The Pennsylvania State University

The Graduate School

EFFECTS OF RUMEN-PROTECTED CAPSICUM OLEORESIN ON FEED INTAKE,

GROWTH PERFORMANCE, HEALTH STATUS, AND DIGESTIBILITY IN

GROWING BEEF CATTLE FED GRAIN-BASED DIETS

A Thesis in

Animal Science

by

Mariana Fontana Westphalen

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Master of Science

August 2020 ii

The thesis of Mariana Fontana Westphalen was reviewed and approved by the following:

Tara L. Felix Assistant Professor and Beef Extension Specialist Thesis Co-Advisor

Alexander N. Hristov Distinguished Professor of Dairy Nutrition Thesis Co-Advisor

William Burton Staniar Associate Professor of Equine Science

Terry D. Etherton Distinguished Professor of Animal Nutrition Head of the Department of Animal Science

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Abstract

Increasing concerns related to antimicrobial resistance derived from the use of antibiotics in the animal industry have raised the demand for alternative products.

Capsaicinoids are substances found in fruits of Capsicum plants, commonly known as hot peppers. Capsaicinoids have been reported to have antimicrobial properties and were initially studied in ruminants as a potential modifier of ruminal fermentation. Further than antimicrobial properties, capsaicinoids have been reported to have direct effects on the host. For example, in poultry and swine the supplementation with capsaicin reduced susceptibility disease, prevented disease symptoms and improved intestine health. It has been reported to regulate appetite in and stimulate digestive enzymes (lipase, amylase, trypsin and chymotrypsin) in rodents. And to have immunoregulatory effects, decrease inflammatory response and increase milk production in dairy cows when fed in a rumen-protected form. Effects of rumen-protected

Capsicum supplementation and its potentially beneficial host-related responses have not been widely investigated in beef cattle.

In two experiments, the present study investigated the effects of rumen-protected

Capsicum (RPC) supplementation on feed intake, growth performance, health status, digestibility and ruminal pH in beef cattle. The first experiment investigated the effects of RPC supplementation on feed intake, performance, nutrient utilization, health status and immune response of beef calves fed grain-based diets for 100 days. Cattle were stratified by sex (steers

= 24; heifers = 12) and body weight (BW; heavy or light) and assigned to treatments: Control

(no additive), or 15RPC (15 mg of RPC/kg of diet dry matter (DM)). Cattle were transitioned over 21 days to a final diet of 80% grain mix and 20% corn silage (DM basis). Blood samples were collected on days 1, 22, and 98 for analysis of parameters related to immune function and nutrient utilization, and health status and medical treatments were recorded. Dry matter intake

(DMI), average daily gain (ADG) and feed efficiency were not different (P ≥ 0.24) between iv treatments throughout the 100 days, but cattle fed 15RPC gained (P = 0.07) 23.3% more weight during the first 50 days of the trial. Blood parameters were not different (P ≥ 0.17) between treatments except for hemoglobin (P = 0.03). Analysis of the health records indicated that 90% of sickness occurrences happened within the two first weeks of the experimental period, and that a greater (P = 0.09) percentage of light weight animals fed Control had fever 2 or more times during the trial when compared the animals fed 15RPC (76.5 vs. 21.8%). In experiment two, effects of RPC supplementation were investigated on total tract digestibility and ruminal pH of cannulated steers fed a 90% grain mix and 10% grass hay. Treatments were Control (no additive) and RPC at 3 levels of dietary inclusion: 5 , 10 , or 15 mg of RPC/ kg of diet DM. In this experiment, ruminal pH and total tract apparent digestibility of DM, OM, CP and NDF were not different (P ≥ 0.32) between treatments. The improvement in ADG during the first 50 days of calves fed 15RPC on the first experiment is not related to DMI and seem to be unrelated to nutrient metabolism and digestibility or ruminal fermentation parameters as indicated by results of the second experiment. Changes in health status could have influenced the observed changes in ADG but unaltered blood parameters related to immune response do not corroborate with this result. Lack of response in blood parameters likely happened due to limitations imposed by blood sampling time points. More frequent blood sampling during the critical phase of transition may contribute to detect earlier differences in immune status of cattle, and more data is necessary to confirm the validity of the results.

Keywords: beef cattle, Capsicum, digestibility, feedlot, immune status v

Table of Contents

List of Figures ...... vi List of Tables ...... vii Aknowlegment ...... vii Chapter 1: Introduction ...... 1 Chapter 2: Review of literature ...... 5 Introduction to Capsicum ...... 5 Active compounds that elicit the effects of Capsicum ...... 5 Creating products for use - Capsicum oleoresins ...... 7 Absorption and metabolism of capsaicinoids...... 7 Functionality of capsaicinoids ...... 10 Capsaicinoids antimicrobial activity and potential use as a rumen modifier ...... 11 Mechanism of animal responses to capsaicinoids ...... 15 Summary ...... 30 References ...... 32 Chapter 3:Effects of feeding rumen-protected Capsicum oleoresin on growth performance, health status, and total tract digestibility of growing beef cattle ...... 40 Abstract ...... 40 Introduction ...... 41 Materials and Methods ...... 43 Experiment 1...... 43 Experiment 2...... 47 Results ...... 50 Experiment 1...... 50 Experiment 2...... 51 Discussion ...... 51 Conclusion ...... 57 References ...... 58 Chapter 4: Conclusion...... 73

vi

List of Figures

Figure 3-1. Effects of rumen protected Capsicum (RPC) on average daily gain (ADG) of cattle fed grain-based diets over time in Exp. 1. Cattle were fed the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). There was a trend of increased (P = 0.13; SEM=0.09) ADG for animals fed 15RPC from d1 to 21 when compared to animals fed Control and ADG was greater (P = 0.07; SEM = 0.08) for animals fed 15RPC from d1 to 50 when compared to animals fed Control. There was no difference (P = 0.57) in ADG between treatments from d51 to 100 ………………………………………………………………….…69 Figure 3-2. Effects of rumen protected Capsicum (RPC) on dry matter intake (DMI) of cattle fed grain-based diets over time in Exp. 1. Cattle were fed the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). There were no effects of treatment on DMI (P > 0.10) ………………………………………70 Figure 3-3. Effects of rumen protected Capsicum (RPC) on gain to feed ratio (G:F) of cattle fed grain-based diets over time in Exp. 1. Cattle were fed the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). Gain to feed ratio was greater (P = 0.07; SEM = 0.010) for animals fed 15RPC from d1 to 50 when compared to animals fed Control. There was no difference (P > 0.10) in G:F between treatments for remaining intervals………………………….….71 Figure 3-4. Effects of Rumen Protected Capsicum (RPC) on ruminal pH in steers fed corn- based diets in Exp 2. Steers were fed the treatments Control (●) , 5RPC (■), 10RPC (♦) and 15RPC (▲), containing 0, 5, 10 and 15 mg of RPC/kg of diet DM, respectively. There was an effect of hour of sampling (P < 0.01) on ruminal pH, however there was no linear (P = 0.85) or quadratic (P = 0.65) effect of treatment nor treatment x hour interaction (P = 0.33). Standard error bars depict the variation associated with the interaction of treatment × hour (SEM = 0.19). One steer fed 15RPC was removed from period 3 due to low intake unrelated to treatment…………………………………………………………………….72

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List of Tables

Table 3-1. Basal diet used to transition cattle to final feedlot diets with 0 (Control) or 15 mg of RPC/kg of diet DM (RPC1) in Exp. 1……………………………………….61 Table 3-2. Composition of diets fed to cattle in Exp. 2……………………………………62 Table 3-3. Effects of rumen protected capsicum (RPC) inclusion on growth performance of feedlot cattle fed for 100 days in Exp. 1……………………………….……….63 Table 3-4. Effects of rumen protected Capsicum (RPC) supplementation on cell blood count of feedlot cattle in Exp. 1………………………………………………………64 Table 3-5. Effects of rumen protected Capsicum (RPC) supplementation on blood metabolites of feedlot cattle in Exp. 1…………………………………………65 Table 3-6. Absolute counts of medical treatments that occurred in Exp 1.……………….66 Table 3-7. Health status of cattle in Exp. 1………………………………………………..67 Table 3-8. Effects of Rumen protected Capsicum inclusion on intake and total tract apparent nutrient digestibility of steers in Exp. 2…………………………………….….68

viii

Acknowledgment

I would like to express my gratitude to the people who guided and assisted me throughout these studies. First, I would like to thank my advisor, Dr. Tara Felix, for accepting me as a student, provide me guidance and valuable advice, and for all the patience, time and effort invested. I would like to thank my co-advisor, Dr. Alexander Hristov, for the research advice and the great opportunity to start a master’s at Penn State. And thank Dr. Burt Staniar for being a valuable member of my committee and the wise advices throughout this project.

Thank you to everyone in Felix’s and Hristov’s lab groups for their help and support with the research projects. I thank the fellow graduate students Ali, Camila, Audino, Sergio,

Hannah, Irene and Jasmine for their time to help with sample collection. I am especially thankful to my lab mate Pedro Carvalho, for the innumerous hours spent helping me at the farm and the always encouraging conversations, and Susanna for teaching me performing determined analysis and running equipment. I appreciate the help of undergraduate students

Katherine Elder and Clayton Wagner who helped me with sample collection and lab analysis.

And would also like to thank Wendall, Brooke, Nadine, Travis and the beef center and dairy barn staff for their support at the farm.

My gratitude extends to the friends I encountered during graduate school, in especial

Margie with her wise life advices, and Karisa with all her kindness. Finally, I am immensely grateful to my parents, Mauricio and Marindia, and my brother Matheus, that even far away have being always loving, supportive and encouraging. 1

Chapter 1

Introduction

Growing concerns related to antimicrobial resistance have raised the demand for alternatives to the use of antibiotics in animal production (Lillehoj et al., 2018; Wong, 2019).

Antibiotics have been used for decades in animal production not only to treat disease and reduce mortality, but also to promote growth and to improve feed utilization (Kirchhelle,

2018). In ruminants, the use of ionophores, a class of antibiotics used to modulate ruminal fermentation, is a common practice. Ionophores inhibit the growth of gram-positive bacteria, favoring the selection of the gram-negative, leading to a decrease in the production of acetate, methane, and ammonia (Bergen and Bates, 1984; Duffield et al., 2012). These changes enhance the energy and nitrogen utilization in cattle fed ionophores, making them more efficient. However, with increased public concern about antimicrobial resistance, alternative supplements are being explored to replace antibiotics.

One such alternative being investigated is capsaicin. Capsaicin is the main active compound present in the fruits and extracts, or oleoresins, from plants of genus Capsicum, commonly known as hot peppers. Capsaicin has been reported to have antimicrobial properties against gram positive and gram negative bacteria (Deans and Ritchie, 1987), and for such reason have been investigated as a potential modifier of ruminal fermentation in cattle (Calsamiglia et al., 2007; Oh et al., 2017c). However, studies in beef cattle reported no or subtle changes in ruminal fermentation parameters of heifers supplemented with Capsicum oleoresin (Cardozo et al., 2006; Fandiño et al., 2008); and no effects were reported in dairy cows (Tager and Krause, 2011; Oh et al., 2015).

While the effects of Capsicum oleoresin supplementation on ruminal fermentation have been negligible, studies in non-ruminants associated the positive effects of Capsicum 2 supplementation to a host response rather than direct antimicrobial effects. Supplementation of Capsicum alone, or in combination with other plant extracts, has been reported to reduce susceptibility to disease or prevent disease symptoms (McElroy et al., 1994; Orndorff et al.,

2005; Lee et al., 2013; Liu et al., 2013) and improved intestine health (Shahverdi et al., 2013;

Liu et al., 2014a; Liu et al., 2014b) in both poultry and swine.

In dairy cows, the supplementation of Capsicum oleoresin has been reported to alter immune response and increase milk yield without altering rumen parameters (Oh et al., 2013;

Oh et al., 2015), supporting the hypothesis of benefits related to a host response. However, to exert host-related beneficial effects only, compounds must be functional post-rumen. Thus, supplementation of rumen-protected forms of Capsicum oleoresin is necessary. Feeding rumen-protected Capsicum oleoresin increased milk yield and feed efficiency, and decreased insulinemic and inflammatory responses in dairy cows (Stelwagen et al., 2016; Oh et al.,

2017a; Oh et al., 2017b).

Because they are fed more grain-based diets, beef cattle may be more susceptible to chronic inflammation due to greater ruminal accumulation of short chain fatty acids and lipopolysaccharides originated from bacteria shedding than dairy cattle (González et al.,

2012). It is known that inflammatory processes represent energy expenditure, energy that could have been directed towards production (Chioléro et al., 1997). While reducing the amount of grain fed may reduce susceptibility to inflammation, it negatively impacts gain.

Alternatively, strategies to optimize the post-ruminal digestion and absorption of feed and maintain effective immune status can potentially lead to improved performance (Celi et al.,

2019).

Based on the evidence from studies with non-ruminants and dairy cows, the supplementation of a rumen-protected form of Capsicum oleoresin could potentially exhibit 3 beneficial post ruminal responses in beef cattle as well. However, the post ruminal effects of

Capsicum spp., have not been widely explored in beef cattle. Therefore, there is a dearth of information regarding the effects of a rumen-protected form of Capsicum oleoresin as feed additive on feed intake, growth performance, digestibility, and immune status of beef cattle.

References Bergen, W. G., and D. B. Bates. 1984. Ionophores: Their Effect on Production Efficiency and Mode of Action. J. Anim. Sci. 58:1465–1483. Calsamiglia, S., M. Busquet, P. W. Cardozo, L. Castillejos, and A. Ferret. 2007. Invited review: Essential oils as modifiers of rumen microbial fermentation. J. Dairy Sci. 90:2580–2595. Cardozo, P. W., S. Calsamiglia, A. Ferret, and C. Kamel. 2006. Effects of alfalfa extract, anise, capsicum, and a mixture of cinnamaldehyde and eugenol on ruminal fermentation and protein degradation in beef heifers fed a high-concentrate diet. J. Anim. Sci. 84:2801–2808. Celi, P., V. Verlhac, E. Pérez Calvo, J. Schmeisser, and A. M. Kluenter. 2019. Biomarkers of gastrointestinal functionality in animal nutrition and health. Anim. Feed Sci. Technol. 250:9–31. Chioléro, R., J.-P. Revelly, and L. Tappy. 1997. Energy metabolism in sepsis and injury. Nutrition. 13:45–51. Deans, S. G., and G. Ritchie. 1987. Antibacterial properties of plant essential oils. Int. J. Food Microbiol. 5:165–180. Duffield, T. F., J. K. Merrill, and R. N. Bagg. 2012. Meta-analysis of the effects of monensin in beef cattle on feed efficiency, body weight gain, and dry matter intake1. J. Anim. Sci. 90:4583–4592. Fandiño, I., S. Calsamiglia, A. Ferret, and M. Blanch. 2008. Anise and capsicum as alternatives to monensin to modify rumen fermentation in beef heifers fed a high concentrate diet. Anim. Feed Sci. Technol. 145:409–417. González, L. A., X. Manteca, S. Calsamiglia, K. S. Schwartzkopf-Genswein, and A. Ferret. 2012. Ruminal acidosis in feedlot cattle: Interplay between feed ingredients, rumen function and feeding behavior (a review). Anim. Feed Sci. Technol. 172:66–79. Kirchhelle, C. 2018. Pharming animals: a global history of antibiotics in food production (1935–2017). Palgrave Commun. 4:96. Lee, S. H., H. S. Lillehoj, S. I. Jang, E. P. Lillehoj, W. Min, and D. M. Bravo. 2013. Dietary supplementation of young broiler chickens with Capsicum and turmeric oleoresins increases resistance to necrotic enteritis. Br. J. Nutr. 110:840–847. Lillehoj, H., Y. Liu, S. Calsamiglia, M. E. Fernandez-Miyakawa, F. Chi, R. L. Cravens, S. Oh, and C. G. Gay. 2018. Phytochemicals as antibiotic alternatives to promote growth and enhance host health. Vet. Res. 49:76. 4

Liu, Y., T. M. Che, M. Song, J. J. Lee, J. A. S. Almeida, D. Bravo, W. G. Van Alstine, and J. E. Pettigrew. 2013. Dietary plant extracts improve immune responses and growth efficiency of pigs experimentally infected with porcine reproductive and respiratory syndrome virus. J. Anim. Sci. 91:5668–5679. Liu, Y., M. Song, T. M. Che, D. Bravo, C. W. Maddox, and J. E. Pettigrew. 2014a. Effects of capsicum oleoresin, garlic botanical, and turmeric oleoresin on gene expression profile of ileal mucosa in weaned Pigs. J. Anim. Sci. 92:3426–3440. Liu, Y., M. Song, T. M. Che, J. J. Lee, D. Bravo, C. W. Maddox, and J. E. Pettigrew. 2014b. Dietary plant extracts modulate gene expression profiles in ileal mucosa of weaned pigs after an Escherichia coli infection. J. Anim. Sci. 92:2050–2062. McElroy, A. P., J. G. Manning, L. A. Jaeger, M. Taub, J. D. Williams, and B. M. Hargis. 1994. Effect of Prolonged Administration of Dietary Capsaicin on Broiler Growth and Salmonella enteritidis Susceptibility. Avian Dis. 38:329. Oh, J., F. Giallongo, T. Frederick, J. Pate, S. Walusimbi, R. J. Elias, E. H. Wall, D. Bravo, and A. N. Hristov. 2015. Effects of dietary Capsicum oleoresin on productivity and immune responses in lactating dairy cows. J. Dairy Sci. 98:6327–6339. Oh, J., M. Harper, F. Giallongo, D. M. Bravo, E. H. Wall, and A. N. Hristov. 2017a. Effects of rumen-protected Capsicum oleoresin on immune responses in dairy cows intravenously challenged with lipopolysaccharide. J. Dairy Sci. 100:1902–1913. Oh, J., M. Harper, F. Giallongo, D. M. Bravo, E. H. Wall, and A. N. Hristov. 2017b. Effects of rumen-protected Capsicum oleoresin on productivity and responses to a glucose tolerance test in lactating dairy cows. J. Dairy Sci. 100:1888–1901. Oh, J., A. N. Hristov, C. Lee, T. Cassidy, K. Heyler, G. A. Varga, J. Pate, S. Walusimbi, E. Brzezicka, K. Toyokawa, J. Werner, S. S. Donkin, R. Elias, S. Dowd, and D. Bravo. 2013. Immune and production responses of dairy cows to postruminal supplementation with phytonutrients. J. Dairy Sci. 96:7830–7843. Oh, J., E. H. Wall, D. M. Bravo, and A. N. Hristov. 2017c. Host-mediated effects of phytonutrients in ruminants: A review. J. Dairy Sci. 100:5974–5983. Orndorff, B. W., C. L. Novak, F. W. Pierson, D. J. Caldwell, and A. P. McElroy. 2005. Comparison of Prophylactic or Therapeutic Dietary Administration of Capsaicin for Reduction of Salmonella in Broiler Chickens. Avian Dis. 49:527–533. Shahverdi, A., F. Kheiri, M. Faghani, Y. Rahimian, and A. Rafiee. 2013. The effect of use red pepper (Capsicum annum L) and black pepper (Piper nigrum L) on performance and hematological parameters of broiler chicks. Eur. J. Zool. Res. 2:44–48. Stelwagen, K., E. H. Wall, and D. M. Bravo. 2016. 1395 Effect of rumen-protected capsicum on milk production in early lactating cows in a pasture-based system. J. Anim. Sci. 94:675–675. Tager, L. R., and K. M. Krause. 2011. Effects of essential oils on rumen fermentation, milk production, and feeding behavior in lactating dairy cows. J. Dairy Sci. 94:2455–2464. Wong, A. 2019. Unknown Risk on the Farm: Does Agricultural Use of Ionophores Contribute to the Burden of Antimicrobial Resistance? B. M. Limbago, editor. mSphere. 4. 5

Chapter 2

Review of literature

Introduction to Capsicum

Capsicum is a genus of plants, originally from the Central and South Americas, that belongs to the Solanaceae family. This genus includes several species of peppers widely variable in their pungency. Although there are over 20 documented species of Capsicum, only five are commonly recognized as domesticated, including C. annuum, C. bacatum, C. chinense, C. frutescens, and C. pubescens (Berke and Shieh, 2012). Fruits of Capsicum have been used as spices since 7000 B.C. by Mexican Indians, while Capsicum cultivation dates back to as early as 5200 B.C. (Heiser, 1969).

During Columbus’ expedition to America in the late 1400s, he observed the use of

Capsicum fruits by natives as condiment and in medicine; and took specimens of Capsicum back to Europe (Govindarajan and Salzer, 1985; Andrews, 1992). By the late 1500s and early

1600s Capsicum cultivation, mainly C. annuum and C. frutescens, had been established throughout Central Europe and spread to countries in Africa and Asia (Andrews, 1992).

Active compounds that elicit the effects of Capsicum

The pungent property of Capsicum fruits is attributed to a group of phenolic substances called capsaicinoids. Capsaicin was the first of the capsaicinoids to be discovered.

First isolated by Thresh (1877), capsaicin’s chemical formula was determined by Nelson and

Dawson (1923). Since then, several other analogs to capsaicin have been discovered, including dihydrocapsaicin, nonivamide, nordihydrocapsaicin, homocapsaicin and homodihydrocapsaicin (Yang and Du, 2018).

Capsaicinoids are synthesized in the epidermal cells of the fruit placenta, the pith where the seeds attach to, in a process mediated by the enzyme capsaicinoid synthase (Aza- 6

González et al., 2011). Capsaicinoid synthase joins a molecule of vanillylamine (derived from phenylalanine) to a branched fatty acid that is synthesized from valine or leucine and contains from 9 to 11 carbons. After synthesis, capsaicinoids are secreted and accumulate inside vesicles on the placenta surface of the fruit. The capsaicinoid profile differs depending on species, cultivar, maturity, and environmental conditions in which the plant grew.

Although the composition varies, capsaicin and dihydrocapsaicin are usually the predominant analogs found in most Capsicum fruits, accounting for 80 to 90% of the total capsaicinoids (Aza-González et al., 2011; Al Othman et al., 2011). Bennett and Kirby (1968) reported a capsaicinoid profile for C. annuum of 69% capsaicin, 22% dihydrocapsaicin, 7% nordihydrocapsaicin, 1% homocapsaicin, and 1% homodihydrocapsaicin. Choi et al. (2006) reported that in eleven different varieties of C. annuum the capsaicinoids concentration in the fruit varied from 1.2 to 121.0 µg capsaicinoids/g of fresh weight. In addition, these authors stated that for the two varieties with highest capsaicinoids content, “Chung yang” and

“Buchon”, the proportions of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin, and homodihydrocapsaicin were 29.4, 54.1, 4.4, 6.3, and 5.3% versus 51.7,

29.9, 8.1, 5.1, and 4.6%, respectively. More recently, Wesołowska et al. (2011) reported C. annuum cultivars with 37% capsaicin, 29 % dihydrocapsaicin, and 2% homodihydrocapsaicin.

The pungency of capsaicin and dihydrocapsaicin is similar (Kosuge and Furuta, 1970) and can be characterized by human perception based on Scoville heat units (SHU; Scoville,

1912). The SHU is a scale that assess the heat sensation derived from capsaicinoids. Weiss

(2002) classifies the levels of pungency into non-pungent (up to700 SHU), mildly pungent

(700 to 3,000 SHU), moderately pungent (3,000 to 25,000 SHU), highly pungent (25,000 to

70,000 SHU), and very highly pungent (>80,000 SHU). According to Todd et al. (1977), capsaicin and dihydrocapsaicin have 16.1 x 106 SHU, homocapsaicin has 6.9 x 106 SHU, 7 homodihydrocapsaicin has 8.1 x 106 SHU, and nordihydrocapsaicin has 9.3 x 106 SHU, making them all very highly pungent compounds.

Creating products for use - Capsicum oleoresins

Aside from the fresh and dried fruits, Capsicum can also be used in the form of oleoresin. Oleoresin is the product obtained by extracting the functional components of the fruits with an organic solvent such as acetone, alcohol, chloroform, ether, or hexane, and then subsequently removing the solvent. Capsicum oleoresin usually consists of a mixture of oils, dyes, sterols, and waxes, has a viscous consistency and dark red color, and contains variable concentrations of capsaicinoids. The quality and composition of the oleoresin depend on the cultivar and method of extraction used (Govindarajan and Salzer, 1986).

Govindarajan and Salzer (1986) report a range of 1.4 to 8.6% capsaicinoid concentration in Capsicum oleoresins, on a dry matter basis. More recently a study by

Gudeva et al. (2013) evaluated two different methods of oleoresin extraction from 4 cultivars of C. annuum. Gudeva et al. (2013) reported a wide range in capsaicin concentration in the oleoresins obtained, from 2.13 to 18.88% (on a DM basis).

Absorption and metabolism of capsaicinoids

The Capsicum extracts or the purified capsaicin has been used in a variety of pharmaceutical applications. In fact, the absorption of capsaicin in topical, subcutaneous, and oral administration has been well documented in humans rodents (Rollyson et al., 2014).

However, for the purpose of this review, solely the mechanisms involved in oral administration will be addressed.

Previous studies report a rapid absorption of capsaicinoids in the gastric stomach and intestines after oral administration. In a in vivo study by Kawada et al. (1984), fasted rats were fed a diet containing 3 mg of a capsaicin mixture (85% capsaicin and 15% 8 dihydrocapsaicin) through a stomach tube. The rats were euthanized at various time-points post feed administration and the concentration of capsaicin and dihydrocapsaicin in the gastrointestinal (GI) tract was determined. Rapidly absorption of capsaicinoids was observed in the stomach and small intestine. At 3 h post administration, just 15% of the original dose continued in the GI tract of the rats. No metabolite of capsaicin or dihydrocapsaicin was found in the GI contents. After 48 h, the amount of capsaicinoids left in the feces was less than 10% of the dose fed, indicating that more than 90% of the administered dose had been absorbed.

Suresh and Srinivasan (2010) corroborated the rapid absorption of capsaicinoids by evaluating the in vivo tissue distribution and subsequent elimination of capsaicin after oral administration (30 mg/kg of BW) in rats. Groups of rats were euthanized at different times after administration, and capsaicin concentration was analyzed in the liver, kidney, intestine, serum and blood, feces, and urine. The average concentration of capsaicin found across all tissues at 1 hour post administration was 24.4% of the initial dose. After 24 hours this percentage decreased to 1.2% and after 48 hours it further decreased to 0.057%. Blood and intestine had the highest concentration of capsaicin at 1 hour, while liver and kidney had the highest in 3 hours and 6 hours, respectively. No capsaicin was detected in tissues four days after oral administration. In addition, 6.3% of the initial dose of capsaicin remained in the feces, indicating that nearly 94% of dosed capsaicin was absorbed. Capsaicin was not detected in the feces after day 4. Only 0.095% of the capsaicin administered was excreted in the urine, no longer being detected in the urine after day 2. Suggesting that capsaicin is rapidly metabolized in the above tissues once absorbed.

Rollyson et al. (2014) reported that capsaicinoids are largely metabolized by the liver and in other tissues to a lesser extent(Rollyson et al., 2014). Kawada et al. (1984) demonstrated dihydrocapsaicin-hydrolyzing enzyme activity in jejunal tissue of rats. Through 9 the use of radioactive markers, these authors observed that 85% of the dihydrocapsaicin absorbed into the portal vein was detected in its original form, while about 15% was detected as 8-methylnonoanoic acid, a dihydrocapsaicin metabolite. The metabolism of dihydrocapsaicin following oral administration (20 mg/kg BW) was further studied in rats by

(Kawada and Iwai, 1985). Although previous reports have suggested little urinary excretion of capsaicin (Suresh and Srinivasan, 2010), analysis of urine samples revealed 8.7% of unchanged dihydrocapsaicin in the urine within 48 hours of administration (Kawada and

Iwai, 1985). Kawada and Iwai (1985) identified that the metabolites in the urine within 48 hours were vanillyl alcohol (37.6%), vanillic acid (19.2%), vanillylamine (4.7%), and vanillin

(4.6%), in their free forms or as their glucuronides. The authors also observed that the activity of dihydrocapsaicin hydrolyzing enzyme was maximal in the liver, followed by the kidney, lungs, small intestine, stomach, and was very low in the brain. Donnerer et al. (1990) also examined the metabolism and absorption of capsaicinoids in anesthetized rats through intragastric administration and reported that the concentration of intact capsaicin and dihydrocapsaicin was decreased in the gastrointestinal lumen compared to the concentration in portal vein blood. In addition, these authors reported that dihydrocapsaicin was almost completely metabolized in a liver tissue culture 30 minutes after incubation with dihydrocapsaicin.

More recently, Kuzma et al. (2015) analyzed the intestinal absorption and metabolism of capsaicin and dihydrocapsaicin in rats using ex-vivo perfusion of capsicum extract solution

(30 μg/mL) through the proximal jejunum. Analysis of capsaicinoid concentration in the perfusate indicated that capsaicinoids were rapidly absorbed in the jejunum. Glucuronide metabolites of capsaicin and dihydrocapsaicin were also identified in the perfusate. These results indicate that following intestinal metabolism of capsaicinoids by UDP- glucuronyltransferase enzymes, metabolites are secreted back into the intestinal lumen. 10

Reilly and Yost (2006) further described the metabolism of capsaicinoids by P450 enzymes in the liver. The metabolism by P450 enzymes occur through metabolic processes that involve alkyl dehydrogenation and oxygenation, aromatic hydroxylation, and O- demethylation of the capsaicinoid molecule. Chanda et al. (2008) also reported the metabolism of capsaicin by P450 enzymes in the liver. These authors detected five primary metabolites after incubation of microsomes and S9 fraction originated from liver tissue of dogs, rats, and humans in medium with concentrations of 1 to 10 μM of capsaicin. Those metabolites included 16-hydroxycapsaicin, 17-hydroxycapsaicin, 16,17-dehydrocapsaicin, vanillylamine, and vanillin. However, the rate and proportion of the metabolites generated was different among the species. In dog microsomes, for example, 30 minutes after incubation at 1 μM, 23% of capsaicin was still present, while in rat microsomes no capsaicin had been detected. Also, vanillylamine and vanillin were identified in the rat liver, but not in dogs or humans.

The rat has been the species most commonly used to study the metabolism of capsaicinoids, but taken together, these metabolism data suggest that capsaicinoid metabolism is variable among species (Chanda et al., 2008). Thus, the metabolism of capsaicinoids in the GI tract or liver of ruminants remains unknown.

Functionality of capsaicinoids

In addition to the organoleptic properties, the capsaicinoids have therapeutic and antimicrobial properties. These therapeutic and antimicrobial properties have made capsaicinoids target of recent research for their potential use in human and veterinary medicine (Calsamiglia et al., 2007; Hayman and Kam, 2008; Fattori et al., 2016; Srinivasan,

2016; Oh et al., 2017c; Adaszek et al., 2019). 11

Capsaicin, in particular, has been studied for use as antioxidant (Kempaiah et al.,

2005; Hassan et al., 2012), analgesic (Frias and Merighi, 2016) and anti-inflammatory (Deal et al., 1991; Kim et al., 2003; Lee et al., 2007; Liu et al., 2012; Allemand et al., 2016), anti- carcinogen (Zhang et al., 2008; Jeon et al., 2012), to reduce cholesterol and obesity (Ludy et al., 2012; Yu et al., 2012), and to protect gastric mucosa (Kang et al., 1995; Holzer and Pabst,

1999; Mózsik et al., 2005). Capsaicinoids have also been tested for their antimicrobial properties to reduce susceptibility to disease caused by pathogenic bacteria in poultry and swine (McElroy et al., 1994; Orndorff et al., 2005; Liu et al., 2013a; Liu et al., 2013b), and as a potential modulator of ruminal fermentation in beef and dairy cattle (Calsamiglia et al.,

2007; Tager and Krause, 2011; Oh et al., 2013; Oh et al., 2017c). While not discussed fully, some of the properties of capsaicinoids are outlined below to demonstrate that they are, without a doubt, far reaching according to previous literature.

The extensive nature of these effects may be related to the mode of action of capsaicinoids. The effects elicited by capsaicinoids may be directly on the animal, also known as host-related responses (Caterina et al., 1997), or they may be related to antimicrobial effects (Calsamiglia et al., 2007). Both of these modes of action will be explored further.

Capsaicinoids antimicrobial activity and potential use as a rumen modifier

As mentioned, capsaicinoids are a compound that has been under investigation for its antimicrobial properties. The extract of Capsicum fruits has been reported to have antibacterial effects on gram-positive and gram-negative bacteria in vitro (Deans and Ritchie,

1987; Cichewicz and Thorpe, 1996). In addition, the isolated compounds, like capsaicin and dihydrocapsaicin, elicit these antimicrobial effects on their own (Mokhtar et al., 2017).

Capsaicinoids antimicrobial properties are related to the presence of a functional phenolic ring (Patra, 2012). The phenol group of capsaicinoids acts on the bacteria cell 12 membrane. Due to the hydrophobic characteristic of the phenol group, it can accumulate on the lipid bilayer of the bacteria, altering the conformation of the bacterial cell membrane.

That alteration results in membrane damage and ion leakage across the membrane. While the bacteria attempt to maintain ion balance, the damage results in suppression of growth, or death, due to energy loss (Lucchini et al., 1990; Calsamiglia et al., 2007).

The antibacterial properties of capsaicinoids first led to the hypothesis that capsaicinoids could be used as a feed additive for ruminants to modify the ruminal microbial population and affect rumen fermentation parameters, inducing changes in short chain fatty acid (SCFA) and ammonia production. While no data has been reported on the effects of capsaicinoids on ruminal microbiota, some studies report the effects of capsaicinoids on ruminal fermentation parameters. An in vitro study by (Cardozo et al., 2005) tested the effects of Capsicum oleoresin (0, 0.3, 3, 30 and 300 mg/L) on rumen fluid from beef heifers fed a 10:90 forage:concentrate diet maintained at two different pH values, 5.5 and 7.0. The

300 mg/L dose greatly inhibited fermentation, regardless of pH, but the results for other doses were less drastic. At pH 7.0, authors reported a decrease of 9 to 11% in total SCFA concentration (mM) for doses 0.3, 3 and 30 mg/L when compared to control (0 mg capsaicin/L). These changes in total SCFA occurred while the propionate proportion decreased 16 to 26% and the acetate proportion increased from 2.7 to 8%, again, for doses

0.3, 3 and 30 mg/L when compared to control. At pH 5.5 there was a 22% increase in total

SCFA concentration and 27% increase in propionate proportion, while acetate proportion decreased from 9 to 22% for those same doses when compared to the non-supplemented control. Calsamiglia et al. (2007) suggested that the difference observed between the two pH values could possibly be due to increased antibacterial activity of the capsaicinoids at pH 5.5, that increased the hydrophobic nature of the capsaicinoid molecule, making it more active in certain types of cell membrane. These effects of pH on antimicrobial activity have not been 13 validated for the capsaicin molecule, but it has been reported in other phenolic compounds.

For example, caffeic acid antimicrobial activity decreased at pH values close to 7.0, with best results being reported at pH 5.0 (Almajano et al., 2007), as result of increased dissociation of acid molecules at higher pH values.

These in vitro studies provide some guidance to the actions of capsicum; but, the in vitro results have not always translated to in vivo studies responses in beef cattle. In beef heifers, feeding 1g Capsicum oleoresin/d with a 90% concentrate and 10% forage diet (DM basis) did not alter pH or total SCFA production when compared to heifers that were not fed the oleoresin. However, heifers fed Capsicum had a 5% reduction in the acetate proportion

(mol/100 mol) in the rumen when compared to heifers that were not supplemented (Cardozo et al., 2006). Similarly, the supplementation of Capsicum oleoresin (0.5g/d) to beef heifers fed a similar diet (90:10 concentrate:forage on a DM basis) did not alter ruminal pH or total

SCFA production, but decreased acetate proportion in about 2.5% when compared to heifers that were not fed capsicum (Fandiño et al., 2008). However, when beef heifers fed a 90:10 concentrate to forage ratio (DM basis) were supplemented with 3 different doses of Capsicum oleoresin (0.125, 0.250, and 0.500 g/d) total SCFA concentration tended to linearly increase in the rumen and pH tended to linearly decrease with increasing dose (Rodríguez-Prado et al.,

2012). Thus, there is some evidence to suggest that the impacts of Capsicum oleoresin, even though small, may be dose dependent in beef heifers.

However, contrary to the results observed in beef cattle, in vivo studies in dairy cows reported no ruminal effects of Capsicum supplementation (Tager and Krause 2011; Oh et al.,

2015). Tager and Krause (2011) supplemented Capsicum oleoresin (0.25 g/d; in a solid form) to dairy cows fed a 58:42 concentrate to forage ratio (DM basis), and observed no differences on ruminal pH, total SCFA concentration or its fractions (mmol/L). According to them, the different results might be attributed to the relatively low dose amount used when 14 compared to those of beef cattle studies. But higher doses also did not affect ruminal fermentation, as reported by (Oh et al., 2015). They reported that supplementing Capsicum oleoresin (0.25, 0.5 or 1 g/d) to dairy cows fed a 49:51 concentrate to forage ratio in the diet

(DM basis) did not affect ruminal pH or SCFA concentrations. One important difference to note between the beef and dairy studies is the concentrate to forage ratio fed in the respective studies. The mechanistic action of capsicum on bacterial cell membranes would support the suggestion that the responses to capsicum should be greater in cattle fed more grain, and this appears to be the case.

In addition to changes in fatty acid production, Capsicum oleoresin could be expected to reduce ammonia production by inhibiting growth of hyper-ammonia producing bacteria

(Patra, 2012). However, the effects of Capsicum oleoresin on ruminal ammonia concentration are inconsistent. Cardozo et al. (2005) observed a reduction in in vitro ammonia concentration when treating cultures with Capsicum. However, these in vitro results have not always been observed in vivo. Two reports suggested no effect on ruminal ammonia concentration when capsicum was fed to beef heifers (Cardozo et al.;2006; Fandiño et al.,

2008). However, Rodríguez-Prado et al. (2012) reported a tendency for increased ruminal ammonia concentration in heifers supplemented with Capsicum oleoresin (0.125, 0.250, and

0.500 g/d) when compared to heifers not supplemented. In dairy cows changes in ruminal ammonia concentrations also have not been observed (Tager and Krause, 2011; Oh et al.,

2015). According to (Calsamiglia et al., 2007) the subtle effects, or lack of effect, of

Capsicum oleoresin on ruminal fermentation parameters of dairy and beef cattle could be result of a low number of hydroxyl and carboxyl groups substitutions on the phenolic ring of the capsaicin molecule, which makes it a weaker anti-microbial when compared to other phenolic compounds. 15

Due to the negligible effects on the ruminal fermentation parameters (Calsamiglia et al., 2007; Oh et al., 2017c), and the positive results in studies using non-ruminant animals. It has been suggested that the host response may have a greater impact on livestock production than the antimicrobial effect of capsaicinoids. This hypothesis was recently tested in dairy cows and suggests that improvements in animal health and performance may be related to a host response rather than antimicrobial effects of capsaicinoids in the rumen (Oh et al., 2015;

Oh et al., 2017a; Oh et al., 2017b).

Mechanism of animal responses to capsaicinoids

Further investigation of the host related response to capsaicin suggest that capsaicin and its analogs exert their effects in the host by binding to and activating the Transient receptor potential cation channel subfamily V member 1 (TRPV-1; Caterina et al., 1997).

This receptor is a non-selective cation channel widely expressed in the body. It is present mainly in sensory neurons of several tissues such as brain, lungs, liver, spleen, intestines, kidneys, stomach, bladder, and reproductive tract; but also found on mucosal epithelial cells, epidermis, vascular endothelium, and some cells of the immune system (Nilius and Owsianik,

2011). TRPV-1 can be activated not only by capsaicinoids, but also by low pH (≤5.9), noxious heat (≥43.0 °C), mechanical stress, and other chemical irritants like ethanol (Omari et al., 2017). The activation of TRPV-1 results in an influx of ions with a preference for Ca2+.

The increase in intracellular calcium ultimately results on depolarization of the cell membrane and the release of neuropeptides, such as calcitonin-gene-related-peptide (CGRP) and substance P (Frias and Merighi, 2016).

The activation of TRPV-1 receptors present in nociceptive neuron cells, cells that relay the perception of pain, generates a signal that is perceived as a sensation of pain (Frias and Merighi, 2016). As agonists of TRPV-1 receptors, capsaicinoids can activate those ion channels generating a pain signal. In contrast, long-lasting or acute stimulation of TRPV-1 16 receptors by capsaicinoids may also inactivate the receptors. The inactivation of TRPV-1 in nociceptive nerves promotes a dose-dependent analgesic effect, as consequence of loss of function, or desensitization, that can be temporary or permanent. The desensitization is derived from the depletion of neuropeptides, and retraction of nerve fiber terminals due to

Ca2+ overload (Anand and Bley, 2011). This dose-dependent analgesic effect is the mechanism of topical application of capsaicinoids for pain relief, and the desensitization of neurons also provides approaches to neurological studies (Anand and Bley, 2011).

Because TRPV-1 receptors are present on a large number of cells and tissues, the potential of capsaicinoids for stimulating or desensitizing those receptors have been target of investigation not only on the treatment of pain, but also regarding their role on thermoregulation, gastrointestinal functions, feed intake regulation (Sharma et al., 2013), and their contribution to the initiation and modulation of inflammation (Richardson and Vasko,

2002). Those functions will be further discussed, but in general they are largely influenced by the release of substance P and CGRP. The inflammatory response initiated by the binding of those neuropeptides to their receptors on endothelial and smooth muscle cells is characterized by vasodilation, plasma extravasation, and hyperalgesia, that results in redness, swelling, and pain (Richardson and Vasko, 2002). Epithelial cells, mast cells, and some immune cells also respond to substance P and CGRP, releasing cytokines that contribute to a sustained inflammatory state.

Effects on the gastrointestinal tract

The GI tract is rich in TRPV-1 receptors. These receptors are expressed in epithelial cells and neural cells present on the mucosa, muscle layers, and blood vessels within the GI wall (Ward et al., 2003). Because of the ability of capsaicinoids to activate TRPV-1 17 receptors, their impacts on GI functions have been investigated in both humans and rat models (Abdel-Salam et al., 1995; Mózsik et al., 2007).

Abdel-Salam et al. (1995) investigated the effects of Capsicum supplementation on the protection of the gastric mucosa. They observed that the treatment with low doses (0.1 μg capsaicin/kg of BW) of capsaicin reduced severity of stomach lesions induced by aspirin, ethanol, and hydrochloric acid in rats. In human subjects, Mózsik et al. (2005) reported that intragastric administration of capsaicin through a nasogastric tube (100, 200, 400, and 800

μg) linearly decreased secretion of gastric acid and reduced induced gastric micro bleeding.

The protective effects of capsaicin administration can be associated to TRPV-1 activation on mucosa and sensory nerve terminals, with subsequent secretion of CGRP. Calcitonin gene- related peptide is a potent vasodilator that upregulates gastric blood mucosal flow through the activation of ATP-sensitive potassium channels (Peskar et al., 2002). The increase of blood flow can promote a protective effect on gastric mucosa that helps to neutralize and wash away the gastric acid. Additionally, CGRP has been reported to inhibit gastric acid secretion by stimulating the secretion of somatostatin, and increasing the secretion of mucus and bicarbonate (Martinez and Taché, 2006). On the other hand, large amounts of capsaicin may desensitize the GI tract sensory neurons and compromise the protective effects of capsaicin.

For example, stomach lesions induced by aspirin, ethanol, or HCl were aggravated in rats fed a high dose of capsaicin (10 mg/kg of BW) when compared to rats fed low dose (0.1 μg/kg of

BW) (Abdelsalam et al., 1995).

Capsaicinoids have also been reported to affect the small intestine ultra-structure, permeability and release of digestive enzymes (Srinivasan, 2016). Rats fed Capsicum peppers

(3.0% of diet) or capsaicin (0.1% of diet) for 8 weeks had increased fluidity and passive permeability of microvilli membrane of enterocytes, and increased microvilli length when compared to control, which resulted in an increased absorptive surface of the small intestine 18 in those rats fed either pepper of capsaicin (Prakash and Srinivasan, 2010). Rats fed capsaicin

(0.015% of diet) for 8 weeks had increased activity of several pancreatic (lipase and acid phosphatase) and enteric enzymes (sucrase and maltase; Platel and Srinivasan, 1996). In a similar trial, feeding capsaicin (0.015% of diet) to albino rats for 8 weeks also increased the enzymatic activity of the pancreatic enzymes lipase, amylase, trypsin and chymotrypsin

(Platel and Srinivasan, 2000). These data from rats suggest that capsaicinoids could increase digestive efficiency in livestock animals by increasing absorptive surface area or digestive enzymes. But, there are currently no data available on enzyme secretion in response to capsaicin supplementation in livestock.

However, when investigated in ruminants, no effects of dietary Capsicum oleoresin

(0.25 g/d) were observed on total tract digestibility of OM, DM, NDF, ADF, CP or starch of dairy cows (Tager and Krause, 2011). Oh et al. (2015) also did not observe differences in any digestibility parameter or nitrogen utilization in cows fed Capsicum oleoresin (0, 0.25, 0.50, or 1 g/d). In these previous reports, the lack of effect may be because Capsicum oleoresin was administrated in a non-protected form and degraded by ruminal microbes before reaching the intestine. However, the post-ruminal supply of Capsicum oleoresin (2g/d) through abomasal infusion also did not affect digestibility parameters nor N metabolism of cows (Oh et al.,

2013). These results conflict with later reports feeding lower doses of capsaicin. In dairy cows supplemented with a rumen-protected form of Capsicum oleoresin at 0.1 or 0.2 g/d a linear increase in apparent total tract digestibility of DM, OM, and CP was observed in response to supplementation (Oh et al., 2017a). These data corroborate previous reports that have suggest a dose-dependent response of capsaicin in animal diets (Abdelsalam et al., 1995,

Rodríguez-Prado et al., 2012).

While the supplementation of rumen-protected Capsicum oleoresin increased total tract digestibility in dairy cows (Oh et al., 2017a), the reasons for this response is unclear. 19

Previous studies with unprotected Capsicum oleoresin reported no differences in ruminal fermentation, indicating that this is likely not the cause of improved digestibility. Although no histological or digestive enzyme activity and secretion date were collected by Oh et al.

(2017a), studies in rats (Platel and Srinivasan, 2000; Prakash and Srinivasan, 2010) indicate that increased enzymatic secretion and activity and villi surface area could possibly play a role in increasing digestibility. The possible increase digestive enzymes activity and secretion as observed in non-ruminant species could be especially relevant in feedlot beef cattle fed grain-based diets due to the increased amounts of starch being digested in the small intestine in comparison to animals fed less grain (Huntington et al., 2006). However, the effects of rumen-protected Capsicum oleoresin supplementation on nutrient digestibility in beef cattle are not known.

Effects on immune response

In addition to altering digestion in the host in some instances, capsaicinoids can stimulate the immune system both directly and indirectly. Capsaicinoids directly stimulate the immune system by their binding to TRPV-1 receptors present on immune cells - including macrophages, neutrophils, T lymphocytes, and B lymphocytes. Once bound, capsaicinoids can either stimulate (through the release of substance P) or inhibit the production of cytokines and antibodies (through the release of Calcitonin gene-related peptide, or CGRP) that will regulate the immune response, as explained below. In addition to the direct effects, capsaicinoids can alter the immune response indirectly by neural stimulation. As stated previously, the binding of capsaicinoids to TRPV-1 receptors on neuron cells causes its activation with subsequent release of neuropeptides CGRP and substance P (Omari et al.,

2017). 20

Calcitonin gene-related peptide (CGRP), one of the neuropeptides released by TRPV-

1 activation of neural cells, can also be produced by activated immune cells (Wang et al.,

2002). The CGRP is a potent anti-inflammatory mediator that can modulate immune responses through direct effects on T lymphocytes, by inhibiting the production of pro- inflammatory cytokines such as tumor necrosis factor α (TNFα) and interferon- γ (IFN-γ).

These actions of CGRP ultimately reduce inflammation (Minter et al., 2001). Substance P, similar to CGRP, is released upon TRPV-1 activation in neural cell, and can also be produced by activated immune cells, such as macrophages, eosinophils and lymphocytes. Substance P is known to stimulate lymphocyte proliferation and immunoglobulin production, as well as the secretion of inflammatory mediators such as pro-inflammatory cytokines, oxygen radicals, and histamine, by lymphocytes, monocytes, macrophages, and mast cells, thus amplifying the inflammatory response (O’Connor et al., 2004). Eglezos et al. (1990) reported that a subcutaneous injection of capsaicin (50 mg/kg of BW) in rats resulted in depletion of substance P, with subsequent reduction of the number of B lymphocyte cells by 75%.

Once again, however, the effects of capsaicin on immune response may be dependent on the dose administered. For example, Demirbilek et al. (2004) reported that 1 mg of capsaicin/kg BW increased CGRP by about 55%, while a dose of 150 mg of capsaicin/kg BW decreased CGRP in nearly 30% in blood of septic rats when compared with non-treated septic rats. The reduction of CGRP in rats fed the higher dose may be caused by desensitization of the sensory nerves. As a result of CGRP reduction, pro-inflammatory cytokines TNFα and interleukin-6 (IL-6) increased (by 12.5 and 7.7%, respectively) in septic rats fed the 150 mg of capsaicin/kg of BW when compared to septic rats not fed capsaicin, indicating increased inflammation at the high dose supplementation.

Another example of the dose dependent response was reported by Nevius et al.

(2012). They tested the effects of capsaicin in type I diabetic mice. In type I diabetes, 21 pancreatic cells that produce insulin are attacked by T lymphocytes from the immune system.

The study reported that the oral administration of 10 μg of capsaicin had increased the anti- inflammatory cytokine interleukin 10 (IL-10) and decreased the T lymphocyte proliferation in the pancreas, resulting in suppression of diabetic symptoms for 20 weeks. In the same study, other doses of capsaicin (0, 0.1, 1, 25 or 50 μg) did not suppress the diabetic symptoms.

In food producing animals, Liu et al. (2012) reported that Capsicum oleoresin affected immune regulation in swine macrophages cultured in vitro. Capsicum oleoresin was dissolved to concentrations of 0, 25, 50, 100, and 200 μg/mL into a solution that was then added to the culture medium at a concentration of 0.05%. Increasing concentration of

Capsicum linearly decreased the pro-inflammatory cytokines TNFα, IL-1β, and TGF-β produced by the macrophages that were challenged with LPS. However, IL-1β concentrations were quadratically increased in macrophages treated with increasing concentrations of

Capsicum oleoresin that were not challenged with LPS, indicating that Capsicum the may have the potential to enhance immune responses in both immune challenged and “normal” conditions. These data are consistent with the follow up study by Liu et al. (2013), in which pigs challenged with porcine reproductive and respiratory syndrome virus were supplemented with 10 mg of Capsicum oleoresin/kg of diet DM. The authors reported a decrease in the concentrations of the pro-inflammatory cytokines TNFα and IL-1β, by 18.5 and 63.7% respectively, and an increase of 92% in the anti-inflammatory cytokine IL-10, in the virus- challenged pigs that were fed the Capsicum oleoresin when compared to those pigs that were not supplemented.

Liu et al. (2014a, 2014b) reported that supplementing Capsicum oleoresin (10 mg/kg of diet DM) to pigs upregulated gene expression profiles related to integrity of membranes in ileal mucosa of healthy pigs and pigs infected with Escherichia coli, indicating enhanced gut 22 mucosa barrier. In poultry, Prieto and Campo (2010) investigated the effects of capsaicin supplementation (50 mg/kg of diet DM) on neutrophil:lymphocyte of chickens under heat stress. They reported that supplemented chickens under heat stress had a 66% decrease in the neutrophils:lymphocyte compared to non-supplemented chickens under stress, suggesting that capsaicin have the potential to alleviate the effects of heat. The neutrophil:lymphocyte ratio is a reliable indicator of stress in chickens.

The efficacy of Capsicum oleoresin to modulate immune responses in ruminant animals is less studied than in other animals. However, Oh et al. (2013) reported that dairy cows receiving abomasal infusions of Capsicum oleoresin (2g/d) had a 2.6% increase in the proportion of lymphocytes, and a 5.9% increase on the proportion of CD4+ type of T lymphocyte when compared to cows on control treatment (no Capsicum). The CD4+ T lymphocyte produces cytokines that activates cells of the innate immune system, such as macrophages, and stimulates the production of antibodies by B lymphocytes. However, authors were not able to characterize the observed increase in lymphocytes as pro or anti- inflammatory, due to unaltered levels of cytokines TNFα, IFNγ, and IL-6.

In a follow-up experiment, dairy cows that were fed Capsicum oleoresin at 0.25, 0.5 and 1 g/d, had a linear increase in total white cells, neutrophils, and eosinophils (Oh et al.;

2015). Oh et al. (2015) suggested the increase may have been a response to neuropeptides secreted by neurons activated by capsaicin. These authors also reported a linear decrease in lymphocyte proportion as doses of Capsicum increased. This disagrees with the last study, and the authors suggest that the difference in route of supplementation and dose could have elicited this response.

Recently, supplementation of rumen-protected Capsicum oleoresin (0, 100, or 200 mg/d) decreased inflammatory responses of cows subjected to LPS challenge (Oh et al., 23

2017b). There was no difference in white cell count, rectal temperature, or cortisol between treatments, but cows fed the rumen-protected Capsicum presented a reduction of about 52% in haptoglobin, an acute phase protein whose levels increase in response to inflammation and scavenges free hemoglobin that can damage cellular constituents (Bicho et al., 2013). The increase in haptoglobin observed by Oh et al. (2017b) suggested that rumen-protected

Capsicum oleoresin may partially alleviate inflammatory responses induced by bacterial infection in dairy cows.

Despite the previous evidence that suggests Capsicum supplementation could potentially reduce susceptibility to disease and have anti-inflammatory effects, thus decrease intensity of immunological responses to disease, the effects of Capsicum oleoresin supplementation on immunological parameters of beef cattle have not yet been investigated.

Approaches to alleviate inflammation symptoms could be of especial relevance in cattle transitioning into feedlot, since transition to grain-based diets may induce metabolic and inflammatory conditions that hinder health and production (Chishti et al., 2020).

Effects on oxidative stress

In addition to immunological stressors, oxidative stress also plays a role in inflammation processes. Oxidative stress is caused by an accumulation of reactive oxygen species (ROS). Reactive oxygen species are produced by normal metabolic processes such as the process of cell respiration (Poyton et al., 2009). However, when exposed to stressful situations, such as elevated production levels or extreme environmental conditions, ROS may accumulate. Overfeeding may also result in an increase of ROS production due to increased oxidation of energy substrates, leading to oxidative stress as consequence of exceeding the oxidative capacity (Fang et al., 2002). The accumulation of ROS initiates an inflammatory process and secretion of pro-inflammatory cytokines. Accumulation of ROS over prolonged 24 periods of time may cause irreversible damage to cells and tissues and make animals more susceptible to disease (Hussain et al., 2016). Additionally, the accumulation of ROS may result in loss of energetic efficiency associated with increased energetic cost of inflammatory responses, neutralization of ROS to prevent oxidative damage and replacement of tissues that have suffered damage (Russel et al. 2016).

Capsaicin has been reported to have antioxidant properties, protecting animal tissues against lipid peroxidation (Asai et al., 1999; Okada and Okajima, 2001). This antioxidant property is attributed to the capacity of the phenol groups in capsaicinoids to scavenge a wide range of ROS. Manjunatha and Srinivasan (2006) observed the effects of dietary capsaicin

(0.015% of diet) in rats submitted to iron-induced lipid peroxidation. They observed a reduction of 28 and 33% on the concentration of thiobarbituric acid reactive substances

(TBARS) in serum and liver tissue, respectively, of rats fed capsaicin versus rats fed control.

TBARS are products of lipid peroxidation, thus, authors suggest their reduction indicated that capsaicin inhibited lipid peroxidation in rats. In another study, subcutaneous administration of capsaicin (1 mg/kg) in rats with sepsis attenuated the increases of plasma nitrite/nitrate

(NOx) concentration by about 20% and lung and liver tissue malondialdehyde by about 23% and 12%, respectively (Demirbilek et al., 2004). Nitrate/nitrites are byproducts of nitric oxide metabolism, and malondialdehyde is a product of lipid peroxidation (Nielsen et al., 1997;

Shiva, 2013).

In addition to its own antioxidant effect, it has been reported that capsaicin may affect the concentration of other endogenous antioxidants, even though this mechanism has not yet been described. In a study with rats subjected to liver injuries, Hassan et al. (2012) reported that oral administration of capsaicin (20mg/kg of BW) increased the content of glutathione

(GSH) in injured liver tissue when compared with non-treated liver tissue. Glutathione is an antioxidant that reduces hydrogen peroxide to water (Sen and Chakraborty, 2011). In dairy 25 cows, neither abomasal infusion of Capsicum oleoresin (2g/d; Oh et al., 2013) nor feeding

0.25, 0.5 or 1 g/d of Capsicum oleoresin (Oh et al., 2015) affected the oxidative stress markers. However, when submitted to a LPS challenged, cows supplemented with a rumen- protected form of Capsicum oleoresin had a lower concentration of TBARS at 24 h post-LPS challenge, suggesting that lipid peroxidation was decreased at the later stages of LPS challenge (Oh et al., 2017b), indicating that the supplementation of Capsicum ameliorate the oxidative stress associated with inflammation and disease.

Russell et al. (2016) suggested that feed efficiency in cattle may be influenced by the energetic cost associated with ROS neutralization and replacement of damaged tissues. Thus, the antioxidant property of capsaicinoids could potentially ameliorate the oxidative balance to prevent loss of performance; however, the effects Capsicum supplementation on oxidative stress have not been investigated in beef cattle.

Effects on feed intake

In addition to effects on inflammatory response, capsaicinoids have also been studied on the regulation of feed intake. Both oral and gastrointestinal exposure to capsaicinoids have been reported to increase satiety and reduce feed intake (Westerterp-Plantenga et al., 2005;

Smeets and Westerterp-Plantenga, 2009). The mechanisms of regulation are not fully elucidated; but, evidence suggests that capsaicinoids may influence intake by affecting the release of hormones that have an influence on appetite, such as Glucagon-like peptide 1

(GLP-1) and ghrelin, or because of palatability (Whiting et al., 2014). GLP-1 is mainly secreted at the small intestine. This hormone regulates appetite, having a satiety effect, by stimulating insulin release, inhibiting gastric emptying and reducing the flow rate of nutrients for intestinal absorption. The activation of the TRPV-1 receptor stimulates the secretion of

GLP-1 (Wang et al., 2012). 26

Westerterp-Plantenga et al. (2005) investigated the satiety effects of capsaicin on food intake in human subjects. Individuals were subjected to the consumption of 0.9 g of red pepper, 30 minutes before meals, orally with juice or in a capsule to be released in the stomach. When compared to individuals given a placebo, satiety increased, and energy intake decreased for participants who ingested pepper. Energy intake was 10% lower for individuals that ingested the pepper in a capsule and 16% for those who ingested pepper in the juice when compared to those receiving the placebo. The increased satiety was attributed to the additional sensory perception of capsaicin in the oral cavity. Another study reported increased levels of GLP-1 and a tendency of decreased levels of ghrelin in humans after the consumption of a meal containing capsaicinoids (Smeets and Westerterp-Plantenga, 2009).

The hormone ghrelin is secreted predominantly by endocrine cells of the stomach and that stimulates appetite.

In ruminants, Cardozo et al. (2006) reported a 9% increase in DMI of beef heifers supplemented with Capsicum oleoresin (1g/d) and nearly 26% increase in water intake.

Similarly, Rodríguez-Prado et al. (2012) reported decrease of DMI during the first 2 hours after feeding for heifers supplemented with Capsicum oleoresin (0.125, 0.25 or 0.5 g/d), but a linear increase in total DMI when compared to control, and a strong correlation between water intake and DMI (R2=0.98). In both trials, the additive was offered once a day mixed in a small amount of concentrate for guaranteed consumption. Authors suggested that the observed increase in feed intake might be related to pungency of capsaicin and increased water consumption. Fandiño et al. (2008) observed a 10% increase in DMI of beef heifers supplemented with 0.5 g daily dose of Capsicum oleoresin. In this case, however, the additive was provided in an encapsulated form, and positive effects on intake seemed to be unrelated to palatability. 27

Conversely, studies with dairy cattle reported no alterations in DMI of cows supplemented with Capsicum oleoresin. Tager and Krause (2011) reported that feeding

Capsicum oleoresin (0.25 g/d) had no effect on DMI of dairy cows, but reduced length of the first meal when compared to the those fed control. (Oh et al., 2015) reported no differences on DMI between dairy cows supplemented with Capsicum oleoresin at 0, 0.25, 0.50 or 1 g/d.

A more recent study evaluating the supplementation of a rumen-protected source of

Capsicum oleoresin (0, 0.1 or 0.2 g/d) also reported no differences in DMI of dairy cows (Oh et al., 2017a). Similarly, the supplementation of rumen-protected Capsicum oleoresin to beef cattle also did not affect DMI (Eidsvik et al., 2019).

The differences in response on feed intake observed between studies with dairy and beef cattle seem to be a result of the smaller doses of Capsicum (as mg/kg of diet DM) used in the dairy studies in comparison to the beef studies. For example, in the beef studies the doses used ranged from 2.5 to 10.6 mg/kg of DMI (Rodríguez-Prado et al., 2012), while the doses used in the dairy studies ranged from 1.8 to 7.5 mg/kg of DMI (Oh et al., 2015).

Effects on glucose and insulin

The secretion and regulation of insulin and glucagon are controlled by the nervous system (Osundiji and Evans, 2013). Thus, damage to the nervous system or pancreas innervation could impair normal pancreatic function. Therefore, the activation or ablation of the TRPV-1 receptors present on sensory afferent neurons by capsaicin has been used to investigate pancreatic hormone regulation (Rodriguez-Diaz and Caicedo, 2014). The TRPV-1 receptors participate in the regulation of glucose-induced insulin secretion through the release of the neuropeptide CGRP (Zhong et al., 2019). This neuropeptide can stimulate or inhibit insulin secretion depending on its concentration and glucose concentrations. High concentrations of CGRP will inhibit insulin secretion, while desensitization will remove the 28 inhibition. Another mechanism involves the secretion of GLP-1 (Wang et al., 2012). The activation of TRPV-1 receptors by dietary capsaicin in the ileum stimulate the release of

GLP-1, that on its turn, stimulates insulin secretion in mice.

Koopmans et al. (1998), for example, investigated insulin sensitivity in adult rats that underwent sensory nerve ablation by a subcutaneous capsaicin injection (50 mg/kg of BW) at birth. When compared to normal rats, the desensitized rats had a 21% increase in whole body glucose uptake at similar plasma insulin levels of approximately 90 mU/l. The increased insulin sensitivity was attributed to the inhibition of CGRP release due to sensory nerve desensitization. Similarly, van de Wall et al. (2005) studied the role of sensory nerves on glucose tolerance of rats submitted to nerve desensitization by subcutaneous capsaicin injection (50 mg/kg of BW). These authors reported that desensitization of sensory neurons, induced by capsaicin led to decreased insulin secretion, likely due to inhibition of CGRP release, while glucose levels remained the same, suggesting capsaicin increased insulin sensitivity.

In rats fed capsaicin (0.01 or 0.02% of diet), dietary capsaicin decreased glucose and insulin levels and AUC within six weeks of treatment (Song et al., 2017). In humans subjected to an oral glucose test, oral administration of 5 g of fresh Capsicum fruit (26.6 mg capsaicin) decreased circulating glucose and increased circulating insulin concentrations compared to those not treated with capsaicin (Chaiyasit et al., 2009). Contrastingly, humans who orally consumed capsaicin (400 μg) had increased levels of glucose and glucagon and unaltered insulin levels after an oral glucose tolerance test (Dömötör et al., 2006).

In sheep supplemented with Jalapeno powder (67 mg of capsaicin/kg of diet DM) or a rumen-protected Capsicum oleoresin (75 mg of capsaicin/kg of diet DM), no alterations in glucose and insulin levels of treated animals were reported (Alford et al., 2016). But, in dairy 29 cows, the dietary supplementation of rumen-protected Capsicum oleoresin (0, 100 or 200 mg/d) decreased serum peak insulin without affecting peak glucose concentration in a dose independent manner during a glucose tolerance test (Oh et al., 2017a). Decreasing serum insulin without altering glucose concentration implies increased insulin sensitivity of tissues which can be associated with increased glucose availability to the mammary gland and, thus, milk production in dairy cows.

The effects of Capsicum supplementation on insulin and glucose response have not been investigated in beef cattle. However, insulin resistance can be associated with increased subcutaneous fat deposition (Trenkle and Topel, 1978). Radunz et al. (2012) reported that insulin resistance develops during the finishing phase in the feedlot as beef cattle are fed high grain diets for long periods of time. Therefore, strategies that improve insulin sensitivity are relevant to beef production because the system is in the business of promoting lean tissue accretion and heavily discounts excess fat that must be trimmed from a carcass.

Effects on animal performance

The effects of supplementation of Capsicum oleoresin on the growth performance of livestock are limited. A few studies reported no observed effect on performance (Tager and

Krause, 2011; Oh et al., 2013), but other studies have reported beneficial effects of Capsicum supplementation (Liu et al., 2013a; Oh et al., 2015; Stelwagen et al., 2016; Wall and Bravo,

2016; Oh et al., 2017a).

Dietary inclusion of Capsicum oleoresin (10 mg/kg) increased average daily gain

(ADG) and reduced diarrhea in weaned pigs (Liu et al., 2013a). Dietary Capsicum oleoresin supplementation had no effect on productivity in dairy cows (Tager and Krause, 2011), and abomasal infusion of Capsicum oleoresin (2 g/d) in fact decreased milk production in dairy cows by 2.2 kg/d (Oh et al., 2013). However, Oh et al. (2015) reported that the 30 supplementation Capsicum oleoresin (250 and 500 mg/d) tended to increase milk yield and increased energy corrected milk yield by up to 6.2%. Studies with rumen-protected Capsicum oleoresin also reported positive results in dairy cows. Stelwagen et al. (2016) reported that the supplementation of rumen-protected Capsicum oleoresin (100 or 200 mg/d) increased milk yield by 6.8%. Wall and Bravo, (2016) reported a 9% increase in milk production of cows supplemented with 100 mg/d of rumen-protected Capsicum oleoresin. When feeding rumen- protected Capsicum oleoresin (100 or 200 mg/d), Oh et al. (2017b) reported that the supplementation increased feed efficiency in dairy cows.

In beef cattle, the supplementation of rumen-protected Capsicum oleoresin (100 or

330 mg/d) did not affect ADG or feed efficiency of steers fed grain-based diets, but the lowest dose tended to decrease marbling score (Eidsvik et al., 2019). However, the evaluation of Capsicum oleoresin supplementation on beef cattle performance is limited and requires further study.

Summary

Capsicum is a genus of plants from the Solanaceae family. It is comprised of several species of peppers that vary in pungency. Capsaicinoids are the active compounds responsible for the pungency in the fruit of Capsicum plants. Capsaicin and dihydrocapsaicin are the predominant analogs and usually account for about 90% of total capsaicinoids.

Capsaicinoids are readily absorbed in the gastrointestinal tract and metabolized in the liver to a large extent.

Although Capsicum has been used in medicine for its antimicrobial properties and was investigated as a potential modifier of rumen fermentation, studies have shown that dietary Capsicum supplementation had little or no effect on rumen fermentation parameters in 31 dairy and beef cattle. However, Capsicum has been reported to exert beneficial health effect in other animals through direct host responses.

Host related effects of Capsicum are largely associated with the activation of TRPV-1 receptors by the capsaicinoids. These receptors are present in many cells of the peripheral nervous system. Capsaicin has been reported to have dose-dependent beneficial impacts on

GI tract mucosal tissue and increase activity of pancreatic enzymes in rats and humans. In addition, capsaicin has also been reported to have immunoregulatory effects, antioxidant activity, and to alter insulin sensitivity.

Capsicum oleoresin has been shown to increase feed intake in beef cattle, but no effects on feed intake were observed in dairy cows. Differences between dairy and beef studies may be related to supplement doses between the beef and dairy studies. More recent dairy studies suggest that the supplementation of Capsicum in a rumen-protected form could increase availability of capsaicinoids post-rumen, improving productivity responses and health in dairy cows. However, the effects of rumen-protected Capsicum supplementation, and its potentially beneficial host-related responses, have not been investigated in beef cattle.

In addition, there is little information regarding the effects of rumen-protected Capsicum on beef cattle growth performance and, to the authors knowledge, no evaluation of the effects of rumen protected Capsicum on immunological, oxidative stress, and glucose metabolism responses in growing beef cattle. 32

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40

Chapter 3

Effects of feeding rumen-protected Capsicum oleoresin on growth performance, health

status, and total tract digestibility of growing beef cattle

Abstract

The objectives were to investigate the effects of supplementing rumen-protected

Capsicum oleoresin (RPC) on feed intake, performance, immune and oxidative stress response, and total tract digestibility of beef cattle fed grain-based diets. Exp. 1: Cattle were stratified by sex (steers = 24; heifers = 12) and body weight (BW; heavy, 240 ± 38 kg; or light, 181 ± 32 kg) and assigned to treatments: Control (no additive), or 15RPC (15 mg of

RPC/kg of diet dry matter (DM)). Cattle were transitioned over 21 days to a final diet of 80% grain mix and 20% corn silage. Cattle were weighed on test (d0 and 1), at end of adaptation

(d22), at midpoint (d49 and 50), and off test (d99 and 100). Blood samples were collected on days 1, 22, and 98 for analysis of parameters related to immune function and energy status.

Health treatments were recorded. Performance were analyzed using the MIXED procedure of

SAS. Blood variables were analyzed using the MIXED procedure of SAS with repeated measures. Pen was the experimental unit. Health data were analyzed using the GLIMMIX procedure; animal nested within pen was the experimental unit. Exp. 2: Total tract apparent digestibility was tested in a replicated 4x4 Latin Square with 8 ruminally-fistulated steers, 4

Angus and 4 Holsteins. Each experimental period consisted of 21 days of diet adaption followed by 6 days of collection. The experimental treatments were the Control (no additive) and RPC at 3 levels of dietary inclusion: 5 , 10 , or 15 mg of RPC/ kg of diet DM. In Exp. 1, there were no treatment effects on DMI (P = 0.54) or final BW (P = 0.42). From d1 to 22, average daily gain (ADG) tended to be greater (P = 0.13) for cattle fed 15RPC and was

23.3% greater (P = 0.07) for cattle fed 15RPC from d1 to 50. Gain to feed ratio (G:F) was 41 also greater (P = 0.07) for cattle fed RPC from d1 to 50 when compared with the control group. There was no interaction (P > 0.15) of treatment and sampling day, nor main effect of treatment (P > 0.10) on red blood cells, white blood cells, haptoglobin, glutathione, glucose, insulin and blood urea nitrogen. Hemoglobin was greater (P = 0.03) on animals fed control, but there was no day by treatment interaction (P = 0.39). Analysis of the health records indicated that 76.5% of light weight animals fed Control had fever 2 or more times (body temperature above 39.5°C) while the same was true for only 21.8% of the light weight animal fed 15RPC (P = 0.09), and 90% of sickness occurrences were registered during the first two weeks in the feedlot. In Exp. 2, intake, ruminal pH and total tract apparent digestibility of

DM, OM, CP and NDF were not different (P > 0.32) between treatments. In summary, supplementing RPC improved early feedlot ADG; however, ADG improvement was not related to DMI and seem to be unrelated to nutrient metabolism and digestibility or ruminal fermentation parameters. Changes in health status could have influenced ADG, but blood parameters measuring health status were not impacted. More frequent blood sampling during the critical phase of diet adaptation may contribute to detect earlier differences in immune status of cattle.

Key words: beef cattle, Capsicum, digestibility, feedlot, immune status

Introduction

Capsicum fruits, commonly known as hot peppers, contain a group of active compounds called capsaicinoids that are responsible for their characteristic pungent flavor.

Capsaicinoids have also been reported to have antimicrobial properties (Deans and Ritchie,

1987). For that reason, the dietary supplementation of Capsicum oleoresin was initially investigated for its potential to modify rumen fermentation in beef (Calsamiglia et al., 2007) 42 and dairy cattle (Tager and Krause, 2011); however, the impacts of Capsicum on rumen modulation are negligible. Despite this, it has been suggested that the supplementation of

Capsicum oleoresin may improve animal performance by affecting immunological and physiological responses (Oh et al., 2017c). Capsicum oleoresin supplementation has been reported to affect the small intestine ultra-structure, permeability and release of digestive enzymes in rats (Prakash and Srinivasan, 2010; Platel and Srinivasan, 1996), and to increase intestinal integrity (Liu et al., 2014a, 2014b) and decrease inflammatory response in virus- challenged swine (Liu et al. 2013). In dairy cows, the supplementation of Capsicum oleoresin did not affect ruminal parameters, milk yield and composition, or nutrient digestibility

(Tager and Krause, 2011). Authors hypothesized that this lack of effect may be due to degradation of Capsicum in the rumen. However, when supplied post-ruminally, through abomasal infusion, Capsicum oleoresin increased the proportion of lymphocytes in circulation relative to the total white blood cells, and the CD4+ type of T lymphocyte cells

(Oh et al. 2013), indicating benefits of the post-ruminal delivery of capsaicinoids. Further studies evaluating supplementation of rumen-protected forms of Capsicum oleoresin reported increased milk yield and feed efficiency, and decreased insulinemic and inflammatory responses in dairy cows (Stelwagen et al., 2016; Oh et al., 2017a; Oh et al., 2017b).

The reports indicate that the supplementation of rumen-protected Capsicum oleoresin has potential to improve animal immune function and performance. However, the studies evaluating the supplementation of Capsicum in beef cattle are limited. Eidsvik et al. (2019) reported that the supplementation of rumen-protected Capsicum did not alter DMI or ADG of finishing feedlot steers. But, there are no reports evaluating the supplementation effects on digestibility and immunological parameters in beef cattle that may be immunocompromised, like young, recently weaned cattle brought in to a feedlot. 43

Therefore, it was hypothesized that the post-ruminal supply of capsaicin through the supplementation of a rumen-protected source of Capsicum oleoresin would ease the transition of recently weaned beef calves from pasture to a grain-based feedlot diet and positively impact growth performance. Furthermore, it was hypothesized that these improvements in performance would be caused by increased feed intake, reduction in inflammation associated with immune response and oxidative stress, improved insulin sensitivity, or improved nutrient digestibility. The objectives of the present study were to investigate the effects of supplementing rumen-protected Capsicum oleoresin on feed intake, performance, immune and oxidative stress response, and total tract digestibility of beef cattle fed grain-based diets.

Materials and Methods

All procedures involving the use of animals were approved by The Pennsylvania State

University Institutional Animal Care and Use Committee (protocol ID: PROTO201800194 and

PROTO201800614) and followed the guidelines recommended in the Guide for the Care and

Use of Agricultural Animals in Research and Teaching (FASS, 2010).

Experiment 1

The experimental design was a randomized complete block with 2 treatments. Thirty- six Angus and Angus cross calves were weaned and brought from pasture into the feedlot on d 0. Cattle were weighed on trial over 2 consecutive days (d 0 and d1), blocked by sex (24 steers and 12 heifers) and stratified body weight into heavy (240 ± 38 kg of BW; 181 ± 31 d of age) or light (181 ± 32 kg of BW; 153 ± 15 d of age) groups within sex and allotted to 1 of

12 pens. There were 3 animals per pen and 6 pens per treatment, 3 heavy and 3 light weight pens, with 2 pens of steers and 1 pen of heifers in each BW block. Each pen was randomly 44 assigned to 1 of 2 treatments: no additive (Control), or rumen-protected Capsicum oleoresin fed at the inclusion rate of 15 mg of RPC/kg of diet DM (15RPC).

Cattle were fed for 100 days. During the first 21 days, cattle were adapted to the final grain-based diet (Table 3-1). Feed was offered initially at 1.5% of BW and increased by 10% every 2 d if bunks were slick until bunk score equaled 0.5, to allow ad libitum intake with approximately 5% refusals. The basal diet was fed as a total mixed ration (TMR) offered twice daily (0700 h and 1300 h). The RPC (Nexulin, 1.1% capsaicinoids, Pancosma,

Switzerland) was mixed into a fraction of the daily allotment of soybean meal prior to addition to the TMR, to obtain a homogeneous distribution throughout the feed, and fed to cattle on the 15RPC treatment beginning on d 1. Feed offered was recorded daily and refusals were measured weekly to determine pen dry matter intake (DMI). Samples of feed ingredients were collected weekly for adjustment of ration DM and composited across all weeks for wet chemistry analysis. Composite samples of feed and weekly refusals were dried for 72 hours at 55°C in a forced-air oven, and ground in a Wiley mill (A. H. Thomas Co.,

Philadelphia, PA) through a 1 mm screen before analysis for DM (100°C for 4 h), OM

(600°C for 4 h), amylase-treated NDF (method 6, Ankom A200 fiber analyzer; Ankom

Technology, Macedon, NY), and N (Costech ECS 4010 C/N/S elemental analyzer by Costech

Analytical Technologies Inc., Valencia, CA). Content of Ca and P (AOAC International,

2000) of the diet was analyzed by Cumberland Valley Analytical Services (Maugansville,

MD). In addition, NEm and NEg were back-calculated based on animal performance (NRC,

1996).

Cattle were weighed at the end of the diet adaptation period (d 22), at the midpoint on two consecutive days (d 49 and d 50), and off test on two consecutive days (d 99 and d 100).

Weights were always taken 3 h after morning feeding. The average of the 2-d BWs was used to calculate average daily gain (ADG) of the steers. 45

Blood samples were collected from the jugular vein on days 1, 22 and 98 of the trial 3 h post-feeding. Blood samples for hematology analysis were collected into 10 mL EDTA- coated vacutainers (Becton, Dickinson and Co., Franklin Lakes, NJ). Hematology analysis included red blood cell count, hemoglobin, platelet count, and total white blood cell count. A second set of blood samples was collected into 10 mL red top vacutainer tubes (Becton,

Dickinson and Co., Franklin Lakes, NJ) and serum was obtained by centrifugation at 2,000 x g for 15 min at 4°C. Serum samples were analyzed for blood glucose and blood urea nitrogen

(BUN) using a chemistry analyzer (Idexx VetTest® analyzer, Idexx Laboratories, Inc.,

Westbrook, ME). Blood samples for hematology and serum samples for glucose and BUN were submitted to the Centralized Biological Laboratory (Pennsylvania State University,

University Park, PA) and analyzed on the same day. A third set of samples was collected into

10 mL EDTA-coated vacutainer tubes (Becton, Dickinson and Co., Franklin Lakes, NJ) and plasma was obtained by centrifugation at 1,500 x g for 15 min at 4°C. Plasma samples were frozen and stored at -80°C until analyzed for insulin (Bovine insulin ELISA, 80-INSBIO-

E01, Alpco, Salem, NH), haptoglobin (Immunoperoxidase assay, E-10HPT, Immunology

Consultants Laboratory, Portland OR), and total GSH (Colorimetric assay kit, no. 703002,

Cayman Chemical).

Detailed health records were maintained by the herd manager to monitor signs of morbidity and medical treatments. If cattle had a fever (defined as body temperature equal or greater than 39.5°C) they received a dose of antibiotic (Nuflor®, Merck Animal health) and anti-inflammatory (Banamine®, Merck Animal health). If cattle had drastically reduced intake the entire pen received a dose of probiotics (Probios®, Santa Cruz Animal Health). Cattle were pulled from pens based on visual evaluation and treated at discretion of the herd manager. For analysis, cattle were listed as, treated ever, or treated 2 times or more with 46 probiotic or the combination of antibiotic and anti-inflammatory, and with reported temperature above 39.5°C ever or and 2 or more times.

Statistical Analysis

The experimental design for this study was a randomized complete block design with

2 treatments. Performance were analyzed using the MIXED procedure (SAS Inst. Inc., Cary,

NC). The model used was:

Yijkl = µ + Si + Wj + Tk + eijkl

where Yijkl = response variable; µ = mean; Si = fixed effect of sex (steer or heifer), Wj = fixed effect of BW block (light or heavy), and Tk = the fixed effect of treatment, and eijkl= the experimental error. Pen was the experimental unit.

The blood variables were analyzed using the MIXED Procedures of SAS as repeated measures to evaluate effect of treatment over time. The model was:

Yijklmno = µ + aj(i) + Sk + Wl + Tm + Dn + TDmn+ eijklmno

where Yijklmno = response variable; µ = mean, aj(i) = the random effect of animal nested within pen, Sk = fixed effect of sex (steer or heifer), Wl = fixed effect of BW block (light or heavy),

Tm = the fixed effect of treatment, Dn = the fixed effect of sampling day, TDmn = the fixed effect of the interaction of treatment and day, and eijklmno = the experimental error. The covariance structure, compound symmetry, was selected based on the lowest Bayesian

Information Criterion. Pen was the experimental unit.

The health data were analyzed using the GLIMMIX procedure of SAS with a binomial distribution. The model was:

Yijklmn = µ + aj(i) + Sk + Wl + Tm + eijklmn 47

where Yijklmn = response variable; µ = mean, aj(i) = the random effect of animal nested within

pen, Sk = fixed effect of sex (steer or heifer), Wl = fixed effect of BW block (light or heavy),

Tm = the fixed effect of treatment, and eijklmn = the experimental error. Animal nested within

pen was the experimental unit.

For all statistical analyses, means were separated using the LSMEANS statement with

PDIFF option. Significance was declared at P ≤ 0.10 and trends discussed at 0.10 < P < 0.15.

Experiment 2

Four Angus (513 ± 3 days of age; 540 ± 54 kg of BW) and 4 Holstein (459 ± 8 days of age; 530 ± 27 kg of BW) steers, previously fitted with rumen cannulae, were assigned to 1 of

4 treatments: 0 mg of RPC/ kg of diet DM (Control), 5 mg of RPC/ kg of diet DM (5RPC),

10 mg of RPC/ kg of diet DM (10RPC), or 15 mg of RPC/ kg of diet DM (15RPC).. Each experimental period consisted of 21 days of diet adaption followed by 6 days of collection.

Feed was delivered once daily (0700) and steers were fed for ad libitum intakes. Diets (Table

3-2) were hand mixed and fed ad libitum to achieve approximately 5% refusals. The RPC

(#6570, 1.7% capsaicinoids, Pancosma, Switzerland) was previously added and mixed into the soybean meal to obtain a homogeneous distribution of it throughout the concentrate. Steers were housed in individual metabolism stalls at the Beef Nutrition Research Lab, State College,

PA. Stalls (2.5 x 1.5 m) were floored with rubber mats (Ani-mat Inc., Sherbrooke, QC, Canada) and equipped with individual feed bunks and non-siphoning automatic water bowls.

Feed offered and refusals were measured daily to determine individual DMI. Feed

samples were collected at the beginning of each period to adjust diet DM. From d 23 to d 27,

approximately 100 g samples of each feed ingredient and 10% subsample of the refusals (wet

basis) were collected daily, composited by period and stored at -20°C for later analysis. Only

DMI data collected during each collection phase was used in the statistical analysis. Total

fecal output was collected from d 23 to d 27 of each experimental period for calculation of 48 apparent nutrient digestibility of DM, OM, NDF, and CP. Feces were collected in fecal bags attached to the steers and subsamples (10% of weight, wet basis) were composited by period and stored at -20˚C until analyzed. Feeds, refusals, and fecal composite samples were dried for 72 hours at 55°C in a forced-air oven, and ground in a Wiley mill through a 1 mm screen before analysis. All samples were analyzed for DM, OM, and amylase-treated NDF and CP using the methods previously described for experiment 1. Total tract apparent nutrient digestibility was calculated as [(Nutrient intake – Nutrient fecal output)/Nutrient intake] ×

100. Rumen contents were collected on d 22 of each period, at 0, 2, 6, 12, and 18 h post- feeding and strained in cheese cloth. The rumen fluid (up to 200 mL) obtained was immediately analyzed for pH using a digital pH meter (F20, FiveEasy series, by Mettler-

Toledo AG analytical, Schwerzenbach, Switzerland).

Prior to the beginning of Exp. 2 and is situ incubation was performed to determine the ruminal disappearance rate of the RPC used on Exp. 2. Two Angus steers fitted with rumen cannulae were fed a typical feedlot diet containing 10% forage and 90% concentrate (DM basis). Polyester bags (5 × 10 cm, 50 μm porosity; Ankom Technology, Macedon, NY) containing 1 g RPC/bag were incubated in triplicates in each steer for 0, 0.5, 1, 2, 6, 12 and

24 h. Bags were inserted in the rumen sequentially and removed simultaneously. After the incubation, the bags were washed with cold tap water and placed in oven at 45°C for 72 h.

The residues were analyzed for capsaicinoid concentration using a colorimetric ELISA essay

(Cat. # 20-0027, Beacon Analytical Systems Inc. Saco, ME, 04072). Ruminal degradability of capsaicinoids in RPC was calculated as:

D = a + ( b x c ) / ( c + k )

where a was the fraction of soluble capsaicinoids (%), b was the fraction of potentially degradable capsaicinoids (%), c was the fractional rate constant for the 49 disappearance of fraction b, and k was the passage rate from the rumen. Ruminal degradability of capsaicinoids in RPC was estimated for a hypothetical passage rate of 0.08/h.

Statistical Analysis

The experimental design was a 4×4 replicated Latin square. Data were analyzed using the MIXED procedure (SAS Inst. Inc., Cary, NC). The model used was:

Yijklm = μ + si + aj(i) + Pk + Tl + eijklm

where Yijklm = the response variable; μ is the mean; si = the random effect of square; aj(i) = the random effect of animal nested within square; Pk = the fixed effect of period; Tl = the fixed effect of the treatment; eijklm = the experimental error. Orthogonal contrasts were used to test the linear and quadratic effect of treatment. Means were separated using the LSMEANS statement with PDIFF option.

Ruminal pH data were analyzed using the Mixed Procedure of SAS as repeated measures to evaluate the effect of treatment over time. The model was:

Yijklm = μ + si + aj(i) + Pk + Tl + Hm + THlm + eijklm

where Yijklm = the response variable; μ is the mean; si = the random effect of square; aj(i) = the random effect of animal nested within square; Pk = the fixed effect of period; Tl = the fixed effect of the treatment; Hm = the fixed effect of sampling hour, THlm = the fixed effect of the interaction of treatment and hour; eijklm = the experimental error. Orthogonal contrasts were used to test the linear and quadratic effect of treatment. Means were separated using the LSMEANS statement.

For all statistical analyses, steer was the experimental unit. Significance was declared at P ≤ 0.10 and trends discussed at 0.10 < P < 0.15.

50

Results

Experiment 1

There were no differences (P = 0.99) in initial BW of the animals (Table 3-3), indicating an equal distribution of BW among treatments, as planned. Despite a numeric difference of 10 kg, final BW was also not different (P = 0.42) between treatments. There were no differences (P ≥ 0.24) in ADG, DMI, or G:F between treatments (Table 3-2) over the

100 days. However, ADG tended to be greater (P = 0.13) for cattle fed RPC during the adaptation phase (d1 to 21) when compared to cattle fed control. In addition, ADG was

23.3% greater (P = 0.07) for cattle fed RPC from d 1 to 50 of the trial when compared with the control group (Figure 3-1). Similar to ADG, gain to feed ratio (G:F) was greater (P =

0.07) for cattle fed RPC from d1 to 50 when compared with the control group (Figure 3-3).

There were no interactions between treatment and day of sampling for any of the blood parameters measured (P > 0.10); therefore, main effects of treatment and day are discussed separately. When compared to the control treatment, supplementing cattle with

15RPC did not affect (P ≥ 0.29) red blood cell count, platelet count, total white blood cell count or its fractions, or neutrophil to lymphocyte ratio (Table 3-4). All these variables were affected (P ≤ 0.02) by day of sampling except for eosinophils and basophils. Overall analyzed hemoglobin was less (P = 0.03) for cattle fed 15RPC when compared to cattle fed control

(Table 3-4) and was also affected by day of sampling (P < 0.01). Glucose, insulin, BUN, and haptoglobin were not affected (P ≥ 0.17) by experimental treatments (Table 3-5); however, they were affected (P ≤ 0.10) by day of sampling. Glutathione was not affected by experimental treatment (P = 0.75) or day of sampling (P = 0.29).

Descriptive numbers of medication administered and fever occurrences are shown on

Table 3-6. Most treatments (90%) occurred within the first 2 weeks of the trial. There was an 51 effect of BW block (P = 0.02) on the percentage of animals that were treated with antibiotic and anti-inflammatory 2 or more times, with 43.6% of light weights being treated versus

7.7% of heavy weights (Table 3-7), but no treatment and BW interaction (P = 0.41). There was a treatment and BW interaction (P = 0.09) on cattle that had a fever 2 or more times

(Table 3-7); while 76.5% of light weight cattle fed Control treatment were reported to have fever, only 21.8% of light weight cattle fed 15RPC treatment were.

Experiment 2

From the in situ incubation it was determined that the rapidly soluble fraction of capsaicinoids on the RPC used was of 13.7%, and rate of degradation of capsaicinoids was of

4.1%/h. Considering the hypothetical passage rate of 0.08/h, degradability of capsaicinoids was 42.9%. Resulting in an estimated amount of 31.3 to 92.4 mg/d of RPC escaping the rumen in Exp. 2. In the digestibility trial, the supplementation of RPC at 5, 10 or 15 mg/kg of diet DM did not affect (P > 0.59) intake or apparent total-tract digestibility (P > 0.32) of DM,

OM, CP or NDF of steers when compared to the control treatment (Table 3-8). Ruminal pH was affected by of hour of sampling (P < 0.01; Figure 3-4). However, even though there was partial degradation of RPC in the rumen as indicated by the results of the in situ, there were no linear (P = 0.84) or quadratic (P = 0.65) effects of treatment nor treatment by hour interactions (P = 0.37) for ruminal pH.

Discussion

The lack of effect of RPC supplementation on DMI in both experiments is contrary to previous studies evaluating the supplementation of unprotected Capsicum oleoresin in beef cattle, that observed a 9 to 26% increase in DMI (Cardozo et al., 2006; Rodríguez-Prado et al., 2012). However, the results of the current trial agree with the only other feedlot 52 experiment that fed protected Capsicum oleoresin. Eidsvik et al. (2019) reported no effects on

DMI of beef cattle when feeding RPC (at 77 to 330 mg RPC/d). Similarly, several studies in dairy cows that supplemented both unprotected (Tager and Krause, 2011; Oh et al., 2015) or protected forms of Capsicum oleoresin (Wall and Bravo, 2016; Oh et al., 2017a) observed no effects of Capsicum in DMI. Authors suggested that the increased DMI observed in beef heifers fed unprotected Capsicum was related to increased water consumption due to pungency of Capsicum oleoresin (Cardozo et al., 2006; Rodríguez-Prado et al., 2012).

However, the doses used in those trials were greater in comparison with the doses used in

Exp.1 and 2. For example, while the rate of application of oleoresin in Rodríguez-Prado et al.

(2012) ranged from 12.7 to 53.2 mg of Capsicum oleoresin/kg of DM, in the current study the highest dose was of 15 mg of protected Capsicum oleoresin/kg of DM. In addition, in the aforementioned studies, the additive was supplied mixed in a small portion of concentrate feed instead of mixed in a TMR, which might have also contributed to increased perception of pungency and water intake.

Similar to DMI, G:F and ADG were not altered overall the 100 days in Exp. 1.

Although there were no overall differences in performance, cattle fed 15RPC had increased

G:F from d1 to 50, reflecting the increased ADG in cattle fed 15RPC for the same period when compared to those fed Control, at a similar DMI. Considering that feed represents the majority of the cost of a beef cattle operation, improvements in feed efficiency can greatly reduce production costs. While the improvement in ADG from d1 to 50 is unrelated to DMI, that did not differ between treatments, it is also not likely related to improvements in nutrient total tract apparent digestibility because RPC supplementation did not alter DM, OM, CP or

NDF total tract apparent digestibility in Exp. 2. There are currently no other studies evaluating effects of RPC on digestibility in beef cattle. In dairy cows, the supplementation of

RPC (0, 100 or 200 mg/d) linearly increased (P ≤ 0.03) digestibility of DM, OM and CP and 53 tended to increase (P ≤ 0.10) NDF and ADF digestibility (Oh et al., 2017a). Even though digestion enzymatic activity was not measured, authors suggested that digestibility improvements could be related to increase in pancreatic and intestinal enzymes secretion or activity, as observed in rats (Platel and Srinivasan, 2000; Prakash and Srinivasan, 2010). The lack of response in the current experiment could be related to differences in DMI or diet composition, which both could alter rate and extent of digestion as well. Thus, it is difficult to make direct comparisons between dairy trials and beef trials regarding the efficacy of RPC.

Even though studies evaluating Capsicum oleoresin as a modifier of ruminal fermentation often presented small or negligible results both in beef (Cardozo et al., 2006;

Fandiño et al., 2008) and dairy cattle (Tager and Krause, 2011; Oh et al. 2015), Calsamiglia et al. (2007) suggested that the more acidic ruminal pH induced by grain-based diets, commonly fed to feedlot cattle, could increase the antimicrobial activity of capsaicinoids by increasing the hydrophobic nature of the molecules. In support of this hypothesis, an in vivo study by Rodriguez-Prado et al. (2012) reported that ruminal short chain fatty acid (SCFA) concentration tended to linearly increase and pH tended to linearly decrease with increasing doses of Capsicum oleoresin (0.125, 0.250, and 0.500 g Capsicum oleoresin/d) when fed to beef heifers consuming a 90:10 concentrate to forage ratio (DM basis). Despite previous studies indicating little efficacy of capsaicinoids to promote major alterations in ruminal fermentation, the association with lower pH (Figure 3-4) induced by grain-based diets could potentially alter fermentation patterns (Calsamiglia et al. 2007) and animal performance. The in situ incubation performed to estimate ruminal degradability of the RPC product utilized on

Exp. 2 indicated that, despite labeled as a rumen-protected, 42.9% of capsaicinoids were degradable in the rumen if assuming a passage rate of 0.08/h. But ultimately, RPC supplementation did not alter ruminal pH. 54

Regarding the blood parameters, supplementation with RPC did not alter blood glucose and insulin. Unaltered glucose and insulin indicate no alteration in insulin sensitivity between treatments over 100 days. In dairy cows, glucose and insulin metabolism were evaluated during glucose challenge (Oh et al., 2017a). Oh et al. (2017a) reported that basal glucose and insulin levels prior to challenge were not different (P > 0.36) between control and RPC (100 or 200 mg/d) treatments, neither was the glucose response post infusion (P >

0.17), but supplementation with RPC tended (P = 0.07) to decrease peak insulin and decreased (P = 0.04) insulin area under the curve by an average of 25%, indicating better insulin sensitivity of insulin-dependent tissues, but insulin sensitivity was not measured in that study. A glucose challenge was not conducted in the current trial and blood samples to determine insulin and glucose were taken 3 h post-feeding, thus current results and the results obtained by Oh et al. (2017a) are not directly comparable. Eidsvik et al. (2019) reported that

RPC supplementation did not affect backfat thickness of feedlot steers when compared to control treatment. Increased subcutaneous fat deposition is associated with insulin resistance

(Trenkle and Topel, 1978), and similar fat deposition could indicate similar glucose and insulin metabolism among treatments. However, in Eidsvik et al. (2019), cattle were fed for only 69 d, according to Radunz et al. (2012), days on feed may affect insulin sensitivity.

Regardless, more research is necessary for more conclusive results regarding the effects of

RPC supplementation on glucose and insulin metabolism in beef cattle fed grain-based diets.

Blood urea nitrogen did not differ (P = 0.89) between treatments Control and 15RPC in Exp. 1. We had hypothesized that feeding RPC would increase nutrient availability in the small intestine; however, as nutrient digestibility was not altered in Exp. 2, the lack of effect on BUN corroborates these findings. To our knowledge there are no studies evaluating protected Capsicum oleoresin on BUN concentration in ruminants. Supplementation of unprotected Capsicum oleoresin (250, 500 or 1000 mg/d) linearly increased BUN by 8% in 55 dairy cows; however, authors of this study offered no hypothesis for the change in BUN because ruminal ammonia, urine urea nitrogen and milk urea nitrogen were similar among treatments (Oh et al. 2015).

One other possible explanation for the differences observed in ADG may be related to the health status of the calves. In Exp. 1 of the current study, the overall percentage of calves that received medication or had fever (body temperature at or above 39.5°C) was not different between treatments, but a greater proportion (P = 0.09) of light weight animals fed

Control had fever twice or more (76.5 versus 21.8%) when compared to light weight animals fed 15RPC (Table3-7). Reports of a relationship between body weight and susceptibility to disease are not always consistent in literature (Taylor et al., 2010), yet, some studies suggest that heavier calves are at lower risk of incidence of bovine respiratory disease than lighter cattle (Sanderson et al., 2008; Taylor et al., 2014). For example, Taylor et al. (2014) reported that lighter arrival weights were associated with the need for treatment of bovine respiratory disease symptoms, and indicated that for each 45 kg decrease in body weight at the entry of the feedlot there was an increased risk for need of medical treatment by 60%. Regardless, according to Bradford and Ylioja (2018), resting metabolic rate can be estimated to increase from 10 to 40% in result of immune activation depending on the severity of the response, thus affecting performance. Busby et al (2004) for example, reported greater ADG (3.06 lb/d;

P < 0.0001) in calves that did not receive health treatment when compared to those treated one (2.93 lb/d) or two or more times (2.87 lb/d) for disease symptoms. Therefore, the improved ADG in cattle fed 15RPC during the early days in the feedlot, may be related to health status of those lightweight cattle in Exp. 1.

Regarding the blood parameters, there was no treatment by day interactions for hemoglobin; however, mean hemoglobin concentrations were reduced (P = 0.03) when cattle fed RPC were compared to cattle fed Control (Table 3-4). A different result was observed by 56

Oh et al. (2017b), where dairy cattle fed RPC did not have altered hemoglobin concentrations when compared to cattle fed no RPC. Similarly, Capsicum oleoresin (2 g/d) abomasal infusion did not alter hemoglobin values (Oh et al., 2013), but an increase (P < 0.01) in hemoglobin concentrations was observed when feeding unprotected Capsicum oleoresin

(250, 500 or 1000 mg/d) to dairy cows (Oh et al., 2015). Despite the mean difference in the current trial, hemoglobin concentrations were within normal levels of 8.4 to 14 g hemoglobin/dL (Roland et al., 2014).

Even though the number of cattle treated differed, white blood cell counts, were within normal range – which is 4.9x103 to 13.3x103 white blood cells/µL; 1.0x103 to 6.3x103 neutrophils/µL; 1.6x103 to 8.1x103 lymphocytes/µL; 0.1x103 to 0.8x103 monocytes/µL;

0.1x103 to 1.5x103 eosinophils/µL; and 0 to 0.3x103 basophils/µL (Roland et al., 2014) - indicating no alterations in immune response, with no effect of treatment nor interaction of treatment by day for any of the above variables. Similarly, in dairy cows, RPC supplementation did not affect white blood cells after a LPS challenge (Oh et al., 2017b).

While there were differences in neutrophils and lymphocytes between d 1 and d 22 in the current experiment, the values remained within a normal range, but may indicate a stress response from animals on day 22 (Tornquist, 2010).

No alterations in oxidative stress among treatments were indicated by total GSH.

Similarly, no differences (P = 0.17) were observed for haptoglobin, despite the 36% change in analyzed haptoglobin concentrations between treatments. The difference in haptoglobin concentration between treatments seems large when examined as percentage, but it is in fact not as relevant considering that haptoglobin levels in calves can be highly variable.

According to Cooke and Arthington (2012) for example, plasma haptoglobin levels in beef calves increased from basal levels of 0.11 μg haptoglobin/mL up to 1053.25 μg haptoglobin/mL only 3 days after vaccination. In Oh et al. (2017b), when compared to the 57 control treatment, supplementation of RPC decreased haptoglobin levels post LPS infusion, indicating that Capsicum could potentially alleviate sickness symptoms in calves. However, even though in the current trial some animals were reportedly sick and pyretic - conditions that are associated with increase in acute phase proteins (Tóthová et al., 2013) and oxidative stress markers - the lack of effects observed in the current trial may be due to the majority

(90%) of health challenges being recorded during the first 2 weeks after feedlot entry, while blood sampling occurred only on days 1, 22, and 98, indicating that the blood collections likely did not overlap with the most critical phase through which the calves were under most stress. Thus, more data are necessary to ascertain the validity of the differences on the sickness symptoms observed during the adaptation period in Exp. 1 and these authors would recommend more frequent sampling to detect possible differences earlier, especially during the first 15 days post feedlot entry where the majority of sickness occurrences were registered. While calves fed RPC had improved ADG and G:F from d1 to 50 in Exp. 1 – likely related to health status - suggesting RPC supplementation can be beneficial during the early transition phase into feedlot, it is also important to note that a limited number of cattle were used in the present trial and additional validation is necessary, yet, these data warrant further investigation in to the health benefits of feeding RPC to recently weaned cattle in the receiving phase of the feedlot.

Conclusion

Supplementation of rumen-protected Capsicum oleoresin increased ADG of calves during the initial period on feedlot. Improvements in ADG seem to be unrelated to changes in nutrient digestibility and nutrient metabolism but could be related to improvement in health status observed during the adaptation phase. However, more rigorous monitoring of health indicators and increased frequency of sampling are recommended to validate this indication. 58

References

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Taylor, J., Holland, B., Step, D., Payton, M. & Confer, A. Nasal isolation of Mannheimia haemolytica and Pasteurella multocida as predictors of respiratory disease in shipped calves. Res. Vet. Sci. 99, 41–45 (2014). Tornquist, SJ, Rigas, J: 2010, Interpretation of ruminant leukocyte responses. In: Schalm’s veterinary hematology, ed. Weiss, DJ, Wardrop, KJ, 6th ed., pp. 307–313. Wiley, Ames, IA. Tóthová, C., Nagy, O., & Kováč, G. (2013). The use of acute phase proteins as biomarkers of diseases in cattle and swine. In S. Janciauskiene (Ed.), Acute phase proteins (pp. 103– 138). Rijeka, Croatia: Tech Publisher. Trenkle, A., and D. G. Topel. 1978. Relationship of Some Endocrine Measurements to Growth and Carcass Composition of Cattle. J. Anim. Sci. 46:1604–1609. Wall, E. H., and D. M. Bravo. 2016. 1554 Supplementation with rumen-protected capsicum oleoresin increases milk production and component yield in lactating dairy cows. J. Anim. Sci. 94:755–755. 61

Table 3-1. Basal diet used to transition cattle to final feedlot diets with 0 (Control) or 15 mg of RPC/kg of diet DM (15RPC1) in Exp. 1. Step 1 Step 2 Step 3 Final Ingredient d0 to 7 d8 to 14 d15 to 21 d22 to end % of diet DM Corn silage 60.0 45.0 30.0 20.0 Dry rolled corn 13.0 33.0 48.0 62.0 Dry distillers’ grains with solubles 10.0 10.0 10.0 10.0 Soybean meal 15.0 10.0 10.0 6.0 Mineral supplement2 2.0 2.0 2.0 2.0 Analyzed composition3 DM, % 64.3 70.9 77.4 81.9 NDF, % DM 28.2 24.6 20.9 18.5 CP, % DM 16.0 14.3 14.3 12.9 Ca, % DM 0.74 0.71 0.70 0.67 P, % DM 0.42 0.40 0.40 0.39

4 NEm, Mcal/kg 1.81 1.91 1.99 2.15

4 NEg, Mcal/kg 1.19 1.27 1.35 1.74 1Rumen protected Capsicum (RPC) was previously mixed to the soybean meal at adequate rates to provide RPC quantities relatives to the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). 2 Mineral supplement contained: 25% Ca as CaCO3; 16% NaCl; 2% Mg as MgSO4 and CaMg(CO3)2; 3.1% K as KCl and K2SO4; 177 mg of Cu/kg as CuSO4; 980 mg of Zn/kg as ZnSO4; and 47.4 KIU of vit A/kg. 3 DM: dry matter; NDF: neutral detergent fiber; CP: crude protein, NEm: Net energy for maintenance; NEg: Net energy for gain. All diets were formulated to meet requirements for energy and protein according to the NRC (2016). 4 NEm and NEg were back calculated using animal performance from the trial (NRC, 1996).

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Table 3-2. Composition of diets fed to cattle in Exp. 2. Control1 5RPC 10RPC 15RPC Ingredient, % of diet DM Orchard grass hay 10.0 10.0 10.0 10.0 Cracked corn 81.5 81.5 81.5 81.5 Soybean meal 6.00 5.9995 5.9990 5.9985 Mineral supplement2 2.0 2.0 2.0 2.0 Urea 0.5 0.5 0.5 0.5 Rumen Protected Capsicum 0.00 0.0005 0.001 0.0015 1Rumen Protected Capsicum oleoresin (RPC) was previously mixed to the soybean meal at adequate rates to provide RPC quantities relatives to the treatments Control: no RPC; 5RPC: 5mg of RPC/kg of diet DM; 10RPC: 10 mg of RPC/kg of diet DM; and 15RPC: 15 mg of RPC/kg of diet DM. 2Mineral supplement contained: 25% Ca as CaCO3; 16% NaCl; 2% Mg as MgSO4 and CaMg(CO3)2; 3.1% K as KCl and K2SO4; 177 mg of Cu/kg as CuSO4; 980 mg of Zn/kg as ZnSO4; and 47.4 KIU of vit A/kg. 3Analyzed composition: diets for all treatments contained 87.28% dry matter; 15.99% neutral detergent fiber; and 13.68% crude protein.

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Table 3-3. Effects of rumen protected capsicum (RPC) inclusion on growth performance of feedlot cattle fed for 100 days in Exp. 1. Item Control RPC1 SEM P-value n, pen 6 6 - - Initial BW2, kg 200 200 7 0.99 Final BW, kg 348 358 9 0.42 ADG3, kg 1.48 1.58 0.06 0.24 DMI, kg/d 7.03 7.24 0.24 0.54 G:F 0.213 0.220 0.005 0.36 1Rumen protected Capsicum (RPC) was previously mixed to the soybean meal at adequate rates to provide RPC quantities relatives to the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). 2Initial body weight (BW) is an average of 2-day BW taken on d0 and d1; and final BW is an average of 2-day BW taken on d99 and d100. 3ADG: Average daily gain; DMI: dry matter intake; G:F: gain to feed ratio.

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Table 3-4. Effects of rumen protected Capsicum (RPC) supplementation on cell blood count of feedlot cattle in Exp. 1. Item Control 15RPC1 SEM P-value2 n, animal (pen) 18 18 - - Overall White blood cells, 103/µL3 11.66 11.61 0.45 0.93 Neutrophils 4.21 4.18 0.20 0.94 Lymphocytes 6.03 5.83 0.31 0.66 Monocytes 1.44 1.40 0.07 0.67 Eosinophils 0.15 0.20 0.07 0.61 Basophils 0.003 0.004 0.001 0.33 White blood cells, % of total Neutrophils 35.77 36.46 1.84 0.66 Lymphocytes 50.55 49.57 1.75 0.53 Monocytes 12.46 12.46 0.92 0.99 Eosinophils 1.19 1.47 0.72 0.63 Basophils 0.03 0.04 0.01 0.29 Neutrophil:Lymphocyte 0.75 0.78 0.06 0.47 Red blood cells, 106/µL 9.55 9.36 0.20 0.41 Hemoglobin, g/dL 12.65 12.16 0.20 0.03 Platelets, 103/µL 441.78 463.19 31.87 0.49 1Rumen protected Capsicum (RPC) was previously mixed to the soybean meal at adequate rates to provide RPC quantities relatives to the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). 2P-value represents the main effect of treatment (Control vs 15RPC). There was an effect of day (P < 0.02) for red blood cells, white blood cells, hemoglobin, platelets, neutrophils, lymphocytes, and monocytes (as total and as % of white cells), and neutrophil:lymphocyte. There were no interactions (P > 0.28) of day and treatment for any of the analyzed variables. 3Blood components normal ranges: 4.9x106 to 10x106 red blood cells/µL; 8.4 to 14 g hemoglobin/dL; 160x103 to 800 x103 platelets/µL; 4.9x103 to 13.3x103 white blood cells/µL; 1.0x103 to 6.3x103 neutrophils/µL; 1.6x103 to 8.1x103 lymphocytes/µL; 0.1x103 to 0.8x103 monocytes/µL; 0.1x103 to 1.5x103 eosinophils/µL; and 0 to 0.3x103 basophils/µL (Roland et al., 2014).

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Table 3-5. Effects of rumen protected Capsicum (RPC) supplementation on blood metabolites of feedlot cattle in Exp. 1. Item Control 15RPC1 SEM P-value2 n, animal (pen) 18 18 - - Overall Glucose, mg/dL 86.07 87.03 2.57 0.70 Insulin, ng/mL 0.43 0.44 0.06 0.97 Blood urea nitrogen, mg/dL 7.88 7.93 0.64 0.89 Haptoglobin, ng/mL 1.64 1.20 0.39 0.17 Glutathione, µmol/dL 0.33 0.34 0.01 0.75 1Rumen protected Capsicum (RPC) was previously mixed to the soybean meal at adequate rates to provide RPC quantities relatives to the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). 2P-value represents the main effect of treatment (Control vs 15RPC). There was an effect of day (P < 0.10) for glucose, insulin, blood urea nitrogen, and haptoglobin. There were no interactions (P > 0.10) of day and treatment for any of the analyzed variables.

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Table 3-6. Absolute counts of medical treatments that occurred in Exp 1. Item Control 15RPC1 Probiotic2 Light3 194 13 Heavy 1 6 Antibiotic/anti-inflammatory Light 17 9 Heavy 2 3 Fever Light 19 10 Heavy 2 4 1Rumen protected Capsicum (RPC) was previously mixed to the soybean meal at adequate rates to provide RPC quantities relatives to the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). 2Probiotic: total amount of times probiotic was administered throughout the trial; Antibiotic/anti-inflammatory: total amount of times antibiotic/anti-inflammatory was administered throughout the trial; Fever: total amount of cases of fever registered throughout the trial. 3Light: light weight block; Heavy: heavy weight block. 4Absolute counts of times probiotic or antibiotic/anti- inflammatory was administered, or fever was registered.

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Table 3-7. Health status of cattle in Exp. 1

2 P-value 1 Control 15RPC SEM T W T*W n, animals 9 9 Probiotic ever, %3 4 Light 100.0 (9/9) 65.6 (6/9) 16 .3 0.98 0.97 0.98 Heavy 10.5 (1/9) 32.1 (3/9) Probiotic 2+, % Light 76.5 (7/9) 53.3 (5/9) 17 .2 0.98 0.9 7 0.98 Heavy 0.0 (0/9) 20.3 (2/9) Antibiotic/anti-inflammatory ever, % Light 100.0 (9/9) 66.7 (6/9) 16 .2 0.98 0.97 0.98 Heavy 11.1 (1/9) 22.2 (2/9) Antibiotic/anti-inflammatory 2+,% Light 62.6 (6/9) 26.3 (3/9) 17 .7 0.41 0.02 0.41 Heavy 7.7 (1/9) 7.7 (1/9) Fever ever, % Light 100.0 (9/9) 66.7 (6/9) 16 .2 0.98 0.97 0.98 Heavy 11.1 (1/9) 22.2 (2/9) Fever 2+, % Light 76.5 (7/9) 21.8 (3/9) 16 .2 0.41 0.02 0.09 Heavy 5.8 (1/9) 12.9 (2/9) 1Rumen protected Capsicum (RPC) was previously mixed to the soybean meal at adequate rates to provide RPC quantities relatives to the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). 2T: main effect of treatment (Control vs 15RPC); W: effect of body weight block (light or heavy); T*W: interaction between treatment and body weight. 3Probiotic ever: percentage of animals that received probiotic at least once throughout the trial; Probiotic 2+: percentage of animals that received probiotic two or more times throughout the trial; Antibiotic/anti-inflammatory ever: percentage of animals that received antibiotic/anti- inflammatory at least once throughout the trial; Antibiotic/anti-inflammatory 2+: percentage of animals that received antibiotic/anti-inflammatory two or more times throughout the trial; Fever ever: percentage of animals that presented fever at least once throughout the trial; Fever 2+: percentage of animals that presented fever two or more times throughout the trial. 4Light: light weight block; Heavy: heavy weight block.

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Table 3-8. Effects of Rumen protected Capsicum inclusion on intake and total tract apparent nutrient digestibility of steers in Exp. 2.

P-value2

Control1 5RPC 10RPC 15RPC SEM L Q n, steers 8 8 8 74 - - -

Intake, kg/d DM3 10.83 10.97 10.65 10.80 0.46 0.75 0.66 OM 10.41 10.53 10.23 10.38 0.44 0.75 0.67 CP 1.50 1.53 1.48 1.50 0.07 0.68 0.73 NDF 1.69 1.73 1.67 1.68 0.08 0.59 0.62

Digestibility, % DM 77.92 78.20 76.78 78.16 1.65 0.59 0.53 OM 78.92 79.20 77.89 79.24 1.65 0.59 0.59 CP 72.22 72.27 71.03 72.14 2.11 0.85 0.66 NDF 45.51 44.07 46.25 47.62 3.37 0.75 0.32 1Experimental diets: Control: no added Rumen Protected Capsicum (RPC); RPC 5: 5mg of RPC/kg of DM; RPC 10: 10 mg of RPC/kg of DM; RPC 15: 15 mg of RPC/kg of DM. 2 L: linear effect of treatment; Q: quadratic effect of treatment. 3DM: dry matter; OM: organic matter; NDF: neutral detergent fiber; N: nitrogen. 4One steer fed RPC 15 was removed from period 3 due to low intake unrelated to treatment.

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2.50

2.00

1.50 *

1.00 ADG, ADG, kg

0.50

0.00 d1 to 21 d1 to50 d51 to 100 Control 15RPC

Figure 3-1. Effects of rumen protected Capsicum (RPC) on average daily gain (ADG) of cattle fed grain-based diets over time in Exp. 1. Cattle were fed the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). There was a trend of increased (P = 0.13; SEM=0.09) ADG for animals fed 15RPC from d1 to 21 when compared to animals fed Control and ADG was greater (P = 0.07; SEM = 0.08) for animals fed 15RPC from d1 to 50 when compared to animals fed Control. There was no difference (P = 0.57) in ADG between treatments from d51 to 100. 70

10.00

8.00

6.00

DMI, kg DMI, 4.00

2.00

0.00 d1 to 21 d1 to50 d51 to 100 Control 15RPC

Figure 3-2. Effects of rumen protected Capsicum (RPC) on dry matter intake (DMI) of cattle fed grain-based diets over time in Exp. 1. Cattle were fed the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). There were no effects of treatment on DMI (P > 0.10). 71

0.400 0.350 0.300 * 0.250

0.200 G:F 0.150 0.100 0.050 0.000 d1 to 21 d1 to50 d51 to 100 Control 15RPC

Figure 3-3. Effects of rumen protected Capsicum (RPC) on gain to feed ratio (G:F) of cattle fed grain-based diets over time in Exp. 1. Cattle were fed the treatments: 0 mg of RPC/kg of DM (Control), or 15mg of RPC/kg of DM (15RPC). Gain to feed ratio was greater (P = 0.07; SEM = 0.010) for animals fed 15RPC from d1 to 50 when compared to animals fed Control. There was no difference (P > 0.10) in G:F between treatments for remaining intervals. 72

6.90

6.70

6.50

6.30

6.10 Ruminal pH Ruminal 5.90

5.70

5.50 0 2 6 12 18 Hours after feeding

Control 5RPC 10RPC 15RPC

Figure 3-4. Effects of Rumen Protected Capsicum (RPC) on ruminal pH in steers fed corn- based diets in Exp. 2. Steers were fed the treatments Control (●) , 5RPC (■), 10RPC (♦) and 15RPC (▲), containing 0, 5, 10 and 15 mg of RPC/kg of diet DM, respectively. There was an effect of hour of sampling (P < 0.01) on ruminal pH, however there was no linear (P = 0.85) or quadratic (P = 0.65) effect of treatment nor treatment x hour interaction (P = 0.33). Standard error bars depict the variation associated with the interaction of treatment × hour (SEM = 0.19). One steer fed 15RPC was removed from period 3 due to low intake unrelated to treatment. 73

Chapter 4

Conclusion

Increasing concerns related to antimicrobial resistance derived from the use of antibiotics in the animal industry have raised the demand for alternative products.

Capsaicinoids are substances found in fruits of Capsicum plants, commonly known as hot peppers. Capsaicinoids have been reported to have antimicrobial properties and were initially studied in ruminants as a potential modifier of ruminal fermentation. Further than antimicrobial properties, these substances have been reported to have direct effects on the host. For example, in poultry and swine the supplementation with capsaicin reduced susceptibility disease, prevented disease symptoms and improved intestine health. It has been reported to regulate appetite in and stimulate digestive enzymes in rodents and to have immunoregulatory effects, decrease inflammatory response and increase milk production in dairy cows when fed in a rumen-protected form. Effects of rumen-protected Capsicum supplementation and its potentially beneficial host-related responses have not been widely investigated in beef cattle.

Therefore, the effects of supplementing rumen-protected Capsicum oleoresin (RPC) to beef cattle were investigated in 2 experiments. The objectives were to 1) determine the impact of RPC supplementation at 15mg/kg of dry matter (DM) on feed intake, average daily gain, feed efficiency, health status, immune responses, blood metabolites of newly received feedlot cattle and 2) determine the impact of increasing doses of RPC on in situ degradation of RPC, ruminal pH, and nutrient digestibility in ruminally fistulated steers.

In the first experiment, supplementation of RPC at 15 mg/kg of DM did not alter feed intake, but improved average daily gain (ADG) and feed efficiency of recently weaned calves during the first 50 days in feedlot when compared to calves fed no supplemental RPC. The 74 increased ADG is likely unrelated to nutrient utilization, because there were no differences in glucose, insulin, or blood urea nitrogen between the 2 treatments.

As evidenced in the second experiment, RPC supplementation at 0, 5, 10, or 15 mg/kg of DM did not alter total tract apparent digestibility of DM, OM, CP or NDF, nor ruminal pH of steers. Thus, nutrient digestibility and ruminal fermentation are also unlikely to have increased the ADG of steers early in the feedlot period.

Previous investigations have suggested that RPC can alter immune status and it is known that health status represents an energy drain, ultimately impacting ADG. These factors likely contributed to the results noted in Exp. 1. Fewer light weight animals developed fever, indicating illness, when fed the RPC than those fed control. Again, these data suggest that

Capsicum supplementation may be alleviating illness symptoms of cattle affording more energy to ADG in those cattle fed RPC.

That said, there were no differences between blood concentrations of haptoglobin or total glutathione, that are indicators of inflammation and oxidative stress, respectively, neither on white blood cells, that are important agents of the immune system. Given that the majority (90%) of cases of sickness were registered within two 2 from the beginning of the experiment, and blood sampling took place on days 1, 22 and 98, the critical period for analyzing those parameters may have been missed, and more frequent sampling would be needed in future research trials to confirm the hypothesis regarding alterations of immune status.