PHARMACOLOGICAL AND DIETARY METHODS TO INFLUENCE THE 
INFLAMMATORY STATE OF DAIRY CATTLE 

By 

Joseph Fehn 

A THESIS 

Submitted to 
Michigan State University 
in partial fulfillment of the requirements   
for the degree of 

Animal Science – Master of Science 

2024 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ABSTRACT  

The replacement rate of dairy cattle is, on average, 35%, while cattle will leave the herd for 

reasons other than health events; this rate is not sustainable. Replacement rate can be defined as 

the percentage of lactating cows that leave the herd and are replaced by new primiparous cows. 

Societal pressure has led to the implementation of a veterinary feed directive (VFD), established 

in 2017 to restrict supplementing  antibiotics in feed or water to livestock without written consent 

from a veterinarian.  Then in 2023 further legislation was introduced requiring veterinarian 

consent to administer any prescription  drugs. While this had little impact on the dairy industry 

when first implemented, the aim of the VFD is to lessen the likelihood of further microbial 

antibiotic resistance  in livestock production. To help improve the resilience  of cows to 

pathogens, research has been done to investigate naturally occurring compounds that may help 

decrease the need for antibiotic use. β-Caryophyllene (BCP) has been demonstrated to possess 

anti-inflammatory properties. In this experiment, 20 mid or late lactation dairy cows, enrolled 

using a randomized complete block design, received two lipopolysaccharide (LPS) 

intramammary  challenges  28 days apart and received 1 of 2 treatments for 42 days: 1) CON 

(basal TMR);  2) BCP, consisting of 5 mg/kg of body weight, as liquid BCP delivered as a top 

dress treatment and mixed in with the top 20% of the ration. No changes in dry matter intake, 

body weight, milk yield or components were observed between treatment groups. BCP 

supplementation resulted in a lower haptoglobin concentration during the first LPS challenge and 

a lower somatic cell count (SCC) prior to the second LPS challenge. Plasma haptoglobin 

concentrations were lower in the second LPS challenge and a higher peak in SCC was observed 

in BCP cows. I conclude that supplementation of BCP during a LPS intramammary challenge 

lessens the systemic inflammatory response experienced by late lactation dairy cows.  

 
ACKNOWLEDGEMENTS 

I would like to first thank Dr. Barry Bradford for all his patience and never-ending 

support while I finished this degree. I would also like to thank Dr. Laman Mamedova for all the 

support, surprises,  and remedies for any illness  I had as she was always a bright spot through 

some dull times. I would also like to thank my committee members, Dr. Abuelo, Dr. 

Templeman,  and Dr. Zhou for all their support while I’ve completed  my program.  

I’d also like to acknowledge all my fellow lab mates who all provided support whenever 

needed. Also, a special thanks to the undergraduates who helped me during my research trial, 

and the MSU dairy farm and staff for allowing us to conduct research.  

Above all, I would like to give a special thanks to Brian Heman and AgOasis. If it 

weren’t for Brian allowing me to work at one of his dairies after dropping out of school, I don’t 

think that I would be here today. Thanks to my experience at the dairy, I discovered my passion 

for dairy cattle, and my desire to help and teach others.  

Overall, I am thankful to everyone who has been in my life during my graduate studies – 

my parents, fellow graduate students, other Animal Science faculty, and friends from all around. 

I would also like to give a special  thanks to Isabelle Bernstein for all of her undying support and 

helping me survive grad school. You all have made this experience such a wonderful and 

memorable  time. Although many of my biggest supporters have not been in Michigan, or always 

understand what I’m studying, they have continued to provide undying support and I couldn’t 

have done it without them.  

iii 

 
 
 
 
 
 
 
 
 
TABLE OF CONTENTS 

CHAPTER 1: REVIEW OF THE LITERATURE......................................................................... 1 

CHAPTER 2: EFFECTS OF β-CARYOPHYLLENE SUPPLEMENTATION ON DRY 
MATTER INTAKE AND PRODUCTIVITY OF LATE-LACTATION DAIRY COWS 
THROUGH REPEATER LIPOPOLYSACCHARIDE CHALLENGES..................................... 18  

CHAPTER 3: IMPLICATIONS AND CONCLUSIONS............................................................ 34 

BIBLIOGRAPHY......................................................................................................................... 37 

APPENDIX................................................................................................................................... 48 

iv 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CHAPTER 1: 

REVIEW OF THE LITERATURE 

1 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Introduction 

In the U.S. dairy industry, there is an average lactating cow replacement rate of 35% 

(Overton and Dhuyvetter, 2020). Replacement rate can be defined as the percentage of lactating 

cows that leave the herd and are replaced by new primiparous cows. Often cows are replaced for 

reasons other than health events, but regardless of the cause, this replacement rate is far from 

optimal in terms of economic, social,  or environmental  sustainability  (Murat Tatar et al., 2017). 

Mastitis and metritis are common infections experienced by dairy cattle and can often require 

antibiotic treatment (Cheng and Han, 2020). Due to societal pressure, a veterinary feed directive 

(VFD) was established in 2017 to restrict supplementing antibiotics in feed or water to livestock 

without written consent from a veterinarian, then further legislation was passed in 2023 to 

require a veterinarian  prescription for any antibiotics. While this had little impact on the dairy 

industry when first implemented, the aim of the VFD is to lessen the likelihood of further 

microbial  antibiotic resistance in livestock production. To help improve the resilience  of cows to 

pathogens, research has been done to investigate naturally occurring compounds that may help 

decrease the need for antibiotic use. This review of the literature will examine the mechanisms  of 

inflammation and common pharmaceutical  and dietary methods to modulate the inflammatory 

state of the dairy cow.  

Inflammation during an immunological challenge 

Inflammation is part of the innate immune system that is commonly associated with 

localized heat, swelling, pain, and a reduction in appetite (Vane and Botting, 1987). 

Inflammation consists of three phases: alarm,  mobilization,  and resolution. The acute 

inflammatory response is activated when pattern recognition receptors (PRR) detect either 

pathogen-associated molecular  patterns (PAMP) or damage-associated molecular  patterns 

2 

 
 
 
 
 
(DAMP). PAMP consist of microbial  nucleic acids, lipoproteins, and carbohydrates (Mogensen, 

2009). PRR are found on both tissue-resident immune cells like macrophages,  mast cells, and 

dendritic cells, as well as on leukocytes and many non-immune cells.  Upon PRR activation, there 

is an increase in expression of pro-inflammatory mediators, including cytokines and chemokines, 

aiding in the recruitment of leukocytes and production of complement proteins, adhesion 

molecules,  histidine, and eicosanoids (Newton and Dixit, 2012). Transcription factors such as 

nuclear factor kappa B (NFκB) and activator protein-1 (AP-1) are found in the cytoplasm of cells 

and are activated thorough extracellular signaling  (Adcock and Caramori, 2009). These 

transcription factors coordinate the activation of many genes, including proinflammatory 

cytokines (Schottelius et al., 1999; Hoffmann and Baltimore, 2006). Depending on the pathogen 

load, NFκB and/or AP-1 will trigger positive or negative feedback, thus allowing the body to 

mount an appropriate response to the pathogen load (Adcock and Caramori, 2009; Sung et al., 

2014). Inflammatory cytokines include tumor necrosis  factor α (TNFα), interleukin (IL)-1 and 

IL-6 (Takeuchi and Akira, 2010). Histidine and adhesion molecules work together with the 

increased blood flow to allow for neutrophils to enter the site of irritation. This passage of 

neutrophils across the vascular epithelium is the cause for heat, swelling, and pain associated 

with inflammation (Sordillo, 2018). If the acute response is adequate, the body switches from 

producing prostaglandins (pro-inflammatory  mediators) to lipoxins that aid in the recruitment of 

macrophages and begin tissue repair (Medzhitov, 2008). If the initial inflammatory response is 

unable to resolve the irritation, the neutrophil cascade is accompanied by macrophages  and T-

cells (Medzhitov, 2008). Once the body has successfully eliminated the pathogen, it will switch 

from pro-inflammatory  to resolving signaling  by producing cytokines such as IL-4, IL-10, IL-11 

and IL-13. Interleukins IL-4 and IL-10 aid in the attenuation of inflammatory transcription 

3 

 
 
 
 
 
factors (Schottelius et al., 1999; Iribarren et al., 2003). If the body cannot eliminate the pathogen, 

it is possible that granulomas and tertiary lymphoid tissues can form (Medzhitov, 2008). The 

inflammatory response is the first step in a immune response and may either be isolated and 

resolved or may require other branches of the immune system to intervene to help clear the 

pathogen.  

Oxidative status  

Oxidants consist of substances able to oxidize other molecules  (Abuelo et al., 2015). Free 

radicals are atoms or molecules  that have one or more unpaired electrons in their outer electron 

shell, and are oxidizing agents (Sordillo and Aitken, 2009). Reactive oxygen species (ROS) are a 

class of free radicals that are produced from superoxide anions as a byproduct of cellular 

metabolism,  and produced by activated neutrophils and macrophages (Sordillo and Aitken, 2009; 

Abuelo et al., 2015). These superoxide anions can interact with other molecules to generate 

various ROS (Sordillo and Aitken, 2009). ROS aid in the host immune response by promoting 

the expression of cytokines and eicosanoids, as well as through the destruction of phagocyted 

pathogens (Abuelo et al., 2015). Antioxidants may be synthesized within the host, or may be 

consumed in the diet, and are generally described as any substance that prevents, delays, or 

removes oxidative damage (Sordillo and Aitken, 2009). Examples of antioxidants include 

vitamin E and selenium (Miller  et al., 1993; Finch and Turner, 1996). Antioxidants and ROS 

work together to maintain the oxidative status of the host. However, when abundant ROS are 

produced and there are insufficient antioxidants present, the condition called oxidative stress 

develops (Abuelo et al., 2015). Oxidative stress can cause tissue damage and deteriorate cell 

membrane  integrity and may even result in cell death (Abuelo et al., 2015; Bradford et al., 2015). 

Oxidants in themselves are harmless,  but if the oxidative status shifts towards a high 

4 

 
 
 
 
 
concentration of ROS damage can occur to the host. In order to balance the oxidative status of 

cattle, it is important to limit ROS production, and or provide sufficient antioxidants to offset 

ROS production.  

Inflammation during the transition  period 

The transition period poses an interesting challenge to both dairy producers and 

nutritionists, leading to it being studied extensively. During the last week preceding parturition, 

the dairy cow begins to back off feed intake while initiating colostrogenesis. Parturition is 

associated with tissue damage, and an immediate increase in energy demands due to lactogenesis 

(Bradford et al., 2015). The combination of already expressing feed adverse behavior and new 

tissue damage following parturition provides the perfect storm for other insults to the immune 

system. This  could explain why this period often accounts for most illness events on a dairy 

farm, especially  for mastitis and metritis (Barragan et al., 2021). This  predicament has led to the 

search for methods to influence the inflammatory state during this period so that the cow may 

return to consuming adequate feed more quickly than a cow with no intervention. Studies have 

shown mixed results when attempting to alter the inflammatory state during the transition period; 

some show an increase in milk yield (Bertoni et al., 2004; Barragan et al., 2021). Others 

observed an increase in the rate of dystocia when flunixin was administered pre-calving and an 

increase in retained placentas when administered post-calving (Newby et al., 2017). While 

inflammation during the transition period is responsible  for most illness events on a dairy, 

influencing the inflammatory state is more complex than just reducing inflammation around 

calving.  

5 

 
 
 
 
 
 
 
 
Mastitis   

Mastitis, which consists of inflammation of the mammary gland, results in roughly 2 

billion  dollars lost annually in the U.S dairy industry (Cha et al., 2011). Mastitis is either 

contagious or environmental (coliform)  but both result in lower fluid milk production, milk 

component concentrations and an increase of milk immunoglobulins  due to increased 

permeability  of the blood-milk barrier,  and may cause tissue damage (Ogala et al., 2007). 

Mastitis cases can be clinical  or sub-clinical.  A clinical case of mastitis results in the clinical 

signs of inflammation in the udder accompanied by watery milk with visible flakes and clots, 

whereas sub-clinical  mastitis demonstrates no visible signs of udder inflammation, but a decrease 

in milk  production and increase in the somatic cell count (SCC) (Cheng and Han, 2020). 

Symptoms experienced during clinical  mastitis may be local, with only heat and redness in the 

infected quarter, or may be more severe, causing systemic fever and a reduction in feed intake 

(Cheng and Han, 2020).  

Coliform bacteria are able to survive in near anaerobic conditions and are able to utilize 

lactose as a fuel source, allowing them to proliferate within the mammary gland (Hogan and 

Smith, 2003). The low oxygen concentration in the mammary  gland can inhibit production of 

ROS like hydrogen peroxide, used by neutrophils to help kill/breakdown pathogens, thus 

compromising  the host’s immune  defense (Sordillo and Streicher, 2002). Contagious bacteria 

may originate from the bedding area or on milk machines  as a result of poor milking hygiene 

(Smith et al., 1985). Once a cow is infected with contagious bacteria, the udder becomes the 

major reservoir  and the bacteria is primarily  spread between cattle while milking; these 

pathogens tend to result in chronic sub-clinical  infections with occasional flares of clinical  cases 

(Abebe et al., 2016). Factors such as age, breed, and udder structure can increase a cow’s 

6 

 
 
 
 
 
susceptibility to mastitis and paired with reduced immune function due to low oxygen 

concentrations, the mammary  gland may be unable to clear the infection on its own, thus 

requiring  antibiotic intervention (Cheng and Han, 2020). Mastitis, regardless of type, results in 

profit loss to the producer through lowered milk production and sometimes treatment costs. 

Thus, management practices to limit the spread of environmental infections or other anti-

infection interventions should be used to help limit milk withdrawal times to help minimize  loss 

of profit by producers.  

Methods to modulate the inflammatory state in dairy cattle 

Inflammation is commonly associated with illness, but it is not inherently bad for the 

host. Often, inflammation is necessary, like during parturition or when fighting off infection 

(Medzhitov, 2008; Bradford et al., 2015). By inhibiting specific enzymes or pathways using anti-

inflammatory drugs, not only are the clinical  signs of inflammation lessened, but the response to 

pathogens can also be delayed. It has been demonstrated that inhibiting key functions of 

inflammation pre-partum can result in increased stillbirth rate as well as an increased odds of 

clinical  signs of inflammation, metritis, retained placenta, and a decrease in milk production 

(Newby et al., 2017). As different methods to modulate inflammation are discussed in this 

chapter, it is important to keep in mind what the potential adverse effects of blindly blocking 

pathways may be.  

Trained immunity. One method for immunomodulation is innate memory, where innate 

immune cells  demonstrate either an enhanced or suppressed response to a repeated immune 

challenge (Byrne et al., 2020). Innate immunity can be thought of as the instant and non-specific 

response arm of the immune system, which has traditionally been considered to be incapable of 

developing an immunological  memory.  Adaptive immunity, in contrast, is able develop a 

7 

 
 
 
 
 
memory response to pathogens, allowing for a more targeted and heightened immune response. 

This greater immune response is achieved through the production of memory B and T cells that 

allow for long-term protection against the same pathogen (Chaplin, 2010). Trained immunity 

works as a protection against reinfection independent of memory B and T cells. Instead, trained 

immunity works through other cells, such as natural killer cells,  and is achieved through 

epigenetics (altering gene expression  without DNA sequence modulation) and metabolic 

programming  (Byrne et al., 2020). Adaptive immunity can be distinguished from trained 

immunity by the pathogen specificity and duration of immunity. Adaptive immunity is highly 

specific to the pathogen and provides long-term immunity (years-lifetime);  conversely, trained 

immunity is often non-specific and provides short term altered immunity (Netea, 2013). LPS is 

one of the best-known innate priming agonists able to alter the immune system, and has been 

demonstrated to alter the immune system response to repeated challenges in a dose-dependent 

manner (Byrne et al., 2020). When LPS is administered in low doses, tolerance is observed 

(Yoza and McCall, 2011); however, administration of even lower concentrations results in 

heightened immune responses (Ifrim et al., 2014). In a bovine mastitis model, repeated LPS 

challenges  with an interval of 14 days resulted in a lower concentration of immune indicators in 

the plasma as well as milder local signs of inflammation  (Suojala et al., 2008). In another study, 

cattle received an intramammary  infusion of LPS and were later challenged with Escherichia 

coli. Cows who had received the LPS infusion demonstrated lesser signs of systemic and local 

inflammation (Petzl et al., 2012). Other forms of innate priming agonists include β-glucans and 

bacilli  Calmette-Guérin (BCG), a live attenuated vaccine for tuberculosis (Byrne et al., 2020). 

One study demonstrated that monocytes isolated from calves who received the BCG vaccine 

produced more pro-inflammatory cytokines when stimulated with LPS and Pam3CSK4, 

8 

 
 
 
 
 
compared to cells isolated from non-vaccinated calves (Guerra-Maupome  et al., 2019). Trained 

immunity poses as a short-term method to lessen the inflammatory response to non-specific 

inflammatory markers  such as LPS.  

Selective  and non-selective  cyclooxygenase inhibitors.  One class of anti-inflammatory 

drugs are the cyclooxygenase (COX) inhibitors, also commonly known as nonsteroidal anti-

inflammatory drugs (NSAID). COX inhibitors are commonly used in the dairy industry and are 

readily available  as over-the-counter (OTC) drugs. The COX enzymes (COX 1 & COX 2) 

catalyze the rate-limiting  step in biosynthesis of many eicosanoids, including prostaglandins 

(Chandrasekharan and Simmons, 2004). COX-1 is expressed at a relatively constant rate and 

plays a role in homeostasis,  whereas COX-2 expression is associated with inflammation and is 

stimulated by LPS, IL-1, IL-2, and TNFα (Vane et al., 1998). In the gastrointestinal tract, 

prostaglandins from COX-1 aid in the production of the mucosal lining. The available  COX 

inhibiting drugs are either selective to COX-1 or COX-2 or nonselective, inhibiting both 

enzymes. Examples of selective COX inhibitors include carprofen, meloxicam,  and tolfenamic 

acid; non-selective COX inhibitor examples  include flunixin and ketoprofen (Breen, 2017). 

COX-2 inhibition during an immune  challenge reduces the fever response, along with mitigation 

of negative effects on dry matter intake (DMI) during an immune challenge, dependent upon the 

half-life of the drug administered (Yeiser et al., 2012).  

COX inhibitors have been studied as potential tools for resolution of acute coliform 

mastitis cases. Orally administered non-selective  inhibitors such as aspirin, ketoprofen, and 

flunixin meglumine  should be used with caution, as excessive COX-1 inhibition around 

parturition may increase  the risk of retained placenta, culling, or metritis  (Trimboli  et al., 2020). 

9 

 
 
 
 
 
COX inhibitors have been shown to lower the SCC in cattle experiencing mastitis (Anderson and 

Hunt, 1989), reduce body temperature (Morkoç et al., 1993), and improve the recovery from 

clinical  mastitis cases (Shpigel et al., 1994). When considering usage of COX inhibitors, there is 

a meat and milk withhold time of 24 hours or more (Smith et al., 2008). COX inhibitors may 

blunt the inflammatory response and its effects on DMI and production. When administering 

COX inhibitors, selective COX-2 inhibitors should be considered as excessive COX-1 inhibition 

may have deleterious effects around parturition.  

Glucocorticoids  and prostaglandins.  Glucocorticoids (GC) are a class of anti-

inflammatory mediators synthesized in the adrenal cortex. Adrenal GC production is regulated 

by the hypothalamic-pituitary-adrenal  axis (Timmermans  et al., 2019). During an immunological 

challenge, cytokines stimulate the hypothalamus, causing the release of corticotropin-releasing 

hormone; in turn, corticotropin is released from the anterior pituitary. Corticotropin then acts on 

the adrenal cortex to trigger synthesis and secretion of cortisol, which is then converted to 

cortisone (Rhen and Cidlowski, 2005). GC secretion inhibits inflammation via the 

downregulation of chemokine and cytokine activity, inhibition of eicosanoid production from 

macrophages, and inhibition of endothelial production of intercellular and vascular adhesion 

enzymes (Cain and Cidlowski, 2017). GC activate glucocorticoid receptors and can inhibit 

prostaglandin production through the inhibition of annexin I, MAPK phosphatase 1, and COX-2 

(Rhen and Cidlowski, 2005). Inhibition of these pathways ultimately results in anti-inflammatory 

and analgesic effects. 

There are many synthetic GC that are widely available, such as prednisone, prednisolone, 

and dexamethasone. When treating acute cases of mastitis, administration of GC such as 

dexamethasone can result in lower body temperatures as well as less milk  production loss than 

10 

 
 
 
 
 
an untreated cow with mastitis (Lohuis et al., 1988). Prednisone may decrease the production of 

pro-inflammatory  cytokines and decrease SCC, depending on the type of infection (Wall et al., 

2016). Because treatment with GC may lessen immune responses, it may impair the immune 

system’s ability to resolve the infection due to impaired immune cell migration  (Roth, 1982). GC 

supplementation during an immune challenge  may reduce the inflammatory response, but should 

be used with caution as the reduction in inflammatory response may also lessen the host’s ability 

to clear the pathogen thus prolonging the infection.  

Omega-3 fatty acids. During an immune  challenge, immune cells  can utilize FA, 

glutamine, or glucose as fuel sources (Sordillo, 2016). Immune cells utilize these energy sources 

at varying rates depending on severity of immune challenge and DMI (Calder, 2013). Oxylipids 

are synthesized from substrates including omega-6 linoleic  and arachidonic acids and omega-3 

eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA; Sordillo and Raphael, 2013). 

Commonly, omega-6 FAs activate NFκB and enhance inflammatory responses, whereas omega-

3 FAs tend to inhibit NFκB and promote resolution of inflammation (Serhan, 2009; Calder, 

2012). DHA and EPA demonstrate anti-inflammatory functions by inhibiting NFκB activation 

indirectly by altering the concentrations of inflammation mediators such as prostaglandin F 2α 

(Badinga and Caldari-Torres; Lee et al., 2010; Calder, 2012). It has been demonstrated that 

increasing  the omega-3 FA content of endothelial cells can lessen their pro-inflammatory 

response (Contreras et al., 2012). By altering the omega-6 to omega-3 ratio of FA supplemented 

to dairy cattle, it may be possible to lessen the inflammatory state during times such as the 

periparturient period or during mastitis (Bradford, 2020). When feeding diets differing in the 

ratio of omega-6 and omega-3 acids, increasing omega-3 supply led to an increase in DMI and 

milk component yields as well as a decrease in the acute phase response to an intramammary 

11 

 
 
 
 
 
LPS challenge. However, increasing the amount of omega-3 in the diet did not attenuate the 

decrease in DMI during an immune challenge (Greco et al., 2015). By altering the FA supplied 

in the diet to a higher concentration of omega-3 acids, the body may produce less pro-

inflammatory mediators during an infection. Feeding omega-3 acids serves as a preventative 

measure to lessen the inflammatory state, but during an active infection, other interventions may 

be required to help clear  up the cause of infection.  

Phytogenics. As demonstrated by the VFD of 2017, the use of feed antibiotics in 

livestock has become a greater consumer  concern, leading to the interest in naturally occurring 

compounds that may support immune system function to reduce the need for antibiotic treatment 

of disease and limit the risk of antibiotic resistance in livestock. Phytogenics refers to a broad 

array of secondary metabolites produced by plants (Swartz and Bradford, 2023). Phytogenics 

have been studied as a dietary method to decrease methanogenesis (Sinz et al., 2019). Ku-vera et 

al. (2020) demonstrated that some tannins, saponins, free phenolics, and flavonoid compounds 

inhibit methane production from ruminal fermentation (Ku-Veraet al., 2020).This review is 

going to discuss primarily  one class of these naturally occurring compounds, polyphenols,  which 

exert antioxidative and immunostimulatory effects. Polyphenols are a group of over 8,000 

compounds, all containing phenol rings. Polyphenols can be further divided into flavonoids and 

other phenolic acids. It is worth noting that the concentration of plant polyphenols is highly 

dependent on the maturity and part of the plant that is fed (Miranda et al., 2014). Because plant 

secondary metabolites are more difficult to patent than synthetic compounds, there has been 

limited private sector investment in these compounds. The limited literature, in turn, means that 

for most polyphenols, there is no standard dose recommendation, and publications vary widely in 

doses administered (Soltan et al., 2023). Phytogenics pose as a natural solution to combat 

12 

 
 
 
 
 
inflammation while avoiding the adverse effects of animal product withhold times due to 

treatment.  

Flavonoids. Flavonoids are a class of polyphenols known to have anti-inflammatory and 

antioxidative functions. Flavonoids are made up of a three-ring core and are divided into five 

subgroups: flavanols, anthocyanidins, flavones, flavanones, and chalcones  (Middleton et al., 

2000). Even though there are thousands of known flavonoids, they almost all promote broad anti-

inflammatory effects on different cell types (Gonzálezet al., 2011). Classically, flavonoids have 

been studied in ruminants as a tool to decrease methane production (Sinz et al., 2019; Ku-Vera et 

al., 2020). While research is limited, groups have set out to analyze flavonoid supplementation 

during the transition period and mastitis challenges (Olagaray and Bradford, 2019).  

Morin is a flavonoid isolated from the mulberry/fig family of plants (Moraceae)  and has 

been demonstrated to help lessen the inflammatory response to bovine mammary  epithelial  cells 

during LPS-induced mastitis in a cell culture model. Morin acts through inhibition of the NF-κB 

pathway (Wang et al., 2016). Supplementing silymarin  (extracted from milk thistle)  pre- and 

post-partum tended to lower the SCC in cows during the first 21 DIM as well as lead to increased 

milk production and no noticeable change in milk  composition (Tedesco et al., 2004; Garavaglia 

et al., 2015). Another group looking at supplementing a cocktail of flavonoids to lactating cattle 

with moderate or high SCC found that supplementation lowered SCC in cows with high SCC but 

had no effects on moderate SCC. An increase in DMI, fluid milk, and feed efficiency was also 

observed (Hashemzadeh-Cigari et al., 2014). Flavanoid supplementation may help reduce the 

inflammatory state, but further research needs to be done looking at the effects during an active 

infection.  

13 

 
 
 
 
 
Other phenolic acids. Another group of phytogenic compounds are other phenolic acids, 

including free phenolics,  essential oils (EO), lignans, saponins, terpenes, quinones, and alkaloids 

(Swartz and Bradford, 2023; Worku and Ismail, 2020). Essential oils are a liquid and volatile 

form of secondary metabolites. Supplementation of non-flavonoid polyphenols in diabetic 

murine and human models decreased the concentration of pro-inflammatory molecules  (Miranda 

et al., 2014). EO found in ginger have been used as folk medicine for the treatment of pain and 

inflammation (Worku and Ismail, 2020). Eugenol is an EO found in clove oil, nutmeg, 

cinnamon, and basil which has demonstrated anti-inflammatory effects (Fujisawa et al., 2002; 

Carrasco et al., 2009). Phenolic compounds found in plants may be polymerized into larger 

molecules  such as proanthocyanidins (condensed tannins) and lignins (Waghorn and McNabb, 

2003). Feeding birdsfoot trefoil (a forage with high concentrations of tannins) to lactating dairy 

cows improved feed efficiency, increased milk yield and milk protein yield, but also decreased 

milk fat concentration (Woodward et al., 1999; Harris et al., 1998). Hydroxycinnamic acids are 

free phenolic acids with the most common ones including caffeic acid, ferulic acid, p-coumaric 

acid, sinapic acid, and chlorogenic acid (Chen and Ho, 1997). Using inflammatory bowel disease 

as a model of inflammation, Zielińska  et al. (2021) observed that caffeic acid exerts anti-

inflammatory properties by targeting COX-2 and PGE2 (Zielińska et al., 2021). Supplementation 

of phenolic acids may serve as a preventative measure  to inflammatory responses, but much like 

omega-3 supplementation, other methods may be required to clear a pathogen in the event of an 

active immune  challenge.  

Endocannabinoid system. Endocannabinoids, closely related to oxylipids, are a class of 

lipid mediators (Walker  et al., 2022). Endocannabinoids are derived from arachidonic acid and 

synthesized in the body, while phytocannabinoids are synthesized in plants and are only active in 

14 

 
 
 
 
 
mammals  after ingestion (Chayasirisobhon,  2021). Phytocannabinoids have properties similar  to 

cannabinoids and/or interact with the cannabinoid receptors (Gertsch et al., 2010). Examples of 

endocannabinoids include anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), and 

examples of phytocannabinoids include β-caryophyllene  (BCP), cannabidiol (CBD) and Δ9-

tetrahydrocannabivarin (THCV) (Bolognini et al., 2010; Gertsch et al., 2010). The 

endocannabinoid system (ECS) is activated by two receptors - CB1 and CB2 - and is involved in 

metabolism,  energy homeostasis,  and inflammation (Zachut et al., 2018). The CB1 receptor is 

associated with the central nervous system and is responsible for the psychoactive effects such as 

catalepsy and memory/cognitive impairment  reported with some cannabinoids (Pertwee and 

Ross, 2002a; Aly et al., 2020). In contrast, CB2 activation is associated with peripheral and 

immune cell  responses (Pertwee and Ross, 2002). Both the CB1 and CB2 receptors can be found 

in immune  cells (macrophages,  dendritic cells and B cells), but the CB2 receptors are 10-100 

times more abundant than CB1 in these cell types (Ziring et al., 2006; Chiurchiù et al., 2015; 

Rahaman and Ganguly, 2021). 

CB2 activation has been demonstrated to exert anti-inflammatory effects in vitro and vivo 

(Gertsch et al., 2008). During infection, endocannabinoid production is increased, paired with a 

downregulation of the AEA-degrading enzymes (Klein, 2005). Depending on both the 

concentration and timing, endocannabinoids may serve as pro- or anti-inflammatory  signals 

(Basu and Dittel, 2011; Walker et al., 2022b). CB2 aids in reducing the inflammatory state by 

inhibiting the release of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-α (Correa et 

al., 2009; Capozzi et al., 2022). During LPS activation of the immune system, CB2 agonists 

inhibit the secretion of TNF-α and IL-1β (Gertsch et al., 2008; Jürg Gertsch and Gertsch, 2017). 

Endocannabinoids function as lipid mediators, thus increasing the dietary supply of 

15 

 
 
 
 
 
endocannabinoids may act as a preventative method to the inflammatory response, but once 

again, other interventions may be required in the event of an immune challenge. 

β-caryophyllene.  BCP is a sesquiterpene phytochemical that is relatively abundant in 

plants such as black pepper, oregano, and cinnamon (Gertsch et al., 2008). BCP selectively 

activates the CB2 receptor and has been shown to have analgesic, anti-inflammatory  (lower 

production of TNF-α, IL-1β, IL-6 and NF-κB), and other immunomodulatory effects (Klauke et 

al., 2014; Francomano et al., 2019; Scandiffio et al., 2020). BCP stimulation of the CB2 receptor 

has been demonstrated in cell culture models stimulated with LPS, and when cells were treated 

with a CB2 antagonist the positive effects of BCP were reverted (Picciolo et al., 2020). In 

another study, researchers found BCP treatment exerted anti-inflammatory effects and decreased 

the oxidative status of cells exposed to a conditioned medium from activated monocytes 

(Mattiuzzo et al., 2021). In murine models, oral supplementation of BCP demonstrated a 70% 

reduction of carrageenan-induced paw edema (Gertsch et al., 2008). In another murine model, 

oral BCP supplementation suppressed the expression pro-inflammatory  cytokines in the context 

of antiretroviral drug-induced neuropathic pain (Aly et al., 2020). BCP, a dietary cannabinoid, 

may help blunt the response to an immune challenge. Before BCP can be recommended for 

commercial  use, further studies need to be conducted to define an effective dose, as well as 

confirm that there are no residuals found in meat or milk products.   

Conclusions  

Inflammation is an essential part of homeostasis, but too much inflammation can impede 

lactation performance (Bradford et al., 2015). Although compounds have been identified which 

can influence immune responses, there are few tools that are proven effective for enhancing 

resilience  to disease challenges.  Supplementation of phytogenic compounds such as BCP may 

16 

 
 
 
 
 
 
potentially decrease pro-inflammatory signals  (Klauke et al., 2014) so that a healthy degree of 

inflammation is still allowed while not completely eliminating  its effects. BCP selective 

activation of the CB2 receptor is of primary interest, as this avoids potential psychotropic effects 

while providing all of the anti-inflammatory effects (Scandiffio et al., 2020). While BCP has 

been shown effective in murine and cell culture models  (Gertsch et al., 2008; Mattiuzzo et al., 

2021), the effect of BCP supplementation on dairy cattle in an LPS mastitis model is pertinent to 

further understanding its effects on inflammation.  

17 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CHAPTER 2: 

EFFECTS OF β-CARYOPHYLLENE SUPPLEMENTATION ON DRY MATTER INTAKE 

AND PRODUCTIVITY OF LATE-LACTATION DAIRY COWS THROUGH REPEATER 

LIPOPOLYSACCHARIDE CHALLENGES 

18 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Abstract 

Inflammation greatly impacts  the health and performance of dairy  cows during lactation. 

The objectives of this study were to evaluate the effect of β-caryophyllene (BCP) supplementation 

on  somatic  cell  score  (SCS)  and  inflammatory  blood  markers  in  late  lactation  dairy  cattle 

experiencing  an  acute  mastitis  challenge,  as  well  as  carryover  effects  of  a  second  mastitis 

challenge.  Twenty  late  lactation  Holstein  cows  (240  ±  60  DIM;  33  ±  6.9  kg/d  of  milk)  were 

enrolled  in  a  randomized  complete  block  design experiment  lasting  70  d. Cows were  assigned 

treatments of no top-dress (CON, n = 10) or BCP (n = 10) consisting  of 5 mg/kg BW liquid BCP 

added as a top-dress as a top dress treatment and mixed in with the top 20% of the ration. All cows 

received  the  same  diet.  Cows  were  challenged  in  the  right-rear  quarter  (RR)  with  10  µg  of 

lipopolysaccharide  (LPS) on d 28 and d 56. Quarter-level milk  (RR) was collected at 0, 8, 16, 24, 

48, 72, 96, 120, 144, and 168 h after each LPS challenge. Treatment effects were determined using 

linear  repeated mixed models in  SAS, including fixed effect of treatment, time, parity, and their 

interactions  and random  effects of block  and cow. Before  LPS, BCP did not affect DMI,  milk 

yield,  milk  composition,  SCS, or  plasma  glucose  concentration  (all  P >  0.10).  After each  LPS 

challenge,  BCP did not affect DMI, milk  yield or composition, but reduced composite milk  SCS  

(P  <  0.01)  A  treatment  ×  time  interaction  (P  <  0.001)  for  SCS  revealed  a  tendency for more 

complete resolution  in BCP-treated cows at d 52 (P = 0.06). Conversely,  SCS in  the RR quarter 

was greater for BCP vs. CON (P = 0.02) at 16 h after challenge  2 (treatment × time: P < 0.001). 

Plasma haptoglobin was significantly reduced by BCP in the 5 d following challenge 1 (P = 0.02) 

but not challenge 2 (P > 0.10). Overall, results suggest that BCP supplementation may reduce the 

inflammatory  response  observed from acute mastitis.  Further work is required to understand the 

underlying mechanisms  of the changes observed in this study. 

19 

 
 
 
 
 
Introduction 

Nutritional methods to influence the inflammatory state of dairy cattle have been a strong 

area of interest in the research community. Improving the understanding and delivery of 

nutraceuticals has the ability to further dairy production while working towards economic and 

environmental sustainability.  The use of nutraceuticals may reduce the need for other 

pharmaceuticals,  thus lowering the quantity of dairy products that are unusable due to drug 

withdrawal times from products.  

Phytocannabinoids, such as β-caryophyllene  (BCP), are secondary plant metabolites that 

have similar  properties to endocannabinoids and/or interact with the cannabinoid receptors 

(Gertsch et al., 2010). BCP has been proposed as a nutrient potentially capable of reducing 

inflammation through its selective activation of the CB2 receptor (Francomano et al., 2019). 

BCP is also known to have antioxidant, neuroprotective, anxiolytic, anti-cancer, and analgesic 

effects (Aly et al., 2020). The CB2 receptor is widely found among immune cells, modulating 

cytokine release  and immune cell migration (Pertwee, 2006). Unlike the CB1 receptor, it 

provides these results without psychoactive effects. Activation of the CB2 receptor through 

agonists can reduce inflammation by inhibiting the release of inflammatory cytokines such as IL-

6, IL-8, IL-1β and TNF-α in LPS-stimulated monocytes (Capozzi et al., 2022; Correa et al., 

2009; Gertsch et al., 2008; Jürg Gertsch & Gertsch, 2017). BCP supplementation, therefore, may 

help lessen the inflammatory state experienced by a dairy cow during an immunological 

challenge through its relationship  with the CB2 receptor.  

While not previously studied in dairy cattle, BCP supplementation has been studied in 

multiple murine  models. Oral supplementation in mice models has demonstrated suppression of 

pro-inflammatory  cytokines (Aly et al., 2020) and a decrease in paw edema (Gertsch et al., 2008) 

20 

 
 
 
 
 
in different inflammatory models. Poulopoulou & Hadjigeorgiou (2021) suggest that the rumen 

microbiome  takes 1 week to adjust to BCP supplementation; after adaptation, the mean ruminal 

degradation rate was just 4.3% per h (compared to 0.1% in PBS solution) using an ovine model. 

In comparison to expected ruminal liquid outflow rates in dairy cattle of ~19% per h (Taylor et 

al., 2005), these findings point to a strong potential for BCP delivery to the small intestine.  

Trained immunity is the process in which innate immune cells  undergo epigenetic 

changes resulting in either an enhanced or suppressed response to a repeated immune challenge 

(Byrne et al., 2020). Unlike adaptive immunity, trained immunity provides short term, often non-

specific alteration of immune function (Netea, 2013). The concept of trained immunity raises  a 

question: if innate immunity can be trained from a primary  challenge, can what is consumed 

during a primary  infection impact the body’s trained response to a repeated challenge?  

The objectives of this experiment were to evaluate if BCP supplementation reduces the 

SCS and inflammatory blood markers in late lactation dairy cattle experiencing  an acute mastitis 

challenge. Additionally, we investigated carryover effects of BCP supplementation on SCS and 

inflammatory blood markers during a second acute mastitis challenge once BCP supplementation 

ceased, as an initial assessment  of potential BCP impacts on immune training. We hypothesized 

that BCP supplementation would lower the SCS, inflammatory blood markers, and improve the 

resolution of mastitis.  

21 

 
 
 
 
 
 
 
 
 
Materials  and Methods 

Cows, treatments, and experimental design. All procedures were approved by the 

Michigan State University Institutional Animal Care and Use Committee (protocol no. 

PROTO202200231). The experiment was conducted at the Michigan State University Dairy 

Cattle Teaching and Research Center (Lansing, MI) between August 2022 and November 2022. 

Twenty mid or late-lactation Holstein dairy cows (240 ± 60 days in milk [DIM]; 6 primiparous, 

14 multiparous),  averaging 33 ± 6.9 kg/d of milk yield and free from clinical mastitis, SCS < 4, 

were enrolled in a randomized complete block experiment. Using data from a previous 

intramammary  LPS challenge study (Swartz et al., 2023), we conducted a power analysis based 

on body temperature. In the previous study the peak mean body temperature occurred at 5 h post-

challenge, with a standard deviation of 0.34 degrees C. Based on these data, 10 cows per 

treatment are sufficient to detect a mean treatment effect of 0.44 °C in body temperature at 5 h 

post-challenge with 80% power at α = 0.05. This  is sufficiently powerful to detect a meaningful 

strategy to reduce LPS-induced inflammation.  

The cows were enrolled in two cohorts of 10 cows each, two weeks apart. Cows were 

blocked, in blocks of two, by parity, bovine leukemia virus (BLV) status, and DIM, in that order, 

at start of challenge, within each cohort. BLV status was used as a blocking factor as it has been 

demonstrated that cows which are BLV positive have impaired immune response to stimuli 

(Erskine et al., 2011). Treatments were randomly assigned within each block. The experiment 

consisted of two treatment groups; 10 cows were assigned to receive the control (CON; no 

treatment) and 10 cows were assigned to receive 5 mg/kg of bodyweight (BW) per d of BCP 

(#W225207 Sigma-Aldrich).  This dose was selected in part due to anti-inflammatory effects of 

this dose in mice (Gertsch et al., 2008) and because this is the recommended greatest dose in 

22 

 
 
 
 
 
livestock feed under a European Union approval (https://eur-lex.europa.eu/legal-

content/EN/TXT/HTML/?uri=CELEX:32017R0065).   

Cows were housed in a tie-stall barn, milked three times daily (0500, 1300, and 2100 h) 

in a milking parlor,  and fed a total mixed ration (TMR) once per day (1000 h) for a 10% refusal 

rate. Cows had ad libitum access to water. Upon morning feeding (1000 h), BCP treatments were 

administered as a top-dress from d 0 until d 42; cows were monitored until d 70. Prior to the first 

mastitis challenge, quarter-level  milk samples  from the right rear (RR) quarter were taken to 

screen for an active mastitis case in that quarter. The 5 cows within each treatment group in each 

cohort (out of 6) with the lowest quarter-level somatic cell count (SCC) were selected for 

mastitis challenge (total n = 10 per treatment). Both CON and BCP groups included 5 BLV-

positive and 5 BLV-negative cows.  

Data collection  and sample analysis.  TMR samples  were collected (prior to BCP 

administration) every week throughout the experiment and composited at two-week intervals 

prior to analysis (Table  1). TMR samples  were evaluated for dry matter (DM), crude protein 

(CP), starch, ethanol-soluble  carbohydrates, neutral detergent fiber (NDF), lignin, crude fat, ash, 

and minerals using NIR spectroscopy (Cumberland Valley Analytical Services).   

Bodyweight was measured once per week and body condition score (BCS) was assessed 

once per week by 3 trained investigators on a 1-5 scale (Wildman et al., 1982). Milk yield and 

milk conductivity were measured at each milking  in the milking parlor  with an electronic 

monitoring system (AFIMILK, Kibbutz Afikim, Israel). Composite milk samples  (~50 mL) were 

collected on d 14, 23, 24, 30, 37, 38, 45, and 52 in a sealed tube with a preservative (bronopol) 

and stored at 4°C for milk component (milkfat, protein, lactose) and SCC analyses (Bentely 

FTM/FCS, Bentely Instruments Inc.; CentralStar DHI). Energy-corrected milk (ECM) yield was 

23 

 
 
 
 
 
 
calculated using the formula: ECM = (0.327 × milk  kg) + (12.95 × fat kg) + (7.65 × protein kg). 

Somatic cell  score (SCS) was calculated using the formula: SCS = log₂ (SCC/100,000) + 3 (Ali 

and Shook, 1980). Quarter-level milk  samples were collected from the RR quarter at the evening 

milking  (2100 h) on d 24 and 0, 8, 16, or 24 h after each challenge. Samples during the first 24 h 

after challenge were diluted 1:10 in PBS for flow cytometric analysis. Quarter level samples 

were then taken once daily for the next 6 d following each LPS challenge; these samples were 

not diluted in PBS. All quarter-level milk samples  were analyzed for SCC as described above.  

Body temperature was collected every 10 min from 24 h prior  to each LPS challenge 

until 72 h after each LPS challenge. The body temperature was collected using a thermometer 

(iButton DS1921H; Embedded Data Systems) which was placed on a blank controlled internal 

drug release device (CIDR; Zoetis). Prior to insertion of the CIDR, the vulva of the cow was 

cleaned with water and disinfected with betadine solution. Then, a lubricant was applied to the 

CIDR and vulva then the CIDR containing the iButton inserted into the vagina of each cow. 

Blood samples were collected on d 14, 24, 28-34 and d 56-62 after the 2100 h milking 

(~2200 h). Blood samples were also collected 8 h and 16 h after each LPS challenge. Blood was 

collected by coccygeal venipuncture with tubes containing K 2EDTA; samples were immediately 

placed on ice and transported to the lab where they were centrifuged for 15 min at 1,500 × g to 

collect the plasma. Two aliquots of plasma were kept at -20°C for later analysis.  Plasma was 

analyzed for glucose by enzymatic methods (kit #997-03001; Fujifilm, Osaka, Japan), insulin 

using a bovine specific ELISA (no. 10–1201–01; Mercodia AB, Uppsala, Sweden), haptoglobin 

using a bovine specific ELISA (Hp; kit # HAPT-11; Life Diagnostics, West Chester, PA), LPS 

binding protein by ELISA (LBP; cat # CKH113; Cell Scienes, Newburyport, MA), and β-

hydroxybutyrate by enzymatic assay (BHB; kit no. H7587-58; Pointe Scientific Inc., Canton, 

24 

 
 
 
 
 
MI). The inter-assay coefficients of variation (CV) were 5.81, 6.48, 7.22, 5.26, and 5.23 for 

glucose, insulin, Hp, LBP and BHB, respectively. Intra-assay CV were 5.17, 5.11, 6.55, 5.02, 

and 5.37 5.81, 6.48, 7.22, 5.26, and 5.23 for glucose, insulin, Hp, LBP, and BHB, respectively. 

The Mass Spectrometry and Metabolomics Core at Michigan State University conducted 

the analysis of BCP in feed and milk using gas chromatography-mass spectrometry. For 

quantification in the TMR, approximately 3.0 – 4.5 g of feed (frozen and thawed, not dried) was 

transferred into 40 mL amber vials  capped with Teflon septa. Samples were loaded onto a 

sampling  tray set at 40°C and equilibrated for 30 min before headspace was sampled with an 

autosampler equipped with a solid phase microextraction (SPME) syringe and fiber (30 mm 

divinylbenzene/Carboxen 50 μm polydimethylsiloxane, Milipore-Sigma).  The headspace 

extraction time was 10 min and volatiles were desorbed at 250°C with splitless mode on an 

Agilent 7890B GC with a 7010b triple quadrupole mass spectrometer. Separation was achieved 

on an Agilent J&W VF5ms (30 m × 0.25 mm × 0.25 mm; Agilent) using the following 

temperature profile:  40°C for 4 min; 20°C min-1 to 250°C; 250°C for 2 min. Ionization employed 

70 eV electron ionization and the mass spectrometer was operated in selected ion mode for m/z 

93 and 204 for BCP. 

Milk samples  were prepared by pipetting 500 μL of milk sample, 100 μL 0.05 ng/μL of 

tetradecane, and 9.4 mL of Milli-Q water to 40 mL amber vials capped with Teflon septa. 

Samples were loaded onto a purge and trap unit (Atomx XYZ). The sample cup was held at 50°C 

and volatiles were purged in the sample vials  for 11 min with 40 mL/min of nitrogen gas onto a 

#9 trap held at room temperature. The trap was heated to 250°C for 2 min and volatiles were 

desorbed with a 1:5 pulsed split mode on an 7890A GC with a 5975 single quadrupole mass 

spectrometer (Agilent). Separation was achieved on an Agilent J&W VF5ms (30 m × 0.25 mm × 

25 

 
 
 
 
 
0.25 mm; Agilent) using the following temperature profile: 40°C for 4 min; 20°C min-1 to 

250°C; 250°C for 2 min. Ionization employed 70 eV electron ionization and the mass 

spectrometer was operated in selected ion mode for m/z 93 and 204 for BCP and m/z 198 for 

tetradecane. 

Lipopolysaccharide  challenges. Lipopolysaccharide (LPS; E. coli O11:B4, Millipore 

Sigma, St. Louis, MO) challenges were conducted at the final milking (2100 h) on d 27 and d 55 

of the experiment. The challenges were all conducted in the RR quarter of each cow. To conduct 

the challenge, cows were milked according to farm protocols. After milking was completed , the 

teat end was scrubbed with an alcohol swab to remove bacteria. Then, 5 mL of PBS containing 

100 µg of LPS (20 µg/mL) were infused into the RR quarter. The solution was then massaged 

upward into the mammary gland. The post-milking  procedure was then completed according to 

farm protocols. 

Statistical  analysis. All data were analyzed using linear mixed models (GLIMMIX; SAS 

9.4, Cary NC). Dry matter intake (DMI), milk yield, milk composition, and milk component 

yields were analyzed using repeated measures mixed linear models for pre-challenge,  post-

challenge 1, and post-challenge 2 data. Change in BW and BCS (ΔBW, ΔBCS) were evaluated 

as repeated measures and included fixed effects of treatment, time, their interaction, BLV status, 

and parity. Random effects included cohort and cow. For ΔBW, ΔBCS, SCS, and body 

temperature, the spatial power covariance structure was used due to unequal spacing of 

measurements.  Normality of residuals was visually appraised. Observations with a Studentized 

residual ≥ 4 or ≤ -4 were removed from the data set for each variable and the models were refit if 

observations were removed.  

26 

 
 
 
 
 
Quarter-level somatic cell score (SCS; [log2(somatic  cell count/100,000) + 3]) was 

analyzed as a repeated measure using a compound symmetry covariance  structure due to equal 

spacing of measurements.  The model included the fixed effects of treatment, time post challenge, 

and challenge (1st vs. 2nd). All two and three-way interactions were included in the initial model 

and removed if P >0.10. For plasma markers,  the model used was the same except for the use of 

autoregressive covariance  structure. Cow was included as a random effect in each model. Model 

assumptions were evaluated as described previously.  

Body temperature and blood analytes were analyzed as an area under the curve (AUC; 

ΔC° × min)  using the trapezoidal rule. Baseline body temperatures were calculated by averaging 

the temperature of each cow for 12 h prior to LPS infusion. The model included treatment, 

challenge (challenge  1 vs. challenge  2) and their interaction. AUC was evaluated as a repeated 

measure using the compound symmetry covariance  structure. Cow was included as a random 

effect in the model. 

Statistical significance was declared at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10 for 

each variable  and model that were run. 

Results and Discussion 

Baseline period & BCP analysis. Prior to LPS challenge, no treatment effects on DMI 

were observed (Table  2), similar  to results reported by Oliveira (2018) using BCP as an oral 

supplement in mice models. Treatment did not significantly affect milk yield or energy-corrected 

milk (ECM; Table 2). Furthermore, the ΔBW and ΔBCS were not affected by treatment (Table 

5), consistent with both the lack of DMI differences and findings in mice (Oliveira et al., 2018). 

BCP was detected in purified samples used for treatment (95% BCP according to the LC-MS 

analysis),  consistent with specifications from the supplier, but we found no evidence of BCP in 

27 

 
 
 
 
 
milk or any basal presence in the TMR. Milk  and TMR samples were stored in a -20°C freezer 

for 6 months, then thawed prior to analysis; storage and handling of the milk and TMR samples 

may have affected our ability to detect BCP due to its volatile nature (Gertsch et al., 2008). The 

volatile nature of BCP also raises the possibility of all of it evaporating prior to ingestion thus 

explaining  our inability to detect it in the milk and TMR.  

Prior to LPS challenges, our findings suggest that BCP supplementation had no apparent 

adverse or beneficial effects on late-lactation dairy cattle. This could possibly be due to rumen 

degradation of BCP. Poulopoulou and Hadjigeorgiou (2021) evaluated terpene degradation in 

rumen fluid using an ovine model and found that BCP degradation rate was greatest after 1 week 

of oral supplementation (approximately  4.3% per hour, dosed at 1 g). These baseline period 

results, paired with our inability to detect BCP in milk collected from treated cows, suggests no 

risks of meaningful quantities of BCP getting into the food chain, thus setting the stage for 

further research on BCP supplementation in dairy cattle.  

One potential explanation for the inability to detect BCP in milk samples during this 

study is BCP’s low oral bioavailability  and high sensitivity to oxidation when exposed to oxygen 

and light (Liu et al., 2013; Di Sotto et al., 2018). Bioavailability of a molecule depends on its 

chemical  and physical properties, and typically reflects water solubility  and membrane 

permeability  (Di Sotto et al., 2018). It is important to note that BCP has high lipophilicity, which 

subsequently limits its bioavailability  and absorption. Assuming the BCP is not degraded in the 

rumen, it would be taken up in the small intestine (Di Sotto et al., 2018). In a human study by 

Mödinger et al. (2022) using similar  dosage of 100 mg per subject, using a self-emulsifying  drug 

delivery system to orally supplement BCP significantly increased the BCP AUC0-12h and AUC0-

24h concentrations by 2.2-fold and 2.0-fold, respectively. They also found a 3.6-fold increase in 

28 

 
 
 
 
 
maximum  plasma concentration and time to maximum blood concentration was 1.43 vs. 3.04 h 

for BCP delivered as a neat oil. Therefore, the form of supplementation in this experiment may 

have affected its uptake. Little information is available on rumen degradation of BCP other than 

results from Poulopoulou and Hadjigeorgiou (2021) using a sheep model, who found that rumen 

degradation of BCP was approximately 13%/h on average for the first week of supplementation 

and then dropped to approximately 4.3%/h in the following week. Further work should be done 

to evaluate the degradation of BCP using a bovine model, as well as determining the efficacy of 

formulating BCP to self-emulsify through oral and abomasal supplementation. 

LPS challenge 1. During an intramammary  LPS challenge, dairy cattle experience a drop 

in DMI and milk production while also having an increase  in body temperature and somatic cell 

count in milk (Johnzon et al., 2018). Temporal  patterns in this study were similar to those 

reported previously, with a noticeable rise in body temperature beginning roughly 2 h post-

challenge, peaking (~41°C) at 6 h post-challenge, and returning to baseline 12 h post-challenge 

(Figure 1). There  was no detectable impact of treatment on these body temperature patterns. 

After challenge, DMI and milk production were not significantly affected by BCP 

supplementation (Table  2). On the other hand, BCP supplementation significantly reduced SCS 

(Figure 3) on d 52 of the study (10 days after BCP supplementation ended), potentially pointing 

to more complete resolution of challenge-induced inflammation in the mammary  gland. 

Treatment did not affect plasma glucose, insulin, BHB or lipopolysaccharide binding protein 

(LBP) concentrations in the 16 h after the first LPS challenge (Table 3). However, BCP 

supplementation significantly lowered haptoglobin concentrations (Table  3) over the 16 h post-

challenge. 

29 

 
 
 
 
 
The reduction in plasma haptoglobin and the greater drop in SCS 10 d after challenge 

may be in part due to BCP’s ability to decrease the expression of pro-inflammatory signals, 

allowing for a more complete resolution (Aly et al., 2020; Johnzon et al., 2018). When looking at 

the results following the first of the LPS challenge, we did not observe sufficient evidence to 

support our hypothesis that BCP would lessen the inflammatory response. A recent study by 

Jermann et al. (2023) varying the dietary supply of macronutrients found no difference in the 

systemic inflammatory response to an intramammary  LPS injection based on macronutrient 

profile, but they did observe a significant increase in acute phase proteins (APPs) in milk before 

APP changes were detectable in plasma. This discovery raises  the question of whether 

inflammatory mediators from the challenged quarter cause the systemic reactions observed, or if 

LPS itself is entering the bloodstream. Haptoglobin is a positive APP synthesized by the liver; it 

is found in low concentrations in healthy animals but is quickly released into the bloodstream 

during an infection. It is a protein with antioxidant, antimicrobial, and anti-inflammatory 

properties through its ability to scavenge free hemoglobin and bind to neutrophil integrins 

(Gulhar et al., 2023). It results in the release of other anti-inflammatory mediators such as IL-10 

(Quaye, 2008; Ceciliani et al., 2012). While we didn’t detect any improvements in feed intake or 

milk yield in this study, lower concentrations of plasma haptoglobin suggests that cows 

experienced less inflammation  and hepatic stress as a result of BCP supplementation. The lower 

SCS observed at 24 d post-challenge in cows previously supplemented with BCP could also be 

explained by the anti-inflammatory effects of BCP potentially blunting the severity of the APP 

response in the affected quarter. The decrease in SCS and haptoglobin that was observed 

provides evidence that further research needs to be conducted looking at supplementing BCP to 

dairy cattle in a challenge setting. However, without any significant changes in any other acute 

30 

 
 
 
 
 
phase proteins, body temperature, or milk production, it is unclear if inflammation was mitigated 

during this challenge as a result of BCP supplementation. 

LPS Challenge 2. Repeated exposure to the same pathogen or pathogen-associated 

molecule  can alter the immune system through innate training, resulting in the host having either 

a heighted or a suppressed innate immune  response (Byrne et al., 2020). BCP treatment 

administration ended 2 weeks after the initial LPS challenge and 2 weeks prior to the second LPS 

challenge, to evaluate whether BCP supplementation influenced trained immunity. The 2 weeks 

prior to the second LPS challenge was to allow for BCP to leave the guy/body, however since we 

didn’t collect adipose samples there is a possibility  that BCP may be stored in adipocytes.  

In accordance with our hypothesis, BCP treatment appeared to have an effect on response 

to repeated LPS challenge. BCP supplementation resulted in a significantly greater quarter-level 

SCS score 16 hours after the second LPS challenge (Figure 4); there was no significant effect at 

any other time point. This  could be due to increased sensitivity after the first LPS challenge 

(Byrne et al., 2020), but more work needs to be conducted into nutritional impacts on innate 

training to explain this response.  After the second LPS challenge no treatment effects were 

observed for plasma glucose, insulin, haptoglobin, BHB, or LBP (Table 3). BCP 

supplementation has not been previously studied in cattle, so while the lack of difference in DMI 

and BW changes are supported by murine models, further research needs to be conducted to 

elucidate BCP effects on performance responses in dairy cattle.  

We expected to see a lesser acute phase response when comparing challenge 2 to 

challenge 1, as demonstrated in other studies looking at innate training (Ifrim et al., 2014; 

Sullivan et al., 2023). For haptoglobin, there was indeed a significant reduction in plasma AUC 

for the second challenge compared to the first (Table 4). However, the body temperature 

31 

 
 
 
 
 
response for the second LPS challenge was similar  to that of the first challenge (Figures 1 & 2). 

When comparing  the AUC for other plasma metabolites between challenges, we expected to see 

a lower AUC value for the second challenge but instead glucose, insulin, BHB, and LBP were 

not significantly different. Our findings when comparing between challenges was unexpected as 

other groups that have looked at repeated LPS challenges observed a lesser fever response due to 

innate training (Petzl et al., 2012).  

In contrast to other plasma analytes, we observed a lower concentration of plasma 

haptoglobin after challenge 2 compared to challenge 1. This lesser  concentration in the second 

challenge points to the possibility of tolerance to LPS following the first challenge, but this idea 

conflicts with the increased SCS response 16 hours after challenge, pointing to increased 

sensitivity as opposed to the tolerance observed with the haptoglobin AUC. This increased 

sensitivity in the mammary  gland may be due to the anti-inflammatory effects of BCP that were 

present during the first LPS challenge. It is possible that BCP limited the local inflammatory tone 

of cows during the first challenge, “training” the intramammary  immune cells in BCP-treated 

cows compared to CON cows and thus leading to, increased immune cell migration and a higher 

SCS in the BCP cows during the second challenge. This raises  a question of whether an 

observable difference in the inflammatory response would be expected, potentially impacting 

body temperature, feed intake, and milk responses, if this altered immune training was monitored 

while BCP supplementation was maintained. To test this, future studies could be conducted 

looking at repeated LPS challenges with BCP being supplied through both LPS challenges.  

While these findings are interesting, without other evidence of altered systemic responses 

– such as a lower body temperature or SCS – we are unable to clearly state that we successfully 

trained the innate immune system through repeated LPS challenges. 

32 

 
 
 
 
 
Conclusions 

Supplementation of BCP to late lactation dairy cattle did not cause any detrimental 

effects to production or homeostasis during a basal period. When looking at BCP 

supplementation during the first LPS challenge, a decrease in SCS and a reduction in 

haptoglobin concentrations suggest that BCP could help mitigate the inflammatory responses to 

an intramammary  LPS challenge. During the second LPS challenge, results suggest that BCP 

supplementation did not have an impact on innate training except for when looking at plasma 

haptoglobin concentrations compared to challenge 1. One measurement that was not collected in 

this study but would be advisable for future research is to confirm BCP supplementation was 

effective by testing its plasma concentration. The few significant results observed could be due 

to BCP’s low bioavailability  and uptake, which we are unable to determine with this experiment. 

We conclude that BCP supplementation appears to be safe at a rate of 5 mg/kg BW without 

disrupting the function of late lactation dairy cattle, however, we are unable to conclude that it is 

a useful treatment for inflammation. Although BCP treatment significantly decreased SCC and 

haptoglobin concentrations at particular timepoints after intramammary  LPS challenge, future 

research needs to be conducted to fully elucidate the function of BCP as an inflammatory 

modulator in lactating dairy cattle.  

33 

 
 
 
 
 
 
 
 
 
 
CHAPTER 3: 

IMPLICATIONS AND CONCLUSIONS 

34 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Implications  and Conclusions 

The average lactating cow replacement  rate in the U.S. dairy industry is 35%, while cows 

are  often replaced  due to  health  events,  this  replacement  rate  is  far  from  optimal  in  terms  of 

economic,  social,  or  environmental  sustainability  (Murat  Tatar  et  al.,  2017;  Overton  and 

Dhuyvetter, 2020). Due to societal pressure,  a veterinary feed directive (VFD) was established in 

2017 to reduce the use  of antibiotics  in  livestock  production.  To  help  improve  the resilience  of 

cows to pathogens, further research needs to be done to investigate naturally occurring compounds 

that may help decrease the need for antibiotic use. By studying compounds such as BCP, we may 

better understand ways in which nutrition can improve the immune function of dairy cattle.  

The goal of this work is to access how BCP supplementation changes the inflammatory 

response in late lactation dairy cattle experiencing repeated intramammary LPS challenges. The 

study evaluated the effect of BCP supplementation on the inflammatory responses from late 

lactation Holstein cows as well as any carryover effects of BCP supplementation on a subsequent 

LPS challenge. Parameters such as BHB, glucose, haptoglobin, insulin,  LBP, and body 

temperature were evaluated to measure the inflammatory response. BCP supplementation 

significantly reduced haptoglobin during the first LPS challenge and led to a significant decrease 

in SCC 10 days after supplementation ended, but had no effect on DMI, milk or component 

yields, or any of the other analytes measured. BCP supplementation resulted in a higher peak for 

SCC at 16 h after the second challenge, but similarly had no impact on DMI, milk or component 

yields, or any of the other analytes measured. When comparing the first challenge to the second 

challenge there was a tendency for cows to have lower levels of haptoglobin during the second 

challenge. Considering the anti-inflammatory effects of BCP it may have lowered the 

haptoglobin levels  following the first challenge, while trained immunity may be the reason for 

35 

 
 
 
 
 
 
the lower levels of haptoglobin when comparing the two challenges, as well as the higher peak 

for SCC observed in the second LPS challenge.  

This study concludes that further work should be done to determine if BCP 

supplementation would alter the inflammatory response upon repeated LPS challenges. Work 

investigating BCP supplementation for both of the LPS challenges is warranted. Evaluation of  

BCP supplementation on the inflammatory response will potentially help the current problem of 

susceptibility to some infectious diseases further lessening the need of antibiotics. With this 

study, we conclude that BCP affects haptoglobin and SCC of dairy cows experiencing an 

intramammary  LPS challenge.  

36 

 
 
 
 
 
 
 
 
 
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47 

 
 
 
 
 
 
 
TABLES AND FIGURES 

APPENDIX 

50.70 
15.76 
25.50 

SD 
2.455 
0.802 
1.298 

Table 1. Nutrient composition of the total mixed ration fed to lactating dairy cows throughout 
the experiment1. 
Item, %DM  Mean  
DM 
CP 
Starch 
ESC2 
NDF 
Lignin  
Crude Fat  
Ash  
Ca  
P  
Mg 
 K 
S 
Na  
Cl 

4.64 
32.92 
3.63 
3.79 
6.45 
0.81 
0.38 
0.34 
1.23 
0.19 
0.35 
0.28 

1.369 
2.061 
0.347 
0.256 
0.483 
0.219 
0.109 
0.095 
0.275 
0.011 
0.083 
0.025 

1n = 5 composite samples  analyzed 
2ESC = ethanol soluble carbohydrates  

48 

 
 
 
 
 
 
 
 
 
Table 2. Effect of β-Caryophyllene (BCP) on feed intake, milk production, and milk 
composition prior to, during, and after intramammary challenges  with lipopolysaccharide (LPS).  

    Treatment  

BCP 

CON 

SEM 

P -values  

Trt   Day   Trt × Day   Lactgrp2 

Item  

Pre-challenge   

DMI, kg/d  
Milk, kg/d 
ECM, kg/d 
Fat, kg/d 
Protein, kg/d 
SCS1 
Post -challenge 1 
DMI, kg/d  
Milk, kg/d 
ECM, kg/d 
Fat, kg/d 
Protein, kg/d 
SCS1 
Post -challenge 2 

24.40 
35.30 
34.97 
1.39 
1.14 
0.52 

24.84 
30.51 
35.40 
1.39 
1.11 
2.12 

24.90 
37.00 
34.67 
1.43 
1.22 
1.07 

25.74 
33.31 
35.30 
1.46 
1.17 
2.85 

1.10 
2.22 
0.60 
0.25 
0.07 
0.35 

0.20 
3.03 
0.80 
0.10 
0.11 
0.36 

0.68  <0.01 
0.50  <0.01 
0.68  <0.01 
0.01 
0.67 
0.75 
0.39 
0.17 
0.14 

0.51  <0.01 
0.41  <0.01 
0.90  <0.01 
0.09 
0.55 
0.54  <0.01 
0.06  <0.01 

0.26 
0.76 
0.89 
0.81 
0.72 
0.71 

<0.01 
0.84 
0.72 
0.92 
0.33 
<0.01 

0.81 
<0.01 

<.0001 

0.02 
0.06 
0.28 
0.10 
0.02 
0.96 

0.03 
0.46 
0.96 
0.73 
0.28 
0.63 

0.77 
0.44 
0.63 

Milk, kg/d 
ECM, kg/d 
SCS1 

2.71 
0.95 
0.45 
1Somatic cell score (SCS) = log2 (SCC/100,000)+3 
2Lactgrp = lactation group, primiparous  vs. multiparous   

26.86 
35.73 
8.23 

30.00 
35.12 
7.82 

0.38  <0.01 
0.53  <0.01 
0.29 

49 

 
 
 
 
 
 
  
  
  
  
  
  
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
Table 3. Effect of β-caryophyllene (BCP) on acute phase proteins after intramammary 
challenges  with lipopolysaccharide (LPS).  

Item  

    Treatment   SEM  P -values  

BCP  CON 

Trt  

Time    Trt × 

Lactgrp1  BLV  

Time    

LPS Challenge 1  

BHB, mmol/L 
Glucose, mg/dL 
Haptoglobin, mg/L 
Insulin, ng/mL 
LBP, mg/L 
LPS Challenge 2 

0.05 
0.59 
0.52 
72.98  75.06 
3.02 
79.87  117.88  12.52 
0.21 
1.33 
1.48 
1.06 
6.49 
6.41 

BHB, mmol/L 
Glucose, mg/dL 
Haptoglobin, mg/L 
Insulin, ng/mL 
LBP, mg/L 

0.51 
0.49 
77.29  76.58 
52.38  68.84 
1.28 
1.38 
7.45 
6.28 

0.03 
4.40 
14.19 
0.19 
0.80 

0.16 
0.50 
<0.01 
0.50 
0.95 

0.42 
0.87 
0.26 
0.47 
0.16 

<0.01  <.04 
0.96 
<0.01 
0.22 
<0.01 
0.78 
<0.01 
0.11 
<0.01 

<0.01 
0.39 
<0.01 
<0.01 
<0.01 

0.79 
0.39 
0.38 
0.13 
0.67 

0.92 
0.56 
0.01 
0.76 
0.49 

0.60 
0.53 
0.80 
0.29 
0.03 

0.26 
0.13 
0.63 
0.42 
0.87 

0.28 
0.55 
0.82 
0.51 
0.66 

1Lactgrp = lactation group, primiparous  vs. multiparous 

Table 4. Effect of β-caryophyllene (BCP) on the area under the curve of acute phase proteins 
and body temperature after intramammary challenges  with lipopolysaccharide (LPS). All 
reported values are relative to the basal period. 

LPS Challenge  

P -values  

Item  

First  

Second  

SEM 

Trt   Lactgrp  BLV   Challenge  
0.84 
-1.75 
-3.17 
BHB  
-140.61  0.64 
-28.61 
Glucose  
0.06 
45739 
543291 
Haptglobin  
0.95 
5.06 
18.29 
Insulin  
0.75 
28955 
496314 
LBP 
     Body Temp  
0.29 
50.65 
538.88 
1Lactgrp = lactation group, primiparous  vs. multiparous 

-1.42 
112.00 
353952 
19.22 
444139 
552.52 

0.37 
0.57 
<.01 
0.86 
0.75 
0.29 

0.41 
0.02 
<.01 
0.78 
0.35 
0.11 

0.3 
0.71 
0.64 
0.39 
0.96 
0.9 

50 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 5. Effect of β-caryophyllene (BCP) on bodyweight and body condition score change in 
late lactation cows challenged with intramammary  lipopolysaccharide  (LPS).  

Item  

  Treatment  

BCP  CON 

SEM 

P -values  

Trt   Week   Trt × Week   Lactgrp2  BLV  

BW, kg/d 

692.81  718.64 

28.33 

0.37  <0.01 

BCS, units/wk1 
1Measured on a 1-5 scale (Wildman et al., 1982) 
2Lactgrp = lactation group, primiparous  vs. multiparous 

3.33 

3.29 

0.04 

0.81 

0.18 

0.75 

0.46 

<0.01 

0.48 

0.8 

0.32 

51 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1. Effect of β-caryophyllene (BCP) on intravaginal body temperature after the first 
lipopolysaccharide  challenge. The solid vertical line indicates the time of the lipopolysaccharide 
challenge. Supplementing BCP did not affect body temperature after the first lipopolysaccharide 
challenge (P = .83). 

52 

 
 
 
 
 
 
 
 
 
 
Figure 2. Effect of β-Caryophyllene (BCP) on body temperature after the second 
lipopolysaccharide  challenge. BCP was not supplemented during the second lipopolysaccharide 
challenge. the solid vertical line indicates the time of the lipopolysaccharide challenge. 
Supplementing BCP did not affect body temperature after the second lipopolysaccharide 
challenge (P = .81).  

53 

 
 
 
 
 
 
 
 
 
Figure 3. Composite somatic cell score (SCS = log2 [SCC / 100,000] + 3) of dairy cows fed β-
caryophyllene  (BCP) or a no-treatment control (CON). Cows were challenged with 
lipopolysaccharide  in their right rear mammary  gland, and results are shown for the challenged 
quarter. Treatment tended to affect SCS (P = 0.06), and there was a significant treatment × time 
interaction (P < 0.01), reflecting a tendency for BCP to reduce SCS relative to CON on d 24 
post-challenge (P = 0.06). 

54 

 
 
 
 
 
 
 
 
 
Figure 4. Quarter level somatic cell score somatic cell score (SCS = log2 [SCC / 100,000] + 3) 
of dairy cows fed β-caryophyllene (BCP) or a no-treatment control (CON). Cows were 
challenged a second time with lipopolysaccharide  in their right rear quarter. Treatment  did not 
affect SCS (P = 0.43). There was a significant effect of BCP supplementation (P < 0.01) at 16 
hours post challenge (P < 0.01). 

55