beginning of each rack, a control high (CH) and a control low (CL) were also placed by triplicate, High and low controls are plasma samples from animals known to have high and l ow ceruloplasmin concentration. A diagram (Figure 3 1) of the disposition can be seen below with C being control, B being blank, and S, sample. That sample in which the reaction was stopped early after the beginning of the analysis process in considered as used as a baseline for later calculations. Plasma (50 ) was added to each of the corresponding samples tubes in the three racks. Room temperature buffer solution (500 ) was then added to all 180 tubes. Buffer solution was prepared in advance in a large beaker by addition and mixing of 13.6 g of sodium ace tate, 900

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80 mL of distilled water and 50 mL of 0.1 M acetic acid. The solution was adjusted to pH 5.95 with 0.1 M NaOH and distilled water was added to complete a liter as final volume. After this point and for the remainder of the assay, as minimum as possi ble light, was used. Plasma samples were also place in the water bath for 5 minutes at 37 ° C before 500 of PPD solution was added to each tube. Alarms timing was followed. After 5 min, 500 of remaining in the bath. Finally, all samples were incubated 30 more mi nutes. At the completion of this period, 500 of sodium azide solution was added to the remaining tubes stopping existent reaction. The content of each tube was poured into reading cuvettes (Fisherbrand disposable plastic Cat. No. 14 955 127) and absorba nce was measured at 530nm in a spectrophotometer (Genesys 20, Thermo Spectronic®, Model 4001/4, Cat 4004, SN 3SGG043018). Distilled water was used to zero balance the device prior to use. Calculation was reported as mg/100 mL based on the following equati on: mg/100 mL = ((Sample 1 + Sample 2) /2 Blank)*1000*0.06 After running all samples, coefficient of variation (cv) between replicates was observed and all samples showing a cv equal or higher than 11 were re run. In total 32 samples were re runed. Intra assay CV was 3.5%. Haptoglobin a ssay Quantitative determination of bovine serum haptoglobin was run as per (Makimura and Suzuki, 1982) . The chemicals and reagents used were O Dianisidine, sodium EDTA (Na2EDTA), sodium phosphate monobasic (NaH2PO4 H2O), distilled water, phosphoric acid or 1M NaOH,

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81 methemoglobin and H 2 O 2 . Supplies needed for this assay were 16x100 mL disposable glass culture tubes (Fisherbrand ® ), flat bottom 96 well plate, (Cat. No. 3997, Costar ® ), 3 different pipette tips: micro 0.1 10 (clear), 1 200 (yellow), and 101 1000 (blue), and two adaptable syringes (Eppendorf ® ), one of 1.25 mL (1 =25 ) and the other one of 5.0 mL (1 = 100 ). Before initiation of the sample process, O Dianisidine solution and Methemoglobin solution were prepared. The first solution is stable for several weeks when stored in an amber jug at room temperature and the second reagent needs to be kept in the refrigerator and only brought out to be used. O Dia nisidine solution was prepared by mixing 2.4 g of O Dianisidine, 2.0 g of disodium EDTA (Na2EDTA), and 55.2 g of sodium phosphate monobasic (NaH2PO4 H2O) with the amount of distilled water needed to complete a volume of 4 liters. This amount of solution is enough to process 542 tubes. Preparation of the solution requires overnight stirring before being filtered off 3 times to remove un dissolved particles. Finally pH was carefully set to 4.1 by addition of phosphoric acid or 1M NaOH. Methemoglobin solutio n was prepared by adding 0.03 g methemoglobin or hemoglobin from bovine blood into 100 mL distilled water; this makes enough solution for 4,000 tubes. A 156 mM H 2 O 2 solution was prepared fresh prior to it usage by adding 391 of 30% H 2 O 2 into 25 mL of distilled water. At the end of each run, remaining solution was disposed of. Different proportions of control high and control low were used to build a standard curve in order to interpret the obtained values. Table 3 1 shows t he proportions used for each case. All plasma samples were run in duplicate. Tubes were set up in 48 cell racks filled with tubes as shown in the diagram below (Figure 3 2). The first and third rows were filled only from

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82 the seventh space and the second an d fourth rows were completely filled. The distribution of tubes on each rack was even, but rack A was filled with 12 control tubes to build the standard curve with different proportions of high and low plasma control (see Table 3 1), and in rack B places w ere filled with trial plasma samples. In total 72 tubes were analyzed per run, 2 racks per run. The number of racks to be analyzed that day were prepared fresh that morning and maintained at 4 ° C until needed. To each tube 25 of methemoglobin solution w as added. After that, 5 control sample were pipetted into the control tubes. Same volume (5 ) of distilled water was then added to the blank tubes and also 5 of test plasma samples were added in their respective tubes in the order presented in the ra cks. After pipetting each single tube, it was moved to a second rack in order to decrease risk of confusion. After this step, labeled racks were kept at 4 ° C until further processing within 4 hs. The process continued by moving the pair of racks out of the fridge and maintained at room temperature for 12 minutes after which each tube was filled with 7.5 mL of O Dianisidine solution. Racks were incubated in a water bath for 45 minutes at 37 ° C. At the completion of this time, 100 of 156 mM H 2 O 2 was added to each tube, homogenized by vortex, and then incubated for 1 hour at room temperature. The following step consisted of pipetting 200 from each culture tube into two wells of a flat bottom 96 well plate (Figure 3 3), and pos terior analysis by using a microplate Tek Instruments®) setted at 450 nm, and the KC4® software. Every plate was read twice, the second time by flipping the plate in order to improve accuracy of the results. Data col lected from the analysis was saved in the software files and after

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83 that, summarized in an excel file for posterior statistical analysis. Final results were presented as mg of Hp/ mL . Variables The analyzed variables were as dichotomous: parity, induction o f parturition, calving related problems (twins, stillbirth, abortion), retained fetal membranes, down cow syndrome, metritis, ketosis, mastitis, digestive issues (DA, scours, indigestion, constipation), lameness, and cull rate; as continuous variables: hap toglobine concentration and cerulaplasmin concentration; as categorical data was lactation number. Statistical A nalysis For baseline comparison statistical analyses refer to Chapter 2. The MIXED and the GLIMMIX procedure of SAS (SAS Inst. Inc., Cary, NC) was used in order to make a statistical inference about the effectiveness of the PAP LPS treatment. The MIXED procedure fits a variety of mixed linear models to the data allowing for modeling not only the mean but also the data variances and covariances (SAS Institute, ) . A first order autoregressive covariate structure was used for all models and dependent variables in the analyses. Models were built up using stepwise regression. This procedure was performed by adding the explanatory variables with p > 0.05 according to Wald statistics criterion. The GLIMMIX Procedure fits models known as generalized linear mixed models to data with correlations or nonconstant variability and where the response is not necessarily normally distributed. This procedure was used to compare incidence of health disorders as ketosis, metritis, mastitis, and digestive issues between groups. Culling of cows was also compared using this method. Pe a rson product mome nt coefficient of correlat ion is a method used to measure the strength of the association between two variables. This measure is denoted by r and range s from

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84 1 to +1. V alues near z ero indicate lit tle association but negative or positive numbers far from z ero, a negative (when on a increase s the other decrease s ) or positive (when one increase s the other also increase s ) association. The Pe a rson correlation coefficient wa s used to measure association between APP and production indexes.

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85 CH B CH CH CL B C L CL S1 B S1 S1 S2 B S2 S2 S3 B S3 S3 S4 B S4 S4 S5 B S5 S5 S6 B S6 S6 S7 B S7 S7 S8 B S8 S8 S9 B S9 S9 S10 B S10 S10 S11 B S11 S11 S12 B S12 S12 S13 B S13 S13 S14 B S14 S14 S15B S15 S15 S16 B S16 S16 S17 B S17 S17 S18 B S18 S18 Figure 3 1. Di agram of sample s disposition in the rack of Cp assay : CH: control high; CL: sample number in each case. Table 3 1. Preparation of Controls for Cp assay of Standard Sample % of Standard Sample HC LC 100 120 0 80 96 24 60 72 48 40 48 72 20 24 96 0 0 120

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86 Rack A: Places not used 100 100 80 80 60 60 40 40 20 20 0 0 Blank Blank CH CH CL CL Places not used 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 Rack B: Places not us ed Blank Blank 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 Places not used 18 18 19 19 20 20 21 21 22 22 23 23 24 24 CH CH CL CL Figure 3 2. Racks set up and labeling of tubes for Hp assay. 12 tubes for the standard curve (100, 80, 60, 40, 20, and 0), 4 tubes for blanks (blank) and 8 tubes for controls: control high (CH) and control low (CL). Numbers from 1 to 24 ar e the plasma samples processed. 1 2 3 4 5 6 7 8 9 10 11 12 A Not Used B 100 100 100 100 80 80 80 80 60 60 60 60 C 40 40 40 40 20 20 20 20 0 0 0 0 D Blank Blank Blank Blank CH CH CH CH CL CL CL CL E Not Used F 1 1 1 1 2 2 2 2 3 3 3 3 G 4 4 4 4 5 5 5 5 6 6 6 6 H 7 7 7 7 8 8 8 8 9 9 9 9 1 2 3 4 5 6 7 8 9 10 11 12 A Not Used B Blank Blank Blank Blan k 10 10 10 10 11 11 11 11 C 12 12 12 12 13 13 13 13 14 14 14 14 D 15 15 15 15 16 16 16 16 17 17 17 17 E Not Used F 18 18 18 18 19 19 19 19 20 20 20 20 G 21 21 21 21 22 22 22 22 23 23 23 23 H 24 24 24 24 CH CH CH CH CL CL CL CL Figure 3 3. Flat bott om 96 well plate for Hp Assay, same distribution as racks. Each tube was analyzed in duplicate. Standard curve (100, 80, 60, 40, 20, and 0), control s: high control (CH) and low control (CL). Numbers 1 to 24 indicate plasma sample number.

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8 7 Results Descripti v e S tatistics Descriptive statistics were reported in the previous chapter and no differences were found between the observed variables. Acute Phase Proteins Blood samples were collected from 29, 25 and 27 of the PAP LPS, PLC and CTL respectively, and 17, 37 and 32 % of the cows from respective groups were removed from further analysis due to then being diagnosed diseased during the sampling period. There was a concern about incursion in a type 2 error due to lost power for statistical analysis. This conce rn was addressed by using Statistics ® 10 analytical software (Tall ahassee, FL). Final output showed that the sam ple size used was strong enough ( 24, 17 and 17 PAP LPS, PLC and CTL animals , respectively were used for the analysis ). No treatment ( p = 0.22) o r treatment by DIM interaction ( p = 0.85) was observed i n plasma Haptoglobi n (Hp) concentration. Figure 3 4 shows the trends defined by the Hp concentration. Although there was no significant difference between treatments, all sampled animals showed low Hp concentration on calving day (0.38 ± 0.09 mg/ mL ), with an increased concentration on day 6 (0.75 ± 0.09 mg/ mL ), returning to low concentration on day 14 (0.42 ± 0.09 mg/ mL ). An effect of parity on Hp concentration ( p = 0.0041) was observed w i th higher val ues for primiparous than multiparous cows . No significant effect of treatment was observed on ceruloplasmin (Cp) assay ( p = 0.24), nor was an interaction of treatment by days in milk found ( p = 0.55). The concentration curve for all 3 groups s howed the sa m e trend (Figure 3 5 ) with low concentration on day of parturition (23.7 ± 0.65 mg/ d l), an increased concentration on day 6 (29.5 ± 0.65 mg/ d l) with values

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88 remaining high on day 14 (28.8 ± 0.65 mg/ d l). A parity effect was also seen ; primiparous cows showed higher concentration than multiparous cows (29.06 vs 26.24 mg/ d l). Pearson correlation coefficient analysis was run. The inflammatory markers at day 14 (Hp and Cp concentration), total milk production and average production during the first 98 day of lact ation were compared (Table 3 2) . Hp concentration was correlated with Cp concentration ( p = 0.0002, R=0.405) but no other correlation was found with production indexes. Health I ssues Overal l results are shown in Table 3 3 . Health issues of 40 0 cows in th eir first 98 days of lactation were analyzed. No significant differences were found between treatments. Overall incidence of ketosis was 13.5 %, 12 % for metritis, 7 % for digestive related issues, and 12 % for mastitis. Of the enrolled cows, 6.5 % were so ld or died within the observational period. Discussion To our knowledge, this is the first experiment where PAP LPS is used in a commercial herd for preventing SARA consequences. Our aim was to evaluate APP and incidence of health disorders in order to su pport enhancement of overall health, and consequently improvement on milk yield in transition dairy cows. In se veral opportunities acute phase proteins had been used to assess inflammatory processes in animals. Within them, haptoglobin, ceruloplasmin, seru m amyloid A, LPS binding protein (LBP) and C reactive protein had been used in different trials (Gozho et al., 2005; Gozho et al., 2006 ; Gozho et al., 2007; Emmanuel et al., 2008; Khafipour et al., 2009a) . In our experiment we decide d to observe Hp and Cp as markers of inflammation to help explain possible differences in health and milk production. In ruminants, although unspecific, Hapt oglobin (Hp) has been reported as one of the mayor APP (Ceciliani et al., 2012) . In the literature it has been proposed to be used as marker of

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89 inflammatory processes such as metritis (Huzzey et al., 2009) , enteritis (Eckersall and Bell, 2010) , and others. Hp has also been used to assess inflammation when SARA was induced (Gozho et al., 2005; Khafipour et al., 2009a) . In our experiment no treatment or treatment by day effect was observed on Hp concentration. Others reported no increase in Hp concentration during successful alfalfa pellet induce SARA (Khafipour et al., 2009a) . Moreover, they observed a linear decrease in Hp concentration every consecutive week when higher proportion of pelleted alfalfa were included, although fr ee ruminal LPS was increased. O n the other hand, when SARA was gra in induced, the same researchers found a significant increase in Hp and free LPS concentration in rumen and i n peripheral blood (Khafipour et al., 2009b) . Another group reported no difference between treatments on Hp concentration when SARA was induced , although free ruminal LPS counts were increased with increased proportion of barley grain (Emmanuel et al., 2008) . In the same trial, other inflammatory markers, different than Hp were increased during the induction period. A similar scenario was observed by Gozho et al., (2007) where free ruminal LPS and SAA were increased but Hp concentration was not different between groups although the concentration observed was higher than t he one from healthy cows reported by others (Skinner et al., 1991) . To clarify their findings, Gozho et al., (2007) also reported that, since both had ruminal pH < 5.6 for more than 3 hours, both groups were sufferin g SARA. Gozho et al. (2005), in a grain induce SARA experiment using steers observed an increased in Hp concentration 3 days after induction initiation. Free ruminal LPS and SAA were also increased. They stated that concentrations of Hp in response to SARA are lower than the one find in response to bacterial or virus infections (Gozho et al., 2005) . Similar findings were reported by Gozho et al. (2006) although Hp concentration was increased 2 to 3 fold.

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90 Comparing the o bserved Hp concentration curve with that of previous publications (Huzzey et al., 2009) , it could be inferred that the observed group of sampled cows in our experiment, mimic the curve of what could be defined as heal thy cow s . It was also proposed that a mild increase in Hp concentration is normal i n first week postpartum animals (Humblet et al., 2006; Drillich et al., 2007; Trevisi et al., 2012) . Huzzey et al. (2009b) compared Hp concentration of cows suffering severe or mild metritis with healthy cows and reported that the healthy cows had low concentration at calving, peaking by 3 DIM and then start ed diminishing until normal levels were reached on days 12 to15. In cows severely affected with metritis, Hp concentrations peak ed at 6 DIM. In our analysis, cows diagnosed with metritis, mastitis, digestive issues or ketosis, were removed from the analysis to be able to detect any other ongoin g inflammatory process. The values that we reported are similar to values of healthy cows presented by Huzzey et al. (2009b), but we only collect blood samples on days 1, 6 and 14, thus sampling time was a probable reason why we observed a delayed peak. An other difference with Huzzey et al . (2009b) is the actual values, ours being 0.38 ± 0.09, 0.75 ± 0.09 and 0.42 ± 0.09 mg/ mL for 1, 6 and 14 DIM, respectively, which are between the reported values of their healthy and mild metritis groups. This may be expl ain ed by the fact that the spectrophotometer used during our assay was not calibrated to perform clinical diagnostics analysis. The purpose of our a nalysis was to compare groups. It was reported that virtual absence of Hp in serum is characteristic of heal thy animals (Eckersall and Bell, 2010) . Iqbal et al. (2014) orally exposed pre partum dairy cows to LPS an d report no difference in APP after parturition, but they observed increased serum IgG, IgM and IgA in the c ontrol group suggesting that a systemic primary humoral response was mounted due to later exposure to free ruminal LPS.

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91 As we observed, others also reported that healthy primiparous cows had higher Hp concentration than healthy multiparous cows (Humblet et al., 2006) . They suggest that cows could undergoes a more severe parturition process in the first compared to Others reported no influence of parity on Hp concentration (Drillich et al., 2007) . Ceruloplasmin (Cp) concentration provides an objective measure of cattle health status and thus it can be used as a marker of animal health and welfare (Hussein and Staufen biel, 2012) . In cattle, Cp has been used to assess inflammatory responses in different scenarios such as mastitis (Tabrizi et al., 2008) , weaning and stress situations (Arthington et al., 2005; Arthington et al., 2008; Carroll et al., 2009) , and vaccination responses (Arthington et al., 2013) . Cp has been shown to be an important player on iron metabolism and act s by reducing the available iron for bacteria to survive during infections since Fe is a limiting nu trient for bacterial growth. It i s known that Cp facilitates the oxidation of iron molecules in order to be bound to transferrin (Carroll et al., 2009; Eid et al., 2014) . In our experiment, we were not able detect differences between treatments. The three groups behaved equal and we observe a rapid increase in Cp concentration within the first week after parturition that was maintained during the second week of the treatment period. Cp concentration has been observed throughout the lactation of high yielding healthy dairy cows and a higher concentration was observed early after parturition (Hussein and Staufenbiel, 2012) . Similar values to ours (295 ± 6.5 mg/l) were reported by the s e researchers (356 ± 80.4 mg/ l) i n this first week after parturition. They suggested that spec ial attention s hould be taken when interpreting Cp concentration after parturition because it could be difficult to distinguish between physiologic acute response of parturition and pathological processes. Others reported similar curves and related the slope of the decre ase with the heath condition of the cows, with cows

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92 suffering more clinical diseases having higher risk for persistent ly increased Cp levels (Bertoni et al., 2008) . Since no publications were found analyzing Cp status as marker of gastrointestinal inflammation in cattle, Cp might have not been the ideal APP to be used in this trial in order to assess SARA during early postp artum. A significant correlation between Hp and Cp was observed on day 14 (r = 0.405, p = 0.000 2) but no correlation with production indexes was found ( p > 0.05). Others reported a significant correlation (r = 0.23, p = 0.001) , although weak, between Hp and Cp (Bertoni et al., 2008) . They also report an associat ion between level of inflammation and milk production, observing that the group of cows more severely diseased and with higher inflammatory markers levels, produced 3 to 4 kg of milk at peak less than the other groups. They also suggest a lower DMI due to increased cytokine release during inflammatory processes (Finck et al., 1998) although SARA is not mentioned. Vaccination with gram ne gative bacterins of toxoids should be considered when designing a trial involving LPS since the target animals will have certain immunity. Also diseases in early lactation such as metritis and mastitis, might be a confoundin g issue leading to misunderstanding of the process. Probably more specific APP as SAA or LBP might have help to tr uly identify other inflammatory ongoing processes in this period. Health issues i n the first 98 days on lactation were analyzed and no significant difference s between treatments were found. Overall incidence of ketosis was 13.5%. Duffield et al. (1998) rep orted incidences as low as 12.1 % and up to 29 % on commercial dairy farms (Duffield et al., 1998) . Others reported incidence i n dairy herds ranging from 26 % to 56 % (McArt et al., 2011) . Oetzel (2007) based on his own clinical data, proposed an alarm level of 10% on herd base ketosis testing (Oetzel, 2007) . This suggests that the incidence found in our

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93 trial was that of a well mana ged herd. Overall, 12% of the enrolled cows were diagnosed with metritis and no difference was seen between treatments. Incidence of metritis range widely and several factors affect this result. In a publication an incidence of 21.1 % was reported with ran g e from 11 to 39.4 % depending on season and parity (Benzaquen et al., 2007) . In another experiment, 25 % overall incidence was reported, ranging from 23 in multiparous to 28 % i n primiparous cows (Galvao et al., 2010) . Within the digestive d is orders not only nutritional , but also infectious , diseases were included. Seven percent was the overall incidence and no differences between treatments were found. It is not easy to com pare incidence of mastitis because differe nt research groups measured it i n differen t ways . Barkema et al. (1999) reported an average incidence of 0.263 case per 365 cow /days in a report involving 274 dairy herds. We observed our cows for the first 98 days of lactation so if we would transfor m his index they would have had an incidence of 0.072 cases per 100 cow/days and our herd incidence rate would be 0.0012 cases per 100 cow/day . Only 6.5% of the enrolled cows were sold or died within the observational p eriod and this was not different between groups. Conclusion In our trial we were not able to find significant differences in the APP (Hb or Cp) between treatments. The slope or the curves for Hp and Cp were similar to those of healthy cows reported by othe rs. It has also been suggested that constantly low exposure to LPS levels in peripheral blood, as the ones proposed on SARA may not induce an acute inflammatory response, but a metabolic endotoxemia triggering a low grade inflammation. The APP evaluated in this experiment may have not been the best inflammatory markers to answer the formulated question and LBP and SAA should be considered for future research. The postpartum per iod is often compromised by various health disorders and although there was no di fferences between

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94 groups in incidence of diseases, higher basal concentration in haptoglobin were found letting a door open for further research to clarify the effect of PAP LPS as preventat ive of SARA consequences.

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95 Figur e 3 4 . Last squares means of plasma haptoglobin (Hp) concentration curves from 1 14 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS (PLC) and those not treated with dry egg yolk (CTL). No difference between PAP LPS, PLC or CTL was observed.

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96 Figure 3 5 . Last squares means of plasma ceruloplasmin (Cp) concentration curves from 1 14 days in lactation in cows treated with avian anti LPS antibodies (PAP LPS), inactivated PAP LPS ( PLC) and those not treated with dry egg yolk (CTL). No difference between PAP LPS, PLC or CTL was observed.

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97 Table 3 2. Pearson Correlation Coefficient for APP and milk production. Numbers found in table P value and number of observat ions. Haptoglobin Cer u loplasmin Total Milk Prod 98DIM Average Milk Kg 98d Haptoglobin 1.000 79 Cer u loplasmin 0.405 1.000 0.000 79 79 Total Milk Prod 98DIM 0.070 0.109 1.000 0.539 0.338 79 79 80 Average Milk K g 98d 0.075 0.126 0.982 1.000 0.507 0.268 <0.0001 79 79 80 80

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98 Table 3 3 . Incidence of health disorders and cull cow in the first 98 days of lactation Treatment Groups Variable level All Cows PAP LPS 1 PLC 2 CTL 3 n ( % ) n ( % ) n ( % ) P value Ketosis Yes 54 ( 1 3. 5) 21 ( 17 ) 15 ( 10 ) 18 ( 14 ) 0.58 No 346 104 ( 83 ) 128 ( 90 ) 114 ( 86 ) Metritis Yes 49 (12 ) 11 ( 9 ) 20 ( 14 ) 18 ( 14 ) 0.48 No 351 114 ( 91 ) 123 ( 86 ) 114 ( 86 ) Digestive Yes 27 (7 ) 10 ( 8 ) 10 ( 7 ) 7 ( 5 ) 0.57 No 373 115 ( 92 ) 133 ( 93 ) 125 ( 95 ) Mastitis Yes 48 (12 ) 14 ( 11 ) 18 ( 13 ) 16 ( 12 ) 0.95 No 352 111 ( 89 ) 125 ( 87 ) 116 ( 88 ) Cull Cows Yes 26 (6.5 ) 8 ( 6 ) 9 ( 6 ) 9 ( 7 ) 0.95 No 374 117 ( 94 ) 134 ( 94 ) 123 ( 93 ) 1 PAP LPS received activated polyclonal antibodies preparation anti LPS from day 1 14 of lactation. 2 PLC received inactivated PAP LPS from day 1 to 14 of lactation. 3 CTL did not receive any treatment.

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99 CHAPTER 4 GENERAL DISCUSION AND CONCLUSION The transition period in general, and initiation of lactation in particular, are delicate moments in the productive life of a dairy cow during which a nimals are metabolically a nd im munologically challenged. It has been proposed that production drives dry matter consumption (Ingvartsen and Andersen, 2000) and also that milk production may be improved by increasing yield at peak producti on (Hutjens, 2008) . Subacute ruminal acidosis is one of the production diseases challenging the cow during the transition period (Donovan et al., 2004) . Although the etio pathology seems to be well identified, increases in APP have been observed in several studies (Gozho et al., 2006; Gozho et al., 2007; Emmanuel et al., 20 08; Khafipour et al., 2009b) which consequently suggest the inclusion of such inflammatory process in the pathophysiology of the disease. To our knowledge, this is the first time that PAP LPS has been used experimentally in a commercial herd for preventi ng the consequences of SARA in early lactation dairy cows. The randomized clinical trial herein reported was designed to evaluate the effect of oral daily administration on early lactation of anti LPS polyclonal antibodies on health, milk production and mi lk components. In several s tudies acute phase proteins had been used to assess inflammatory processes in animals (Gozho et al., 2005; G ozho et al., 2006; Gozho et al., 2007; Emmanuel et al., 2008; Khafipour et al., 2009a) . We observed haptoglobin (Hp) and ceru loplasmin (Cp) concentrations at 3 different times as markers of inflammation to help explain possible differences in health and p roduction. Daily milk production was weekly averaged and no differences were detected between treatments although treatment by week interaction was found favoring PAP LPS on weeks 3, 4 and 10 compared to control cows and, in week 6 compared with both, con trol and placebo. Milk

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100 production was also analyzed as total yield over the first 98 days of lactation and PAP LP S was higher than both other groups. Although only a trend was found between placebo and PAP LPS (96 kg), a significant difference of 112 kg wa s seen when PAP LPS and control were compared. When milk was corrected by MF % and MP % (ECM), a treatment effect showed a difference in milk yield of 1.5 and 0.85 kg in favor of PAP LPS when compared with CTL or PLC, respectively. This finding supports ou r previous observation in total milk production although it ECM when SARA was induced although their control group was also undergoing SARA. It is doubtful to state that PAP LPS effectively prevent ed consequences of SARA since the disease was not diagnosed , although i ncrease d free rumen LPS concentrations were also found in non SARA situations leading to suggest that the same mechanism of action might apply . Decreas ed milk production and DMI was reported in several studies when SARA was induced, although an acute phase response was not always detected. Khafipour et al. (2009a) gradually increased the proportion of alfalfa pellets to induce SARA and reported a linear increase in DMI but a linear decrease in milk yield. No translocation of LPS from the rumen or increase in inflammatory markers was reported. When grain induced SARA was studied by the same group (Khafipour et al., 20 09b) a decrease in DMI and consequently a trend for decreased milk yield were observed proposing endotexemia and increased rumen accumulation of propionic acid as possible hypophagic causes. Krause and Oetzel (2005) reported decreased milk yield and DMI b ut they were not able to explain the drop in milk production only by the decreased DMI. No difference in DMI or milk yield was detected by Gozho et al. (2007) but they reported SARA in both groups although the induced one shown numerical decreased on both parameters and also had increased serum amyloid A as an indicator of inflammation. Another

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101 experiment induced SARA by replacing barley grain with barley silage and reported increased DMI and milk production but also increased LPS in blood (Emmanuel et al., 2008) . A hypothesis for the increase d milk yield observed is that PAP LPS had a protective effect on rumen mucosa. The suggested mechanism of action of PAP LPS is through neutralization of free LPS, which are incre ased during SARA (Gozho et al., 2005; Emmanuel et al., 2007; Gozho et al., 2007; Khafipour et al., 2009a) but also when bacterial population is increased due to consumption of high amount of organic matter . Free ruminal LPS may be acting locally, at the rumen epithelium, or may be absorbed inducing the release of cytokines which decrease DMI. We believe that PAP LPS may be blocking this pathway, and consequ ently maximizing milk production. Research in ruminants is lacking ; in mice challenged with LPS an s to decreased DMI either through increase of leptin concentration or by direct effect of the cytokine on the central nervous system (Finck et al., 1998; Ingvartsen and Andersen, 2000) . It is also known that increase free LPS concentration in blood could lead to decrease rumen motility (Jacobsen et al., 2005) consequently reducing the abso rption of volatile fatty acids, and also decreasing passage rate producing increased retention time and reduction of DMI. A previous experiment using PAP LPS on steers showed increased DMI and daily body weight gain (data not published, DiLorenzo & Galyean ).Unfortunately, DMI was not measured in our experiment because it was performed in a commercial dairy herd. No enhancement of health status or difference on milk yield was observed on the first 2 weeks. This finding motivates us to suggest a second hypot hesis: PAP LPS may work by accumulation reason why no differences were found in the first two weeks but in the 3 rd , 4 th , and 6 th , being neutralization of LPS the mechanism of action. This hypothesis may be explore d by feeding the antibodies prior to partur ition.

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102 A third alternative stimulated by the lack of difference in the first two weeks (this is very not limiting but after two weeks, DMI start being l imiting factor and milk production is depressed (Allen et al., 2009) . This is seen in control cows but not in the treatment group because these cows has the cytokine pathway blocked, or because PAP LPS diminish local inflammation allowing increased absorption of VFA. Allen et al . (2009) states that between days 10 to 21 after parturition is when regulation of voluntary DMI switch from a metabolic control (hepatic oxidation control) to a mechanic control (gut fill). Th e difference in milk yield between PAP LPS and placebo was only seen in week 6. The lack of difference on weeks 3 and 4, compared to the control group may be explained by the production process of the placebo. Placebo anti LPS PAP were produced by inactiva ting dried egg from immunized hens by acid shock. Unfortunately we had no access to quality control test to assess 100% success of this process. There is evidence that inactivation of purified IgY, the main antibody in the egg, is time and pH dependent (Shimizu et al., 1992) . Antibody activity was decreased when it was incubated at pH 3.5 or lower for 7 h. Our findings suggest that placebo way have still had some protective action. As commented previously, the experime nts that induce SARA by restriction protocols are difficult to compare with ours, however the aim of those experiments was to evaluate acute inflammatory responses during SARA (Gozho et al., 2007; Khafipour et al., 2009a) . We also measured APP although no differences between treatments were found in haptoglobin (Hp) or ceruloplasmine (Cp). Discrepancies are found between different publications on Hp concentration when SARA was induced. Although eve rybody reported increased free ruminal LPS concentration, only one reported increased Hp and blood LPS concentrations (Khafipour et

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103 al., 2009b) . Others observed an increased (Gozho et al., 2005; Gozho et al., 2006) or no difference (Gozho et al., 2007; Emmanuel et al., 2008) in Hp concentration while only one reported no difference but linear decrease in Hp (Khafipour et al., 2009a) . No reports evaluating Cp concentrations during SARA were found. The curves observed for both Hp and Cp concentrations were similar to the ones reported for healthy dairy cows by other s. A mild increase in Hp concentration is normal during the first week postpartum (Humblet et al., 2006; Drillich et al., 2007; Galvao et al., 2010; Trevi si et al., 2012) . Our reported values are similar to those of healthy cows presented by Huzzey et al. (2009b) although one difference is the actual values, laying ours (0.38 ± 0.09, 0.75 ± 0.09 and 0.42 ± 0.09 mg/ mL for 1, 6 and 14 DIM) between the report ed values of their healthy and mild metritis groups. In our analysis, cows diagnosed with metritis, mastitis, digestive issues or ketosis were not considered for statistical analysis in order to detect any other ongoing inflammatory process. The difference s in values could be explained by the fact that the spectrophotometer used to analyze the samples was not validated by an external one for diagnostic analysis. Gozho et al. (2005) stated that concentrations of Hp in response to SARA are lower than those fo und in response to bacterial or viral infections, although if this were the case all the cows would be affected from day 1, which is not likely to happen judging by the numbers of sick cows diagnosed. As we observed, others reported that healthy primiparou s cows had higher Hp concentration than healthy multiparous cows (Humblet et al., 2006) suggesting that primiparous cows could suffer more ill defined inflammatory processes after parturition than multiparous cows. The Cp curve observed here mimics the one seen by Hussein and Staufenbiel (2012) who analyzed Cp concentration throughout the lactation of high yielding healthy dairy cows. A higher

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104 concentration was observed early after parturition. These researchers meas ured Cp to be 356 ± 80.4 mg/l in this first week we observed a peak of 295 ± 6.5 mg/l in the first 2 weeks postpartum. Since no publications were found analyzing Cp status as a marker of gastrointestinal inflammation in cattle, Cp might have not been the i nflammatory marker to use in this trial for assessing inflammation produce by LPS during early postpartum. Gozho et al. (2007) was able to observe inflammatory differences on SARA affected cows by measuring serum amyloid A as an indicator of inflammation. LBP has also been shown to be specific as marker of LPS presence (Emmanuel et al., 2008) . Milk fat percentage (MF %) was not affected by treatment. In the other hand milk protein percentage (MP %) on PAP LPS group w as significantly lower than the other two groups although differences between values were so small that biological value of this could be meaningless (2.74 ± 0.01% on PAP LPS, 2.77 ± 0.01 % on ithers, difference: 0.03 %). In a grain induced study of SARA, Khafipour et al. (2009b) observed a trend difference in MF % values of 2.93 % and 3.30% for SARA and control groups, respectively, and a significant difference in MP % values of 3.42 and 3.29 for the same groups, respectively. Some points have to be consid ered: the differences experienced by the experimental groups in other trials are biologically important (0.13% for MP %), but also when one of those is affected the other one (MF %) is also skewed (Stone, 1999; Khafipour et al., 2009b) , which is not the case in our trial. Another consideration is the percentage of animals potentially suffering SARA. Since we did not assess SARA incidence, the presence of the disease is another doubtful point that c ould be affecting this outcome. A significant difference in fat yield was observed between PAP LPS and CTL although it was of minimal amount (60 g/d, 4 %). This difference could be explained mainly by the

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105 numerical difference in milk yield between both groups (milk yield was not included in the model). Krause and Oetzel, (2005) observed decreased fat yield after SARA was induced and Khafipour et al. (2009a) reported a linear decrease in fat yield (11 %) as a consequence of a reduction in milk yield. This finding supports our theory that PAP LPS act locally inducing an increase or preventing a decrease, in DMI when SARA is present and thus enhancing milk production. Health disorders in the first 98 days in lactation were analyzed and no significant differe nces between treatments were found. Overall incidence of diseases are in the lower side of the ranges presented in the literature (Duffield et al., 1998; Oetzel, 2007; Benzaquen et al., 2007; Galvao et al., 2010; McArt et al., 2011) suggesting that managerial practices in the herd were finely regulated and proposing this, that SARA incidence may had also run the same luck since we did not diagnose or monitored the disease during the experimental period. This might have also decreased our chances of detecting statistical differences. Although we observed a difference in ECM, several considerations were discussed in order to explain our findings. SARA was not measured , but bearing in mind the health performance observed , it is unlikely that the disease was present in a high incidence rate . Nonetheless, the main effect of the product might have been through neutralization of f ree rumen LPS and blockage of the cytokine pathway. This could not be support ed due to lack of differences of APP concentration , but for further research we would recommend measuring SAA, LBP or TN . Another analysis that could be done in order to asses d ifferences in DMI is blood glucose concentration. To clarify the doubt about the activity of the placebo treatment , dried egg from non immunized hens might be used. As a final comment, the challenge on a

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106 different stage of lactation would be interesting to remove the ongoing effect of other diseases that also present bacterial LPS in their physiopathology.

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119 BIOGRAPHICAL SKETCH Lucas Ibarbia was born in Buenos Aires. He spent his childhoo shadowing his father, who was also involved in the cattle industry. Growing up on a farm created a strong foundation of knowledge, which would later inspire him to pursue a career in food animal systems. Lucas attended Universidad Nacional del Centro de la Provincia de Buenos Aires (UNICEN) in Tandil, Argentina, where he received a DVM degree in 2008. In addition to his student obligations, he took on side jobs at local farms, which would serve to pique his interest in dairy farmin g. He began practicing and providing consultation on herd health to commercial dairy farms as an associate at West Vets, a private practice in Buenos Aires province. After graduating in 2008, Lucas moved to Boulder, Colorado in order to pursue an internsh ip with Aurora Organic Dairy Company. His objectives were to learn about dairy production medicine in confinement dairy systems, which he accomplished by working year p eriod with Aurora, he performed specific duties concerning animal health in organic systems such as maternity, calf raising, and milking procedures and reproduction. With the conclusion of the Aurora internship, Lucas returned to Argentina and joined Dair y Health Systems to continue working on dairy production medicine as a private practitioner. For the next two years he was able to implement new concepts learned from his ansition cow health and surgery, milk quality assurance, reproduction, and foot health. During his time with DHS, Lucas recognized the importance of management and employee training related to the success of dairy farming. Due to this realization, Lucas d ecided to continue his education in order to improve his knowledge and successfully contribute to the dairy industry.

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120 In 2011, Lucas returned to the United States and joined the Food Animal Reproduction and Medicine Service at the College of Veterinary Me dicine at the University of Florida. His role as a resident provided him extensive training on herd health and management, reproductive management, mastitis control, neonatal health and management, foot health and nutrition management. He was also able to increase his knowledge of design, implementation, and evaluation of health monitoring protocols. An additional important component of his residency included the training of farm employees in both Spanish and English on nutrition management, transition cow disorders, milk quality, calf raising, lameness and animal welfare. During the last year of his residency, he also accomplished a M aster of S cience in Veterinary Medicine at the same University, focusing his research on nutrition of dairy cows. Lucas sees his future closely associated with the dairy industry, especially in nutrition and management, working with the people that make this intense job their every day life.