DISSERTATION. Paige Nicole Gott, M.S. Graduate Program in Comparative and Veterinary Medicine. The Ohio State University. Dissertation Committee:

Transcription

1 Impact of milk cessation method on intramammary infections at calving and milk yield and quality in the subsequent lactation DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Paige Nicole Gott, M.S. Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2015 Dissertation Committee: Gustavo M. Schuenemann, Advisor Päivi J. Rajala-Schultz, Co-Advisor Joseph S. Hogan Kathryn L. Proudfoot

2 Copyright by Paige Nicole Gott 2015

3 Abstract A large field study investigating the impact of milk cessation method (abrupt or gradual) on udder health and productivity in the subsequent lactation was conducted in nine Ohio dairy herds of varying size and management styles between May 2012 and October Gradual cessation of milking was implemented on farms as once daily milking for the final week of lactation while abrupt cessation cows kept their farm s normal milking schedule until dry-off. All cows that were due to be dried off within a week were assigned to the same group to facilitate management; group assignment alternated weekly between gradual and abrupt cessation treatments. The overall goal of the study was to assess the impact of different dry-off methods on udder health as well as milk yield and quality in today s high producing dairy cows. The central hypothesis was that once daily milking during the final week of lactation would improve udder health and improve milk production and quality in the subsequent lactation. The results from the study are presented as three subsets of data and analyses. For the first set of analyses, the objective was to evaluate the impact of milk cessation method (abrupt and gradual) and daily milk yield prior to dry-off on intramammary infections (IMI) at calving. Data from 1086 quarters of 285 cows from five Ohio dairy herds with milk meters in their milking parlor were analyzed. Aseptic ii

4 quarter foremilk samples were collected at the time of enrollment, the final milking before dry-off (DRY), and within 7 d of calving. Cows in the gradual cessation group were observed for milk leakage during the period of once daily milking. In the only herd that did not use internal teat sealants after dry off, milk leakage was recorded for both abrupt and gradual treatment groups. Gradual cessation decreased milk production by 33.4% during the final week of lactation, causing milk yield at DRY to be lower for these cows compared to abrupt cessation cows (13.2 kg/d vs kg/d; P < ). No differences (P > 0.05) were observed in proportions of quarters with new, cured, or persistent IMI between milk cessation groups from DRY to calving. Logistic regression models were used to model the probability of a quarter being infected at calving with any pathogen, accounting for clustering of quarters within cows and cows within herds. The model investigating the probability of IMI at calving was stratified by parity (primiparous and multiparous). The following factors were associated with a higher risk of IMI at calving among quarters of primiparous cows: abrupt cessation of milking and milk leakage after dry-off. For quarters of multiparous cows, on the other hand, gradual cessation of milking, presence of IMI at DRY, and thrice daily milking during lactation increased the odds of IMI at calving. Gradual cessation of milking successfully decreased milk production prior to dry-off, however, the impact of milk cessation method on the odds of IMI at calving differed between primiparous and multiparous cows. These results indicate that implementation of differing management practices near dry-off for different parity groups may improve mammary health within a herd. In addition, a simultaneous change to a reduced energy iii

5 diet and gradual cessation of milking may further reduce milk production and help reduce the risk of milk leakage for high producing cows. The objective of second set of analyses was to assess the impact of milk cessation method (abrupt and gradual) and milk yield near dry-off on major and minor intramammary infections (IMI) at calving, using Dairy Herd Improvement Association (DHIA) test day data from before dry-off. Data from 1573 quarters of 410 cows from eight Ohio dairy herds were analyzed. Aseptic quarter foremilk samples were collected at the time of enrollment, the final milking before dry-off (DRY), and within 7d of calving. Logistic regression models were used to model the probability of a quarter being infected at calving with minor pathogens or major pathogens, accounting for clustering of quarters within cows and cows within herds. Milk cessation method was not associated with the odds of either major or minor IMI at calving. However, major IMI at DRY and increasing milk yield on the DHIA final test day were associated with a higher risk of major IMI at calving. Minor IMI at DRY increased the odds of minor IMI at calving. Milk cessation method was not shown to impact IMI status at calving in the current study, but quarterlevel infection status at dry-off was significantly associated with both major and minor IMI at calving. Parity at dry-off was not significantly associated with major or minor IMI at calving, but confounded the association of milk cessation method with major IMI at calving. Managers of herds with high-producing cows approaching dry-off should consider management practices which decrease milk yield prior to dry-off, such as gradual cessation of milking, as a potential means to improve udder health in the subsequent lactation. iv

6 The objective of the final set of analyses was to assess the impact of milk cessation method (abrupt or gradual) at dry-off on milk yield and somatic cell score (SCS) up to 120 days in milk during the subsequent lactation using Dairy Herd Improvement Association test day records. Data from 428 cows from eight Ohio dairy herds were analyzed. Multiple factors influenced DHIA test day milk production and SCS in the current study. However, milk cessation method was not associated with either outcome of interest. When test day milk yield was modelled, increasing test day SCS by 1 unit decreased test day milk yield by 0.8 kg. In contrast, when test day SCS was the outcome, every 1 kg increase in test day milk yield was associated with 0.05 unit decrease in test day SCS, which may indicate the presence of a dilution effect in healthy udders. The current results suggest that IMI status of the cow may be crucial to understanding the relationship between these two factors. In addition, cows producing greater amounts of milk around dry-off were more likely to have increased milk yield and higher SCS in the following lactation. Cows in their fourth or greater lactation had higher test day milk yields and higher SCS than cows in their second or third lactation. Both SCS prior to dry-off and the presence of an IMI around calving increased test day SCS during the first 120 DIM. Additionally, both an extended previous lactation and increasing dry period length were associated with increased test day milk yield in the subsequent lactation. Finally, Jersey cows were shown to produce lower volumes of milk than Holstein cows across test days. The udder health and productivity of dairy cows are influenced by many factors, but milk cessation method did not appear to impact either of these traits when test day milk yield and SCS were investigated up to 120 DIM in the v

7 following lactation. Gradual cessation of milking can be considered as a viable dry-off method without concerns of negatively impacting milk quality and production in the next lactation when compared with abrupt cessation of milking. In conclusion, gradual cessation of milking, as demonstrated using once daily milking for the final week of lactation, is a viable method to decrease milk production prior to dry-off. Gradual cessation of milking may have different impacts on udder health at calving for different parity groups, but further research is needed to confirm this association. Other than the finding that cows in their second or greater lactation which underwent gradual cessation of milking had an increased risk of IMI at calving, gradual cessation was not shown to affect the udder health, milk quality, or milk yield of cows differently than abrupt cessation. The similarities to abrupt cessation with the added benefit of decreasing milk yield prior to dry-off suggests that gradual cessation of milking is a practical and effective method to dry-off cows which should be considered by herd managers. Further research investigating the impact of milk cessation method on cow behavior and comfort is warranted to identify the best practices which will assure good animal welfare. vi

8 Acknowledgments I would like to thank my academic advisors and committee members for their guidance and shared knowledge, my family and friends for their love and support, everyone who volunteered to help collect samples during the study, and all of the producers and farm personnel which participated in the study for allowing me to disrupt their normal routines in the pursuit of knowledge. This project was supported by Agriculture and Food Research Initiative Competitive Grant no from the USDA National Institute of Food and Agriculture. vii

9 Vita August 4, Born - Ashland, Ohio June Mapleton Local Schools, Ashland, Ohio June B.S. Animal Sciences, The Ohio State University December M.S. Animal Sciences, The Ohio State University 2012 to present...graduate Research Associate, Department of Veterinary Preventive Medicine, The Ohio State University Publications Gott, P.N., J.S. Hogan, and W.P. Weiss Effects of various starch feeding regimens on responses of dairy cows to intramammary lipopolysaccharide infusion. J. Dairy Sci. 98: Eastridge, M.L., A.H. Lefeld, A.M. Eilenfeld, P.N. Gott, W.S. Bowen, and J.L. Firkins Corn grain and liquid feed as non-fiber carbohydrate sources in diets for lactating dairy cows. J. Dairy Sci. 94: Fields of Study Major Field: Comparative and Veterinary Medicine viii

10 Table of Contents Abstract... ii Acknowledgments... vii Vita... viii Publications... viii Fields of Study... viii Table of Contents... ix List of Tables... xiii List of Figures... xv CHAPTER 1: Review of Literature... 1 Definition of Mastitis... 1 Diagnosis of Intramammary Infections... 4 Importance of the Dry Period... 8 Dry-Off Methods Introduction to Current Study REFERENCES CHAPTER 2: Intramammary infections and milk leakage following gradual or abrupt cessation of milking ix

11 ABSTRACT INTRODUCTION MATERIALS AND METHODS Study Population Sample Collection and Milk Microbiology Milk Yield Milk Leakage Statistical Analysis RESULTS Descriptive Statistics Milk Yield and Intramammary infections Models for Intramammary Infections at Calving Descriptive Statistics Milk Leakage Models for Milk Leakage DISCUSSION CONCLUSIONS REFERENCES CHAPTER 3: Impact of gradual or abrupt cessation of milking on major and minor intramammary infections at calving ABSTRACT x

12 INTRODUCTION MATERIALS AND METHODS Study Population Sample Collection and Milk Microbiology DHIA Test Day Milk Yields and SCC Statistical Analysis RESULTS Study Population Final Test Day Data Intramammary Infections DISCUSSION CONCLUSIONS REFERENCES CHAPTER 4: Effect of gradual or abrupt cessation of milking at dry-off on milk yield and somatic cell score in the subsequent lactation ABSTRACT INTRODUCTION MATERIALS AND METHODS Study Population xi

13 DHIA Test Day Milk Yields and SCC Sample Collection and Milk Microbiology Statistical Analysis RESULTS Study Population Intramammary Infections Test Day Milk Yield in the Subsequent Lactation Test Day SCS in the Subsequent Lactation DISCUSSION CONCLUSIONS REFERENCES CHAPTER 5: General Discussion, Future Directions, and Overall Conclusions Future Directions Overall Conclusions REFERENCES BIBLIOGRAPHY xii

14 List of Tables Table 2.1. Descriptive statistics for herds enrolled in a study investigating the impact of milk cessation method at the end of lactation on IMI status at calving...53 Table 2.2. Descriptive statistics for cows dried off via gradual cessation and abrupt cessation of milking at the end of lactation...54 Table 2.3. Microbiological culture results from aseptic quarter foremilk samples 7-14 d prior to dry-off (PRE), at the final milking (DRY), and within 7 d of calving (CALV), presented by milk cessation group...55 Table 2.4. Quarter-level intramammary infection (IMI) status at dry-off (DRY) and within 7 days of calving (CALV) and IMI status change from DRY to CALV by parity group (primiparous vs. multiparous; designated at dry-off) Table 2.5. Final logistic regression model for primiparous cows explaining the association of milk cessation method and other factors with the odds of quarter-level IMI at calving. Herd was included in the model as a random effect...57 Table 2.6. Final logistic regression model for multiparous cows explaining the association of milk cessation method and other factors with the odds of quarter-level IMI at calving. Herd was included in the model as a random effect...58 Table 3.1. Descriptive statistics for herds enrolled in a study investigating the impact of milk cessation method at the end of lactation on IMI status at calving...86 Table 3.2. Distribution and characteristics of cows in gradual and abrupt cessation of milking groups Table 3.3. Descriptive statistics of gradual cessation and abrupt cessation cows...88 Table 3.4. Microbiological culture results from aseptic quarter foremilk samples 7-14 d prior to dry-off (PRE), at the final milking (DRY), and within 7 d of calving (CALV), presented by milk cessation group...89 xiii

15 Table 3.5. Final logistic regression model explaining the association of milk cessation method and other factors with the odds of quarter-level major IMI at calving. Herd was included in the model as a random effect...90 Table 3.6. Final logistic regression model explaining the association of milk cessation method and other factors with the odds of quarter-level minor IMI at calving. Herd was included in the model as a random effect...91 Table 4.1. Distribution and characteristics of cows in gradual and abrupt cessation of milking groups Table 4.2. Descriptive statistics of gradual cessation and abrupt cessation cows Table 4.3. Final model estimating the effects of milk cessation method on test day milk yield (kg/d) in the subsequent lactation, up to 120 DIM. Herd was included as a random effect Table 4.4. Final model estimating the effects of milk cessation method on test day SCS in the subsequent lactation, up to 120 DIM. Actual SCC values were converted to SCS according to the formula by Shook (1993). Herd was included as a random effect xiv

16 List of Figures Figure 2.1. Daily milk yields for gradual and abrupt milk cessation groups, presented as mean ± standard error of the mean (SEM) at PRE (average milk yield of days 8 and 9 before dry-off) and at DRY (average milk yield of days 2 and 3 before dry-off)...52 Figure 4.1. Least squares means of test day milk yield (kg/d) by study group from the final model are presented for stage 1 through stage 9. Stages represent of 14-d periods based on cows DIM when the test day occurred, up to 120 DIM Figure 4.2. Least squares means of test day SCS by study group from the final model are presented for stage 1 through stage 9. Stages represent of 14-d periods based on cows DIM when the test day occurred, up to 120 DIM xv

17 CHAPTER 1: Review of Literature Definition of Mastitis Mastitis, which is defined as an inflammation of the mammary gland, can be caused by physical trauma or chemical irritation, but is most commonly caused by bacterial intramammary infection (IMI) (Watts, 1988; Bannerman, 2009). The terms mastitis and IMI are often used interchangeably, but the presence of an IMI is not required for mastitis to occur (Smith and Hogan, 2001). Bovine mastitis is a highly prevalent disease of dairy cows which leads to substantial economic losses and increased costs for dairy producers (DeGraves and Fetrow, 1993; Seegers et al., 2003; Halasa et al., 2007). Economic losses and expenses associated with mastitis accrue from decreased milk production, discarded milk due to clinical abnormalities or antimicrobial residues following treatment, drug costs, veterinary fees, diagnostic testing fees, premature culling or death of infected cows, and increased labor costs (DeGraves and Fetrow, 1993; Fetrow et al., 2000; Seegers et al., 2003; Halasa et al., 2007). The negative impact mastitis has on milk quality can also cause producers to lose premiums milk processors offer for low somatic cell count (SCC) milk (Smith and Hogan, 2001). Processors want to purchase high quality milk in order to increase the yields and shelf life of manufactured products as well as to ensure their consumers of the safety and wholesomeness of the products they market (Shook, 1993; Smith and Hogan, 2001). 1

18 Mastitis is a complex, multifactorial disease. The epidemiologic triangle is a model frequently used by scientists to describe the complex interplay between the host factors, environmental factors, and agent factors involved in the development of disease. When mastitis is the disease of interest, the host is the cow and the agent is the causative microorganism. Husbandry and management practices greatly influence these interactions and should be taken into account when attempting to prevent and control mastitis (Hogan, 2014). A wide spectrum of bacteria have been found in secretions from infected mammary glands, but a relatively small array of species are frequently isolated (Bannerman, 2009). Commonly isolated Gram-positive bacteria include Staphylococcus aureus, coagulase-negative staphylococci (CNS), Streptococcus dysgalactiae, and Streptococcus uberis. Important Gram-negative organisms include Escherichia coli, Klebsiella spp., Serratia marcescens, Pseudomonas aeruginosa, and Enterobacter spp. (Hogan and Smith, 2003; Bannerman, 2009). Bacteria from the Genera Escherichia, Klebsiella, and Enterobacter are often collectively referred to as coliforms due to the fact they are Gram-negative bacilli which ferment lactose and are natural inhabitants of the intestinal tracts of warm-blooded animals (Eberhart et al., 1979; Hogan and Smith, 2003; Willey et al., 2008). One way to classify cases of mastitis is based on the degree of inflammation present in the mammary gland (Harmon, 1994; Philpot and Nickerson, 2000). Clinical mastitis is when overt, visible changes and signs are present in milk and/or udder. Some cases of mastitis can results in systemic signs also at the cow-level. Clinical cases are 2

19 often rated as mild, moderate, or severe depending on the number and severity of signs present. Clinical changes of the milk include flakes or clots, watery or serum-like secretions, or the presence of blood (Harmon, 1994; Philpot and Nickerson, 2000; Blowey and Weaver, 2011). Changes in the mammary gland can also occur including swelling or pain, hardening of the quarter, or the presence of heat or a reddish discoloration (Schalm et al., 1971; Philpot and Nickerson, 2000; Blowey and Weaver, 2011). Systemic signs including fever, lethargy, and anorexia can occur during cases of severe clinical mastitis (Harmon, 1994; Philpot and Nickerson, 2000). In contrast, subclinical mastitis is when inflammation is present, but milk and the gland appear normal (Schalm et al., 1971; Harmon, 1994; Philpot and Nickerson, 2000). Although subclinical cases are more difficult to identify, they can be detected by testing the milk for the presence of inflammation. The California Mastitis Test (CMT) is a cow-side test used to detect inflammation by indirectly measuring the somatic cell count (SCC) in milk. True somatic cell counts are determined by laboratory techniques such as automatic electronic cell counters or by microscopic examination (Schalm et al., 1971; Philpot and Nickerson, 2000). Subclinical infections can also be identified via microbiological milk culture and the causative organisms of clinical mastitis are often identified using milk culture techniques as well (NMC, 2004). Milk from cows exhibiting clinical signs of mastitis is not saleable, so clinical cases are an immediate concern to producers, but subclinical mastitis is 15 to 40 times more prevalent than clinical mastitis (Philpot and Nickerson, 2000) and contributes to the vast economic losses related to decreased milk production and reduced milk quality (Philpot and Nickerson, 2000; Zhao and Lacasse, 3

20 2008). Both manifestations of mastitis negatively impact the productivity and success of dairy herds. Although many factors influence the economic losses and increased costs related to mastitis, decreased milk production is considered to have the greatest economic impact (DeGraves and Fetrow, 1993; Fetrow et al., 2000; Seegers et al., 2003). Cows which had a clinical case of mastitis did not reach their pre-mastitis milk yield again during the lactation (Rajala-Schultz et al., 1999), indicating that mastitis can have a long-lasting impact on milk production. Presence of a single quarter IMI for one lactation was reported to reduce a cow s milk production 10 to 12% (Elvinger and Natzke, 1992). Additionally, a one-point increase in somatic cell score (SCS), a frequently used transformation of SCC, is associated with a 400 lb decrease in milk yield per lactation at all levels of SCS (Shook, 2008). Mastitis is estimated to cost the U.S. dairy industry $2 billion annually with the average cost per cow estimated at $171 (Jones and Bailey, 2009). The cost of mastitis alone provides ample motivation for dairy producers to improve mammary health within their herds, but mastitis is also an animal welfare issue (Leslie and Petersson-Wolfe, 2012) and a food safety concern (Smith and Hogan, 2001). Diagnosis of Intramammary Infections The criteria used to diagnose IMI, especially when identifying cases of subclinical mastitis, are debated among mastitis experts (Dohoo et al., 2011a). Many experts rely on a combination of results from microbiological milk cultures, including the number of times a pathogen was isolated from a quarter, the number of colonies which grew from a 4

21 specific volume of milk, whether the organism was recovered in pure or mixed culture, and some indicator of inflammation, such as SCC, to diagnosis an IMI (Andersen et al., 2010; Dohoo et al., 2011a,b). A series of studies investigated the sensitivity and specificity of various sampling schemes and criteria used to diagnose IMI from microbiological cultures (Anderson et al., 2010; Dohoo et al., 2011a,b; Reyher and Dohoo, 2011). No universal criteria to diagnose IMI has been agreed upon, but Dohoo et al. (2011a) suggest that the criteria and definition used should depend on the objectives of those who will utilize the results and that the sensitivity (ability to detect an IMI) and specificity (ability to correctly classify non-infected quarters) of the procedures should be taken into consideration (Reyher and Dohoo, 2011). Microbiological culturing of milk samples is an expensive element in mammary health studies (Dohoo et al., 2011b) and this high cost may influence the sampling scheme and IMI definition criteria used. With the added interest in developing genome-based molecular techniques for mastitis detection (Zadoks et al., 2011; Taponen and Simojoki, 2014), further consideration will be made into how to diagnose IMI. Although the diagnosis of IMI is not straightforward, especially when interpreting microbiological milk culture results, the use of SCC as an indicator of inflammation and a tool to monitor the presence of IMI at the herd, cow, and quarter-level is commonly used (Harmon, 1994; Laevens et al., 1997; Smith and Hogan, 2001). Somatic means derived from the body, but somatic cells in milk are primarily white blood cells from the immune system (Hillerton, 1999; Philpot and Nickerson, 2000). The three types of white blood cells (also called leukocytes) commonly found in milk from cows include: 1) 5

22 macrophages, 2) lymphocytes, and 3) polymorphonuclear lymphocytes (also called neutrophils ) (Philpot and Nickerson, 2000; Smith and Hogan, 2000). Healthy mammary glands are primarily populated with macrophages, but an immune response including neutrophil recruitment is initiated during bacterial invasion and thus, neutrophils are the main type of white blood cell found during infection (Paape et al., 2000; Sordillo et al., 1997; Sordillo and Streicher, 2002). Although differential cell counts are sometimes used to identify the proportion of each cell type present, the overall SCC is sufficient to identify changes in the IMI status of quarters as the influx of neutrophils is primarily responsible for increased SCC. The presence of an IMI is not required for mastitis to exist, but most mastitis cases are associated with IMI and the major determinant of SCC in milk is the presence of an IMI (Bramley and Dodd, 1984; Smith and Hogan, 2001). Somatic cell counts are determined electronically and can be processed relatively cheaply (Smith and Hogan, 2001). Different SCC thresholds have been used to distinguish infected cows from non-infected cows since SCC were introduced in the late 1970s (Dohoo and Leslie, 1991). Truly healthy individual mammary glands which are free from IMI usually have SCC below 100,000 cells/ml (Smith and Hogan, 2001). Somatic cell counts from individual quarters (Hillerton, 1999; Smith and Hogan, 2001) and composite milk samples (Royster and Wagner, 2015) which have SCC 200,000 cells/ml are associated with the presence or recent clearance of an IMI. There is potential dilution of SCC when milk from an infected quarter is combined with milk from noninfected quarters and this can decrease the sensitivity of IMI diagnosis when composite 6

23 samples are used (Reyher and Dohoo, 2011). However, composite milk samples are frequently used to measure SCC, as in the Dairy Herd Improvement Association (DHIA) testing program. Knowing the type of sample the SCC represents is key to interpreting the results. Mastitis pathogens are categorized multiple ways. The first categorization is based on the reservoir where the pathogen is located that provides exposure to cows. Contagious pathogens are those which survive in infected mammary glands and are spread from cow to cow, often during milking while environmental pathogens are those present in the cow s environment which can gain access to the mammary gland between milkings (Harmon, 1994; Philpot and Nickerson, 2000). Additionally, more than 20 species of staphylococci normally referred to jointly as coagulase-negative staphylococci (CNS) are considered opportunistic pathogens because they are normal flora of healthy skin (Philpot and Nickerson, 2000; Smith and Hogan, 2001; Taponen and Simojoki, 2014). Some common contagious pathogens are Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma spp., and Corynebacterium bovis (Philpot and Nickerson, 2000; Smith and Hogan, 2001). Environmental organisms include the coliforms and environmental Streptococci (Philpot and Nickerson, 2000). Mastitis-causing organisms are also classified as major or minor pathogens based on their effect on milk production and composition, SCC, and clinical cases of mastitis, with major pathogens having greater impact on these factors (Griffin et al., 1977; Harmon, 1994; Reyher et al., 2012). Coagulase-negative staphylococci (CNS) and Corynebacterium species are considered minor pathogens (Griffin et al., 1977; Harmon 7

24 and Langlois, 1986; Taponen and Simojoki, 2014) while other mastitis-causing organisms such as Staphylococcus aureus, species of Streptococci, and coliforms are major pathogens (Griffin et al., 1977; Harmon, 1994). Importance of the Dry Period Dairy cows are managed such that they are simultaneously lactating and pregnant (Capuco and Akers, 1999). Producers stop milking cows prior to the next expected calving date to allow a period of non-lactation called the dry period which encourages turnover of mammary epithelial cells and helps promote optimal milk production during the subsequent lactation (Nickerson, 1989; Capuco and Akers, 1999). Research has been done investigating what dry period length is required to maximize milk production in the following lactation, with d dry periods commonly being recommended (Nickerson, 1989; Sorensen and Enevoldsen, 1991; Watters et al., 2008; Pezeshki et al., 2010). The dry period is commonly described as having three separate functional phases: active involution, steady-state involution, and lactogenesis-colostrogenesis (Smith and Todhunter, 1982; Nickerson, 1989; Smith and Hogan, 2000). Active involution occurs from the time normal milking ceases through 30 days dry (Smith and Hogan, 2000). The duration of steady-state involution depends on the length of the dry period and is considered the time when the udder is most resistant to new IMI (Smith and Hogan, 2000). The period of lactogenesis-colostrogenesis is regulated by hormonal changes and is believed to begin d prior to calving when secretory epithelial cells regenerate 8

25 and differentiate in preparation of a new lactation (Nickerson, 1989; Smith and Hogan, 2000). The importance of the dry period for mastitis prevention and control is well established (Neave et al., 1950; Oliver and Mitchell, 1983; Bradley and Green, 2004). Mammary glands are most susceptible to acquiring new IMI during the periods immediately following dry-off when milk synthesis continues for several days prior to stopping and again prior to calving when the gland transitions back to an active secretory state (Neave et al., 1950; Oliver and Mitchell, 1983; Smith et al., 1985; Bradley and Green, 2004). When normal milking is terminated, regular flushing of the teat canals which can help prevent bacterial colonization no longer occurs and the removal of debris and application of antimicrobial teat dips stops which may allow organic material to accumulate and the concentration of bacteria on the teat skin may increase (Nickerson, 1989). Many physiological changes occur in mammary glands during the dry period which contribute to both the increased susceptibility to IMI at the beginning and end of the dry period, but changes also occur which make the mammary gland a highly antimicrobial environment during steady-state involution (Hurley, 1989; Nickerson, 1989; Smith and Hogan, 2000). It has been reported that many IMI present following parturition and most clinical cases of mastitis that occur up to 120 days in milk (DIM) can be attributed to IMI acquired during the dry period (Smith et al., 1985; Eberhart, 1986; Bradley and Green, 2004; Smith and Hogan, 2000). Dry period IMI are also associated with high SCC during the first third of lactation (Smith and Hogan, 2000). Quarter, cow, and herd-level risk factors all contribute to the susceptibility to new IMI 9

26 during the dry period (Bradley and Green, 2004; Dingwell et al., 2004). Management of cows leading to dry-off, throughout the dry period, and around the time of calving is of the utmost importance, especially when mammary health is considered. Milk synthesis continues for several days after cessation of milking, with the maximum accumulation of fluids in the udder occurring 2 to 3 d after milking is stopped (Nickerson, 1989; Smith and Hogan, 2000). Mammary secretions accumulate in the udder in the absence of regular milking and can lead to increased intramammary pressure following dry-off and again prior to calving during colostrum synthesis (Cousins et al., 1980; Schukken et al., 1993; Tucker et al., 2009). The increased intramammary pressure is believed to induce the process of active involution (Nickerson, 1989; Smith and Hogan, 2000). However, increased intramammary pressure can cause milk to leak from the teat canal ( milk leakage ) which may facilitate bacterial entry into the mammary gland by keeping the teat canal open (Dingwell et al., 2004) and providing a connection between the external environment and internal mammary structures (Schukken et al., 1990). Milk leakage during various periods of the lactation cycle has been associated with an increased risk of clinical mastitis (Schukken et al., 1990; Schukken et al., 1993; Elbers et al., 1998; Waage et al., 2001). In addition to its relation to milk leakage, increasing intramammary pressure causes teat canals to shorten and dilate (Comalli et al., 1984; Nickerson, 1989) which may also ease bacterial entry during this time. One important anatomical change mammary glands undergo during the dry period which aids in resistance to new IMI is the formation of a keratin plug in each teat canal (Cousins et al., 1980; Comalli et al., 1984; Nickerson, 1989; Dingwell et al., 2004). The 10

27 keratin plug functions as a physical barrier which occludes the teat canal, but keratin has also been reported to have antimicrobial properties (Capuco et al., 1992) which may contribute to the importance of this anatomical feature. Delayed formation or a complete lack of development of a keratin plug contributes to an increased risk for IMI (Williamson et al., 1995; Dingwell et al., 2004). Of the clinical mastitis cases which occurred during the dry period, 97% were in open quarters and it has been reported that approximately 5% of quarters were open at 60 d into the dry period (Williamson et al., 1995). In another study, 23% of quarters had not formed a keratin plug after 6 weeks and milk yield prior to dry-off was negatively associated with keratin plug formation as quarters of cows producing more than 21 kg of milk on the day before dry-off were less likely to form a keratin plug (Dingwell et al., 2004). The composition of the mammary secretion changes dramatically throughout the course of the dry period, with a general increase in the concentration of various natural protective factors occurring until lactogenesis-colostrogenesis begins, when the increased secretory activity during this phase leads to a dilution of the natural protective factors (Bushe and Oliver, 1987; Oliver and Sordillo, 1988; Hurley, 1989; Nickerson, 1989; Smith and Hogan, 2000). Lactoferrin and citrate are two compounds present in milk which bind iron (Nonnecke and Smith, 1984; Smith and Hogan, 2000). Lactoferrin, in the presence of bicarbonate, binds iron such that bacteria cannot use the element to meet growth requirements, but coliform bacteria can utilize iron bound to citrate such that microbial growth is supported (Nickerson, 1989; Smith and Hogan, 2000). Following dry-off, the molar ratio of citrate:lactoferrin decreases (Nonnecke and Smith, 1984; 11

28 Kutila et al., 2003; Smith and Hogan, 2000) and the volume of fluid in the udder also decreases, which both contribute to the increasing antimicrobial nature of mammary secretions. Additionally, the concentration of bicarbonate increases following dry-off which aids in iron sequestration by lactoferrin (Nickerson, 1989). The molar ratio of citrate:lactoferrin increases during colostrogenesis-lactogenesis and contributes to the increased susceptibility to IMI acquisition around calving (Nickerson, 1989; Kutila et al., 2003; Smith and Hogan, 2000). The concentration of white blood cells also increases within 3 d following dry-off (Nickerson, 1989; Capuco and Akers, 1999; Smith and Hogan, 2000). Macrophages and neutrophils help remove degenerated secretory epithelial cells, fat, and casein from the glands via phagocytosis (Nickerson, 1989; Smith and Hogan, 2000). These cells might be less effective at engulfing and destroying invading microorganisms during this time and this reduced activity may contribute to the increased susceptibility to new IMI early in the dry period (Nickerson, 1989; Smith and Hogan, 2000). The population of leukocytes is dynamic across the dry period, but levels are generally elevated in comparison with those present in healthy, lactating glands. The rate of synthesis of milk components including fat, casein, lactose, citrate, β- lactoglobulin, and α-lactalbumin decreases within 4 d after dry-off (Smith and Hogan, 2000). Fat and casein present in the mammary secretions are removed by the phagocytic leukocytes in the gland (Nickerson, 1989; Smith and Hogan, 2000). The removal of fat and casein reduces the nutritive value of mammary secretions (Nickerson, 1989; Bradley and Green, 2004), making the environment of the mammary gland less conducive to 12

29 bacterial growth. The reduction of lactose and α-lactalbumin, the major osmoregulators of milk, helps reduce the volume of fluid in the udder as well (Smith and Hogan, 2000). The concentration of all classes of immunoglobulins increase during the dry period (Hurley, 1989; Nickerson, 1989; Smith and Hogan, 2000). Immunoglobulins in mammary secretions function primarily to bind microorganisms and mark them for phagocytosis by leukocytes (Nickerson, 1989). All of these coinciding alterations of the mammary secretions contribute to the antimicrobial environment which is present in fully involuted mammary glands. The mammary gland becomes less resistant to microbial invasion again during the hormonally-driven process of lactogenesis-colostrogenesis prior to calving (Nickerson, 1989; Smith and Hogan, 2000). The concentration of the various protective factors in the udder decreases as colostrum production begins and fluid begins to accumulate in the udder. The citrate:lactoferrin ratio also increases during this time which allows bacteria to access to iron more readily (Nickerson, 1989; Smith and Hogan, 2000). The concentration of the major milk constituents begins to increase 5 d before calving and the volume of fluid in the udder increases markedly 1 to 3 d prior to parturition (Smith and Hogan, 2000). Additionally, the keratin plug may degrade due to the accumulation of colostrum and might permit bacterial entrance into the gland (Nickerson, 1989). With the onset of all of these changes and the potential immunosuppression cows experience near calving (Mallard et al., 1998), the risk of acquiring new IMI increases as the dry period comes to an end. 13

30 Dry-Off Methods With the advent of antimicrobial use in production animal medicine, antimicrobial dry cow therapy (DCT) was developed in an attempt to try to combat IMI acquisition during the dry period. The use of DCT provides an opportunity to eliminate existing IMI and provide protection against new IMI during the early dry period (Smith et al., 1967; Natzke, 1981; Eberhart, 1986). However, use of antimicrobial DCT does not guarantee the clearance of existing IMI or the prevention of new IMI since many factors influence both of these outcomes (Funk et al., 1982; Schukken et al., 2001; Dingwell et al., 2002; Dingwell et al., 2004; Royster and Wagner, 2015). The use of intramammary DCT has been shown to increase the rate of keratin plug formation following dry-off (Williamson et al., 1995). The use of antimicrobial DCT was suggested as the one management practice which could be easily implemented to alter the prevalence of IMI within a herd (Natzke et al., 1975) and blanket DCT is a commonly recommended dry-off management practice in the US. According to the NAHMS 2014 Dairy Study estimates, 80% of dairy operations treat all cows with intramammary DCT (Lombard et al., 2015). Although DCT has successfully reduced the prevalence of IMI caused by contagious pathogens, especially Streptococcus agalactiae, IMI of environmental origin are problematic even in well-managed herds (Bradley and Green, 2000; Bradley and Green, 2004). The efficacy of different DCT products to cure existing IMI (Halasa et al., 2009b) and prevent new IMI (Halasa et al., 2009a) varies, but in general, the preparations currently available for use in the U.S. are less effective against environmental pathogens, 14

31 especially the coliforms (Bradley and Green, 2004). Therefore, additional management practices around the dry period may be needed for optimal udder health. Multiple studies have been conducted to investigate what the best method to dryoff cows is, often with regard to mammary health, but many of the studies were done when milk production per cow was substantially lower than what is seen in most dairies today (Wayne and Macy, 1933; Wayne et al., 1933; Neave et al., 1950; Oliver et al., 1956a,b). Abrupt and gradual milk cessation were two commonly investigated approaches to discontinue milking (Wayne and Macy, 1933; Espe and Smith, 1952; Oliver et al., 1956a,b). Abrupt cessation or stop milking is the practice of terminating normal daily milking on a set day which is typically determined by the expected calving date and goal dry period length. Gradual cessation of milking (also referred to as intermittent milking or reduced milking frequency) is when cows are weaned from milking over a period of days or weeks. Gradual cessation methods more closely resemble the process cows would experience if they were weaning their calf naturally, as is commonly practiced in beef cattle production where calves typically stay with their mother until they no longer require their mother s milk to supply adequate nutrition. The frequency of milking and the duration until dry-off has varied during gradual cessation studies, but a once daily milking schedule for a week or less prior to dry-off has been used in some of the more recent studies (Natzke et al., 1975; Oliver et al., 1990). Gradual cessation of milking has been shown to reduce milk yield prior to complete cessation of milking at dry-off (Oliver et al., 1956b; Bushe and Oliver, 1987; Oliver et al., 1990; Newman et al., 2009; Newman et al., 2010). Additionally, gradual cessation was 15

32 often associated with improved udder health when compared with abrupt cessation, especially for uninfected quarters at the time of dry-off, as measured by fewer IMI during the dry period or lower prevalence of IMI at calving (Oliver et al., 1956a; Oliver et al., 1990; Newman et al., 2010). Gradual cessation of milking has been beneficial to udder health when antimicrobials were used at dry-off (Oliver et al., 1990) and when antimicrobial DCT was not administered (Natzke et al., 1975; Oliver et al., 1990). The greatest decrease in IMI from dry-off to calving was reported in quarters of cows dried-off via gradual cessation, with or without a ration change, during the last week of lactation (Oliver et al., 1990). The benefit gradual cessation of milking has on udder health may be due to the reduction in milk yield prior to complete cessation of milking. Increasing milk yield prior to dry-off has been associated with an increased risk of environmental IMI at calving (Rajala-Schultz et al., 2005) and an increased risk of acquisition of new IMI during the dry period (Dingwell et al., 2002; Dingwell et al., 2004; Odensten et al., 2007), but Natzke et al. (1975) reported milk yield at dry-off had no significant effect on the rate of new IMI. Additionally, increasing milk yield prior to dry-off has been associated with elevated SCC early in the subsequent lactation (Green et al., 2008). It has been suggested that reducing milk volume via gradual cessation of milking may improve udder health by increasing the efficacy of DCT by increasing the concentration of antimicrobials in mammary glands (Bushe and Oliver, 1987). The reduced volume of milk was also hypothesized to reduce the risk of milk leakage during early involution (Bushe and Oliver, 1987). A more recent study reported that abrupt cessation cows leaked milk more frequently than cows which 16

33 were dried-off via gradual cessation, but there was no difference in milk yield prior to dryoff between cows which leaked or did not leak milk (Zobel et al. 2013). Milking cows intermittently while simultaneously feeding a hay-only diet during the final week of lactation increased the concentrations SCC, lactoferrin, and immunoglobulin G (IgG) prior to dry-off and decreased the citrate:lactoferrin during the first 2 to 4 d of the dry period when compared with cows which were fed a total mixed ration and milked intermittently or abruptly dried-off (Bushe and Oliver, 1987). The changes in the concentrations of these natural protective factors in addition to others, suggested that mammary glands of intermittently milked cows fed hay during the final week of lactation involuted more rapidly than cows which were only milked intermittently or were dried-off via abrupt cessation (Bushe and Oliver, 1987). In a more recent study, milk from cows which were gradually dried-off without a ration change also had increased lactoferrin concentrations relative to cows which were dried-off abruptly (Newman et al., 2009). The mechanism(s) by which gradual cessation of milking confers protection to mammary glands and improves udder health have not been thoroughly studied and thus, are not well-understood. Relatively few studies have investigated milk cessation methods and most are outdated considering the high milk yields found in today s dairy cows. Although little is known about the physiological factors which impact mammary health, even less is known about how cow comfort and behavior are impacted by different milk cessation methods. A recent study reported that cows dried-off via gradual cessation were less likely to spend time standing at the gate during regular milking hours following dry- 17

34 off than abruptly dried-off cows and that this behavior indicated that abruptly dried-off cows were motivated to be milked (Zobel et al., 2013). Cows which are abruptly dried-off have been shown to have increased udder firmness, increased udder swelling, reduced dry matter intake, and increased vocalization and such behaviors are believed to indicate stress, pain, and discomfort (Bertulat et al., 2013). High yielding cows which were abruptly driedoff had increased udder pressure and increased concentration of fecal stress hormone, but cows producing lower volumes of milk prior to dry-off did not experience significant changes (Bertulat et al., 2013). Although few studies have tried to determine how milk cessation methods impact animal behavior and well-being, the data available suggests that abrupt cessation of milking in high producing cows can induce stress and discomfort. Introduction to Current Study Most studies investigating milk cessation methods are outdated due to the advances in genetics, nutrition, and other management practices the dairy industry has adopted to increase milk production per cow. A current study comparing the impact different milk cessation methods have on mammary health, milk production, and milk quality is needed to identify what method is most beneficial in high producing dairy cows. The overall goal of the current study was to assess the impact of different dry-off methods on udder health as well as milk yield and quality in today s high producing dairy cows. The central hypothesis was that once daily milking during the final week of lactation would improve udder health and improve milk production and quality in the 18

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