The Effects of a Novel Direct-Fed Microbial on Animal Performance and Carcass Characteristics of Feedlot Cattle

The effects of a novel direct-fed microbial on animal performance and carcass characteristics of feedlot cattle

Tosha Opheim, M.S.

A Dissertation

Animal Science

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Approved

Markus F. Miller, Ph.D. Chair of Committee

Mindy. M. Brashears, Ph.D.

Kristin E. Hales, Ph.D.

Bradley J. Johnson, Ph.D.

Jhones O. Sarturi, Ph.D.

Carlos E. Carpio Ph.D.

Mark Sheridan Dean of the Graduate School

ACKNOWLEDGEMENTS

I would like to sincerely thank Texas Tech for the education I have received, the mentors I have had, the friends I have gained, and the enrichment it has given my life. I cannot imagine how differently my life would be had I not taken a leap of faith and moved to Lubbock five years ago.

To my committee members, I appreciate everything you have done for me both big and small. Dr. Miller and Dr. Brashears, the memories and experiences gained on trips with you are invaluable and will be greatly missed. Dr. Carpio, Dr. Johnson, and Dr.

Sarturi thank you to each of you for your mentorship, knowledge, and involvement in my academic career. Finally, thank you to Dr. Hales for joining in on the fun and taking on another committee.

To all my fellow graduate students and colleagues, thank you for the help, friendship, and patience as we have all navigated our academic paths. The friendships I have made during my time here are invaluable. Many of them have already graduated but have not hesitated in helping to make sure I finish this degree!

Finally, I cannot even put into words how much I appreciate the support my friends and family have given me. Phone calls, texts, and good vibes from the great north have played an integral role in my success and sanity. Without all of you, none of this would be possible. Lastly, I am so thankful my path led me to Lubbock, to continue on and get my Ph.D., but most importantly to my fiancé, Brant. I sincerely doubt I would have survived this degree without you!

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii LIST OF TABLES v LIST OF FIGURES vi ABSTRACT vii I. INTRODUCTION 1 II. REVIEW OF LITERATURE 3 Ruminants 3 Direct-Fed Microbials 6 Bacteria 8 Mechanisms of Action 9 Feeding Applications 12 Antibiotics 16 Tylosin 16 Monensin 18 Literature Cited 22 III. Evaluation of a novel direct-fed microbial on gain performance and carcass characteristics in beef feedlot steers. 32 Abstract 32 Introduction 34 Materials and Methods 34 Growth Performance 35 Carcass Characteristics 39 Statistical Analyses 39 Results 39 Discussion 40 Conclusion 48 Literature Cited 49 IV. Evaluation of the comparative effects on gain performance and carcass characteristics in beef feedlot steers when fed a novel direct-fed microbial or the industry standard subtherapeutic antibiotic with and without an ionophore. 59 Abstract 59

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Introduction 61 Materials and Methods 62 Growth Performance 62 Carcass Characteristics 66 Statistical Analyses 66 Results 67 Discussion 68 Conclusion 73 Literature Cited 74 V. Conclusion 82

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LIST OF TABLES

3.1. Beef cattle finishing diet ingredient inclusion and nutritional composition (DM basis) 53

3.2. Effect of DFM and subtherapeutic antibiotic use on feedlot performance of beef steers 54

3.3. Effect of DFM and subtherapeutic antibiotic use on nutrient intake and apparent total tract nutrient digestibility 55

3.4. Effect of DFM and subtherapeutic antibiotic use on VFA concentrations 55

3.5. Effect of DFM and subtherapeutic antibiotic use on feedlot carcass characteristics of beef steers 56

4.1. Beef cattle finishing diet ingredient inclusion and nutritional composition (DM basis) 77

4.2. Effect of DFM and subtherapeutic antibiotic use on feedlot performance of beef steers 78

4.3. Effect of DFM and subtherapeutic antibiotic use on nutrient intake and apparent total tract digestibility 79

4.4. Effect of DFM and subtherapeutic antibiotic use on feeding behavior on d90 of the feeding period 79

4.5. Effect of DFM and subtherapeutic antibiotic use on feedlot carcass characteristics of beef steers 80

4.6. Effect of DFM and subtherapeutic antibiotic use on the economics of treatments compared to control 81

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LIST OF FIGURES

2.1. Potential mechanisms of action for DFMs 10

3.1 Average Papillae Area cm2 57

3.2 Absorptive Surface Area, % 57

3.3 Average Papillae Number, n/cm2 58

3.4 Absorptive Surface Area, cm2 58

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ABSTRACT

Two beef finishing studies were conducted at the Texas Tech Burnett Center to evaluate a novel direct-fed microbial (DFMs) Lactobacillus Salivarius L28. The newly isolated strain of L. salivarius L28 has demonstrated pathogenic inhibition of Escherichia coli,

Salmonella, and Listeria monocytogenes in-vitro. Additionally, Krehbiel et al. (2003) reported that DFMs have been shown to reduce fecal shedding of Escherichia coli

O157:H7 and have minimal negative effects on growth performance and carcass traits. In the first study, single source crossbred steers (n = 144; BW = 371 ± 19 kg) were used in a randomized complete block design feedlot study to evaluate the effects of L28 on performance and carcass characteristics of finishing steers. Treatments included: no

DFM, no subtherapeutic antibiotics, and no ionophore (CON), monensin sodium

(Rumensin 90; Elanco; Greenfield, NJ; 33 g/ton DM basis) and tylosin phosphate (Tylan

40; Elanco; 11 g/ton DM basis) (MonTy), and monensin and L. salivarius L28 (10⁶ CFU steer/d) (MonPro). In the second study, single source crossbred steers (n = 240; BW =

319 ± 29 kg) were used in a randomized complete block design feedlot study. Treatments were the same as the first study, with the addition of tylosin phosphate (Tylan 40; Elanco;

11 g/ton DM basis) (TY) and L. salivarius L28 (10⁶ CFU steer/d) (PRO). No differences were observed for BW, ADG, or feed efficiency in either study. Cattle fed L28 consistently had a decreased DMI compared to controls in both studies. Both studies had a numerical depression in HCW and decrease in marbling scores for L28 fed cattle.

Apparent total tract digestibility was not improved with the addition of L28. Further research is needed into the mechanism of action in order to determine the most appropriate use of L28 within the agriculture industry.

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CHAPTER I

INTRODUCTION

Consumers understanding of present production technologies such as Beta- adrenergic agonists, steroidal implants, and subtherapeutic antibiotics is limited; therefore, they are continually urging the removal of these technologies from beef cattle production. Although these technologies are beneficial and allow beef production to be more sustainable and efficient, they are still not well perceived. In general, the public perception issues have created a magnitude of problems for the beef industry, but it has created a large opportunity for natural technology developments. Direct-fed microbials

(DFMs) have become increasingly popular in beef cattle production as consumers continue to demand more naturally produced meat. In a survey by Cargill, it was reported that a majority of U.S. and Brazilian consumers prefer beef from antibiotic-free cattle

(Cargill, 2016). Going forward, this presents both opportunities and challenges to the beef feedlot industry.

In 2012, selection and evaluation of lactic acid bacteria (LAB) isolates (200+) from cattle feces and retail meat products began in the Texas Tech experimental sciences laboratories. Positive LAB strains were identified using API 50 CH kit with final confirmation done by DNA sequencing. From there, pathogen inhibition testing began.

Specific pathogen screening included Salmonella, Escherichia coli O157:H7, and

Listeria monocytogenes. As screening continued, strains were narrowed to 30, then four, and finally the L28 strain was selected.

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After successful in-vitro pathogen inhibition, the research scope widened and L28 began in vivo applications in produce, cheese, pet food, and beef cattle.

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CHAPTER II

REVIEW OF LITERATURE

Ruminants

Ruminants are a unique classification of animals; the specific design of their digestive systems separates them from monogastric animals. Cows, sheep, and goats are the primary ruminant animals in livestock production; however, buffalo, deer, and elk are also ruminants. The unique four-chambered stomach, specifically the rumen, allows ruminants to utilize feedstuffs in a manner unlike other animals (Hofmann, 1988).

Ruminant animals can breakdown cellulose and hemicellulose found in plant-based material because of microbial fermentation within the rumen, Animals with monogastric stomachs are unable to break down these fractions. This unique capability allows ruminant animals to graze and utilize rangeland which is typically not suited for farming or the production of other livestock. Additionally, the unique ruminant system has led to the development of many specialized products that alter fermentation within the rumen and subsequent gastrointestinal tract (GIT) microbiota. Beef cattle production extensively uses these products because their specific mechanisms of action within the rumen that positively impact the ruminant animal. This review seeks to address the interactions between direct-fed microbials, antibiotics, the rumen, and the ruminant animal.

The rumen is a large microbial fermentation vat that contains bacteria, protozoa, and fungi. Bacteria compose the greatest proportion of microbes accounting for 109 to

1011 cells/mL (Stewart et al., 1997; Russell, 2002). Protozoa account for 104 to 106

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cells/mL (Bohatier, 1991). Fungi make up 103 to 105 zoospores/mL (Stewart et al. 1995,

Qi et al., 2009, and Krause et al., 2013). The microbes are specialized to be able to ferment and breakdown specific kinds of substrates such as carbohydrates, protein, fat, etc. The microbial population will adapt depending on the diet the animal is consuming.

Because of the extensive microbial population in the rumen, substrates such as fat, protein, carbohydrates are fermented into various products such as volatile fatty acids

(VFAs), CH4, CO2, NH3, and microbial cells (NASEM, 2016). The main fermentation substrate is carbohydrates which results primarily in VFAs. The three principal VFAs are acetate, propionate, and butyrate, in descending order of quantity produced in the rumen.

Traditionally, in beef cattle production two types of diets are fed to growing or finishing animals (NASEM, 2016). Typically, a high-forage (fibrous) diet is fed during the growing phase to allow the animals to reach skeletal maturity before they finish accruing lean tissue. A high-concentrate (starch-based) diet is fed during the finishing phase of an animal. Once the animal has reached skeletal maturity, the animal is fed a high-concentrate diet the remainder of its life to accrue both lean tissue and fat. The more energy dense, high-concentrate diet allows the animal to reach physiological maturity; the point at which the animal is accruing more fat than lean tissue (Bruns et al, 2004).

Depending on the type of diet the animal is consuming, the microbial population adapts to meet the supply of substrates. If the diet is more fibrous, the rumen will contain more fiber-digesting (cellulolytic) bacteria which will result in an increased amount of acetate produced as well as an increased ruminal pH (NASEM, 2016). Notable cellulolytic bacteria include Fibrobacter succinogenes Ruminococcus albus, and R. flavefaciens.

Cellulolytic bacteria prefer a more neutral pH environment; a pH below 5.5 can impact

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the fiber digestibility (Weimer, 1996). Cattle grazing on pasture or receiving high-fiber diets that include a large percentage of hay or silage would typically have an increase in cellulolytic bacteria. In contrast, if the animal is consuming more starch, more starch- digesting (amylolytic) microbes will be present resulting in more propionate and a decreased rumen pH (Weimer, 1996). A high-starch diet is typical of most finishing diets in a feedlot. Notable amylolytic bacteria include Ruminobacer amylophilus,

Succinomonas amylolytica, and S. bovis.

As previously mentioned, carbohydrates are the main rumen fermentation substrate when high-concentrate diets are fed (NASEM, 2016). Carbohydrates consist of several fractions including starch, cellulose, hemicellulose, sugar, etc. Through several pathways and metabolism steps, carbohydrates are broken down into hexose and then two pyruvate (Russell and Wallace, 1988; Miller, 1995). The pyruvate is further broken down into two acetyl CoA, two propionate, and two lactate. The previous steps describe the direct process in which carbohydrates are broken down into propionates. Carbohydrates can eventually be reduced into acetate and butyrate; however, this requires additional steps. Two acetyl CoA must be further broken down to subsequently produce two acetate and one butyrate. The two lactate are further broken down into two more propionate, increasing the total number of propionate to four.

Propionate is the most highly sought after VFA, as it in the only one to contribute to glucose synthesis (NASEM, 2016). Eighty to ninety-five percent of propionate is absorbed through the rumen epithelium which goes to the liver and is then converted in the liver to glucose (Fahey and Berger, 1988; Kristensen and Harmon, 2004). Propionate is a three-carbon chain; therefore, two propionate can make one glucose. The three-

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carbon design allows carbohydrates to easily be broken down into propionate and propionate to be converted into glucose. Additionally, propionate production decreases

H2 production which in turn decreases the amount of CH4 being produced by the animal.

The rumen microbial environment and VFA production can be altered by feeding different diets to the animal, but it can also be altered by feed additives, such as DFMs.

Direct-Fed Microbials

The broad overarching term, probiotic, is used not only in the human health and wellness arena, but also the animal world. Oftentimes the labeling can be vague and not well defined as the supplements may include both pre-and probiotics or other live cultures. Probiotics are considered viable microorganisms that can benefit the health of the host (FAO, 2016). Given the ambiguous and generous use of the term probiotics, the

US FDA has subsequently required animal supplements and feed manufacturers to use the term direct-fed microbial (DFM) instead (Yoon and Stern, 1995). Direct-fed microbials are defined as “a source of live, naturally occurring microorganisms (bacteria or yeast)” (Yoon and Stern, 1995) and are limited to generally recognized as safe organisms. A live microbe intended for human use should be labeled with the scientific definition “live biotherapeutics” (Vaillancourt, 2006) instead of the general term probiotic (Sanders, 2008). Many times, DFMs are used in a manner that simultaneously benefits the health and immune system of the animal, while also reducing the colonization of pathogenic bacteria. The latter effect is beneficial in food animal production systems as a first step towards reducing the amount of pathogenic bacteria that enter the food system. The simultaneous benefit to animals and humans has made

DFMs a very attractive potential supplement in food animal production. The two-fold

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benefit combined with the veterinary feed directive (VFD) changes and consumer desire for less antibiotic use has positioned DFMs in the middle ground of the ongoing consumer/producer dialogue.

The most recent and all-encompassing change to the feed additive sector was the further expansion of the VFD. Prior to 2017, the VFD was already in place as policy to direct the judicious use of antibiotics in food production animals; however, major changes were enacted on January 1, 2017 (Federal Register, 2015). These changes were made with a goal to improve the judicious use of antibiotics, improve the public perception of antibiotic use in the production animal industry, and to potentially reduce antimicrobial resistance. All feed grade antibiotics that were medically important, meaning they are used in both the livestock industry and the human health sector, were placed on the VFD. Examples of a medically relevant antibiotics are penicillins, tetracyclines, and macrolides (FDA, 2012). The VFD now requires the veterinary oversight of the use antibiotics on the VFD. This has resulted in many feed grade antibiotics that were previously sold over-the-counter as being under the sale and distribution of a veterinarian. Non-medically important antimicrobials such as ionophores are not on the VFD and do not require veterinary oversight. An additional change was made that requires all antibiotics to be labeled for the mitigation, treatment, or prevention of diseases and claims of “growth promotion” were to be removed from the label. As mentioned, one of the reasons for the change in the VFD was a result of public perception and public pushback of antibiotic use in production animals. This created even more potential for DFM research and use.

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Bacteria

According to Kanwar et al. (2016) the two most common bacteria (bifidobacteria and lactic-acid bacteria (LAB)) used in both human and animal supplements.

Bifidobacteria are gram-positive obligate anaerobes that are typically found in the GIT of humans and animals. These bacteria are essential to the function of the GIT and largely responsible for immune system tolerance and immunomodulation (Schell et al., 2002).

Bifidobacteria are most commonly used in DFMs in poultry production (Flint and

Garner, 2009). Lactic-acid bacteria are a large homogenous group of bacteria that can be found in a wide range of hosts: humans, animals and plants and are associated with fermented foods, beverages, and probiotics (Makarova et al., 2006). These organisms are microaerophilic, gram-positive organisms that have been popularized because of their ability to produce bacteriocins, improve immune responses, balance gut microbes, and inhibit the colonization of pathogenic bacteria (Messaoudi et al., 2013). Lactic-acid bacteria have been used in an array of different ways in food animal production.

Including but not limited to: innoculates for ensiling feeds (Kang et al., 2009; Schmidt et al., 2009), reduction of opportunistic pathogens in weaned pigs (Maré et al., 2006), improved health and performance markers in suckling pigs (Rondón et al. 2013), competitive exclusion of opportunistic bacteria in poultry (Pascual et al., 1999; Zhang et al., 2007; Sornplang et al., 2015), and the reduction of fecal shedding of Escherichia coli

O157:H7 in feedlot cattle (Brashears et al., 2003; Perterson et al., 2006; Stephens et al.,

2006; Tabe et al., 2007). In addition to the two most common bacteria in all DFM supplements, the most commonly marketed product for ruminants contains

Saccharomyces cerevisiae, a live yeast (Chaucheyras-Durand and Durand, 2009). Several

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studies including Sniffen et al. (2004), Jouany (2006), and Stella et al. (2007) have reported improved performance in dairy cattle, supplemented with S. cerevisiae, including increased dry matter intake (DMI) and milk production. Similarly, Lesmeister et al. (2004) reported improvement in beef cattle parameters such as average daily gain

(ADG), DMI, feed efficiency and final body weight (FBW).

Mechanisms of Action

Direct-fed microbials can have a magnitude of varied responses depending on the strain, dose, and in vivo vs in vitro applications. First, we will discuss the general mechanisms of action for the beneficial bacteria. Followed by a more in-depth look at the ruminant animal and DFMs. The general public is commonly aware that probiotics and/or

DFMs can balance the bacteria within the GIT; this balance can be established in a number of different ways. The microbiota of the host can be balanced via the increase in beneficial bacteria (colonization in the GIT) or the reduction of bacterial pathogens

(inhibiting the virulence or growth) (Sherman et al., 2009). Virulence and growth can be impacted by the production of antimicrobial substances, known as bacteriocins (Corr et al., 2007), influencing pathogenic gene expression (Medellin-Pena et al., 2007), reduced local pH (Fayol-Messaoudi et al., 2005), or antagonistically blocking the binding receptor sites preventing colonization (Johnson-Henry et al., 2007). Less popularized effects take place in the mucosal and submucosal layers of the GIT. Typically, these effects are generalized into the reduced ability of pathogens to bind to the host. Binding can be impacted by increased mucin production (Mack et al., 1999), increased trefoil factors

(antibacterial peptides; Clyne et al., 2004) and defensins (antibacterial cationic peptides;

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Wehkamp et al., 2004), or enhanced epithelial lining in the GIT (Sherman et al, 2005;

Johnson-Henry et al., 2008).

Ruminant animals can benefit from several of the bacteria benefits, as well as benefits specific to that of the rumen. Once such effect is the stabilization of ruminal pH from live yeast products (Bach et al., 2007; Thrune et al., 2007; Marden et al., 2008;

Desnoyers et al., 2009; and De Ondarza et al., 2012). The importance of maintaining a stable pH > 5.6 (Owens et al., 1998; Cooper et al., 1999; Brown et al., 2000; Bevans et al., 2005; Nagaraja and Tigemeyer, 2007) been well documented. In reviews by Owens et at. (1998) and Vasconcelos and Galyean (2008), both have

Figure 2.1. Potential mechanisms of action for DFMs. Adapted from Sherman et al. (2009). slp= surface-layer proteins; PB= probiotics; G= goblet cells; P= pathogen; ZO= zona occludins; NF-휅B= nuclear factor-휅 B; IFN훾= interferon-훾; MAPK= mitogen- activated protein kinases; IL-10= interleukin-10; TBF훽= transforming growth factor-훽; Ig= immunoglobin; TC= T lymphocyte; DC= dendritic cell; PC= Paneth cells

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outlined the negative effects of a low and unstable pH: ruminal acidosis, reduced absorption, variable DMI, diarrhea, laminitis, and liver abscess. It is thought that when live yeast is supplied with ample soluble nitrogen and carbohydrates, microbial growth may be enhanced (Chaucheyras-Durand et al., 2008). This is supported by research that indicates an increase in cellulolytic bacteria. Jouany et al. (1999) proposes this is because the yeasts ability to scavenge free oxygen in the rumen creating a more anaerobic environment. Whereas, Robinson and Ersmus (2009) propose that the both pre-and probiotic nature of yeasts contribute micronutrients to the bacteria.

Pathogen exclusion is a key component for ruminant DFMs. Rumens are large microbial vats; therefore, finding a way to not only increase the good bacteria, but also decrease the bad bacteria is ideal. The reduction of bad bacteria can impact the overall health of the animal and have a positive downstream effect in the reduction of pathogens entering the human food system. As DFM bacteria attach to the intestinal lining, less space is available for pathogen bacteria to adhere and begin colonization. This was first supported by Muralidhara et al. (1977) when more Lactobacilli was counted than E. coli in the intestinal tissue of piglets. Abu-Tarbouch et al. (1996) further supported the adhesion of DFM bacteria in the GIT of young calves. Competitive exclusion and shedding of pathogens such as E. coli O157 have been reported with DFM use in feedlot cattle as well (Elam et al., 2003; Zhao et al., 2003; Callaway et al., 2004; and LeJune and

Wetzel, 2007).

Although we know several DFMs produce bacteriocins; the specific roles of bacteriocins in the ruminant animal are not as well understood. It is believed that most bacteria are capable of producing at least one bacteriocin (Klaenhammer, 1988).

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Bacteriocins produced by Enterococcus faecium CCM4231 inhibited growth of pathogenic bacteria Salmonella, E. coli, and Listeria monocytogenes in vitro (Lauková and Czikková, 1998) and bacteriocins produced by Streptococcus bovis HC5 performed similarly to monensin in vitro (Lee et al., 2002). Because of the complex nature of the rumen microbiota, bacteriocins effects are simply not as clearly defined yet.

Shifts in rumen parameters and VFA production when using DFMs is a complex and ambiguous area. Several theories exist with limited production research to support the claims. According to Yoon and Stern (1995) and Nocek et al. (2002) the use of lactate-producing bacteria may adapt the rumen microbes to increases in lactic acid concentration and subsequently prevent acidosis. Servin (2004) postulated that the production of lactic acid may cause negative interactions for the intracellular pH of competitor bacteria. In contrast, Kung and Hession (1995) found that using a lactate- utilizing bacteria may prevent the accumulation of lactate. S. cerevisiae can utilize lactic acid in vitro but the extent to which is can utilize it in the rumen is undetermined

(McAllister et al., 2011).

Feeding Applications

Although the dairy industry extensively uses DFMs in both pre-ruminant calves and cows for milk production, for the purposes of this review, we will only be covering beef industry applications. For a calf entering the feedlot, the adaptation phase may be one of the most stressful events it will incur. Some calves will be freshly weaned, others will be transitioning off forage or crop residue. Alone these events are stressful, but combined with transporting, vaccinations, commingling, and other traditional processing events (castrating, dehorning, etc.) can result in the animal to be immunocompromised.

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These stressors have been documented to impact the GIT microbiota (Williams and

Mahoney, 1984). This is where DFMs can help play a role in a smoother transition during the adaptation phase at the feedlot and sometimes improve subsequent growth performance.

Fox (1988) did a review of combinations of L. acidophilus, L. plantarum, L casei, and S. faecium at receiving/during adaption for approximately 30 days. The result was a

13.2% increase in ADG, 2.5% increase in DMI, and 6.3% improvement in feed efficiency. The greatest DFM response was during the first 14 days where the most stress was likely incurred (Crawford et al., 1980; Hutcheson et al., 1980). Gill et al. (1987) reported a 9.3% increase in ADG, 9.5% improvement in feed efficiency, and 10.9% reduction in morbidity when feeding a DFM during the 28d receiving period. Dew and

Thomas, 1981; Kercher et al., 1985 & 1986; and Kiesling and Lofgreen, 1981) reported no performance responses for newly weaned or received cattle when given a DFM.

Similarly, Krehbiel et al. (2001) reported no performance responses when comingled sale barn cattle were administered a DFM cocktail during the receiving period. The only response was a reduction in animals that were administered an antibiotic for bovine respiratory disease when the DFM was given. The more recent meta-analysis by

Wagner et al., (2016) reported an increase in ADG and feed efficiency in cattle fed S. cerevisiae product during the receiving phase. In two studies by Keyser et al. (2007), no performance responses were observed during the adaptation period for newly-received heifers that were administered an oral and feed grade level of yeast. In the first study, the percentage treated for bovine respiratory disease was greater in the control cattle; however, there was not a health response in the second study for cattle fed the DFM.

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When sheep were fed a live yeast during the high-concentrate diet adaptation period, rumen lactate concentrations were lower and pH levels were more stable (Michalet-

Doreau et al. 1997). Although there are mixed results on the benefits of DFMs in the receiving phase, Samuelson et al. (2016) reported in the most recent consulting nutritionist survey, that 62.5% of clients surveyed use a DFM in the receiving phase.

This survey represents over 14,000,000 cattle on feed in the U.S.

Similarly, to the receiving study data, there are mixed results on what DFMs can do for growth performance and carcass data in the finishing phase. According to Krehbiel et al. (2003) in a review, DFMs feed to feedlot cattle increased ADG by 2.5-5%, feed efficiency by 2%, and hot carcass weight by 6 to7kg. Consistent with the review by

Krehbiel et al. (2003), Wagner et al. (2016) did a meta-analysis on eight feedlot studies where cattle were fed S. cerevisiae yeast fermentation products. It was reported that cattle on yeast had increased ADG, improved feed efficiency, and had a greater percentage of cattle grade low choice or greater. Additionally, there was an interaction for days on feed and DMI, indicating that the effect on DMI depended on how long the cattle were fed.

This is somewhat in agreement with the previous research reviewed by Krehbiel et al.

(2003) which reported DMI was highly variable from study to study. In recent study

Wilson et al. (2016) fed heifers a combination of L. acidophilus and P. freudenreichii in the finishing diet but found no effect on performance responses or carcass metrics. This is in contrast with an earlier study using a similar combination of P. freudenreichii and L. acidophilus but, reported feed efficiency improvements (Swinney-Floyd et al., 1999).

Tabe et al. (2007) reported the combination of P. freudenreichii and L. acidophilus was able to reduce fecal shedding of E. coli O157:H7 but not Salmonella.

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There have been various results feeding single strain DFMs. Ware et al. (1988) first reported positive ADG and feed efficiency responses for L. acidophilus. Conversely, both Brashears et al. (2003) and Peterson et al. (2006) reported no differences in performance responses; however, both studies found reductions in fecal shedding of E. coli O157:H7. Arthur et al. (2010) fed Bacillus subtilis and found no difference in performance or fecal shedding E. coli. A study conducted on various strains of L. acidophilus observed that strains NP28, NP51, and the combination NP35/NP51 were effective at reducing fecal shedding of E. coli O157:H7 in feedlot steers vs. no DFM or the strain NP35 alone (Stephens et al., 2006). Finally, in a feed supplement study, heifers were fed two treatments: 1) monensin, tylosin, and Bovamine Defend (the feedlot industry standard ionophore, subtherapeutic antibiotic, and DFM, respectively) and 2) a fermented S. cerevisiae product (NaturSafe). There were no differences in growth performance, carcass characteristics, and liver abscess prevalence (Scott et al., 2016).

Further complicating DFM research in feedlot cattle is the ruminal responses because of the complex and competitive microbe community. Dawson et al. (1990) fed L. acidophilus plus yeast and enterococci and reported increased in cellulolytic bacteria counts. Contrastingly, using the same mixed DFM, Dawson and Newman (1988) reported no difference in anaerobic, cellulolytic, and lactobacilli concentrations. Koevering et al.

(1994) fed L. acidophilus and observed lower concentrations of total ruminal lactate.

Beauchemin et al. 2003 fed E. facieum EF212 to feedlot calves and increased propionate and the count of lactic-acid utilizing bacteria. The disparities in DFM research are not easily discernible; there is a severe lack of true replication using the same strain,

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combinations of strains, or analyzing the same metrics. The positive responses observed and the vast amount of bacteria potential leave unlimited opportunity.

Antibiotics

Currently antibiotics can be used therapeutically (to treat an illness), metaphylactically (widespread treatment of a group of animals to prevent an illness), or prophylactically (subtherapeutically treating animals to prevent a disease) (Landers et al.,

2012).

Tylosin

One particular medically-important antimicrobial used in beef cattle production is tylosin phosphate. Tylosin phosphate is a macrolide that is used in the mitigation of liver abscess in cattle. In order to feed tylosin phosphate at the label approved dose of 12.1 mg/kg of diet DM. Tylosin phosphate is fed to reduce liver abscess by reducing

Fusobacterium necrophorum and Truperella pyogenes. F. necrophorum is thought to be the primary cause of liver abscesses in feedlot cattle and can be isolated in as many as 81-

100% of liver abscesses (Nagaraja and Chengappa 1998). T. pyogenes which was originally considered Arcanobacterium pyogenes was recently discovered to be a separate group; this was after a German microbiologist Hans Georg Trüper (Yassin et al., 2011).

Both F. necrophorum and T. pyogenes are naturally occurring in the rumen of cattle.

Tylosin phosphate works to reduce the numbers of these bacteria. Macrolides traditionally target gram-positive bacteria (T. pyogenes), but are also able to reduce the gram-negative bacteria (F. necrophorum). Although these are the main two bacteria present in liver abscess the number of other bacteria being isolated from liver abscesses are increasing and include Salmonella enterococcus and E. coli O157:H7 (Amachawadi

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and Nagaraja, 2016). Liver abscess are typically formed via the rumenitis-liver abscess complex (Nagaraja and Lechtenberg 2007) which was first reported by Jensen et al.

(1954). Damage to the rumen wall such as scratches from roughages or hair, punctures from accidental foreign objects, or epithelial tissue damage from low rumen pH and/or stress can cause opportunistic bacteria such as F. necrophorum and T. pyogenes to adhere to the rumen wall and begin to colonize (Jensen et al., 1954; Nagaraja and Lechtenberg

2007). F. necrophorum utilizes lactate; therefore, it is potentially increased in high- concentrate diets that increase lactate and reduce ruminal pH. After F. necrophorum and

T. pyogenes have colonized they are able to escape the rumen and enter the portal vein.

Ultimately these bacteria end up in the liver. Once in the liver, they recolonized and begin to form a fibrin-encapsulated abscess. The ability of F. necrophorum and T. pyogenes to escape the rumen and colonize in the liver are because of several factors but specifically the virulence factor. F. necrophorum produces a leukotoxic virulence factor that helps it to colonize and prevents phagocytosis and protect T. pyogenes. Additionally,

T. pyogenes is a helper bacterium that aids F. necrophorum in its establishment and transition from an anaerobic to an aerobic environment. T. pyogenes produces lactic acid which F. necrophorum utilizes (Rzequska, et al., 2019).

In a study conducted by Nagaraja et al. (1999) five feedlots fed a control diet without tylosin phosphate and five feedlots included tylosin phosphate in their diet. The incidence of liver abscess was 18.4% in the control and 12.6% when tylosin phosphate was fed. Livers from each treatment group were collected and they all contained F. necrophorum. In a review, Vogel and Laudert (1994) summarized 40 trials which included 6,971 cattle fed, liver abscess incidence was reduced by 73% with the inclusion

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of tylosin. A more recent meta-analysis reported that tylosin phosphate reduced the incidence of liver abscess from 30% to 8% (Wileman et al., 2009)

In an economic analysis by Brown and Lawrence (2010), it was estimated that liver abscess costs the packing plant industry over $22 million dollars in lost sales of livers and GIT each year and a loss of over 25 million kg of carcass weight each year.

Liver abscess costs the overall beef industry millions of dollars, not just on the packing industry side, but also in the reductions of feed efficiency and growth performance, especially in the case of large abscesses which impact the bottom-line of the producer.

With concerns for the future of antibiotic use in production animals, the need for alternatives are evident.

Monensin

Ionophores are feed additive antimicrobials used to alter fermentation in the rumen and improve the overall health of the animal (NASEM, 2016). Ionophores are used extensively in beef cattle production in both the growing and the finishing phase;

93% of clients surveyed in the nutritionist use an ionophore in receiving diets and 97% use it in finishing diets (Samuelson et al., 2016). By improving health in these phases, feed efficiency of the animal can also be improved. There are currently four licensed ionophores marketed and fed in North America; they include Lasalocid, Laidlomycin propionate, Salinomycin, and Monensin.

The ionophore mechanism of action is to bind to cation and facilitate their movement across the cell membrane (NASEM, 2016). Each ionophore works in a slightly different manner by having a greater affinity to different cations. Ultimately the result is the same, regardless of which cations are binding. The ionic gradient is disrupted and

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more cations are pulled across the membrane either increasing the concentration outside the cell membrane or inside. This change in the gradient then causes the intracellular pH to decrease. The decreased pH and imbalanced ionic gradient causes the pump, such as the sodium-potassium pump, to increase the speed in which it is pumping. This results in more ATP being expended and the bacteria depleting its energy stores. Ionophores specifically affect gram-positive bacteria. As they expend energy trying to regain homeostasis, they are unable to reproduce. Gram-negative bacteria are largely unaffected which is thought to be a result of their less permeable cell membrane (NASEM, 2016).

As the gram-positive bacteria become outnumbered, the microbial population shifts which leads to an increase in propionate, and decrease in butyrate and acetate (Bergen and Bates, 1984).

Monensin is a carboxylic polyether compound that has a greater affinity to sodium ions (Russel and Strobel, 1989). When an animal is being fed a diet containing monensin, more sodium ions are carried inside the cell of the gram-positive bacteria.

These bacteria continually expend energy trying to remove the sodium ions and balance the concentration gradient. If they are unable to do this, they will futilely expend more and more energy to increase the speed of the sodium-potassium pump. The addition of monensin not only inhibits gram-positive bacteria growth, but also inhibits amino-acid fermenting bacteria. The inhibition of amino-acid fermenting bacteria results in a protein sparing effect as fewer amino acids are fermented in the rumen allowing the animal to directly utilize the amino acids instead of the microbes (Russel and Strobel, 1989; Krause and Russell, 1996). Additionally, this can result in a decrease in ruminal ammonia concentrations. Other benefits to feeding monensin include decreased methane produced

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because of an increase in propionate produced (Guan et al., 2006). Finally, monensin can decrease the incidence of acidosis, by decreasing meal size and increasing meal frequency (Bergen and Bates, 1984; Goodrich et al., 1984, Cheng et al., 1998, Birkelo,

2003). Lactic acid bacteria are gram-positive; therefore, monensin inhibits their growth, thus decreasing acidosis.

In a meta-analysis conducted by Duffield et al. (2012), 40 peer-reviewed articles plus 24 trial reports were analyzed. Monensin was either fed during the growing or the finishing phase of the animals in the meta-analysis. Several aspects were reviewed, but of particular interest were the growth performance metrics: feed efficiency, ADG, and DMI.

In the meta-analysis, feed efficiency was decreased by 6.4%, DMI decreased by 3.1%, and ADG increased by 2.5%; Duffield et al., 2012). Simply stated, the animals were able to gain more weight more efficiently by consuming less as a proportion of body weight gain. On average, by feeding monensin, DMI decreases by 0.53 kg, ADG increased by

0.029 kg/d, and feed efficiency decreased by 0.53 kg feed/kg body weight gain (Duffield et al., 2012).

Although monensin is an antimicrobial, it falls within a drug class that humans do not use. Ionophores are used to alter the fermentation within the rumen increasing the amount of propionate and decreasing acetate and butyrate. Increasing propionate will increase glucose synthesis and decrease the amount of methane the animal is producing.

By decreasing the amount of methane produced within the rumen, which also decreases the amount of methane eructated. However, the negative perceptions surrounding antibiotic use in animal production has given rise to many new products and many new research trials. A non-antibiotic product that can positively impact animal health, animal

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efficiency, and profitability in beef cattle production in the same way monensin has is yet to be discovered.

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CHAPTER III

EVALUATION OF A NOVEL DIRECT-FED MICROBIAL ON GAIN PERFORMANCE AND CARCASS CHARACTERISTICS IN BEEF FEEDLOT STEERS.

Abstract

Single source crossbred steers (n = 144; BW = 371 ± 19 kg) were used in a randomized complete block design to evaluate the effects of a novel direct-fed microbial (DFM) on performance and carcass characteristics of finishing steers. Steers were blocked by initial

BW and assigned randomly to treatments within block (12 pens/treatment; 4 steers/pen).

Treatments included: no DFM, no subtherapeutic antibiotics, and no ionophore (CON), monensin sodium (Rumensin 90; Elanco; Greenfield, NJ; 33 g/ton DM basis) and tylosin phosphate (Tylan 40; Elanco; 11 g/ton DM basis; MonTy), and monensin and L. salivarius L28 (10⁶ CFU steer/d; MonPro). All treatments were included in a steam- flaked corn-based diet fed once daily using a slick bunk management approach and no β- adrenergic agonist was fed. Prior to treatment initiation cattle were processed using which included the administration of a Synovex Plus implant (200 mg TBA trenbolone acetate + 28 mg estradiol benzoate; Zoetis; Florham Park, NJ). Cattle were weighed at 28 d intervals, alternating between pen and individual body weights. On d 70 – d 74 diet, orts, and fecal samples were collected for apparent total tract digestibility. Steers were shipped in 2 groups after treatment-blind personnel deemed approximately 60% of steers within a block had sufficient external fat cover to grade USDA Choice (group 1 = 140 d; group 2 = 158 d). Ruminal fluid and rumen tissue samples were collected at the slaughter facility for VFA and morphology analysis. Data were analyzed using the GLIMMIX procedure of SAS with pen serving as the experimental unit, treatment was the fixed

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effect, and block was the random effect. There were no differences in carcass adjusted final BW (P = 0.23) or carcass adjusted overall ADG (P = 0.23) across treatments. Dry matter intake decreased for cattle fed MonPro (P < 0.01); however, this did not result in improved overall feed efficiency (P = 0.61) for MonPro. There were no differences across treatment for apparent digestibility of nutrients (P > 0.13) nor were there differences in VFAs upon slaughter. There was a tendency for an increase in papillae number for MonPro cattle. Hot carcass weight, dressing percent, LM area, and yield grade did not differ (P > 0.23) across treatments. Marbling scores were increased for

CON cattle; however, all treatments graded USDA Choice or better. There were no differences in the percent liver abscess across all treatments (P =0.94). These results suggest that L. salivarius L28 does not positively impact growth performance or carcass characteristics when compared to beef cattle fed subtherapeutic levels of antibiotics. The results of this study indicate additional research is warranted elucidate the mechanisms of action of L28 and future of this novel probiotic in the feedlot sector.

Key Words – beef steers, direct-fed microbial

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Introduction

Antimicrobial resistance is a growing concern to public health as continued use of antibiotics can results in increased pools of resistant-gene reservoirs among bacteria. In response to public concerns, as of January 1, 2017, medically important antibiotics have been listed in the Veterinary Feed Directive (VFD; Federal Register, 2015). The VFD requires veterinary oversight and the prescription of feed-through antibiotics for production animals; one such antibiotic is tylosin phosphate. In the United States, tylosin phosphate is fed in beef cattle to reduce the incidence of liver abscesses associated with Fusobacterium necrophorum and Trueperella pyogenes (Nagaraja and Chengappa,1998). The newly isolated strain of Lactobacillus salivarius L28 has demonstrated pathogenic inhibition of

Escherichia coli, Salmonella, and Listeria monocytogenes in- vitro. Additionally, Krehbiel et al. (2003) reported that direct-fed microbials (DFMs) have been shown to reduce fecal shedding of E. coli O157:H7 and can have minimal negative effects on growth performance and carcass traits. According to the 2015 feedlot nutritionist survey

Samuelson et al. (2016), only 59% of clients surveyed use DFMs in finishing diets. The potential benefits combined with the potential market capture (40% of clients not feeding a

DFM), has warranted the further exploration of L. salivarius L28 in a feedlot setting.

Materials and Methods

All experimental procedures were conducted in accordance with an approved Texas

Tech University Institutional Animal Care and Use Committee (protocol number 16054-

06).

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Growth Performance

Single source crossbred steers (n = 144; bodyweight (BW) = 371 ± 19 kg) were used in a randomized block design to evaluate the effects of a novel DFM on growth performance and carcass characteristics. Cattle were received on September 15, 2016 and placed in 12 soil-surfaced pens (16 steers/pen). The cattle had access to fresh water and long-stem grass hay; the following day, cattle received a Sweet Bran-based (Cargill Corn

Milling, Blair NE) receiving diet (65% concentrate) and remained on the receiving diet until treatment initiation. Forty-eight hours after arrival, cattle were processed: individually weighed (Silencer Chute, Moly Manufacturing, Lorraine, KS, mounted on Avery Weigh-

Tronix load cells, Fairmount, MN; readability ± 0.45 kg; before each use, the scale was validated with 454 kg of certified weights), individually tagged with a unique identification number, and vaccinated against Clostridium chauvoei, Clostridium septicum, Clostridium novyi, Clostridium sordellii, Clostridium perfringens types C and D (UltraChoice 7; Zoetis

Animal Health, Florham Park, NJ), Mycoplasma bovis bacterin (Mycoplaz; Vetbio Inc.,

San Angelo, TX), and infectious bovine rhinotracheitis virus, parainfluenza-3 virus, bovine respiratory syncytial virus, and bovine viral diarrhea virus types 1 and 2 (Bovi-Shield Gold

5; Zoetis Animal Health). On September 23, 2016, a sorting BW (unshrunk) was collected and cattle were treated for external parasites (Dectomax Pour-On; Zoetis Animal Health) and on September 26, 2016, the animals were sorted into weight blocks and returned to dirt pens. Thirteen days post arrival, the steers were randomly assigned to treatments within block (12 pens/treatment; 4 steers/pen) and placed in partially slotted concrete pens (2.9 m wide x 5.5 m deep; 2.4 m of linear bunk space). Steers were allowed 7 days to adapt to the concrete pens before trial initiation on October 5, 2016. At trial initiation, an individual

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BW was collected, and each steer was implanted with Synovex Plus (200 mg TBA + 28 mg estradiol benzoate; Zoetis Animal Health). At the time of trial initiation, pens began receiving their respective treatments; over a 21-d period, steers were transitioned from the

65% concentrate diet to a 90% concentrate diet.

Treatments were arranged in a randomized complete block design and included: no

DFM, no subtherapeutic antibiotic, and no ionophore (CON), monensin (Rumensin 90;

Elanco; Greenfield, NJ; 36.4 mg/kg DM) and tylosin phosphate (Tylan 40; Elanco; 12.1 mg/kg DM) (MonTy), and monensin and L. salivarius L28 (106 CFU steer/d) (MonPro).

L. salivarius L28 was diluted into a lactose carrier at 1 x 1010 concentrated culture. The L. salivarius L28 and lactose premixed was subsequently diluted into a ground corn carrier to reach the desired 106 CFU steer/d; adjusted for dry matter intake. The ground corn premix was then added to a total mixed ration. All treatments were included in a steam-flaked corn-based finishing diet (Table 3.1) fed once daily using a slick bunk management approach. No β-adrenergic agonist was fed. Diet samples were taken weekly, dried in a forced air oven for approximately 24 h at 100° C, and the weekly DM value was used to calculate the daily DMI.

Cattle were weighed at 28 d intervals, alternating between pen BW collection and individual BW collection for purposes of corresponding days on feed for the fecal microbial collections for a microbiology counterpart study (data not presented). Individual

BW was collected on d 0, 56, and 112; final BW was collected on d 140 and 158 for the 1st and 2nd slaughter groups, respectively. Steers were transported to a commercial abattoir in

2 slaughter groups after treatment-blind personnel deemed approximately 60% of steers

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within a block had sufficient external fat cover to grade USDA Choice (group 1 = 140 d; group 2 = 158 d). A 4% pencil shrink was used for determination of final BW.

Sample Collections and Laboratory Analyses

Diet, orts, and fecal samples were obtained over a 5-day digestibility collection period. The first diet sample was obtained at 0700 on December 6th, d 70, with the first fecal sample collected at 1600 that day. Diet samples were collected from each bunk immediately after feed delivery; samples were composited 12 pens/treatment. Fecal samples were collected upon defecation from each steer and homogenized by pen; samples were collected at 0700 and 1600 for the five-day duration. A minimum of 2 steers were collected from each pen if fresh fecal samples were not available from all four steers. Orts were collected each morning before the daily feed delivery. Diets were composited by treatment and fecal samples were composited by pen. Sample composites were dried at 60°C in a forced-air oven for 72 h and ground in a Wiley mill (Thomas

Scientific, Swedesboro, NJ) to pass a 1-mm screen.

Acid insoluble ash (AIA) was used as the internal marked for estimation of total fecal output and apparent total tract nutrient digestibility. The 2N HCl method (Van

Keulen and Young, 1977) was performed on all samples in duplicate. Laboratory DM content was determined in a 100°C forced-air oven for 4 h; OM was determined by burning samples at 550°C for 4 h (AOAC International, 2005). Analysis for NDF and

ADF were performed in succession using an Ankom Fiber Analyzer (Ankom Technology

Corp., Macedon, NY) (Van Soest et al., 1991); NDF was performed using sodium sulfite and α-amylase. Hemicellulose (HEM) was calculated as the difference between NDF and

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ADF concentrations. Crude protein concentration was analyzed in a Leco CNS Analyzer

(Leco CNS-2000, St Joseph, Michigan, USA). To determine apparent total tract digestion of DM, OM, CP, NDF, ADF, and HEM, the following equation was used: 100 − 100 ×

([AIA concentration in feed/AIA concentration in feces] × [nutrient concentration in feces/nutrient concentration in feed]).

Individual samples (6 cm2) of ruminal cranial sac epithelium were collected post slaughter (n = 144), immersed in 70% alcohol, and preserved under refrigeration (4℃).

Using the methodology of Daniel et al. (2006), papillae were counted within an area of ruminal tissue that was trimmed to 2 cm2, followed by the random removal of 12 papillae specimens for further electronic scanning to evaluate papillae area, size, and total absorptive area (The University of Texas Health Science Center of San Antonio, Image

Tool).

For the first slaughter group only, individual samples (50 mL) of ruminal fluid (n =

53) were collected following evisceration for VFA analysis. Rumen fluid samples were chilled on ice, transported back to Texas Tech University, and frozen (- 20℃). Samples were thawed, strained through cheesecloth, and centrifuged (10,000 x ɡ; 10 min; 4℃). Four mL of the ruminal fluid supernatant were combined with 0.8 mL of 25% metaphosphoric acid containing 2-ethylbutyrate (0.2005 g in 100 mL) as internal standard (Erwin et al.,1961). Using the methods of Gotsch and Galyean (1983), individual VFAs were analyzed in duplicate utilizing gas chromatography (Shimadzu GC-8A, Shimadzu

Scientific Instruments Inc., Columbia, MD; Supelco SP-1200, 2 m × 5 mm × 2.6 mm glass column, Supelco/Sigma-Aldrich Inc., Bellefonte, PA).

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Carcass Characteristics

Steers were transported 327 km to commercial slaughter facility. Trained Texas

Tech personnel collected data during slaughter and camera carcass data 48 hrs after slaughter. Livers were assessed for abscesses using the Eli Lilly (Elanco) Liver Check

System (✓ = no abscesses, A- = 1 or 2 small abscesses, A = 2 to 4 small active abscesses,

A+ = 1 or more large active abscesses, A+ Adhesion = liver adhered to gastrointestinal (GI) tract, A+ Open = open liver abscess; as described by Brown and Lawrence, 2010. The presence of an abscess (A-, A, and A+) was pooled for percent liver abscess. Dressing percentage (DP) was calculated by dividing the HCW by the shrunk final BW. Carcass- adjusted final BW was calculated as HCW divided by the average dressing percent of each slaughter group, 63.30% and 64.54% for the first and second slaughter groups, respectively.

Statistical Analyses

Data were analyzed using the GLIMMIX procedure of SAS (SAS Inst. Inc., Cary,

NC) with pen serving as the experimental unit, treatment as the fixed effect, and block as the random effect. Binomial proportions were used to analyze quality and yield grade distributions using the GLIMMIX procedure of SAS with block as a random effect. The protected F-test was used and when significant, the least squares means were separated using the LSD procedure of SAS (훼 = 0.05); P-values between 0.05 and 0.10 are discussed as trends.

Results

No differences in interim BW or carcass adjusted final BW were observed (P >

0.23; Table 3.2). However, a tendency for a decrease in final BW for MonPro compared to

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the control and MonTy was observed (P = 0.09). No differences in interim ADG or carcass adjusted ADG was observed (P > 0.23). The MonPro did have a tendency for a decrease in overall ADG for the feeding period (P = 0.10). During the first 56 days of the feeding period, MonPro had a decrease in DMI compared to the control with MonTy being intermediate (P = 0.05). After 56 days, MonPro cattle had a decrease in DMI (P < 0.02) which resulted in a decrease over the entire feeding period (P < 0.01). During the first 56 days, MonTy cattle had a greater feed efficiency (P = 0.03) than the control cattle. The improved feed efficiency did not last throughout the entire feeding period as there were no differences in overall feed efficiency (P > 0.25).

There were no differences in the apparent total tract digestibility of the analyzed nutrients (P > 0.13; Table 3.3). Steers fed MonPro tended (P = 0.09) to have an increased number of ruminal papillae compared to control and MonTy treatments. Combinations of feed additives did not affect papillae area, size, or absorptive area (P ≥ 0.26). Similarly, ruminal VFA profile was not affected (P ≥ 0.12) by dietary treatments.

Dressing percentage, LM area, 12th-rib fat, and YG were not different across treatments (P > 0.31; Table 3.5). Hot carcass weight did not differ across treatments.

MonTy and MonPro had a decrease in marbling scores (P < 0.01) which resulted in a tendency for both treatments to have an increase in percent select (P = 0.06). There were no differences in the percentage of liver abscess across treatments (P = 0.94).

Discussion

This was the first feedlot study evaluating the effects of feeding L. salivarius L28 on growth performance, carcass characteristics, liver abscesses, apparent total tract digestibility, and rumen morphology. There have been several studies conducted feeding

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DFMs, specifically the use of lactic acid bacteria and live yeast (Saccharomyces cerevisiae) both in feedlot cattle and receiving studies. In reviews conducted by Ware et al. (1988) and

Krehbiel et al. (2003), both reported improvements in ADG when DFMs were fed. Ware et al. (1988) combined 8 feedlot trials feeding L. acidophilus and noted a 4% increase in

ADG. Similarly, when Krehbiel et al. (2003) combined 6 feedlot studies using L. acidophilus and Propionibacterium freudenreichii a 2.6% increase in ADG was noted. In contrast, a single study feeding a combination of L. acidophilus P. and freudenreichii reported no improvements in ADG (Vasconcelos et al., 2008). Brashears et al. (2003) and

Peterson et al. (2007) found no improvements in growth performance when feeding L. acidophilus NPC 747 or NP51, respectively. The present study did not detect an improvement in ADG during the first half of the feeding period for the DFM-fed cattle compared to the control and MonTy treatments. As the trial continued, the MonPro cattle tended to decrease in ADG slightly more than the MonTy or control cattle. This resulted in an additional tendency and numerical decrease in ADG for MonPro cattle over the entire feeding period. This decrease in ADG may be explained by a decrease in DMI throughout the trial for this treatment group.

In the first 56 days, cattle fed MonPro exhibited a decrease in DMI compared to the control with MonTy being intermediate. After 56 days, MonPro fed cattle had a lesser DMI compared to the control and MonTy treatment. This is reflected in the numerical decrease in DMI during the digestibility collection. The depression in DMI continued for the

MonPro treatment for the remainder of the study. It is likely monensin played a role in the decreased DMI for treatment cattle, as discussed in Duffield et al. (2012). Although not statistically different, the MonTy treatment had a numerically lower DMI across the entire

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study compared to the control. The depressed DMI for MonPro cattle over the entire feeding period may be explained by an associative effect between the ionophore and DFM.

In a meta-analysis Duffield et al. (2012) reported a consistent decrease in cattle fed monensin; typically, a 3.1% decrease which translates to approximately a 0.53 kg decrease in DMI often resulting in improved feed efficiency. In contrast to the consistent performance responses garnered by feeding monensin, DMI data for cattle fed DFMs is inconsistent (Krehbiel et al., 2003). In 4 studies evaluating various strains of Lactobacillus acidophilus, there were no differences in DMI for cattle receiving a DFM (Galyean et al.,

2000; Brashears et al., 2003; Peterson et al., 2007; Vasconcelos et al., 2008). In contrast, in a 6-study analysis by Krehbiel et al. (2003) when contrasts where used to compare all DFM treatments, various L. acidophilus strains and P. freudenreichii to controls, there was a tendency for increased DMI in DFM fed cattle. When other contrasts were used to elucidate the effects of specific strains of DFMs, no DMI changes were reported. Previous live animal studies have not reported on the potential interaction between monensin sodium and DFMs, or the mode of action of an interaction.

It is probable, specific DFM strains or combinations will impact DMI differently.

Several studies have reported pH stabilization using yeast products (Bach et al., 2007;

Thrune et al., 2007; Marden et al., 2008; Desnoyers et al., 2009; and De Ondarza et al.,

2012) as well an increase in cellulolytic bacteria (Robinson and Ersmus, 2009). Feedlot diets are typically high-concentrate and low-fiber; therefore, the effects of increased cellulolytic bacteria are less pronounced. Nevertheless, cattle fed the MonPro treatment maintained fiber digestibility similar to the control cattle despite the inclusion of monensin.

MonTy fed cattle had numerical decreases for fiber digestibility which could be attributed

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to the application of antibiotics (Mir, 1989). More research into the mechanism of action of specific strains of DFMs coupled with various diets is needed to explain the variation in

DMI data observed in the current study.

The decrease in DMI ultimately did not result in improved feed efficiency for the

MonPro treatment as ADG was 7.1% lower compared to the control. The lack of improvement in feed efficiency is in agreement with other L. acidophilus trials of (Galyean et. al., 2000; Brashears et al., 2003; Peterson et al., 2007). In contrast to the current study, and the study by Peterson et al. (2007), Vasconcelos et al. (2008) observed improvements in overall feed efficiency in cattle fed treatments of L. acidophilus NP51 and P. freudenreichii NP24. Interestingly, as dose of NP51 increased, feed efficiency quadratically increased. It is also notable that Vanconcelos et al. (2008) and Peterson et al. (2007) fed

NP51, but improvements in efficiency were unable to be observed until NP24 was combined with NP51, which could indicate associative effect between different strains of

DFM.

The lack of differences across treatments in the present study suggests that the feed additives had little effect on digestibility of diet. Similarly, in a continuous culture study,

DFMs were tested for digestibility differences (Yang et al., 2004). Treatments were control

(without DFM), Propionibacterium P15, Enterococcus faecium EF212, and E. faecium

EF212 combined with a yeast, S. cerevisiae; and no differences in digestibility were observed for any of the treatments. Beauchemin et al. (2003) conducted 2 studies comparing digestion of a DFM and control diet. When E. faecium EF212 was fed to ruminally cannulated steers, intestinal digestion of NDF and total tract digestibility was decreased compared to control. However, in the second study when S. cerevisiae was

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combined with E. faecium EF212 digestibility did not differ from the control across nutrients and site of digestion. It would appear the addition of the yeast mitigated the negative digestive effects of E. faecium EF212 (Beauchemin et al., 2003).

Overall, there was little impact to the carcass characteristics, except marbling score

(discussed later) when feeding the DFM in the present study. Likewise, Brashears et al.

(2003) and Vasconcelos et al. (2008) also reported no carcass differences when feeding a

DFM. In the present study there was a decrease in marbling score, which contrasts the 6 studies Krehbiel et al. (2003) reviewed. Peterson et al. (2007) reported no difference in marbling score for the DFM fed cattle, but had a decrease in yield grade compared to the control group. Low residual feed intake has been correlated with a decrease in marbling score, indicating that final marbling is a direct result of the excess calories available to the animal to be stored as fat (Basarab et al., 2003; Pethick et al., 2004). Based on this concept, it is likely that the decrease in DMI for the MonPro cattle contributed to their decrease in marbling score. In the present study, there was a numerical reduction in HCW for cattle fed the MonPro treatment. The 9 kg reduction will result in economic losses in a sector with increasingly tight profit margins. This finding contrasts the findings of Krehbiel et al.

(2003) that reported an increase in HCW when feeding a DFM. The numerical reductions of ADG and HCW coupled with the steady decrease in DMI over the feeding period, may indicate L. salivarius L28 is better suited for shorter days on feed because of the improvement in feed efficiency within the first 56 days and fewer days on feed may lessen the negative carcass impacts.

In-vitro, L28 demonstrated pathogenic inhibition of Salmonella, E. coli, and

Listeria monocytogenes by 4.5, 6.5, and 8.5 log10 CFU/ml, respectively, compared to

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controls cultivated without L28 (Ayala et al., 2017). In a review of Lactobacillus salivarius, Chaves et al. (2017) describes multiple animal and human studies where L. salivarius was applied with mixed results depending on the strain and dose. However, decreases in gram-negative bacteria such as Salmonella, Prevotella intermedia, and

Fusobacterium nucleatum and decreases in gram-positive bacteria such as Enterococcus faecalis and Staphylococcus aureus were observed (Chaves et al., 2017). Based on these observations, it can be proposed that L. salivarius has demonstrated antimicrobial effects in various capacities on both gram-positive and gram-negative bacteria. Further research in human and animal applications, as well as in-vitro, are needed to further elucidate the mechanism of action and to what extent the antimicrobial effects are and their potential application in the feedlot industry.

Antimicrobial effects on gram-negative and gram-positive bacteria could significantly modify rumen bacterial populations. It is plausible that L28 may have had an antimicrobial effect on rumen bacteria that ultimately led to the decrease in DMI. Dry mater intake decreases are commonly observed with the antimicrobial monensin (Duffield et al., 2012). However, monensin increases propionate and decreases acetate and butyrate

(Bergen and Bates, 1984). Although the VFA samples are considered a momentary metabolic marker, there were no differences in VFA concentrations in the present study.

The performance responses coupled with the VFA changes allow monensin to sequester improvements in feed energy (NASEM, 2016). These results would suggest L28 was unable to capture these same feed energy improvements.

The MonPro fed cattle had a 0.63 kg reduction in daily DMI compared to the control cattle. This 0.63 kg reduction in feed resulted in a loss of 0.91 Mcal of NEg/d for a

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total loss of 127.4 Mcal of NEg for the first slaughter group and 143.78 Mcal of lost NEg for the second group. This 6% caloric loss can help to explain the 15 kg reduction FBW and 9 kg reduction in HCW when the MonPro cattle are compared to the control cattle. Using the dressed steer average price for 2017 ($186.04/cwt), a 9 kg reduction in HCW would result in a loss of $36.91 (USDA, 2018).

The decrease in total energy consumed, particularly NEg, helps to explain the decrease in marbling scores for MonPro cattle. In a review on marbling, Pethick et al.

(2004) discusses the importance of excess energy consumption for the accretion of intramuscular fat deposition. For increased marbling accretion, increases in propionate and subsequent glucose synthesis is required (Park et al., 2018). Along with decrease in DMI, the proposed antimicrobial effects occurring in the rumen for cattle fed the MonPro treatment may have had a negative effect on glucose synthesis and the subsequent intramuscular fat accretion in the present experiment. Although there were no differences in ruminal VFA concentration at slaughter, it cannot be ascertained if there were differences in VFAs produced throughout the feeding period.

Based on Gaylean et al. (1981), when cattle are fasted acetate concentration peaks at approximately 36 hours into fasting, while propionate and butyrate concentrations plunge compared to their fed, control counterparts. Because the VFA measurements were taken at the plant after a fasting period and transport stress, they are likely a misrepresentation of the VFA concentrations while on their treatment diets. They were also postmortem which has an effect on pH, which in turn, affects how the microbial population performs. Subsequently, this would alter the VFA profile for that particular metabolic snapshot.

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A ruminant adapts to the microbial environment it hosts by developing a capacity to absorb and metabolically utilize that VFA profile. Therefore, upon death, it is likely that there would be an abundance of the VFA’s they were not predisposed to utilize. Therefore, cattle that produced more acetate would have a greater capacity to absorb it prior to death, leaving their paunch with a decreased amount of acetate comparatively. This theoretical inversion would suggest that the acetate:propionate ratio in MonPro cattle would have been numerically greater than the control and MonTy cattle. A shift in the acetate:propionate ratio in favor of acetate results in a more convoluted conversion of energy down-stream.

The inefficiency associated with an increase in the molar proportion of acetate does not support marbling development (Park et al., 2018).

The numerical increase in butyrate for the MonPro cattle may be explained by their increase in papillae density. Lee et al. (2011) recorded that concentrations of VFAs increased over time postmortem. This indicated the residual production of VFAs via microbial activity regardless of the deceased host animals’ inability to absorb them.

Although all treatments underwent the same protocol, the increase in ruminal papillae is indicative that the MonPro steers would have a greater concentration of butyrate compared to the control and MonTy treatments, as it is the primary energy source for gut health and development. Additionally, several studies have indicated that the dietary inclusion of lactobacillus enhanced the epithelial lining in the GIT (Sherman et al, 2005; Johnson-Henry et al., 2008). This improvement in epithelial tissue could allow modifications to feedlot rations. With an increased capacity for roughage utilization, a greater concentrate: roughage ratio could be used in the diet with the DFM, allowing an opportunity to increase diet energy density, potentially mitigating the decrease in growth performance that was

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observed in the current study. More investigations into the mode of action of L28, and its interaction with butyrate-producing bacteria, as well as direct effects on GIT tissue should be conducted.

Conclusion

The results suggest that L28 decreased DMI and marbling; however, carcass- adjusted ADG and HCW did not differ, and were similar to cattle fed monensin and tylosin.

These results need to be confirmed with further research, especially the tendency for decreased DMI to investigate the use of this novel probiotic in a beef finishing capacity.

Additional research is warranted in shorter day feedlot studies to elucidate the future of this novel probiotic and the impact of feeding duration. Based on the difference papillae density and numerical observations in fiber digestibility and VFA concentrations, further research into the mode of action of L28 is warranted, particularly with modified diet compositions in ruminant animals.

Although there was a decrease in performance from a beef cattle perspective in the current study, some of the results indicate that L28 could be beneficial in a dairy setting.

The capacity for the MonPro cattle to numerically maintain fiber digestibility, in spite of the inclusion of monensin, could increase the acetate:propionate ratio, a desirable process in dairy cows, as it increases butter fat content (Van Soest and Allen, 1959). This could allow the dairy industry to have more flexibility with their roughage inclusions and sources, in addition to the hygienic improvements made by the interaction between L28 and common pathogens.

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Federal Register. 2015. Veterinary Feed Directive. https://www.federalregister.gov/documents/2015/06/03/2015-13393/veterinaryfeed- directive.

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Goetsch, A.L., and M.L. Galyean. 1983.Influence of feeding frequency on passage of fluid and particulate markers in steers fed a concentrate diet. Can. J. Anim. Sci. 63:727-730.

Krehbiel, C. R., S. R. Rust, G. Zhang, and S. E. Gilliland. 2003. Bacterial direct-fed microbials in ruminant diets: Performance response and mode of action. J. Anim. Sci. (E. Suppl.2): E120-E132. doi:10.2527/2003.8114_suppl_2E120x

Lee, T. L., D. U. Thomson, and B. W. Wileman. 2011. Stability of Rumen pH Measurements Obtained Postmortem. Bovine Practitioner, 45(1), 52.

Mir, Z. 1989. Monensin, chlortetracycline and tylosin effects on performance and digestion in lambs fed a ground alfalfa diet. Can. J. An. Sci. 69: 505-508

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Park, S., H. Kim, M. Piao, H. Kang, D. Fassah, D. Jung, and S, Yoo. 2018. PSXII-34 Effects of genomic estimated breeding value (GEBV) of marbling score and dietary energy level on growth performance during fattening stage of Korean cattle steers. J. Anim. Sci. 96(3):431-432.

Peterson, R. E., T. J. Klopfenstein, G. E. Erickson, J. Folmer, S. Hinkley, R. A. Moxley, and D. R. Smith. 2007. Effect of Lactobacillus acidophilus strain NP51 on Escherichia coli

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O157:H7 fecal shedding and finishing performance in beef feedlot cattle. J. Food Prot. 70:287-291.

Pethick, D. W., G. S. Harper, and V. H. Oddy. 2004. Growth, development and nutritional manipulation of marbling in cattle: a review. Australian Journal of Experimental Agriculture, 44(7), 705-715.

Samuelson, K. L., M. E. Hubbert, M. L. Galyean, and C. A. Löest. 2016. Nutritional recommendations of feedlot consulting nutritionists: The 2015 New Mexico State and Texas Tech University survey. J. Anim. Sci. 94:2648–2663 doi:10.2527/jas2016-0282

Sherman, P. M., K. C. Johnson-Henry, H. P. Yeung, P. S. C. Ngo, J. Goulet, and T. A. Tompkins. 2005. Probiotics reduce enterohemorrhagic Escherichia coli O157:H7 and enteropathogenic E. coli O127:H6-induced changes in polarized T84 epithelial cell monolayers by reducing bacterial adhesion and cytoskeletal rearrangements. Infect. Immun. 73:5183-5188.

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Van Keulen, J., and B. A. Young. 1977. Evaluation of acid-insoluble ash as a natural marker in ruminant digestibility studies. J. Anim. Sci. 44:282–287. doi: 10.2527/ jas1977.442282x

Van Soest, P. J. and N. N. Allen. 1959. Studies on the relationships between rumen acids and fat metabolism of ruminants fed on restricted roughage diets. J. Dairy Sci. 42(12):1977-1985.

Van Soest, P. J., J. B. Robertson, and B. A. Lewis. 1991. Methods of dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74:3583–3597. doi: 10.3168/jds.S0022-0302(91)78551–2

Vasconcelos, J. T., N. A. Elam, M. M. Brashears, and M. L. Galyean. 2008. Effects of increasing does of live cultures of Lactobacillus acidophilus (Strain NP 51) combined with a single dose of Propionibacterium freudenreichii (Strain NP 24) on performance and carcass characteristics of finishing beef steers. J. Anim. Sci. 86:756-762.

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Yang, W. Z., K. A. Beauchemin, D. D. Vedres, G. R. Ghorbani, D. Colombatto, and D. P. Morgavi. 2004. Effects of direct-fed microbial supplementation on ruminal acidosis, digestibility, and bacterial protein synthesis in continuous culture. An. Feed Sci. Tech. 114:179-193.

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Table 3.1. Beef cattle finishing diet ingredient inclusion and nutritional composition (DM basis) Item % DM Ingredients Steam-flaked corn 63.80 WCGF, Sweet Bran 20.00 Alfalfa hay 4.00 Cottonseed hulls 4.00 Fat (yellow grease) 3.00 Treatment Premix1 1.00 Urea 0.60 Limestone 1.60 TTU Supplement2 2.00 Nutrient Composition3 CP 13.50 Ether Extract 5.50 Ca 0.66 P 0.41 K 0.60 Mg 0.20 S 0.20 NEm, Mcal/kg 2.38 NEg, Mcal/kg 1.45 1Treatment premixes containing 1 of 3 feed additives packages will be mixed with fine ground corn (carrier) to equal 1% of the diet (DM). Expected DMI is 9 kg/d. 2Supplement: NaCl, 15%; CuSO4, 0.12%; MnSO4, 0.08; Se Premix 0.2%, 0.25%; ZnSO4, 0.21%; Vitamin A, 110000 IU/kg; and Vitamin E, 875 IU/kg. 3Based on tabular values for individual feed ingredients (NRC, 2000).

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Table 3.2. Effect of DFM and subtherapeutic antibiotic use on feedlot performance of beef steers. Treatment1 Item CON MonTy MonPro SEM P-value BW, kg Initial BW 370 371 371 5.0 0.97 d 56 497 503 496 8.3 0.39 d 112 597 599 588 10.6 0.39 Final BW2 618 618 603 9.0 0.09 Final BW, Adj.2,3 618 617 604 8.9 0.23 ADG, kg d 0 to 56 2.26 2.37 2.25 0.083 0.39 d 57 to 112 1.79 1.71 1.64 0.075 0.29 d 113 to end2 0.57 0.49 0.36 0.070 0.10 d 0 to end2 1.68 1.67 1.56 0.059 0.10 d 0 to end, Adj.2,4 1.68 1.66 1.58 0.059 0.23 Daily DMI, kg d 0 to 56 10.18a 9.86ab 9.66b 0.202 0.05 d 57 to 112 10.61a 10.49a 9.88b 0.239 0.02 d 113 to end2 9.99a 9.81a 9.11b 0.240 <0.01 d 0 to end2 10.16a 9.96a 9.53b 0.192 <0.01 G:F, kg/kg d 0 to 56 0.222b 0.239a 0.232ab 0.0047 0.03 d 57 to 112 0.168 0.163 0.165 0.0049 0.67 d 113 to end2 0.058 0.050 0.040 0.0074 0.25 d 0 to end2 0.165 0.167 0.164 0.0033 0.61 d 0 to end, Adj.2,4 0.164 0.166 0.165 0.0033 0.93 1CON = No DFM, subtherapeutic antibiotics, or ionophore; MonTy = monensin and tylosin; MonPro = monensin and L. salivarius L28. 2Final BW was taken on d 140 for the first slaughter group and d 158 for the second slaughter group; denoted as ‘end’; final BW shrunk 4%. 3Adjusted final BW equaled HCW divided by the average dressing percent of each slaughter group, 63.30% and 64.54% for the 1st and 2nd groups, respectively. 4Adjusted final BW, initial BW, and days on feed (for each slaughter group) were used to calculate adjusted ADG (d 0 to end). Adjusted G:F was calculated as the ratio of adjusted ADG to average DMI (d 0 to end). a,b,cMeans within rows that do not have a common superscript differ, P ≤ 0.05.

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Table 3.3. Effect of DFM and subtherapeutic antibiotic use nutrient intake and apparent total tract nutrient digestibility. Treatment1 Item CON MonTy MonPro SEM P-value Intake, kg/d DM 10.46 10.34 9.90 0.290 0.12 OM 9.95 9.83 9.42 0.276 0.13 CP 1.44 1.42 1.36 0.040 0.12 NDF 1.86 1.84 1.76 0.052 0.12 ADF 0.72 0.71 0.68 0.020 0.12 HEM 1.14 1.13 1.08 0.032 0.13 Daily fecal output, 1.78 1.90 1.66 0.087 0.17 kg/steer Digestibility, % DM 82.91 81.67 83.09 0.822 0.40 OM 84.03 82.76 84.26 0.805 0.35 CP 75.60 74.26 75.92 1.211 0.56 NDF 53.86 47.86 53.25 2.371 0.13 ADF 55.03 48.79 54.05 2.323 0.13 HEM 53.14 47.28 52.75 2.422 0.13 1CON = No DFM, subtherapeutic antibiotics, or ionophore; MonTy = monensin and tylosin; MonPro = monensin and L. salivarius L28. a,b,cMeans within rows that do not have a common superscript differ, P ≤ 0.05.

Table 3.4. Effect of DFM and subtherapeutic antibiotic use on VFA concentrations. Treatment1 Item CON MonTy MonPro SEM P-value Total VFA, mMol/L 92.9 106.9 102.7 7.46 0.40 C2:C3 1.77 2.06 1.64 0.148 0.14 VFA profile, mMol/ 100 mMol total VFA Acetate 52.5 55.2 48.6 2.51 0.20 Propionate 30.8 27.7 32.0 1.68 0.17 Butyrate 9.4 9.8 11.0 0.75 0.24 Isobutyrate 0.5 0.5 0.5 0.05 0.63 Valerate 2.9 2.7 3.0 0.35 0.75 Isovalerate 3.9 4.1 4.8 0.28 0.12 1CON = No DFM, subtherapeutic antibiotics, or ionophore; MonTy = monensin and tylosin; MonPro = monensin and L. salivarius L28. a,b,cMeans within rows that do not have a common superscript differ, P ≤ 0.05.

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Table 3.5. Effect of DFM and subtherapeutic antibiotic use on feedlot carcass characteristics of beef steers. Treatment1 Item CON MonTy MonPro SEM P-value HCW, kg 396 395 387 4.9 0.23 Dressing percentage 64.06 63.90 64.21 0.321 0.64 12th-rib fat, cm 1.57 1.63 1.50 0.066 0.38 LM area, cm² 80.55 82.73 80.96 1.052 0.31 KPH, % 1.81 1.80 1.77 0.116 0.93 Yield grade 3.75 3.75 3.64 0.101 0.68 Yield grade 2-2.99, % 14.58 14.58 14.58 0.051 1.00 Yield grade 3-3.99, % 47.84 50.03 58.76 0.087 0.55 Yield grade 4-5, % 37.24 35.13 26.70 0.078 0.53 Marbling score3 488a 456b 437b 10.9 <0.01 Select, % 10.42 31.91 27.08 0.068 0.06 Low Choice, % 50.00 42.55 54.17 0.072 0.53 Upper ⅔ Choice & Above, %4 39.56 25.47 18.69 0.073 0.10 Liver Abscess, %5 10.42 12.50 12.50 0.048 0.94 1CON = No DFM, no subtherapeutic antibiotics, or ionophore; MonTy = monensin and tylosin; MonPro = monensin and L. salivarius L28. 2Dressing percentage was calculated by dividing the HCW by the shrunk final BW. 3300 = slight00, 400 = small00, 500 = modest00. 4Upper ⅔ Choice & Above includes choice, high choice, and prime. 5All liver abscesses were pooled and include: A- = 1 or 2 small abscesses, A = 2 to 4 small active abscesses, A+ = 1 or more large active abscesses, A+ Adhesion = liver adhered to GI tract, A+ Open. a,b,cMeans within rows that do not have a common superscript differ, P ≤ 0.05.

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0.25 0.24 P = 0.27 SEM = 0.015 0.24 0.23 0.23 0.22 0.22 0.21

0.21 Average Average Papillae Area, cm2 0.20 0.20 CON MonTy MonPro Treatment

Figure 3.1 Average Papillae Area cm2

92 P = 0.48 92 SEM = 0.62 92 92 91 91 91

91 Absorptive Absorptive Surface Area, % 91 90 CON MonTy MonPro Treatment

Figure 3.2 Absorptive surface area, %

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34 P = 0.09 33 SEM = 1.73 32 31 30 29 28 27

26 Average Average PapillaeNumber, n/cm2 25 CON MonTy MonPro Treatment

Figure 3.3 Average papillae number, n/cm2

15 P = 0.26 SEM = 0.965 14

12 Absorptive Absorptive Surface Area, cm2

12 CON MonTy MonPro Treatment

Figure 3.4 Absorptive surface area, cm2

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CHAPTER IV.

EVALUATION OF THE COMPARATIVE EFFECTS ON GAIN PERFORMANCE AND CARCASS CHARACTERISTICS IN BEEF FEEDLOT STEERS WHEN FED A NOVEL DIRECT-FED MICROBIAL OR THE INDUSTRY STANDARD SUBTHERAPEUTIC ANTIBIOTIC WITH AND WITHOUT AN IONOPHORE.

Abstract Single source crossbred steers (n = 240; BW = 320 ± 28 kg) were used in a randomized block design feedlot study to compare the effects of a novel direct-fed microbial (DFM) and a subtherapeutic antibiotic with and without monensin on performance and carcass characteristics. Steers were blocked by initial BW and assigned randomly to treatments within block (12 pens/treatment; 4 steers/pen). Treatments included: no DFM, no subtherapeutic antibiotics, and no ionophore (CON), monensin (Rumensin 90; Elanco;

Greenfield, NJ; 33 g/ton DM basis) and tylosin phosphate (Tylan 40; Elanco; 11 g/ton

DM basis) (MonTy), monensin and L. salivarius L28 (10⁶ CFU steer/d) (MonPro), tylosin (Tylan 40; Elanco; 11 g/ton DM basis) (TY), and L. salivarius L28 (10⁶ CFU steer/d). All treatments were included in a steam-flaked corn-based diet fed once daily using a slick bunk management approach; no β-adrenergic agonist was fed. Prior to treatment initiation cattle were processed which included the administration of a Synovex

Plus implant (200 mg trenbolone acetate+ 28 mg estradiol benzoate; Zoetis; Florham

Park, NJ). Cattle were weighed at 35 d intervals using a pen scale. Individual BW measurements were taken at initial BW, d 105 (deemed approximate midpoint), and final

BW. Steers were shipped in 2 slaughter groups after treatment-blind personnel deemed approximately 60% of steers within a block had sufficient external fat cover to grade

USDA Choice (group 1 = 181 d; group 2 = 208 d). Data were analyzed using the

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GLIMMIX procedure of SAS with pen serving was the experimental unit, treatment was the fixed effect, and block was the random effect.

There were no differences in carcass adjusted final BW (P = 0.23) or carcass adjusted overall ADG (P = 0.23) across treatments. Dry matter intake decreased for cattle fed

MonPro (P < 0.01); however, this did not result in improved overall feed efficiency (P =

0.61) for MonPro. Apparent total tract digestibility percentages were not different (P >

0.12); except for CP which tended to be lower for the control treatment (P = 0.06).

Carcass weight, dressing percent, LM area, and yield grade did not differ (P > 0.23) across treatments. Marbling scores were increased for CON cattle. There were no differences in the percent liver abscess across all treatments (P =0.94). These results suggest that L. salivarius L28 does not have a positive impact on live performance and may have a negative impact on carcass responses. The results of this study indicate additional research is warranted to better understand the mechanism of action of L28 and how to best use this novel direct-fed microbial.

Key Words – beef steers, direct-fed microbial

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Introduction

In a recent study, the DFM L28 was fed to cattle at the Texas Tech Burnett Center in a randomized block study. In this trial, 3 treatments were applied (n = 12 pen/treatment): no DFM, no subtherapeutic antibiotic, and no ionophore (Con), monensin (Rumensin 90;

Elanco; Greenfield, NJ; 36.4 mg/kg DM) and tylosin (Tylan 40; Elanco; 12.1 mg/kg DM)

(MonTy), and monensin and L. salivarius L28 (106 CFU steer/d) (MonPro). No statistical differences were reported in the growth performance or efficiency metrics. Hot carcass weight was numerically depressed in the MonPro treatment, and the marbling score was lower than the other 2 treatments. Additionally, there was a mitigation of the emergence of

AMR in the commensal microorganisms when the diets were supplemented with the L28 and monensin (English et al., 2018). Supplementation of L28 resulted in reduction of generic E. coli AMR. These results, combined with the data of Krehbiel et al. (2003), show the increased need to continue researching the inhibition of pathogens and the effect of

DFMs, particularly L28, on animal performance. In the pilot L28 study, the ionophore, monensin was included in the diet. Although this is a standard feed additive, it alters the microbial population of the rumen (NASEM, 2016) and could affect the activity of the gram-positive L28. Therefore, this study was designed with and without monensin to assess the DFM individually without possible associative or deleterious effects occurring on rumen parameters.

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Materials and Methods All experimental procedures were conducted in accordance with an approved Texas

Tech University Institutional Animal Care and Use Committee (protocol number 18090-

11).

Growth Performance

Single source crossbred steers (n = 240; BW = 319 ± 29 kg) were used in a randomized complete block design feedlot study to evaluate the effects of a novel direct- fed microbial (DFM) and the industry standard subtherapeutic antibiotic, with and without monensin, on growth performance, carcass characteristics, and liver abscesses. Two- hundred and eighty-six steers were received on November 20, 2018 and placed in 16 soil- surfaced pens (16-18 steers/pen). The cattle had access to fresh water and long-stem grass hay; the following day, cattle received a Sweet Bran-based (Cargill Corn Milling, Blair

NE) receiving diet (65% concentrate) and remained on the receiving diet until treatment initiation. Six days after arrival, cattle were processed: individually weighed (Silencer

Chute, Moly Manufacturing, Lorraine, KS, mounted on Avery Weigh-Tronix load cells,Fairmount, MN; readability ± 0.45 kg; before each use, the scale was validated with

454 kg of certified weights), individually tagged with a unique identification number, treated for external parasites (Noromectin Pour-On; Norbrook Inc., Overland Park, KS) and vaccinated against Clostridium chauvoei, Clostridium septicum, Clostridium novyi,

Clostridium sordellii, Clostridium perfringens types C and D (Vision 7 with SPUR; Merck

Animal Health, Summit, NJ), Mycoplasma bovis bacterin (Myco-B One Dose, American

Animal Health, Inc. Grand Prairie, TX), and infectious bovine rhinotracheitis virus, parainfluenza-3 virus, bovine respiratory syncytial virus, bovine viral diarrhea virus types 1 and 2, and mannheimia haemolytica (Bovi-Shield Gold One Shot; Zoetis Animal Health).

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On December 4th, a sorting bodyweight (BW; unshrunk) was taken and on December 7th, the animals were sorted into weight blocks and returned to dirt pens. Twenty-one days post arrival, the steers were randomly assigned to treatments within block (12 pens/treatment; 4 steers/pen) and then placed in partially slotted concrete pens (2.9 m wide x 5.5 m deep; 2.4 m of linear bunk space). Steers were allowed 7 days to adapt to the concrete pens before trial initiation on December 18, 2018. Upon trial initiation, an individual BW was taken, and each steer was implanted with Synovex Plus (200 mg TBA + 28 mg estradiol benzoate;

Zoetis Animal Health). At the time of trial initiation, pens began receiving their respective treatments; over a 21-d period, steers were transitioned from the 65% concentrate diet to a

90% concentrate diet.

Treatments were arranged in a randomized complete block design and included: no

DFM, no subtherapeutic antibiotic, and no ionophore (CON), monensin (Rumensin 90;

Elanco; Greenfield, NJ; 36.4 mg/kg DM) and tylosin (Tylan 40; Elanco; 12.1 mg/kg DM)

(MonTy), monensin and L. salivarius L28 (106 CFU steer/d) (MonPro), L. salivarius L28

(106 CFU steer/d) (PRO), and tylosin (Tylan 40; Elanco; 12.1 mg/kg DM) (TY). The concentrated L. salivarius L28 culture was diluted into a ground corn carrier to reach the desired 106 CFU steer/d; adjusted for intake. The ground corn premix was then added to a total mixed ration. All treatments were included in a steam-flaked corn-based finishing diet fed once daily using a slick bunk management approach. No β-adrenergic agonist was fed.

Diet samples were taken weekly, dried in a forced air oven for approximately 24 h at 100°, and the weekly DM value was used to calculate the daily DMI. Additional weekly diet samples were taken and composited by 35-d weigh periods. Composited samples were analyzed for proximate analysis by Servi-Tech Laboratories (Amarillo, TX).

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Cattle were weighed at 35 d intervals using the pen scale. Individual BW was taken on d 0 and 105 and respective final BW (d 181 and 208 for the 1st and 2nd slaughter groups). On d105, the approximate study midpoint, steers were re-implanted with Synovex

Plus (200 mg TBA + 28 mg estradiol benzoate; Zoetis Animal Health). Steers were shipped in 2 slaughter groups after treatment-blind personnel deemed approximately 60% of steers within a block had sufficient external fat cover to grade USDA Choice (group 1 = 181 d; group 2 = 08 d). A 4% pencil shrink was used for determination of final BW.

On February 11th, 2019 (55 days into the feeding period) cattle began showing clinical signs of respiratory infection. Animals with depressed intake, elevated temperature, and clinical signs of illness were treated. As the infection continued to spread across the cattle, the Texas Tech clinical veterinarian was consulted, and the decision was made to administer metaphylaxis to all cattle in the study. February 16th - 18th, all pens received chlortetracycline (Aureomycin 100 Granular; 0.5 mg per .454 kg of BW Zoetis Animal

Health) in the feed. Average BW was estimated at 385 kg at the time of treatment.

Even after individual treatment and metaphylaxis, six animals died because of respiratory infection. Additionally, over the duration of the study, two animals were removed from the study because of Mycoplasma bovis, two died because of bloat, one died because of twisted gut, and one died because a cardiac incident. Pens that lost two or more animals were pulled from the study and subsequently the pen was removed from the trial.

Pens removed from the study because of illness included one PRO, one CON, and one TY.

Because of a logistical error, one TY, one CON, and one MonTy pen were also removed from the study because of the misapplication of treatments. The replications for each treatment, CON (n = 10 pens/treatment), MonTy (n = 11 pens/treatment), MonPro (n = 12

Texas Tech University, Tosha L. Opheim, May 2020

pens/treatment), TY (n = 10 pens/treatment), and PRO (n = 11 pens/treatment), were altered as a result of the removal of the pens.

Sample Collections and Laboratory Analyses

On d 90, feeding behavior was visually observed continuously for 24 h (5 min intervals). Trained personnel recorded the feeding behaviors observed during each interval

(chewing, ruminating, eating, resting, active, and drinking) according to Campanilli et al.

(2017).

Diet, orts and fecal samples were obtained over a five-day digestibility collection period. The first diet sample was obtained at 0700 on April 14th, d117 with the first fecal sample collected at 1600 that day. Diet samples were collected from each bunk immediately after feed delivery; samples were composited 12 pens/treatment. Fecal samples were collected upon defecation from each steer and homogenized by pen; samples were collected at 0700 and 1600 for the five-day duration. A minimum of 2 steers were collected from each pen if fresh fecal samples were not available from all 4 steers. Orts were collected each morning before the daily feed delivery. Diets were composited by treatment and fecal samples were composited by pen. Sample composites were dried at 60°C in a forced-air oven for 72 h and ground in a Wiley mill (Thomas

Scientific, Swedesboro, NJ) to pass a 1-mm screen.

Acid insoluble ash (AIA) was used as the internal marked for estimation of total fecal output and apparent nutrient digestibility. The 2N HCl method (Van Keulen and

Young, 1977) was performed in duplicate. Laboratory DM content was determined in a

100°C forced-air oven for 4 h; OM was determined by burning samples at 550°C for 4 h

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(AOAC International, 2005). Analysis for NDF and ADF were performed in succession using an Ankom Fiber Analyzer (Ankom Technology Corp., Macedon, NY) (Van Soest et al., 1991); NDF was performed using sodium sulfite and α-amylase. Hemicellulose was calculated as the difference between NDF and ADF concentrations. Crude protein concentration was analyzed in a Leco CNS Analyzer (Leco CNS-2000, St Joseph,

Michigan, USA). To determine apparent total tract digestion of DM, OM, CP, NDF,

ADF, and HEM, the following equation was used: 100 − 100 × ([AIA concentration in feed/AIA concentration in feces] × [nutrient concentration in feces/nutrient concentration in feed]).

Carcass Characteristics

Steers were transported 169 km to commercial slaughter facility. Trained Texas

Tech personnel collected data during slaughter and carcass data 48 hrs after slaughter.

Livers were assessed for abscessed using the Eli Lilly (Elanco) Liver Check System (✓ = no abscesses, A- = 1 or 2 small abscesses, A = 2 to 4 small active abscesses, A+ = 1 or more large active abscesses, A+ Adhesion = liver adhered to gastrointestinal (GI) tract, A+

Open = open liver abscess; as described by Brown and Lawrence, (2010). The presence of an abscess (A-, A, and A+) was pooled for percent liver abscess. Dressing percentage (DP) was calculated by dividing the HCW by the shrunk final BW. Carcass adjusted final BW was calculated as HCW divided by the average dressing percent of each slaughter group,

63.37% and 62.17% for the 1st and 2nd kill groups, respectively.

Statistical Analyses

Data were analyzed using the GLIMMIX procedure of SAS (SAS Inst. Inc., Cary,

NC) with pen serving as the experimental unit, treatment as the fixed effect, and block as

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the random effect. Because of unequal of an unequal number of pens, the Kenward Rogers degrees of freedom adjustment was used. Binomial proportions were used to analyze quality and yield grade distributions using the GLIMMIX procedure of SAS with block as a random effect. The protected F-test was used and when significant, the least squares means were separated using the LSD procedure of SAS (훼 = 0.05); P-values between 0.05 and 0.10 are discussed as trends.

Results

There were no differences across all treatments for BW, ADG, and feed efficiency

(P > 0.25; Table 4.2). The only performance differences were noted in DMI. In the first

105 days, control cattle had a greater DMI compared to MonTy, MonPro, and PRO, with

TY being intermediate (P = 0.05). From day 106 to the end, there was a tendency for cattle fed PRO to have a 6.3% and a 5.2% decrease in DMI compared to CON and

MonPro cattle, respectively; treatments including tylosin (MonTy and TY) were intermediate (P = 0.07). Cattle fed a subtherapeutic antibiotic or DFM had a 3.7- 5.6% decreased in DMI compared to the control cattle for the entire feeding period (P = 0.03).

There were no differences in digestibility percentages (P > 0.12; Table 4.3) across nutrients, except for CP. Control fed cattle had a tendency for lesser CP digestibility (P =

0.06). There were differences in nutrient intakes across all treatments which was solely a function of DMI differences. Control cattle consumed the greatest amount of feed and

PRO cattle consumed the least with all other treatments falling intermediate (P = 0.04).

Although intake was impacted by treatment, feeding behavior was not (P > 0. 021; Table

4.4).

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No differences were observed for HCW, dressing percentage, 12th-rib fat, and

LM area (P > 0.34; Table 4.4). There was no difference in yield grade (P = 0.22); except for the change in percent yield grade 4 and yield grade 5. Cattle on the MonPro treatment had a lower percent of yield grade 4 and yield grade 5 compared to the control cattle (P =

0.05). Marbling score for cattle fed PRO were lower than all other treatments (P = 0.04).

There were no differences in liver abscess percentages across the treatments (P = 0.35).

Discussion

The current study was designed to evaluate L. salivarius L28 subsequent to a smaller pilot trial, that was also conducted at the Texas Tech Burnett Center. In this discussion, the results from the current study as well as the results from the pilot study will be addressed to begin understanding the effects of L. salivarius L28. The authors acknowledge that the inclusion of monensin in a treatment by itself would likely have allowed for a more robust interpretation of the data. However, due to budget and facility restraints for the current project it was not possible. The authors understand the removal of pens because of illness and logistical error reduced the power of the study and subsequently the sensitivity of the analyses. Gill et al. (1987) discussed that cattle health may affect the measurable response to a DFM, where extremely healthy or extremely sick cattle may show no response. In the projects evaluating L28 at Texas Tech, the cattle in the pilot study required little or no treatment for illness. While in study 2, metaphylaxis had to be administered to the entire herd. Both scenarios could be considered atypical for a commercial feedlot setting. The following discussion seeks to interpret the available data.

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The first L28 study resulted in a tendency for decreased final BW in cattle fed monensin and L.salivarius L8; however, this was not supported in the present study.

Similarly, to the present study, Brashears et al. (2003), Peterson et al. (2007), and

Vasconcelos et al. (2008) also reported no differences in BW for cattle fed DFM. In contrast, Galyean et al. (2000) and Rust et al. (2000) reported cattle fed DFMs were heavier than control cattle. This variability in results is likely due to differences in the species of DFMs used, their activity within the rumen, and their subsequent effect on

ADG.

Neither the current nor previous L28 studies observed improvements in ADG.

This contrasts the reviews of Ware et al. (1988) and Krehbiel et al. (2003) which reported cattle fed DFMs typically experienced a 2.6 - 4% increase in ADG. The species of DFM, the combination of multiple microbial species, and their interactions, as well as additives having anti-microbial properties may be in large part responsible for the differences of

ADG seen in various studies. Vasconcelos et al. (2008) and Brashears et al. (2003) both reported no differences in ADG feeding L. acidophilus NP 51combined with

Propionibacterium freudenreichii NP 24 or L. acidophilus NPC 747 or 750, respectively.

In an earlier study, Peterson et al. (2007) used the same strain of L. acidophilus as

Vasconcelos except without the addition of P. freudenreichii NP 24 and was also not able to incur an increase in ADG for NP51 supplemented cattle. Based on the available studies, the mechanism of action and the inclusion rate of the additive must be considered to evaluate the effect of each microbial population introduced into the rumen environment.

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In the first L28 study, a decrease in DMI for the monesin and L28 treatment was observed over the entire feeding study. It was not clear whether this was because of consistent decreases in DMI for cattle fed monensin, such as those observed by Duffield et al. (2012), a reduction because of the DFM, or an associative effect from the combination. Unfortunately, no further conclusions were ascertained from the present study data, as both antibiotic and DFM treatments had decreased DMI regardless of the inclusion of monensin. Numerically, the PRO cattle in study two performed more similarly to the MonPro cattle from study one than the MonPro cattle from study two.

The combination of monensin with the DFM in study two seemed to mitigate the decreases in FBW, ADG, DMI, HCW, and marbling better than L28 alone, when comparing both treatments to control cattle.

There was a 15 kg numerical decrease in final BW for cattle fed the PRO treatment compared to the control. It is likely this decrease can be attributed to the 0.5 kg reduction in DMI for the PRO cattle. Using the second slaughter group’s days on feed, a difference of 0.5 kg/d resulted in a difference of 104 kgs of feed consumed between the control and PRO cattle. The diet NEg was 1.45 Mcal/kg which results in 150.8 Mcals of lost NEg for the PRO cattle when compared to the control cattle. The PRO cattle had an average intake of 8.40 kg of feed per day during the feeding period. Cattle on the PRO treatment would have needed 13 more days on feed in order to make up for the difference in DMI and energy. Using the 1.67 kg ADG for the PRO treatment and the 13 additional days on feed, cattle would have gained 22 more kg resulting in BW more similar to control treatment. However, the added time required to equalize the energy intake

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between the PRO and control treatment would increase the labor and management costs associated with finishing cattle.

Similar to the previous L28 study, the current L28 study was not able to realize the improvements in feed efficiency that were presented in the Krehbiel et al. (2003) review. Single studies from Galyean et al. (2000), Brashears et al. (2003), and Peterson et al. (2007) were also unable to note feed efficiency improvements. Supporting the 2003 review (Krehbiel et al.), Vasconcelos reported an improvement in feed efficiency for all cattle fed a DFM. The decrease in intake observed for the PRO and MonPro cattle suggested that the L28 microbial interaction affected animal satiety; however, the downstream effect did not mirror the magnitude of anabolism supported by other DFMs.

Similar to the first study, digestibility percentages were not impacted by feed additives. However, CP digestibility tended to be lower for control cattle. This could be attributed to a positive antimicrobial effect. In a digestibility study using lambs, Adams et al. (1981) reported a tendency for CP digestibility to be greater for lambs fed monensin.

The antimicrobial effects of monensin are well known (Bergen and Bates, 1984; Duffield et al., 2012), as monensin inhibits the growth of gram-positive bacteria. Although the mechanism of action for L28 is not fully understood, L. salivarius has decreased gram- positive and gram-negative bacteria in various human and animal studies (Chaves et al.,

2017). Similar to the first study, there were no differences in fiber digestion; however, there were numerical increases in fiber digestibility for the treatments that included the

DFM. Robinson and Ersmus (2009) reported an increase in cellulolytic bacteria when feeding S. cerevisiae to dairy cattle. The high-concentrate diet in the current study likely did not contain enough fiber for the greater percent difference in the fiber digestion.

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There were no feeding behavior differences observed in the behavioral analysis.

Gonzalez et at. (2012) observed a decrease in meal size and increase in frequency of meals when monensin was fed; however, these same behavioral changes were not realized. Direct-fed microbials can have pH stabilization effects (Bach et al., 2007;

Thrune et al., 2007; Marden et al., 2008; Desnoyers et al., 2009; and De Ondarza et al.,

2012) which can be a result of the stabilization of intake. The lack of observed behavior differences indicates that neither antibiotics nor DFMs had any meal frequency or rumination modification. However, the decrease in intake for the MonTy, TY, and Pro cattle suggests that the meal size for those treatments was decreased compared to control cattle.

With the exception of a decrease in marbling score for the PRO treatment (P <

0.04), no differences were observed in carcass characteristics for the present study. This is in agreement with the first study conducted using L28 in feedlot diets. Hot carcass weight was also numerically depressed for the PRO treatment which, along with the decrease in marbling, can be attributed to the reduction in DMI and NEg loss (Basarab et al., 2003; Pethick et al., 2004). According to Bertelsen (2010), marbling develops as the number of days on feed with calories in excess of maintenance increases. When compared to the control cattle, PRO required an additional 13 days on feed to match caloric intake. Numerical differences in liver abscess percentages were also observed; however, this study was not sensitive enough to detect differences, which is partly because of the variability around liver abscess prevalence and the use of small pens.

For PRO cattle compared to control, a 9 kg reduction in HCW during the first slaughter group would result in a loss of $35-45/carcass (Table 4.6). The loss could have

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been somewhat offset as the control group had 20% more yield grade 4 and 5 cattle and could have had an average deduction of $32/hd. Nevertheless, the 34-point reduction in marbling score and the 26% increase in select cattle for the PRO treatment compared to the control would have resulted in a $22 average reduction/hd. All PRO cattle would have had a decrease in profit due to a reduction in HCW, and 26% would have received a choice/select spread discount. Although the PRO treatment had fewer, yield grade 4 and

5 cattle, in this particular market setting, the 20% of control cattle receiving a YG 4 and 5 discount would not have been offset. Although, depending on market conditions, the reduction in DMI could have save $20-24/hd.

It is important to note that markets are extremely volatile and that the size of the discounts as well as the carcass basis fluctuates. To put this into perspective, the 2006 average choice/select discount was $13.49 and the 2009 average was $4.70 (USDA,

2019). When the study cattle were sold, the YG 4 and 5 discount applied to the control cattle did not counterbalance the increase in select; however, with 2009 prices it would have. Although prices change and discounts fluctuate in severity (Table 4.6), the profit or loss of the PRO cattle will depend if the savings in feed cost and reduction in YG 4 and

5s can offset the discount for increased selects and reduced kgs of HCW. In many market conditions, this would be challenging.

Conclusion

The numerical decreases in both HCW and marbling scores for PRO cattle could result in economic losses; therefore, for commercial application, it is critical to further investigate not only L28, but also the combined feeding potential of L28 and conventional subtherapeutic antibiotics such as monensin. The decrease in DMI in both

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studies did not result in feed efficiency improvements; nonetheless, it indicates the

potential for decreased feed costs. Further research is needed to understand the effects on

dry matter intake, carcass characteristics, and economic viability of the L28 DFM.

Literature Cited

Adams, D. C., M. L. Galyean, and H. E. Kiesling. 1981. Influence of viable yeast culture, sodium bicarbonate, and monensin on liquid diluation rate, rumen fermentation and feedlot performance of growing steers and digestibility in lambs. J. Anim. Sci. 53:780- 789.

Bach, A., C. Iglesias, and M. Devant. 2007. Daily rumen pH pattern of loose-housed dairy cattle as affected by feeding pattern and live yeast supplementation. Anim. Feed Sci. Technol. 136:156-163.

Bergen, W. G. and D. B. Bates. 1984. Ionophores: Their effect on production efficiency and mode of action. J. Anim. Sci. 58:1465-1483.

Bertelsen, B. 2010. Quality grade continues to generate largest grid premiums: Recipe for marbling. United States Premium Beef Update. Accessed Nov. 17, 2019. http://www.uspb.com/DocumentItem.aspx?ID=69

Campanili, P. R. B., J. O. Sarturi, M. A. Ballou, S. J. Trojan, J. D. Sugg, L. A. Ovinge, A. U. Alrumaih, L. A. Pellarin, and A. A. Hoffman. 2017. Effects of silage type and inclusion level on ruminal characteristics and feeding behavior of steers fed finishing diets. Journal of Animal Science. 95: 4623- 4637.

De Ondarza, M. B., T. Hall, J. Sullivan, and E. Chevaux. 2012. Effect of live yeast supplementation on milk yield, milk components, and rumen pH in dairy cows. J. Dairy Sci. E-suppl. In press.

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Brown, T. R. and T. E. Lawrence. 2010. Association of liver abnormalities with carcass grading performance and value. J. Anim. Sci. 88:4037-4043. doi:10.2527/jas.20103219

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 intake. J. Anim. Sci. 90:4583-4592.

English, A. R, A. R. Echeverry, J. O. Sarturi, T. L. Opheim, K. K. Nightingale, M. F. Miller, and M. M. Brashears. 2018. Effect of Lactobacillus salivarius L28 used as a feed additive on the antimicrobial resistance of commensal organisms of feedlot cattle. Proc 64th International Congress of Meat Science and Technology, Melbourne, Australia.

Gill, D. R., R. A. Smith, and R. L. Ball. 1987. The effect of probiotic feeding on health and performance of newly-arrived stocker calves. Oklahoma Agric. Exp. Stn. MP-119:202– 204.

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. doi.10.1016/j. anifeedsci.2011.12.009

National Academies of Sciences, Engineering, and Medicine. 2016. Nutrient Requirements of Beef Cattle, Eight Revised Edition. Washington, DC: The National Academies Press. doi:10.17226/9014.

Pethick, D. W., G. S. Harper, and V. H. Oddy. 2004. Growth, development and nutritional manipulation of marbling in cattle: a review. Australian Journal of Experimental Agriculture, 44(7), 705-715.

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Rust, S. R., K. Metz, and D. R. Ware. 2000. Effects of BovamineTM rumen culutre on the performance and carcass characteristics of feedlot steers. Mich. Agric. Exp. Sta. Beef Cattle, Sheep, and Forage Sys. Res. Dem. Rep. 569. 22-26.

Van Keulen, J., and B. A. Young. 1977. Evaluation of acid-insoluble ash as a natural marker in ruminant digestibility studies. J. Anim. Sci. 44:282–287. doi: 10.2527/ jas1977.442282x

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Table 4.1. Beef cattle finishing diet ingredient inclusion and nutritional composition (DM basis) Item Diet Ingredients Steam-flaked corn 63.80 WCGF, Sweet Bran 20.00 Alfalfa hay 4.00 Cottonseed hulls 4.00 Fat (yellow grease) 3.00 Treatment Premix1 1.00 Urea 0.60 Limestone 1.60 Supplement2 2.00 Analyzed composition for the finishing phase3 CON MonTy MonPro TY PRO DM 77.04 76.98 77.4 76.86 77.86 CP 12.76 12.80 12.86 13.10 12.56 ADF 9.14 8.36 8.88 9.18 8.40 NDF 17.94 17.02 17.48 18.10 17.08 Ca 0.56 0.61 0.58 0.63 0.52 P 0.39 0.40 0.40 0.40 0.38 K 0.83 0.84 0.84 0.85 0.81 S 0.17 0.17 0.17 0.18 0.17 Cl 0.33 0.33 0.33 0.30 0.30 NaCl 0.54 0.54 0.55 0.50 0.50 1Treatment premixes containing 1 of 3 feed additives packages will be mixed with fine ground corn (carrier) to equal 1% of the diet (DM). Expected DMI is 9 kg/d. 2Supplement: NaCl, 15%; CuSO4, 0.12%; MnSO4, 0.08; Se Premix 0.2%, 0.25%; ZnSO4, 0.21%; Vitamin A, 110000 IU/kg; and Vitamin E, 875 IU/kg. 3Based on tabular values for individual feed ingredients (NRC, 2000).

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Table 4.2. Effect of DFM and subtherapeutic antibiotic use on feedlot performance of beef steers. Treatment1 Item CON MonTy MonPro TY PRO SEM P-value n, pens 10 11 10 10 11 BW, kg Initial BW 320 320 319 317 317 8.4 0.26 d 105 498 484 487 484 482 10.8 0.35 Final BW2 631 620 625 618 616 7.7 0.36 Final BW, Adj.2,3 630 619 623 623 616 8.5 0.53 ADG, kg d 0 to 105 1.71 1.57 1.59 1.60 1.58 0.054 0.35 d 106 to end2 1.49 1.57 1.56 1.53 1.44 0.056 0.25 d 0 to end2 1.74 1.69 1.71 1.69 1.67 0.038 0.59 d 0 to end, Adj.2,4 1.60 1.55 1.57 1.58 1.54 0.039 0.71 Daily DMI, kg d 0 to 105 7.92a 7.36b 7.34b 7.54ab 7.39b 0.170 0.05 d 106 to end2 9.89a 9.60ab 9.78a 9.50ab 9.27b 0.163 0.07 d 0 to end2 8.90a 8.52b 8.52b 8.57b 8.40b 0.127 0.03 G:F, kg/kg d 0 to 105 0.215 0.212 .0.217 0.212 0.213 0.0047 0.86 d 106 to end2 0.151 0.163 0.159 0.161 0.156 0.0056 0.28 d 0 to end2 0.196 0.199 0.200 0.198 0.200 0.0034 0.50 d 0 to end, Adj.2,4 0.180 0.182 0.184 0.184 0.184 0.0032 0.70 1CON = No DFM, no subtherapeutic antibiotics, or ionophore; MonTy = monensin and tylosin; MonPro = monensin and L. salivarius L28; TY = tylosin; PRO = L. salivarius L28. 2Final BW was taken on d 181 for the first slaughter group and d 208 for the second slaughter group; denoted as ‘end’; final BW shrunk 4%. 3Adjusted final BW equaled HCW divided by the average dressing percent of each slaughter group, 63.37% and 62.17% for the 1st and 2nd groups, respectively. 4Adjusted final BW, initial BW, and days on feed (for each slaughter group) were used to calculate adjusted ADG (d 0 to end). Adjusted G:F was calculated as the ratio of adjusted ADG to average DMI (d 0 to end). a,b,cMeans within rows that do not have a common superscript differ, P ≤ 0.05.

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Table 4.3. Effect of DFM and subtherapeutic antibiotic use on nutrient intake and apparent total tract digestibility. Treatment1 Item CON MonTy MonPro TY PRO SEM P-value n, pens 12 12 12 12 12 Intake, kg/d DM 9.26a 8.67bc 9.10ab 8.60bc 8.50c 0.232 0.04 OM 8.82a 8.26bc 8.67ab 8.20bc 8.09c 0.221 0.04 CP 1.33a 1.25bc 1.31ab 1.24bc 1.22c 0.033 0.04 NDF 1.80a 1.69bc 1.77ab 1.67bc 1.65c 0.045 0.04 ADF 0.65a 0.61bc 0.64ab 0.61bc 0.60c 0.016 0.04 HEM 1.15a 1.07bc 1.13ab 1.07bc 1.05c 0.029 0.04 Daily fecal output, 1.90a 1.68b 1.68b 1.72ab 1.55b 0.085 0.03 kg/steer Digestibility, % DM 79.50 80.60 81.63 80.01 81.85 0.781 0.12 OM 80.91 81.87 82.90 81.33 83.17 0.766 0.13 CP 71.88 75.06 75.00 73.43 75.90 1.070 0.06 NDF 54.69 54.42 57.89 55.55 59.07 2.112 0.35 ADF 51.82 52.09 55.66 52.21 56.14 2.005 0.26 HEM 56.32 55.75 59.16 57.46 60.73 2.221 0.40 1CON = No DFM, no subtherapeutic antibiotics, or ionophore; MonTy = monensin and tylosin; MonPro = monensin and L. salivarius L28; TY = tylosin; PRO = L. salivarius L28. a,b,cMeans within rows that do not have a common superscript differ, P ≤ 0.05.

Table 4.4. Effect of DFM and subtherapeutic antibiotic use on feeding behavior on d90 of the feeding period. Treatment1 Item CON MonTy MonPro TY PRO SEM P-value n, pens 12 12 12 12 12 Time, min/d Resting 1073 1110 1082 1068 1095 17.3 0.25 Ruminating 75 68 74 68 69 6.3 0.82 Drinking 17 19 16 22 18 2.9 0.59 Chewing 249 213 245 235 228 15.0 0.21 Eating 174 145 171 167 158 13.9 0.41 Active 103 99 98 116 100 8.6 0.32 Min/kg of DM Ruminating 8.3 8.7 9.6 8.8 9.2 0.81 0.75 Chewing 28.3 27.1 31.6 20.7 30.6 2.08 0.40 1CON = No DFM, no subtherapeutic antibiotics, or ionophore; MonTy = monensin and tylosin; MonPro = monensin and L. salivarius L28; TY = tylosin; PRO = L. salivarius L28. a,b,cMeans within rows that do not have a common superscript differ, P ≤ 0.05.

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Table 4.5. Effect of DFM and subtherapeutic antibiotic use on feedlot carcass characteristics of beef steers. Treatment1 Item CON MonTy MonPro TY PRO SEM P-value n, pens 10 11 10 10 11 HCW, kg 396 389 391 391 387 6.0 0.53 Dressing percentage2 62.76 62.59 62.63 63.21 62.76 0.346 0.53 12th-rib fat, cm 1.58 1.54 1.41 1.40 1.43 0.081 0.34 LM area, cm² 90.99 92.00 91.88 91.68 92.13 1.636 0.96 KPH, % 4.25 4.13 4.06 3.80 4.15 0.123 0.10 Yield grade 3.72 3.55 3.42 3.36 3.42 0.125 0.22 Yield grade 2- 2.99, % 12.82 32.56 23.91 25.00 24.39 0.479 0.39 Yield grade 3- 3.99, % 48.65 37.10 65.23 57.35 58.45 0.325 0.12 Yield grade 4-5, % 37.14 29.97 10.32 17.95 16.23 0.510 0.05 Marbling score3 452a 448a 444a 458a 418b 14.6 0.04 Select, % 12.35 31.37 24.83 28.43 38.85 0.539 0.16 Low Choice, % 64.10 44.19 54.35 47.50 51.22 0.334 0.46 Upper ⅔ Choice & Above, %4 17.73 17.13 16.06 20.57 6.89 0.653 0.46 Liver Abscess, %5 19.59 10.22 6.70 4.51 23.32 1.084 0.35 1CON = No DFM, subtherapeutic antibiotics, or ionophore; MonTy = monensin and tylosin; MonPro = monensin and L. salivarius L28;TY = tylosin; PRO = L. salivarius L28. 2Dressing percentage was calculated by dividing the HCW by the shrunk final BW. 3300 = slight00, 400 = small00, 500 = modest00. 4Upper ⅔ Choice & Above includes choice, high choice, and prime. 5All liver abscesses were pooled and include: A- = 1 or 2 small abscesses, A = 2 to 4 small active abscesses, A+ = 1 or more large active abscesses, A+ Adhesion = liver adhered to GI tract, A+ Open. 5300 = slight00, 400 = small00, 500 = modest00. a,b,cMeans within rows that do not have a common superscript differ, P ≤ 0.05.

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Table 4.6. Effect of DFM and subtherapeutic antibiotic use on the economics of treatments compared to control. Treatment1 Item CON MonTy MonPro TY PRO Avg Interval Avg Interval Avg Interval Avg Interval Avg Interval DM Cost $/hd2 392 353 – 432 376 338 – 413 376 338 – 413 378 340 – 416 370 333 – 407 Savings $/hd3 17 15 – 18 17 15 – 18 15 13 – 16 22 20 – 24 HCW Profit/hd4 1746 1528 – 1964 1715 1500 – 1930 1724 1509 – 1940 1724 1509 – 1940 1706 1493 – 1920 Loss $/hd5 31 27 – 35 22 19 – 25 22 19 – 25 40 35 – 45 SE Discount $/hd6 11 0 – 22 27 0 – 54 21 0 – 43 25 0 – 49 33 0 – 66 Loss $/hd7 16 0 – 32 10 0 – 21 13 0 – 27 22 0 – 45 YG 4 & 5 Discount $/hd8 32 0 – 65 25 0 – 51 9 0 – 18 15 0 – 31 14 0 – 28 Savings $/hd9 7 0 – 13 24 0 – 47 17 0 – 34 19 0 – 37 1CON = No DFM, subtherapeutic antibiotics, or ionophore; MonTy = monensin and tylosin; MonPro = monensin and L. salivarius L28;TY = tylosin; PRO = L. salivarius L28. 2Calculated with the average treatment DMI for a 200d feeding period. Diet cost for avg = $220.5/metric ton. Interval = $198.5 and 242.6/metric ton, respectively. Solely diet DM cost. No supplement pricing included. 3Difference of DM diet cost compared to CON. 4Calculated as average HCW for treatment multiplied by $200/cwt. Interval = $175 and $225/cwt, respectively. 5Difference of HCW treatment profits compared to CON. 6Calculated using the % Select per treatment. Avg select discount -$10/cwt. Interval $0 and -$20/cwt, respectively. 7The difference in the Select discount for treatments compared to CON. 8Calculated using the % yield grade 4 and 5s per treatment. Avg YG 4 & 5 discount -$10/cwt. Interval $0 and -$20/cwt, respectively. 9The reduced in discount of treatments compared to CON.

Texas Tech University, Tosha L. Opheim, May 2020

CHAPTER V

CONCLUSION

Although most consumers still make purchasing decisions on factors such as price, convenience, and taste, a growing number of consumers are placing emphasis on health, environmental impact, sustainable product practices, and naturally derived products. This is good for niche markets; however, it is impacting traditional agricultural practices such as the use of subtherapeutic antibiotics. As consumers drive the end products, more shifts will be made to more natural alternatives such as direct-fed microbials, natural enzymes, and potentially more specific recommendations on micronutrients such as Zinc and Chromium. Continuing to research back to basics production practices such as improving forage digestibility, starch utilization, protein metabolism with new and ever-changing byproducts and existing feedstuffs will continue to be of value. Advancements in areas of study like epigenetics, nutrient utilization, microbiome, and antimicrobial resistance will be increasingly more robust. Gut health, rumen function, and nutrient utilization will become even more important than it is now.

If natural alternatives can be developed without sacrificing efficiency of production, then it will be a much easier transition. However, if production efficiency is decreased that will result in the retail prices of beef increasing. This will be extremely hard on beef sales as it is already seen as a high-priced protein in comparison to its contemporaries, poultry and pork.

The results of these studies indicate that growth performance was not positively impacted by the use of L. salivarius L28 and could result in reduced HCW and marbling.

Both metrics offer financial incentives for feedlots to capitalize on. Any reduction,

Texas Tech University, Tosha L. Opheim, May 2020

including numerical, could have significant impacts on the profitability to the feedlot producer. Although DMI was reduced, subsequent improvement in feed efficiency were not realized. As more is learned about the mechanisms of action of specific strains, it is foreseeable that a combination of L28 and another strain may result in a more favorable economic outcome. More research needs to be conducted to further elucidate the mechanisms of action of L28. With slight numerical improvements in fiber digestion, it may be more suited to a high-fiber dairy-type diet. Additionally, targeted feeding during specific periods of the feeding study may be beneficial in order to reduce pathogens without sacrificing carcass value.