An investigation of the impacts of supplementing cattle finishing rations with winery by-products on feed intake, meat characteristics, fecal microbiology and pre-harvest pathogen reductions. A Dissertation Presented by Paul E. Moote Submitted to the Graduate School of Thompson Rivers University in partial fulfillment of the requirements for the degree of MASTERS OF SCIENCE July 2013 Environmental Science ! ! © Copyright by Paul E. Moote 2013 All Rights Reserved ! ""! An investigation of the impacts of supplementing cattle finishing rations with winery by-products on feed intake, meat characteristics, fecal microbiology and pre-harvest pathogen reductions. A Dissertation Presented by PAUL E. MOOTE Approved as to style and content by the thesis committee: ! Jonathan D. Van Hamme, Supervisor ! John S. Church, Supervisor ! Wendy C. Gardner, Member ! Edward Topp, Member ! Doug M. Viera, External examiner ! """! Thesis Supervisors: Jonathan Van Hamme, Ph.D and John Church, Ph.D ABSTRACT The production of novel beef products is crucial to ensuring the sustainability of British Columbia’s beef industry as it increases product differentiation in the beef marketplace. This research has evaluated the impacts of supplementing cattle finishing rations with winery by-products (WB), mainly wine lees, at a feedlot in the South Okanagan region of British Columbia. To accomplish this, 69 Angus cross feedlot steers (live weight; 51.3 ± 27.9 kg) were randomly allocated to one of two pens, two per treatment, and fed finishing rations supplemented with either 6-7 % WB (WB; n = 18, 17) or water (control (C); n = 17, 17) for 143 d. Over the course of the study the impacts of WBsupplemented feed rations on the weight and flight speeds of cattle were evaluated, as well as the impacts of this feed on the frequency of fecal samples harbouring antimicrobial resistant Escherichia coli (EC) and the diversity of fecal bacterial communities. Cattle fed WB-supplemented feeds were found to require less feed than C fed steers to reach the same slaughter weight, while producing meats with no changes to the colour or composition than the control, save for the colour of ground steak meat, which was found to be darker in meats from WBfed animals. WB-supplemented feeds did not alter cattle temperament, however cattle became habituated to handling over the feeding period. WB-fed cattle had elevated EC loads compared to C-fed animals, however no changes to the proportions of antimicrobial resistant EC were observed between treatments. Similarly, diet was not found to alter the bacterial communities of cattle feces, however these communities were found to change with time on feed. In conclusion, supplementing cattle feeds with WB can provide economic incentives to producers through reduced feed demands, without altering the loads of fecal pathogens or temperament of cattle. Key Words: Cattle, Wine, Feed, Gains, Antimicrobial Resistance, Escherichia coli ! "#! TABLE OF CONTENTS Page ABSTRACT ...................................................................................................................... iv TABLE OF CONTENTS .................................................................................................. v ACKNOWLEDGMENTS .............................................................................................. vii DEDICATION ...............................................................................................................viii LIST OF FIGURES........................................................................................................... ix LIST OF TABLES............................................................................................................. xi ! 1. Background and conception of “wine finished beef”...........................................1 1.1 Project overview...........................................................................................1 1.2 Project rationale............................................................................................2 1.3 Study location: Southern Plus Feedlots ....................................................2 1.4 An introduction to the BC wine industry................................................4 1.4.1 By-products of wine making and viticulture............................4 1.5 “Wine-finished beef”: what is it?...............................................................8 1.6 Previous research on wine lees and other fermented winery by-products as ruminant feed supplements .........................................8 1.6.1 Potential benefits of condensed tannin containing feeds to ruminants ........................................................................9 1.6.2 Potential benefits of polyphenolic containing feeds to ruminants .....................................................................................10 1.7 Pre-harvest pathogen reduction ..............................................................11 1.8 Antimicrobial resistance in animal agriculture .....................................13 1.9 Resulting meat products ...........................................................................14 1.10 Effects of diet on cattle behaviour .........................................................16 1.11 Summary of research objectives ...........................................................17 ! 2. Effect of fermented winery by-product supplemented rations on beef cattle temperament, feed intake, growth performance and meat quality ..............18 2.1 Introduction ................................................................................................18 2.2 Materials and methods..............................................................................21 2.2.1 Experimental design...................................................................21 2.2.2 Data collection .............................................................................24 2.3.2 Growth and performance ..........................................................28 2.4 Statistical analysis .......................................................................30 2.3 Results..........................................................................................................31 2.3.1 Feed analysis................................................................................31 2.3.2 Feed intake ...................................................................................31 2.3.3 Growth and performance ..........................................................33 2.3.4 Animal mortality.........................................................................33 2.3.5 Flight speed..................................................................................33 2.3.6 Carcass characteristics and meat quality.................................34 2.4 Discussion ...................................................................................................38 2.4.1 Feed analysis................................................................................38 2.4.2 Feed intake ...................................................................................38 ! "! 2.5 2.4.3 Growth and performance ..........................................................39 2.4.4 Flight speed..................................................................................40 2.4.5 Carcass characteristics and meat quality.................................41 Conclusion ..................................................................................................42 ! 3. Effect of fermented winery waste supplemented rations on the diversity and antimicrobial resistance profiles of fecal E. coli loads in beef cattle 43 3.1 Introduction ................................................................................................43 3.2 Materials and methods..............................................................................45 3.2.1 Experimental design...................................................................45 3.2.2 Data collection .............................................................................48 3.2.3 Statistical analyses ......................................................................52 3.3 Results..........................................................................................................53 3.3.1 Fecal EC loads..............................................................................53 3.3.2 Tetracycline and ampicillin resistance.....................................56 3.3.3 Minimum inhibition concentrations (MIC).............................60 3.3.4 Fecal bacterial diversity..............................................................64 3.3.5 E. coli isolate fingerprinting .......................................................68 3.4 Discussion ...................................................................................................71 3.5 Conclusion ..................................................................................................73 ! 4. Project Summary .....................................................................................................74 4.1 Overview.....................................................................................................74 4.2 Concluding remarks ..................................................................................76 ! 5. Literature Cited .......................................................................................................77 ! 6. Supplementary Information...................................................................................90 ! "#! ACKNOWLEDGMENTS Financial support for this research was provided by the Investment Agriculture Foundation of BC (grant #A0624). Paul Moote was financially supported by the Brigadier W.N. Bostock Memorial Grant (BC Cattlemen’s Association), and an Industrial Postgraduate Scholarship (IPS1) provided by the Natural Science and Engineering Research Council of Canada and Southern Plus Feedlots. Logistical support provided by feedlot staff from Southern Plus Feedlots was much appreciated and this research could not have been completed without their help. Appreciation is bestowed to Melissa Leigh-Stewart for her assistance with evaluating the minimum inhibition concentrations of Escherichia coli. ! "##! DEDICATION To my family—both blood and wed ! "###! LIST OF FIGURES Figure 1.1 From Left to Right: outlined circle indicates location of Southern Plus Feedlots in relation to Oliver, BC and image on right indicates layout of holing pens at facility 1.2 3.1 3.2 3.3 3.4 3.5 3.6 Page 3 Reported tonnage of grapes produced by region; data obtained from the British Columbia Wine Institute, 2012 6 Shifts to the loads of fecal E. coli in cattle fed a 143-day diet supplemented with either 6-7% winery by-products (A and B; ! and ") or 6-7% water (X and Y; ! and !). 55 Proportion of fecal samples containing E. coli capable of growth at 4 µg mL-1 ampicillin from cattle fed a 143-day diets containing either 6-7% wine (A and B; ! and ") or 7% water (X and Y; ! and !). 57 Proportion of fecal samples containing E. coli capable of growth at 4 µg mL-1 tetracycline from cattle fed a 143-day diets containing either 6-7 % wine (A and B; ! and ") or 6-7 % water (X and Y; ! and !). * Indicates findings between diets (P < 0.5, n=4). 59 Bacterial Phyla diversity between samples. Letters of sample ID indicate pens and diets that feces originated from: A and B from cattle fed a 143-day diet supplemented with 6-7 % winery byproducts (WB) and X and Y from cattle fed diets supplemented with 6-7 % water as the control (C). The numbers in sample ID indicates sampling event, 1 at d1, 3 at d63 and 6 at d143. 65 Multi-dimensional scaling plots of Rep-PCR patterns created from EC obtained from cattle fed 143-day diets of 6-7 % winery byproducts (Green), or water as the control (Red) at harvest. 69 Multi-dimensional scaling plots of Rep-PCR patterns obtained from Escherichia coli isolates obtained from the feces of cattle fed 143-day diets of 6-7 % winery by-product or 6-7 % water as a control by membrane fecal coliform plates containing no antibiotics (Red), 4 µg mL-1 Tetracycline (Green), or 4 µg mL-1 Ampicillin (Blue). 70 ! "#! A.1 A.2. Principal component analysis of bacterial diversity between fecal samples from cattle fed experimental diets using discrete unweighted UNIFRAC parameters. A. Diversity between cattle fed a diet supplemented with 6-7 % winery by-products (!) and cattle fed a diet supplemented with 6-7 % water (") at d1, 64 and 143 on feed. B. Sample diversity between cattle fed 6-7 % winery byproducts (d1!, d64" and #d143) and cattle fed 6-7 % water as the control (d1!, d64" and d143$). 93 Bacterial diversity and species richness between samples from cattle fed experimental diets. A) Changes between observed species; B) changes to Shannon’s diversity; and C) Chao1 richness index. 94 ! "! LIST OF TABLES Table 1.1 Wastes produced from viticulture and wine-making, their waste management strategies and previous uses as ruminant feeds. 2.1 2.2 2.3 2.4 2.5 2.6 2.7 Page 7 Wet matter composition of experimental diets used during the 143-d feeding program; control diets and experimental diets differed from one another only by their contents of water and winery by-products. 23 Number of samples (#), mean values, standard error of the calibration (SEC), R2 (RSQ), and the standard error in cross validation (SECV) used to prepared the calibration of the NIR hardware and software used for determining the % ash, fat, protein, acid detergent fibre (ADF), neutral detergent fibre (NDF), and starch of cattle feeds containing 6-7 % winery by-products or 6-7 % water as a control. 26 Near infrared analysis of ash, fat, protein, acid detergent fibre (ADF), neutral detergent fibre (NDF), and starch, as well as the amounts of dry matter and calories within the finishing rations of cattle feeds containing 6-7 % winery by-products (WB) or 6-7 % water (C). 27 Impacts of a 143d diet containing 6-7 % winery by-products (WB) or 6-7 % water (C) on the average daily gains (ADG), wet matter feed intake (FI), feed conversion (FC) and flight speeds (FS) of cattle; similar letters within each measurement indicate statistically similar numbers; standard error of measurements are indicated after each value. 32 Half weights of carcasses (kg) and the observed tenderness of resulting steaks (load at maximum; kgf) determined using the shear force test between cattle fed either 6-7 % winery by-products (WB) or 6-7 % water as control (C). 35 Near infrared analysis of % protein, fat, moisture and collagen contained in steak samples from cattle fed either 6-7 % winery byproducts (WB) or 6-7 % water as control (C). 36 Comparison of lightness (L*), red (a*) and yellow (b*) colours in rib eye meats (a), trim fats (b), and ground rib eye steaks from 14d aged meat samples of cattle fed 143-d diets of either 6-7 % winery by-products (WB) or 6-7 % water as control (C). 37 ! "#! 3.1 3.2 3.3 3.4 A.1. A.2. Wet matter composition of experimental diets used during the 143d feeding program; control diets and experimental diets differed from one another only by their contents of winery byproducts and water respectively. 47 Minimum inhibitory concentrations of ampicillin antibiotics for Escherichia coli isolates obtained from fecal samples of cattle fed 143-day diets supplemented with 6-7% winery by-products (WB) or water as a control (C) on membrane fecal coliform agar containing BCIG and supplemented with no antibiotics (mFc) 4 µg mL-1 tetracycline (mFcT) or 4 µg mL-1 Ampicillin Sodium Salt (mFcA); letters indicate statistically similar numbers (P = 0.05), not compared between isolation methods. 61 Minimum inhibitory concentrations of tetracycline antibiotics for E. coli isolates obtained from fecal samples of cattle fed 143-day diets supplemented with 6-7% winery by-products (WB) or water as a control (C) on membrane fecal coliform agar containing BCIG and supplemented with no antibiotics (mFc) 4 µg mL-1 tetracycline (mFcT) or 4 µg mL-1 Ampicillin Sodium Salt (mFcA); letters indicate the presence of similar numbers (P = 0.05), not compared between isolation methods. 63 Bacterial diversity and species richness of fecal samples obtained from cattle fed 143-day diets of 6-7 % WB or 6-7 % water as the control and sequenced using bTEAFAP pyrosequencing as outlined through Chao1, Shannon’s diversity (Shann.) as well as the observed species (Obs. Spec.) indices at 2,518 sequence reads and Principal Component analyses of sequence reads (PCoA) and the change in Phylum and Genera between samples over time (P1 = Firmicutes; P2 = Bacteroidetes; P3 = Tenericutes; P4 = Proteobacteria; P5 = Spirochaetes; P6 = Cyanobacteria; P7 = all other observed Phyla; G1 = Ruminococcus); Letters indicate statistically significant similarities between numbers in columns. 66 Statistical evaluations of the fecal E. coli loads as well as the proportion of samples containing E. coli capable of growth on membrane fecal coliform agar containing the indicator BCIG and supplemented with no antibiotics (mFc), 4 µg mL-1 ampicillin sodium salt (mFcA) or 4 µg mL-1 tetracycline (mFcT) from cattle fed diets supplemented with 6-7 % winery by-products (WB) or 6-7 % water as a control (C). Letters represent statistically similar numbers (95 %), not compared between isolation methods. 91 Sequence numbers between samples from cattle fed winery byproduct (WB) and water (C) supplemented feeds over time. 92 ! "##! A.3 Diversity within bacterial Phyla and Genera populations between cattle fed experimental diets. Numbers in sample ID indicate sampling session: 1 at d1, 3 at d63 and 6 at d143. Letters indicate statistically similar numbers between each Phyla or Genera (P < 0.05). 95 ! "###! ! CHAPTER 1 1. BACKGROUND AND CONCEPTION OF “WINE FINISHED BEEF” 1.1 Project overview This thesis provides a comprehensive analysis of the research conducted at Thompson Rivers University surrounding the production of a “wine-finished beef” product currently available through Okanagan’s Finest Angus Beef in the Southern Okanagan region of British Columbia (BC). To produce “wine-finished beef” cattle are fed diets of winery by-products (WB) for a minimum of 90 d (days), primarily wine lees. Novel beef products, such as this, can positively contribute to the sustainability of the BC beef industry as they improve product differentiation in the beef marketplace. One advantage of novel food products is that their differentiation from traditional products can improve their marketability, potentially increasing consumer demands. Considering “winefinished beef”, the Toronto Sun, MacLean’s, Canadian Manufacturing and others published information about this unique product when it was first being sold in BC (Findlay 2010; LeBlanc 2010; Food In Canada Staff 2010). However, this novelty comes with a cost: regulation and certification. Novel animal feed ingredients require certification by the Canadian Food Inspection Agency (CFIA) before their use, which was not conducted for “winefinished beef” at the commencement of this project. Without certification, the CFIA prohibited the feeding of this ration to cattle in 2010, until the safety and efficacy of this feed additive could be verified. In reply, Southern Plus Feedlots spoke with the Globe and Mail magazine to publicly voice their frustration over this decision. This worked, as the CFIA removed their restriction on the this feeding program shortly after the Globe and Mail article was released (Bailey 2010). To satisfy the requirements of the CFIA, and to verify the safety and efficacy of this product, Southern Plus Feedlots partnered with Thompson Rivers University to evaluate the impacts of this feed on animal performance, meat characteristics, as well as the changes to fecal Escherichia coli (EC) loads in WB-fed animals. This thesis will provide detailed accounts and discussions of the ! "! ! impacts observed when WB-supplemented feeds were fed to cattle. Specifically regarding its effects on: ! Feed efficacy and chemistry, ! Animal gains and feed performance, ! Cattle temperament, ! Fecal EC loads in animals, ! Loads of antimicrobial resistant EC in feces, as well as ! The diversity of EC and bacteria within fecal samples. 1.2 Project rationale To improve the sustainability of the beef industry, one strategy that Canadian cattle producers can utilize to attempt to remain economically viable is to incorporate by-products of other industries into cattle feedstuffs, thereby lowering their overall feed costs. Examples of by-products that have been used efficiently in North America include dried distillers grains, potato processing residues and citrus pulp (Stanhope et al. 1980; Leiva et al. 2000; Gibb et al. 2008). This practice can also be used to create novel beef products to differentiate the beef marketplace. As an example, feeding winery by-products to cattle enables retailers to market the resulting beef products as “wine-finished beef”. This feeding practice is currently being utilized at Southern Plus Feedlots. 1.3 Study location: Southern Plus Feedlots Southern Plus Feedlots is located in Oliver, BC and is owned and operated by Bill Freding (Figure 1.1). This feedlot has a total capacity of over 4,000 cattle, although the average capacity is often much lower, and generally consists of Angus bred cattle, harvested under the “Okanagan’s Finest Angus Beef” brand of Southern Plus Feedlots, as well as custom fed cattle for other ranchers. The feedlot is located in the South Okanagan region of BC, which represents the North-most extension of the Sonoran desert (French 2009). Located around numerous wineries and restaurants, the owners have begun growing grapes and manufacturing their own wine. The close proximity of the feedlot to additional ! #! ! wineries and established restaurants is ideal for the production and distribution of “wine-finished beef”. 2 km 100 m Figure 1.1 From left to right: outlined circle indicates location of Southern Plus Feedlots in relation to Oliver, BC and the image on right indicates layout of holding pens at the facility. ! $! ! 1.4 An introduction to the BC wine industry The economic impact of the wine industry to BC is valued at two billion dollars, or $42 per bottle (British Columbia Wine Institute 2013a). The major regions in BC for wine production are Vancouver Island, the Fraser Valley, the Similkameen Valley and the Okanagan Valley, with the Okanagan valley being home to the majority of the provinces wineries (121 of 215) (British Columbia Wine Institute 2013b). Similarly, the majority of the province’s grapes are grown in the Okanagan Valley with the city of Oliver delivering almost 50% of this (Figure 1.2; British Columbia Wine Institute 2012). With this geographically isolated wine and grape production, many companies are looking to find alternative ways to reduce or dispose of their by-products in an environmentally friendly manner, such as composting. 1.4.1 By-products of wine making and viticulture In Greece, the by-products from wine making and viticulture have been found to account for 30% of the original grape mass, mainly in the form of pomace (also known as grape marc) (15%), stalks (2.5-7.5%), seeds of grapes (36%), as well as the lees produced from fermentation (3.5-8.5%) (Table 1.1) (Nerantzis & Tartaridis 2006). A majority of these wastes are valuable to the pharmaceutical industry as they contain polyphenols and anti-oxidants that can be extracted and sold as supplements. However, when these wastes cannot be sold, a consistent method for their disposal needs to be developed. Ideally, these by-products would be composted on-site and used as fertilizer for grape growing; unfortunately, this requires a large amount of organic matter and space for anaerobic composting, not readily available at most vineyards. As such, wine making wastes such as compressed pomace are commonly provided to feedlots so that they can be mixed with feces as an activator and composted to reduce the dry matter and adjust the nitrogen, phosphorus and potassium contents (Ferrer et al. 2001). However, not all products have the appropriate pH and dry matter required for anaerobic composting, such as wine lees. As an alternative form of disposal, they can be used as animal feeds (Nerantzis & Tartaridis 2006; ! %! ! Arvanitoyannis et al. 2006b). Of note, research with goats has suggested that the condensed tannins and polyphenols in this product are of important research and practical interest for animal producers as they hold nutritive and antimicrobial properties as will be described in section 1.6. ! &! ! )#$ (#$ )#$ %#$ ()*+,-!%./&.0! (123221!"'/450! (#$ 6,78*9827!./&"0! (#$ :,)2;7<7!?<))1!%/%&0! '#$ @,18A<7=!%/".0! !"#$ B*C*)= 0.05< 0.10. ! $K! ! 2.3 Results 2.3.1 Feed analysis In order to evaluate the impacts of a WB-supplemented feedlot finishing ration, changes to the chemical and energy composition of the feed was evaluated by NIRS and bomb calorimetry, respectively. Feed analysis showed no differences (P > 0.10) between the two diets with respect to percentages of ash, crude fibre, fat, moisture, protein, and total dietary nitrogen for each of the total mixed rations. Further, no changes between the WB-supplemented and control rations were observed in the percent dry matter or energy contents of the feeds, which were compared on a dry matter basis (P = 0.4897 and 0.7516, respectively; Table 2.3). The ethanol contents of four separate WB batches were found to be 10.6 % (n=4). 2.3.2 Feed intake Changes to the FI of cattle fed WB and C fed cattle are important determinants of the overall cost to producers. When comparing the mass of feeds delivered to pens between d1 and d 36, 64, 98 and 143, differences were observed between the main and interactive effects of time and diet (P = 0.0130; Table 2.4). Means for FI were found to increase over time (P < 0.001); in addition, FI for cattle fed WB-supplemented feeds was less then cattle fed C-supplemented feeds at each sampling event (P ≤ 0.05). . ! $"! ! Kg $#! -1 C WB C FS ms --- -1 1.5bc ±0.27 0.87bc ± 0.1 1.7a ±0.39 1.88a ± 0.4 0.94 ± 0.1 1.84bc ±0.07 1.77bc ±0.10 1.88 ± 0.4 1.82bc ±0.09 1.82bc ±0.11 ±0.1 4 b 1.5bc ±0.23 1.5ab ±0.31 a 229 ±6.1d e 130 ±5.2e 210.5 ± 3.8 d f 218 ±4.5d 185.4 ± 3.7 e 33 35 64 119 ±4.0e 234.0 ± 2.3 213.2 ± 4.3 33 35 36 NA a 2.14 ±0.1a 2.05 1b NA FC NA WB Kg d NA ADG NA C WB C NA Kg WB Gain NA C NA FI WB 35 1 33 n WB Units C Value Diet d 1.56cd ±0.10 1.67cd ±0.09 0.83 ± 0.1 bcd 0.74d ± 0.1 1.4bc ±0.34 1.4cd ±0.35 337 ±7.7b 319 ±6.7c 276.2 ± 19.8 c 232.6 ± 12.7 31 35 98 Time on Feed (d) 1.61cd ±0.14 1.45d ±0.08 0.86 ± 0.1 bc 0.77cd ± 0.1 1.1e ±0.47 447 a ±9.9 1.3e ±0.29 d 442 ±8.6a 380.7 ± 25.5 a 337.1b ± 34.4 30 34 143 0.9849 0.2825 0.675 0.1388 0.013 NA P(diet) <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 NA P(time) 0.8619 0.6605 0.0864 0.8069 <0.0001 NA P(time ×diet) ! Table 2.4 Impacts of a 143d diet containing 6-7 % winery by-products (WB) or 6-7 % water (C) on the average daily gains (ADG), wet matter feed intake (FI), feed conversion (FC) and flight speeds (FS) of cattle; similar letters within each measurement indicate statistically similar numbers; standard error of measurements are indicated after each value. ! )*<*<$ =>?8:3$9.4$@/>1?>A9.B/$ The ADG and FC results are presented in Table 2.4. No differences (P = 0.1379) were observed between the diet treatments. Overall, the ADG of animals decreased over time, with the ADG of animals fed the WB ration decreasing from 1.54 to 1.25 kg d-1 while the ADG of animals fed the C ration decreased from 1.69 to 1.10 kg d-1 (P=0.0864). Changes in ADG between d1, d36, 64, 98 and 143 illustrated economic advantages between the C and WB feeds. No changes to the FC of treatment cattle were observed by diet (P = 0.2825); however, FC was found to change over time (P < 0.0001). In a post hoc analysis of power, the power with which we were able to detect changes in ADG and FC among 67 samples was determined to be 0.94 and 0.99 respectively. 2.3.4 Animal mortality Surveying the impacts of a WB-supplemented feed on animal health was not an objective of this research. During the experiment, four cattle were removed from the study, three fed C rations (d95, 97, 112) and one fed WB rations (d106). Of these animals, one fatality was observed, and catalogued, from the animals fed the C ration. The cause of death was thought to be bloat by the onsite animal health technician. These samples sizes are too small to draw any valid conclusions. 2.3.5 Flight speed Cattle FS was found to decrease over the course of the experiment (P < 0.0001), however, when comparing flight speeds between cattle fed WB and C diets, no differences were observed (P = 0.9849), and similarly no interactions were observed over time with respect to diet (P = 0.8619; Table 2.4). In our post hoc power analysis, the observed power of our experiment detect changes in flight speed was 0.95 for 66 samples. ! $$! ! 2.3.6 Carcass characteristics and meat quality Carcass data are presented in Table 2.5. No treatment differences (P = 0.4399) were observed in the carcass half weights, indicating that all carcasses chilled similarly and that any changes to meat properties would likely be the result of the feed differences, and not simply as a result of the energy contents of the finishing rations. Tables 5, 6, and 7 summarize the results of changes to chemical, tenderness and colour characteristics of 14 d aged rib eye steaks measured using near infrared spectrophotometric, shear force and calorimetric tests, respectively. Results shown in Table 2.6 illustrate that no differences (P > 0.34) were observed in the protein, fat, moisture or collagen content of WB and C steak samples. Similarly, the tenderness of steaks was not impacted by diet (P = 0.2343) (Table 2.5). When comparing steak meat colour, no differences (P > 0.17; Table 2.7) were observed between the WB or C treatments. However, the colour of ground steak samples was darker (P = 0.0477; Table 2.7) in WB than C finished cattle; the incidence of dark, firm and dry (DFD) meat or pH of meat samples was not evaluated in this study, but no obvious cases of DFD were detected by personnel at slaughter. Further, trends towards significance were observed in the fat colours between meats from treatment diets. Specifically, the fats of steak meats were found to be redder in samples from cattle fed the C feeds (P = 0.0517; Table 2.7). Other than these findings, there were no colour or tenderness changes observed. In post hoc analyses of power, the power with which we were able to detect differences within half carcass weights, as well as steak and trim meat colours was found to be > 0.8825; however, the observed power within our experiment to discern changes to ground meat colour was found to be 0.4514, which was a result of reduced sample size to accommodate tenderness evaluations. ! $%! ! Table 2.5 Half weights of carcasses (kg) and the observed tenderness of resulting steaks (load at maximum; kgf) determined using the shear force test between cattle fed either 6-7 % winery by-products (WB) or 6-7 % water as control (C). Diet Value Units n Mean STDEV t P WB Weights kg 10 146.0 7.18 -0.790 0.4399 10 148.9 9.12 32 2.92 0.77 1.203 0.2343 24 2.71 0.51 C WB C ! Tenderness kgf $&! ! Table 2.6 Near infrared analysis of % protein, fat, moisture and collagen contained in steak samples from cattle fed either 6-7 % winery byproducts (WB) or 6-7 % water as control (C). Diet Item n Mean STDEV t P WB C WB C WB C Protein 5 3 5 3 5 3 21.93 22.11 13.17 16.22 64.90 62.91 0.91 0.24 4.30 3.51 3.24 2.95 -0.325 0.7562 -1.030 0.3429 0.864 0.4210 5 3 1.96 1.95 0.45 0.43 0.044 0.9667 WB C ! Fat Moisture Collagen $'! ! Table 2.7 Comparison of lightness (L*), red (a*) and yellow (b*) colours in rib eye meats (a), trim fats (b), and ground rib eye steaks from 14-d aged meat samples of cattle fed 143-d diets of either 6-7 % winery byproducts (WB) or 6-7 % water as control (C). (a) Meat (b) Fat (c) Ground ! Diet WB C WB C WB C WB C WB C WB C WB C WB C WB C Light L* a* b* L* a* b* L* a* b* n 9 8 9 8 9 8 10 6 10 6 10 6 4 3 4 3 4 3 Mean 39.86 40.34 19.75 20.58 17.40 17.89 62.70 61.46 6.77 8.44 16.39 17.82 49.02 54.59 23.14 2.58 22.04 0.94 $4! STDEV 3.42 1.51 2.33 1.32 1.86 0.90 5.72 3.20 1.33 1.80 1.83 2.17 2.35 3.36 1.20 2.58 0.22 0.94 t -0.361 P 0.7231 -0.881 0.3920 -0.673 0.5109 0.487 0.6340 -2.126 0.0517 -1.416 0.1786 -2.609 0.0477 1.405 0.2189 -1.158 0.2992 ! 2.4 Discussion 2.4.1 Feed analysis The addition of WB into finishing rations was found to not alter the chemical and energy contents of the feed. These results are in agreement with previous studies that show grape pomace is an insufficient sole source of energy (Baumgärtel et al. 2007; Spanghero et al. 2009; Abarghuei et al. 2010). As the diets contained less than 7% WB or water, these results were expected. Additionally, as changes to the % DM of feeds were not found between samples, it was assumed that changes to cattle performance characteristics could be extrapolated between wet and dry matter feed references. 2.4.2 Feed intake Before considering the impacts of WB and C feed on cattle feed intake (FI) it is important to discuss the limitations of this study. As the cattle diet changed from a silage to a chopped hay based diet, the rumen microbes would require a period of time to adapt (Fernando et al. 2010). However, both WB and C diet groups maintained consistent contents of either WB or water throughout the course of the study. The changes to FI in cattle between the main and interactive effects of diet and time are thought to be the result of either the alcohol or the condensed tannins of the WB fraction of feeds because no changes were determined in either the chemical or energy contents of the feeds. In a study evaluating the impacts of alcohol on cattle feed intake and behaviour, Asato et al. (2003) showed that increases to intra-ruminal alcohol concentrations can increase bolus formation, mastication and rumination over time as well as lowering FI for up to 6 h after alcohol infusion. The reasoning for this is that the alcohol in the rumen acts to signal neural receptors stimulating mastication through increases in plasma isopropanol levels (Asato et al. 2003). This may partially explain our modest findings that feed intake decreased through the interaction of time×diet between the two groups. ! $5! ! Another possibility could be that the diminished FI in WB fed steers was caused by the condensed tannin (CT) fraction of these by-products. The impacts of CT on feed intake and palatability are well understood. When supplemented into animal feeds; condensed tannins have been shown to reduce FI and lower animal performance by forming insoluble protein complexes inaccessible to rumen microbes (Reed 1995). To accommodate feeding of high CT containing forages, such as Acacia cyanophylla, to ruminants, Ben Salem et al. (1999) found Polyethylene Glycol (PEG) could deactivate the CT preventing protein bypass. As a research tool, this method provides a way to discriminate the impacts of CT within individual feeds. In lambs, the feeding of Acacia cyanophylla without PEG has been shown to increase weight gains while not impacting FI (Ben Salem et al. 1999, 2000; Priolo et al. 2000). In cattle, it has been reported that when animals are fed high CT containing diets, quebracho and Acacia mearnsii for example, depressions in FI are observed (Landau et al. 2000; Grainger et al. 2009). Further, Landau et al. (2000) found cattle fed quebracho tannins to have more frequent eating bouts over a longer feeding period than animals fed diets not supplemented with quebracho tannins (Landau et al. 2000). The concentration of CT contained in WB feeds was not evaluated as it was beyond the scope of our initial research design; however, their concentration in red wine lees has been reported to be 180.3 g kg-1 DM (Molina-Alcaide et al. 2008). These studies do lend support to the results obtained from this current study, which show that feeding WB-supplemented feeds can alter the FI of cattle. 2.4.3 Growth and performance Supplementing finishing rations with WB was not found to alter the average daily gains in cattle. These results are in agreement with previous studies that show grape pomace is an insufficient sole source of energy (Baumgärtel et al. 2007; Spanghero et al. 2009; Abarghuei et al. 2010). Information on the use of wine lees as a feed for cattle was not previously available, and as such, studies utilizing grape pomace remain the closest comparable feed (Molina-Alcaide et al. 2008). Similar to the resulting gains observed, no changes were observed when comparing the feed conversion rates by diet (P = 0.2825). ! $.! ! These results suggest that the WB volumes provided to cattle may lack the required levels of condensed tannins or alcohol to elicit a change in animal performance. Other benefits of CT-supplemented feeds, not evaluated in this study, include reduced methane production, reduced occurrence of parasites including nematodes, as well as reductions to bloat in cattle (McMahon et al. 2000; Grainger et al. 2009; Novobilský et al. 2011). As the observations of bloat in this study were small it is not possible to state that WB reduced the occurrence of bloat in cattle. Other studies have shown that feeds containing elevated levels of CT can reduce the occurrence of bloat by suppressing the activity of rumen microbes. Specifically, in vivo studies of lamb micro flora found that the inclusion of tannins into feeds increased the numbers of the bacteria Butyrivibrio fibrisolvens, while decreasing the abundance of Butyrivibrio proteoclasticus bacteria (Vasta et al. 2010b). Further, Min et al. (2005) found that supplementing the diets of cattle grazing winter wheat with quebracho CT reduced bloat scores as well as increasing weight gains. Finding diets that can act to reduce bloat in feedlot rations provides an ideal way to reduce the need for bloat preventing antimicrobials, such as monensin. 2.4.4 Flight speed To evaluate claims surrounding changes to animal behaviour made by the marketers of WB finished cattle, the objective and quantifiable evaluation of flight speed was chosen over other more subjective measurements. Although flight speed is not a thorough evaluation of animal behaviour, this method has been found to correlate with other subjective measurements of behaviour including crush scores, exit scores and pen behaviour (Muller & von Keyserlingk 2006; Petherick et al. 2009; Cafe et al. 2011). Further advantages of flight speed include its repeatability and correlation with serum concentrations of cortisol (Curley et al. 2006). This study identified that cattle became habituated to handing during the course of the study, as indicated by increasingly slower cattle flight speeds as the study progressed. This relationship is supported by other studies which found ! %K! ! cattle habituated to handling over time (Petherick et al. 2009; SchwartzkopfGenswein et al. 2012). Overall, diet was not found to alter cattle behaviour in this study and measured by flight speed. Currently, no research has been conducted on the impacts of WB on ruminant behaviour. Previous research on the role of diet on cattle behaviour has found that providing oral supplements of tryptophan to beef cattle produced sedative effects as well as increased lying and eating behaviours (Nakanishi et al. 1998). Supplementing cattle diets with electrolytes, such as Nutricharge®, has also been found to reduce the stress of transport in animals, improving carcass gains and yield grades during transport (Schaefer et al. 1997). As no change to the chemical and energy contents of experimental diets were observed, it is somewhat expected that the diets tested would not alter the flight speeds of cattle. 2.4.5 Carcass characteristics and meat quality The treatment diets in this study did not alter the hot carcass weights. Further, as carcass weights did not differ, changes to meat quality are most likely the result of the micronutrients in the treatment diet and not the chilling properties of the individual carcasses. Our results, observing darker colours in ground steak meats (L*) between WB and C treatments, are consistent with other studies. Guzik et al. (2006) found darker colouration in pigs fed L-tryptophansupplemented feeds. Additionally, Luciano et al. (2009) found similar colour patterns when supplementing lamb diets with quebracho tannins. The colour changes observed in the latter study were found to be caused by increased metmyoglobin formation in animals fed tannin-supplemented feeds. Similar research has also been conducted on the impacts of citrus pulp inclusive diets on lamb meat. Here, researchers found that these diets can lead to slightly less tender meats, as the animals fed the treatment diets produced meats with shear force values that were slightly more than animals fed the control feed (Scerra et al. 2001). Additionally, it has also been demonstrated that citrus pulp inclusive diets can reduce the redness of lamb meats, which was observed in the fats of the WB steaks, albeit at non-significant levels when compared to C animals (Caparra ! %"! ! et al. 2007). Overall, the lack of changes to cattle FS and the chemical properties of treatment feeds indicate that changes to meat colour were likely caused by components not measured in this study, such as CT, and not animal behaviour. 2.5 Conclusion Winery by-products (WB) are not an effective sole energy source for cattle finishing rations as they do not increase the energy or chemical contents of feeds. However, as a co-product WB can potentially compliment high-energy rations through additions of nutritive factors not yet identified, such as condensed tannins. As a co-product, winery by-products were found to reduce the feed intake of cattle and improve their feed efficiency without altering the weights of carcasses at slaughter. Further, ground muscle meats were found to be darker in colour from animals fed this co-product. No differences were observed in the FS between cattle fed the different diets, suggesting that the behaviour of cattle is likely unaffected by the inclusion of WB. Further research in this area should be conducted to repeat and validate this work on a dry matter basis and verify if unidentified nutritive factors exist in the WB-supplemented feed. Understanding the biochemical processes that impacted the colour changes observed in this study is required to ensure consistent production, especially if the overall goal is to improve meat quality. ! %#! ! 3. EFFECT OF FERMENTED WINERY WASTE SUPPLEMENTED RATIONS ON THE DIVERSITY AND ANTIMICROBIAL RESISTANCE PROFILES OF FECAL E. COLI LOADS IN BEEF CATTLE 3.1 Introduction As novel diets are introduced in animal production their impacts on animal welfare, food safety and environmental issues must be evaluated. Of specific interest in animal agriculture is identifying diets that have the ability to reduce fecal pathogens and antimicrobial resistance (AMR) in manure while maintaining growth yields (Nakanishi et al. 1998; Callaway et al. 2003, 2003, 2009, 2010; Gilbert et al. 2005; Ferguson & Warner 2008). The focus of this work was to evaluate the impacts of a grain-based feed supplemented with fermented winery by-products (WB), mainly wine lees, on the proliferation of fecal Escherichia coli (EC), their antimicrobial resistance phenotypes, as well as the bacterial diversity of feces from cattle fed WB-supplemented feeds. This diet is currently being fed to cattle in the South Okanagan Valley of British Columbia, Canada. Understanding the impacts of diet on the spread of antimicrobial resistant organisms from animal agriculture is essential as the use of antibiotics as growth promoters has increased the loads of drug resistant pathogens in fecal wastes, which can be spread through manure to drinking water sources (Duriez & Topp 2007; Alexander et al. 2008; Jokinen et al. 2011). In healthcare, tetracycline and ampicillin are often used as first line drugs to treat infections, as well as being used in agriculture as prophylactics (Hamilton-Miller 1984). The subtherapeutic administration of antimicrobials as prophylactics promotes the prevalence of resistant EC in cattle feces. Additionally, the role that diet plays in promoting this resistance is not understood (Alexander et al. 2008; Sharma et al. 2008). Dibner & Richards (2005) have suggested that consumer trends will force producers to reduce the use of antimicrobials as prophylactics; further they suggest that dietary supplements able to increase animal performance need to be ! %$! ! identified to replace antibiotics. In Europe, the use of antimicrobial prophylactics is restricted, yet producers have found ways to avoid losses to animal performance; however, the EU still allows the administration of ionophore antibiotics, such as salinomycin in poultry and monensin in cattle (Dibner & Richards 2005). While ionophores are used to manage rumen microbes during dietary changes, they are still antimicrobial in nature, however they have been shown to not increase the prevalence of specific serotypes or antimicrobial resistance in fecal pathogens when added to cattle diets (McAllister et al. 2006; Nisbet et al. 2008). Previous research on the use of WB in animal feeds has focused on the addition of grape pomace, also known as grape marc, with limited studies on wine lees, likely due to the availability of by-products (Bravo & Saura-Calixto 1998; Baumgärtel et al. 2007; Alipour & Rouzbehan 2010; Abarghuei et al. 2010). Of note, by-products from wine making are good sources of condensed tannins (CT) and polyphenols, which have been found to reduce fecal pathogen loads and methane production in beef and dairy cattle (Min et al. 2007; Molina-Alcaide et al. 2008; Grainger et al. 2009). Although CT can restrict the growth of bacteria, such as EC and Salmonella spp., and nematodes in cattle intestinal systems, some species have developed resistance to CT through the degradation of the CT molecules (Goel et al. 2005; Min et al. 2007; Wang et al. 2009; Berard et al. 2009; Novobilský et al. 2011). Molina-Alcaide et al. (2008) found higher levels of free CT in feeds supplemented with red wine lees compared to those supplemented with grape pomace, and similar ratios of protein- and fiber-bound CT. Both wine lees- and pomace-supplemented feeds were found to be good sources of protein and energy for ruminants. Further, Bahrami et al. (2010) found that supplementing lamb rations with dried grape pomace increased the crude protein and dry matter contents of feeds while also improving animal gains and feed conversion rates. Unfortunately, grape pomace alone lacks sufficient energy to maintain animal growth and milk production and, as such, it can be only used as a feed supplement; however, it holds promise as a co-product (Hadjipanayiotou & Louca 1976; Baumgärtel et al. 2007; Tsiplakou & Zervas 2008; Molina-Alcaide et al. 2008; Spanghero et al. 2009; Hersom et al. 2010; Abarghuei et al. 2010). ! %%! ! Considering the impacts of diet on fecal pathogen loads, Diez-Gonzalez et al. (1998) found that switching cattle from grain based to forage based rations five days before harvest reduced EC loads 1,000 fold while also reducing their virulence. Wang et al. (2009) evaluated the impacts of a phlorotanninsupplemented feed and found them capable of reducing the viability of EC O157:H7 in vitro. Further, Min et al. (2007) found that chestnut tannins reduced fecal EC loads in cattle feces when supplied directly into the rumen. However, in vivo trials by Berard et al. (2009) have found tannin-supplemented feeds to have limited impacts on reducing the loads of EC O157:H7 in cattle feces. In the case of supplementing cattle rations with WB, we have previously evaluated their impacts on the weight gains, temperament, and feed conversion rates of cattle (Moote et al. 2012). The addition of WB to feeds did not alter their caloric or chemical composition, however it was found to decrease the feed intake and increase feed conversion ratios of cattle, without impacting cattle temperament. In this report, we evaluate the impacts of WB-supplemented feeds on the loads of fecal EC, their antimicrobial resistances, and the bulk changes in the diversity of fecal bacterial communities. 3.2 Materials and methods 3.2.1 Experimental design 3.2.1.1 Animals Before commencing this project, animal use research protocols were reviewed and approved by the Thompson Rivers University Animal Care Committee. The study followed the Canadian Council of Animal Care guidelines for Farm Animals (Canadian Council on Animal Care 2009) and the Canadian Beef Cattle Code of Practice guidelines (Agriculture Canada 1991). The experimental design used for this project followed that of Moote et al. (2012). Specifically, 69 single-sourced Angus-cross steers (351.3 ± 27.9 kg) were transported 678 km to a small custom feedlot near Oliver, BC (Southern Plus Feedlots). On arrival, cattle were acclimated to finishing rations by stepping up the grain ration content from 0 to 50% in the diets over a 30 d period. Once ! %&! ! acclimated, animals were randomly separated into four freshly cleaned pens, two pens per treatment (n = 18, 17, 17, and 17). 3.2.1.2 Housing Cattle were held in pens of 1,600 m2 (40 m × 40 m) and pen walls were made of 2-m wood panels along three sides. The remaining side contained a feed bunk with metal railings above a concrete bunk to prevent cattle from exiting the pen through this area. To minimize the spread of bacteria from animals of varying diets, pens shared water bowls, with an adjacent pen (one bowl per two pens), that contained animals fed the same diets. Pens were cleaned on a monthly basis, or as needed, using front-end loaders, and bedding of either gypsum or wood chips were added after cleaning. 3.2.1.3 Dietary treatments Animals were fed finishing diets, twice daily at 0800 and 1500, containing either 6-7 % (w w-1) winery by-products (WB; 18, 17) or 6-7 % water (C; 17, 17) for a 143 d period (Table 3.1) using industrial feeders equipped with scales (± 4.55 kg) that mixed feeds before delivery. Wine lees were the sole source of the winery by-products for this experiment, except for one batch, which contained improperly fermented wine. For this reason we refer to the wine supplement as WB, as opposed to wine lees. The oats and barley used in the feeds were at least 489 and 618 kg m-3 respectively and rolled on-site. Further, malt contents were fermented cereal grains provided from local breweries and the liquid supplement contained minerals and protein designated for beef cattle. Variation in the feed contents occurred as a result of a dietary change from corn silage to chopped hay when silage supplies diminished (d105) two thirds of the way through the feeding trial. During the experiment the feedlot manager monitored the daily feed weight delivered to each pen through slick bunk demand management, which is routinely utilized at the feedlot. ! %'! ! Table 3.1 Wet matter composition of experimental diets used during the 143d feeding program; control diets and experimental diets differed from one another only by their contents of winery by-products and water respectively. Item % Diet (d1-d105) % Diet (d106-d148) WB C WB C Corn Silage 30 30 0 0 Chopped Hay 0 0 12 12 Fermented cereal grains (Malt) 10 10 31 31 Oats 20 20 20 20 Barley 30 30 28 28 Protein and mineral feed supplement Winery by-products 3 3 3 3 7 0 6 0 Water 0 7 0 6 Percent Dry Matter (%) ± SD 64.3 ± 2.3 4.9 ± 0.4 58.5 ± 8.1 4.19 ± 0.7 63.3 ± 0.8 4.21 ± 0.2 84.6 ± 32.4 Energy content (Kcal gm-1 DM ± SD) ! %4! 4.7 ± 0.5 ! 3.2.2 Data collection 3.2.2.1 Fecal sampling Fecal samples were gathered from the cattle six times during the experiment using sterile double-pronged swabs (StarSwab SO9D; Starplex Scientific Inc., Etobicoke On, Can). Swabs were inserted ~4 cm into the rectum and rotated around the mucosal membrane to ensure an even distribution of feces, a technique referred to as a Rectal Anal Mucosal Swab (RAMS). At each sampling event, whole fecal samples (n = 6 per pen) were gathered from the first six animals to enter the squeeze providing the animal had not recently had a prolapsed rectum. Grab samples were extracted by hand using a fresh sterile nitrile examination glove (High Five Products Inc., Chicago IL) for each sample, and transferred directly into a sterile urine analysis cup (VWR International Inc., Randor PA). All samples were stored on ice for no more than four hours before being processed at the on-site laboratory operating under the Biohazard regulations of Thompson Rivers University (TRU) and approval of Southern Plus Feedlots. RAMS samples were prepared for EC isolation by re-weighing the sampled StarSwab container to determine the mass of fecal sample gathered. Grab samples were processed by adding ~15% sterile glycerol (w w-1) into the urine analysis tubes followed by freezing at -20oC until being transferred back to the main lab where they were stored at -80oC. 3.2.2.2 Escherichia coli (EC) isolation RAMS samples were diluted in 4.5 mL Brain Heart Infusion Broth (BHI) (EMD Chemical Inc., Darnstadt, GER) and vortexed until homogenous slurries were produced. The resulting slurries were then serially diluted up to 10,000 times in BHI broth and duplicate 100 µL samples were spread onto Membrane Fecal Coliform Agar supplemented with 100 mg L-1 5-Bromo-4-chloro-3-indolylß-D-glucuronic acid (BCIG) (mFc agar) (Oxoid Ltd., Basingstoke, Hants, ENG) that contained no antibiotics, 4 µg mL-1 of tetracycline hydrochloride or 4 µg mL-1 ampicillin sodium salt to produce mFc, mFcT and mFcA plates, respectively. The 1 ! %5! ! " 104, 105, 106 dilutions were spread onto mFc plates to enumerate the EC load of selected cattle (n = 6 per pen). All mFc, mFcT and mFcA plates were incubated at 44.5oC overnight, and presumptive EC were identified by the production of a blue pigment indicating the presence of ß-D-glucuronidase. Loads were only determined from the mFc plates if between 30-300 cultures were present. An error was made when starting this experiment as the concentration in mFcA plates should have been 32 µg mL-1 or 50 µg mL-1 as done previously (Alexander et al. 2008). As such, only the changes to the susceptibility of EC in samples to ampicillin can be monitored, where the changes to resistance patterns to tetracycline can be determined through the mFcT plates. From a single mFc, mFcA or mFcT plate, four presumptive isolates were selected and streaked onto fresh Luria Bertani (LB) agar (5 g L-1 tryptone, 10 g L-1 yeast extract, 10 g L-1 NaCl, 15 g L-1 agar) and incubated at 37oC overnight; after, single colonies were plate purified on LB agar. Isolates were then transferred into 96-well microtiter plates (Grenier Bio-One North America, Inc., Monroe, NC) using sterile toothpicks and grown at 37oC overnight; after incubation, 15% (v v-1) glycerol (Sigma-Aldrich, St. Louis MO) was added and the plates were frozen at 20oC for transport to TRU. Presumptive isolates were confirmed as EC by their growth at 44.5oC, their production of ß-glucuronidase, as well as their ability to metabolize lactose as a sole carbon source using Lactose broth (EMD Chemicals Inc., Darnstadt, GER), and produce indole by adding 10 µl of Kovacs’ Indole reagent (EMD Chemicals Inc., Darnstadt, GER) following growth in Peptone broth (Oxoid Ltd., Basingstoke, Hants, ENG). Confirmed EC isolates were transferred into microtiter plates along with the control strain EC K12 MG1655 (provided by Dr. Julian Davies, UBC) being added to one well per plate. 3.2.2.3 Antimicrobial susceptibility of E. coli isolates The minimum inhibitory concentration (MIC) for EC isolates was estimated using 25-cm2 bioassay plates (Corning Inc., Corning NY, USA) with 250 mL Mueller Hinton agar (BD, Sparks, MD, USA) supplemented with either 0, 1, 2, 4, 8, 16, 32, 64, 128 µg mL-1 ampicillin or 0, 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, 128 ! %.! ! µg mL-1 tetracycline. Freshly cultured EC isolates were replicated onto these plates and incubated at 37oC for 16 h. The MIC for each isolate was determined by the absence of growth occurring at the lowest concentration of each antibiotic. MIC values were grouped by diet and compared using a Student’s t-tests, assuming un-equal variances, to determine if changes occurred between diet and over time via JMP statistical software (SAS Institute Inc., Cary NC). 3.2.2.4 DNA extraction for 16S rRNA gene sequencing DNA was extracted from frozen fecal grab samples taken at d1, 64, and 143 using MoBio Ultra Clean Soil Isolation Kits (Carlsbad CA, USA lots SD11E5 and SD11G11) as described by the supplier; deviations to the supplier protocol were that the frozen samples were partially thawed at 37oC before beginning the isolation. DNA concentrations were determined fluorometrically using a Qubit (Invitrogen Inc., Grand Island, NY) as well as visually following gel electrophoresis. Fecal DNA extractions from two cattle per pen were pooled and adjusted to 20 ng µl-1. A total of 12 DNA samples were sent to Research and Testing Laboratories (RTL) (Lubbock, TX) for pyrosequencing. 3.2.2.5 Massively parallel bTEFAP Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) was performed by Research and Testing Laboratory (Lubbock, TX) using the Gray28F (5#TTTGATCNTGGCTCAG) and Gray519r primers (5# GTNTTACNGCGGCKGCTG) (Suchodolski et al. 2009, 2012; Handl et al. 2011). The bacterial DNA tagging and sequencing was performed as described by Dowd et al. (Dowd et al. 2008). A Roche 454 FLX sequencer with Titanium reagents was used to sequence DNA from within the 28F and 519R regions of the 16S rRNA gene. 3.2.2.6 Pyrosequencing and data analyses Data analysis was performed using the macQiime V1.6.0 Software package downloaded through the Werner Lab website, SUNY Cortland ! &K! ! University (Caporaso et al. 2010b). In total, 81,214 raw sequences were filtered based on length, eliminating those <200bp, and quality. From this, a total of 49,578 sequences averaging 313.7 bp were extracted. The resulting sequences were aligned with the UCLUST function in Qiime, to generate an OTU table outlining sequence similarity. The UCLUST function has been shown as an effective way to align generated sequences in Qiime (Edgar 2010; Jami & Mizrahi 2012). In parallel, the taxonomy of generated OTUs was assigned using the Ribosomal Database Project (RDP) to generate alignment seeds against the Greengenes reference database available at blog.qiime.org as “Most recent Greengenes OTUs” (“QIIME News and Announcements” n.d.; Wang et al. 2007). A 97% alignment between OTUs and BLAST results was set to predict the identity to the species level for each sequence. In total, 11,433 OTUs were generated from the 81,214 sequence reads for analysis; further, to reduce biases 2,028 OTUs were evaluated from each sample yielding a total of 7,882 OTUs for analyses. During the same workflow script, the Pynast algorithm was used to calculate sample phylogeny, enabling the generation of a tree through the FastTree algorithm to determine the similarities between the bacterial communities within samples (Price et al. 2009; Caporaso et al. 2010a). To identify similarities between the bacterial communities within samples by time and diet, Principal Component Analyses were generated using the unweighted Unifrac discrete parameters. Species richness and bacterial diversity was evaluated using the Chao1, Shannon’s index and observed species functions in Qiime. Changes between specific Genera and Phyla over time were evaluated using the Student ttest package available in JMP Software V8 (SAS, Carey, NC), incorporating unequal sample variances. 3.2.2.7 BOX-PCR (Rep-PCR) Rep-PCR was performed on confirmed EC isolates using the BOX A1R primer (5#-CTACGGC AAGGCGACGCTGACG -3#) (Alpha DNA, Montreal QC, CAN Lots 399,824 and 444,206) in 25-µl PCR reactions that contained 3 mM MgCl2 1X EconoTaq PCR buffer, 2 µM BOX A1R primer, 600 µM dNTPs and 1U of 5U µL-1 Taq polymerase (EconoTaq, Lucigen Corp., Lot 3672). PCR ! &"! ! amplification was performed using a MyCycler thermal cycler (BioRad Laboratories Inc, Segrate, Italy) as follows: after an initial denaturation step of 94oC for 10 minutes, reactions underwent 34 cycles of denaturation (94oC for 3 seconds, 92oC for 30 seconds), annealing (50oC for 1 minute) and extension (65oC for 8 minutes), followed by a final extension (65oC for 8 minutes). PCR products (10 µl) were then separated on 1.5% agarose gels using 50-cm OWL A3-1 or 30cm A5 gel electrophoresis boxes (Owl Separation Systems Inc., Plymouth NH) at 20 V cm-1 for 16 h using BioRad PowerPack Basic power supplies (BioRad Laboratories Inc, Segrate, Italy). For each gel, 5 µl of VWR 1Kb ladder with fragments ranging from 0.3 to 10 Kb in size (VWR International Inc., Randor PA lot MD03100301) was added in every eighth lane; in addition, 5µl of PCR reactions from positive and negative controls (EC K12MG1655 and water, respectively) were added to each gel. Gels were stained using GelRed (Biotium Hayward, CA) and visualized using a BioRad GelDoc XR imager (BioRad Laboratories Inc, Segrate, Italy). 3.2.2.8 Computer assisted fingerprinting Normalization of gel images and assignment of fingerprints to isolates were done with BioNumerics (version 6.5; Applied Maths, Kortrijk, Belgium). Positions of fingerprints on gels were normalized using the MassRuler DNA ladder as the external standard in the range of 300 bp to 3,000 bp. Similarity coefficients were generated using the curve-based cosine correlation coefficient. Similarity trees were generated using the unweighted-pair group method using average linkage, and a similarity cutoff of 80 % was used in order to determine related fingerprint types. Comparisons of 355 isolates from the d143 sampling event were made using the Multi-Dimensional Scaling (MDS) package available in BioNumerics in order to determine the change in bacterial diversity by diet Figure 3.5 and by isolation method Figure 3.6. 3.2.3 Statistical analyses Statistical analyses were performed using JMP Software V8 (SAS, Carey, NC). Repeated measures mixed model MANOVAs were used to identify the ! &#! ! impacts of WB on changes to fecal EC loads over time. The number of cultures isolates able to grow on antibiotic containing media was evaluated by providing values for samples with resistant cultures as 1 and those without as 0. In this way all samples could be evaluated and compared through the repeated measures and Student’s t-test functions available in JMP. Further, for statistical analyses the pen served as the experimental unit over the five sample periods with the pens being represented in the statistical equation through the use of an ID function. To accomplish this, data tables were organized by sorting determinate values (ex: loads) into one column, and then mixed model MANOVAs were carried out to determine changes to the main factors of time and diet between results, as well as time×diet interactions using the univariate approach outlined by the software manufacturer (S.A.S. Institute Inc 2012). The normality of collected data was determined using the Shapiro-Wilk test in JMP, with results greater than 0.05 being deemed as coming from normally distributed samples. Of the data collected, all were from normally distributed samples with the exception of the analysis of samples containing tetracycline and ampicillin resistant EC as the results were limited to presence/ absence, as well changes to Genera and Phyla diversity. As such, all changes to the minimum inhibition concentrations, as well as Genera and Phyla diversity, were determined using Student t-test functions incorporating un-equal sample variations. For all the statistical analyses, significance was declared at P ≤ 0.05 and trends at 0.05 < P < 0.10. 3.3 Results 3.3.1 Fecal EC loads To evaluate the impacts of WB-supplemented cattle rations on fecal Escherichia coli (EC) loads, the numbers of EC were evaluated on a Log Colony Forming Unit per gram (LogCFU g-1) basis after incubation on selective media. Over the course of experiment, the EC loads per sample averaged 7.01 ± 0.96 LogCFU g-1 (Figure 3.1). Fecal EC loads gradually declined from d1 to d64. From here, a spike in numbers was observed at d98, and these numbers remained consistent until harvest at d143. Repeated measures MANOVAs revealed ! &$! ! changes occurring to the EC loads when monitoring the interactions of time×diet (P = 0.0498); however, no changes were observed to the fecal EC loads at any sampling event (Table A.1). This indicates that over the feeding period cattle fed WB were found to have elevated EC loads over time than cattle fed C diets. ! &%! ! C?D?.E$1?>A-.F$G.-:2$HI?FCJK$LFM$ .! 5! 4! '! &! K! #K! %K! 'K! 5K! "KK! "#K! "%K! "'K! N-A/$?.$1//4$H4M$ M! P! Q! R! Figure 3.1 Shifts to the loads of fecal E. coli in cattle fed a 143-day diet supplemented with either 6-7% winery by-products (A and B; ! and ") or 6-7% water (X and Y; ! and !). ! &&! ! 3.3.2 Tetracycline and ampicillin resistance To understand the impacts of WB on EC resistance to tetracycline and ampicillin, the number of fecal samples harbouring EC able to grow in 4 µg mL-1 of these antibiotics was determined at each sampling event. Resistance to ampicillin was found to be greatest at d1, 98 and 143, with greater than 95.5% of samples containing resistant organisms, while EC were found to be most susceptible to ampicillin at d64 with 81.8 % of samples containing resistant organisms (Figure 3.2). Diet, and the interactions of time×diet, were not found impact the number of samples containing ampicillin resistant EC over time (P > 0.05; Table A.1). ! &'! O9A@D/2$8-:3$>/2-2:9.:$PC$H#M$ ! "K&! .K! 4&! 'K! K! #K! %K! 'K! 5K! "KK! "#K! "%K! "'K! N-A/$?.$1//4$H4M$ M! P! Q! R! Figure 3.2 Proportion of fecal samples containing E. coli capable of growth at 4 µg mL-1 ampicillin from cattle fed a 143-day diets containing either 6-7 % wine (A abd B; ! and ") or 6-7% water (X and Y; ! and !). ! &4! ! Resistance to tetracycline among samples was similarly found to be the greatest at d1, 98 and 143, with 95.5% of samples containing EC resistant to tetracycline. The greatest proportion of samples containing resistant EC were found at d63, with 84.3 % of samples found to be harbouring resistant EC (Figure 3.3). The number of samples containing resistant EC changed over time (repeated measure mixed model MANOVA, P = 0.0062); however, diet was not found to have an impact (P = 0.1008; Table A.1). The proportion of samples harbouring tetracycline resistant EC were different between diets at d64, where 72.7% of samples from C cattle were found to harbour resistant EC compared to 97.0% of samples from cattle fed WB rations. From here, it is important to further evaluate the antimicrobial resistance patters of EC to understand if diet altered the resistance profile of these organisms. ! &5! ! O9A@D/$8-:3$>/2-2:9.:$PC$H#M$ ""K! "KK! .K! 5K! S! 4K! 'K! K! #K! %K! 'K! 5K! "KK! "#K! "%K! "'K! N-A/$?.$J//4$H4M$ M! P! Q! R! Figure 3.3 Proportion of fecal samples containing E. coli capable of growth at 4 µg mL-1 tetracycline from cattle fed a 143-day diets containing either 6-7% wine (A and B; ! and ") or 7% water (X and Y; ! and !). * Indicates findings between diets (P < 0.5, n=4). ! &.! ! 3.3.3 Minimum inhibition concentrations (MIC) The MIC of EC was determined on all isolates through agar-based techniques. Resistance to ampicillin antibiotics was found to be highest in EC isolates from d1 with an average MIC of 202.3 ± 73.3 µg mL-1 (Table 3.2). The lowest MIC values recorded were from d64 with an average of 51.8 ± 11.5 µg mL-1. At d143, differences between diets (P < 0.05) were observed within the MIC values of EC isolated from all thee selection methods (mFc, mFcA and mFcT). Diet was also found to impact the MIC values of EC isolated on d1 from mFc plates and on d36 from mFcA and mFcT plates. No trends were observed between EC isolated on mFc, mFcA or mFcT plates between diets. In general, the MIC of all isolates remained above the resistance threshold of 32 µg mL-1 throughout the experiment. ! 'K! ! Table 3.2 Minimum inhibitory concentrations of ampicillin antibiotics for Escherichia coli isolates obtained from fecal samples of cattle fed 143-day diets supplemented with 6-7% winery by-products (WB) or water as a control (C) on membrane fecal coliform agar containing BCIG and supplemented with no antibiotics (mFc) 4 µg mL-1 tetracycline (mFcT) or 4 µg mL-1 Ampicillin Sodium Salt (mFcA); letters indicate statistically similar numbers (P = 0.05), not compared between isolation methods. Diet Media n WB mFc 28 C mFc 12 WB mFcT 40 C mFcT 27 WB mFcA 28 C mFcA 21 Total STDEV ! 1 mean 231a 55cdfh 240a 211a 231a 247ab 202.3 73.3 n 83 15 51 85 83 50 36 mean 30bfgh 54bcefgh 151bf 40de 30b 121cdef 71.0 52.1 '"! Day 64 n mean 84 62cefh 14 32bfgh 90 49cdf 70 48cdcf 84 62cdef 65 58cdef 51.8 11.5 n 78 25 76 65 78 53 98 mean 74cfh 42bcegh 56cdf 48bcdcf 74cdefg 49cdef 57.3 13.9 143 n mean 103 122d 101 51cefh 114 57e 82 90cdcf 103 122cdefg 89 53def 82.6 33.6 ! The MIC values for tetracycline were found to be the greatest at d1, with an average MIC of 162.9 ± 85.2 µg mL-1 (Table 3.3). The lowest average MIC was recorded on d143 at 25.1 ± 20.0 µg mL-1, the MIC values of EC were observed to differ between diets when isolated on various media at each sampling event. Of note, WB fed cattle consistently yielded EC with elevated MIC values compared to EC from C fed cattle when EC were selected on mFc and mFcA plates. Conversely, EC from WB fed cattle were found to have lower MIC values compared to EC isolated from cattle fed the C diet when isolated on mFcT plates. ! '#! ! Table 3.3 Minimum inhibitory concentrations of tetracycline antibiotics for E. coli isolates obtained from fecal samples of cattle fed 143-day diets supplemented with 6-7% winery by-products (WB) or water as a control (C) on membrane fecal coliform agar containing BCIG and supplemented with no antibiotics (mFc) 4 µg mL-1 tetracycline (mFcT) or 4 µg mL-1 Ampicillin Sodium Salt (mFcA); letters indicate the presence of similar numbers (P = 0.05), not compared between isolation methods. Day 1 36 64 Diet Media n mean n mean n mean a bch WB mFc 29 222 92 52 86 38bcegh C mFc 12 2f 15 16cghi 14 2f WB mFcT 36 141a 55 38bc 80 14bc C mFcT 25 218a 85 8d 67 65d WB mFcA 29 222a 92 52bcdfgi 86 38bcdh C mFcA 20 173e 55 13bcij 61 8bfgi AVG 162.9 36.1 27.5 STDEV 85.2 17.5 23.9 ! '$! n 84 24 75 65 84 53 98 143 mean n mean bcdeh 65 104 49bcdeh 31bcdeghi 97 8i 16b 116 8bc 75d 86 30d 65bbcdhj 104 49ch 20bfgi 89 7bdfj 45.1 25.1 25.9 20.0 ! 3.3.4 Fecal bacterial diversity Sequencing of 16S rRNA gene fragments from fecal DNA, using Gray28F and 519R primers, generated 49,578 sequences with an average length of 313.7 bp after being filtered for length (<200 bp) and quality. On average, 4,131.5 ± 637.5 sequences were observed per sample, generating a total of 11,733 OTUs for community comparisons (Supplemental information, Table A.2). Table 3.4 outlines how species richness and bacterial diversity increased between d1 and 143 through shifts in the Observed Species and Shannon’s indices, respectively (P < 0.05, n=4 per sampling date). No changes to species richness or bacterial diversity were observed between diets, and dominant phyla were not found to change over the feeding period or between diets, however, changes were observed between the less dominant Cyanobacteria and Lentisphaerae phyla over time (P <0.05, n=4, Table A.3). Numbers of Ruminococcus were found to increase over time (n=4, P < 0.05), however not between diets (Table 3.4). The changes are in agreement with the principal component analyses, which showed isolation of the d1 sample by component and conversely, an isolation of the sample from d143 through the analysis of principal component two (Table 3.4). ! '%! ! "KK0! (8I,-! .K0! T)F1*C*9-2A*@>-.:-.F$ To determine if diet impacted the diversity of EC isolated immediately prior to animal harvest (d143), BOX-PCR generated fingerprints were analyzed using Multi-Dimensional Scaling Plots. Fingerprint types from cattle fed WB and C diets were found to cluster together and not in distinct groups (Figure 3.5). Similarly, no distinct clustering was observed between EC isolated from various selection media (Figure 3.6). ! '5! ! Figure 3.5 Multi-dimensional scaling plots of Rep-PCR patterns created from EC obtained from cattle fed 143-day diets of 6-7 % winery byproducts (Green), or water as the control (Red) at harvest. ! '.! ! Figure 3.6 Multi-dimensional scaling plots of Rep-PCR patterns obtained from Escherichia coli isolates obtained from the feces of cattle fed 143-day diets of 6-7 % winery by-product or 6-7 % water as a control by membrane fecal coliform plates containing no antibiotics (Red), 4 µg mL-1 Tetracycline (Green), or 4 µg mL-1 Ampicillin (Blue). ! 4K! ! 3.4 Discussion The impact of diet on the loads of fecal pathogens has been a topic of research for decades (Brownlie & Grau 1967; Callaway et al. 2009). DiezGonzalez et al. (1998) found shifting grain finished animals to hay rations before harvest to be an effective strategy for reducing the loads of EC at harvest. Other approaches that have been evaluated for reducing fecal microbial loads are the inclusion of Sainfoin tannins and direct-fed microbials (probiotics) to feeds, and the use of phages to reduce the loads of specific pathogens in cattle feces (Callaway et al. 2003; Loneragan & Brashears 2005; Sheng et al. 2006; Casey et al. 2007; Mirzaagha et al. 2011). The goal for this research was to evaluate the impacts of supplementing cattle finishing rations with WB on the loads of fecal EC, the resistance of these isolates toward ampicillin and tetracycline antibiotics, and overall fecal microbial diversity. Switching cattle diets from silage (d64) to hay based rations (d98) increased EC loads in feces, and is in agreement with the observations of Brownlie and Grau (1967) (Brownlie & Grau 1967). Conversely, over the feeding period cattle fed the WB feeds were found to have higher fecal EC loads when comparing the interactions of time×diet (P = 0.0498). This is contrary to our original hypothesis as it was thought that the antimicrobial and polyphenol contents of WB could reduce the EC loads in cattle feces. The elevated EC loads observed between diets were not found to increase the number of samples harbouring EC resistant to ampicillin or tetracycline antibiotics (P = 0.4247 and 0.0806 respectively); however, the number of samples harbouring EC resistant to tetracycline increased with time on feed (P=0.0062). Unfortunately, the concentration of ampicillin in plates used in this experiment was inappropriately set to 4 µg mL-1 instead of the breakpoint concentration of 36 µg mL-1 or 50 µg mL-1 as done previously (Mirzaagha et al. 2011). To accommodate this, the numbers of samples containing EC capable of growth in 32 µg mL-1 ampicillin was evaluated at harvest (Results not shown). Diet had no effect on the number of samples harbouring resistant EC when plated on media containing 32 µg mL-1 ampicillin (P = 0.3268). The decision to not correct this mistake was decided upon to maintain consistency throughout the experiment. If ! 4"! ! the limit was set to 50 µg mL-1 it is expected that the number of samples harbouring resistant EC would have increased over time, but not between diets as seen in the tetracycline study. The MIC study identified greater average MIC values in EC when compared to other studies. These changes are likely due to the differing methods, where we evaluated resistance patterns up to 128 µg mL-1 and other studies stopped evaluating for resistance past the 32 µg mL-1 resistance threshold (Alexander et al. 2008, 2011; Mirzaagha et al. 2011). The average MIC values of EC varied greatly over time. In general, the values for both ampicillin and tetracycline decreased with time on feed regardless of the antibiotic used for isolation. This in agreement with Alexander et al. (2009) where the average MIC of EC from animals fed no antibiotics was greatest at d0 and decreased there after. MIC values from EC isolated were heavily impacted by selection method. This was observed as EC from cattle fed WB feeds had elevated MIC values when isolated on mFcT plates, and reduced MIC values when isolated on mFcA plates compared to EC from C fed cattle. Additional trials identifying specific genes present in isolated EC would assist in understanding this polarization. When comparing the changes to the diversity of EC isolates at harvest, the diversity among isolates within both diets and isolation methods was too great to identify any degree of dissimilarity in BOX-PCR patterns. This lack of change is unexpected as other studies utilizing Pulsed Field Gel Electrophoresis have were able to differentiate from cattle fed differing back grounding diets (Su et al. 2011). Many studies have evaluated the bacterial communities in soils, animals and humans using deep sequencing technologies and this present study adds more knowledge to this area of research (Finegold et al. 2010; Gaidos et al. 2011; Nacke et al. 2011; Handl et al. 2011; Goddard et al. 2012; Suchodolski et al. 2012). The Phyla observed in our study are similar to those observed by Durso et al. (2010), as we found that Firmicutes, Bacteroidetes, and Tenericutes, dominated the fecal Phyla at 68.5, 23.6, and 1.6 %, respectively, where Durso et al. (2010) found Firmicutes, Bacteroidetes and Proteobacteria to represent 62.8, 29.5, and 4.4 %, respectively. The similarities between these two studies is expected as overlap occurred with sequencing 16S regions; i.e., Durso et al. (2010) sequenced within the 27 and 1392 bp and we sequenced within the 27 and 519 bp region of the 16S ! 4#! ! gene. When comparing identified Genera with the literature, Rice et al. (2012) similarly found Ruminococcus spp. to dominate cattle feces, similarly their study found that the members of this Genus were impacted by the concentrations of Dried Distillers Grain in diets, which is similar to our results. These reports illustrate how this dominant Genus is highly impacted by cattle diet and stresses further understanding of its impact on cattle health. 3.5 Conclusion Supplementing cattle feeds with winery by-products (WB) was not found to alter the bacterial populations within cattle feces. However supplementing cattle rations with fermented winery by-products (WB) was found to increase the EC loads over time (P = 0.0498). Although greater levels of EC were found, the proportion of samples containing resistant EC was not altered (P > 0.0874). The isolation methods used were found to alter the resistances of EC towards tetracycline, however no consistent link was observed between diet and antimicrobial resistance patterns in EC. In summary, the use of WB as cattle feed supplements did not alter the loads of EC by diet as well as the numbers of samples containing antimicrobial resistant organisms, or the diversity of fecal samples. ! 4$! ! 4. 4.1 PROJECT SUMMARY Overview This thesis has described the impacts of a wine by-product (WB)- supplemented cattle feed on feed intake, meat quality, temperament, as well as the changes to EC loads, associated antimicrobial resistance profiles and the bacterial diversity within cattle feces. Cattle fed WB feeds required less feeds to yield carcass weights similar to cattle fed the control ration. Further, feeding WBsupplemented feeds was not found to change the tenderness, chemistry or colours of meats, with the exception of ground steak meat that was found to be darker in colour in cattle fed WB finishing rations. As such, it is hypothesized that chemical compounds not evaluated in feeds, such as polyphenols or condensed tannins, may have caused the observed changes to feed intake in cattle. Another aspect of this research was to evaluate the impacts that WBsupplemented feeds had on cattle behaviour. At the commencement of this project the marketers of the “wine-finished beef” product made un-validated claims suggesting that feeding WB to cattle altered their behaviour (Findlay 2010). To evaluate these claims, the flight speed of cattle was determined over the feeding period to objectively measure animal temperament. From this research it was determined that cattle became habituated to handling over time, which is in support of other published results (Petherick et al. 2009; Schwartzkopf-Genswein et al. 2012). However, WB-supplemented feeds were not found to alter the flight speeds of cattle. The potential for feeding cattle WB-supplemented feeds to reduce the Escherichia coli (EC) loads in feces was evaluated also evaluated. When comparing the impacts of the WB-supplemented feeds on pre-harvest EC loads, fecal EC were found to increase over the feeding period in WB fed animals over time, when compared to the control. However, no changes between diets were observed when comparing the number of samples containing EC resistant to ampicillin or tetracycline antibiotics. ! 4%! ! To further evaluate changes to antibiotic resistance in feces the minimum inhibitory concentration (MIC) of EC was monitored. The results of this study found that the resistance of EC towards antibiotics decreased with time on feed, which is in support of other published research (Alexander et al. 2008). The isolation method of EC was found to alter the MIC of EC over the experiment as changes between diets were not consistent throughout isolation methods. Of note, many pens were found to contain EC able to withstand ampicillin and tetracycline antibiotics at concentrations over 100 µg mL-1 indicating a great level of resistance in fecal EC even among cattle not fed antibiotics. As such, this stresses the need for further research to identify novel production methods that can reduce the antimicrobial resistance in fecal pathogens pre-harvest. Recent advancements in sequencing technology have revealed that the diversity of fecal bacterial is directly related to animal health, as such it was important to understand if WB can alter the diversity of fecal bacterial (Mao et al. 2012). To accomplish this, repetitive extragenic palindromic-PCR (Rep-PCR) and pyrosequencing were used to evaluate changes to the diversity of EC and the diversity of global fecal bacterial communities respectively. The diversity of EC at harvest, was not found to be impacted by diet or isolation method, through multi-dimensional scaling comparisons. However, the communities of fecal bacteria were observed to change over time, but not between diets. Specifically, the diversity and species richness of samples increased with time on feed, however diversity did not increase after the d64 sampling event. These changes were supported in the principal component analysis of this data, which found the components at d1 to be different from the other sampling events with no changes observed between diets. When comparing the populations of bacteria, the numbers Ruminococcus spp., the dominant bacterial Genus, were found to increase over time, however, not between diets. No changes were observed within the dominant bacterial Phyla over time, indicating that feeding cattle WBsupplemented rations caused minimal changes to the bacterial diversity of their feces. ! 4&! ! 4.2 Concluding remarks In conclusion, feeding cattle winery by-product (WB)-supplemented feeds appears to be a safe way to produce a novel niche beef product “wine-finished beef”. This product offers economic incentives to producers in the form of feed reductions, additionally, the ground steak meat of WB finished animals was found to be darker in colour than animals fed the control, with no other changes observed to the tenderness, chemistry or colour of meats. WB-fed animals were found to have elevated EC loads compared to control animals, however no changes were observed to the antimicrobial resistant populations and their virulence between diets. Global fecal bacterial diversity changed over time, with no changes observed between diets. In conclusion, supplementing cattle rations with WB was not found to alter the behaviour of the cattle as measured by flight speed or antimicrobial resistance in EC while reducing the feed required to meet slaughter weights when compared to animals fed the control ration. The results of this study indicate that the practice of feeding WB supplemented rations to beef cattle to produce “wine-finished beef” appears to have altered the internal meat colour of the final beef product, without negatively impacting either food safety or animal behavior as determined by the measures utilized in this research project. ! 4'! ! 5. LITERATURE CITED Abarghuei, M. J., Rouzbehan, Y. & Alipour, D. 2010. The influence of the grape pomace on the ruminal parameters of sheep. Livestock Science, 132, 73–79. Aerts, R. J., Barry, T. N. & McNabb, W. C. 1999. Polyphenols and agriculture: beneficial effects of proanthocyanidins in forages. Agriculture, Ecosystems & Environment, 75, 1–12. 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SUPPLEMENTARY INFORMATION .K! ! ! ."! 0.0806 96.77a (31) 93.55a (31) 84.85a (33) % 93.94a (33) 72.73b (33) 100.00a (33) 96.77a (35) 90.91a (35) 100.00a (35) 96.97a (35) 84.85a (33) 96.97a (33) 81.82a (33) 93.55a (31) 96.77a (31) 0.1008 0.0062 0.4247 C mFcT WB C C WB mFcA % 97.14a (35) 97.14a (35) 94.29a (35) 97.14a (35) 7.68deh (12) 5.42cfg (7) 5.88bcfg (12) 7.57adeh (12) mFc WB 100a (33) 7.06abcdeh (10) 0.0874 0.0647 <0.0001 0.0498 0.0774 7.65acdeh 7.65adeh (12) (11) 6.69bcfh (12) 7.62adeh (12) Log CFU 6.17bcfh (12) Diet x time Time Diet 98 64 36 1 Units Media Diet Time on feed (d) 143 P (repeated measures MANOVA) Table A.1. Fecal E. coli loads and proportions (%) of fecal samples containing E. coli capable of growth on membrane fecal coliform agar containing with the indicator BCIG with no antibiotics (mFc), 4 µg mL-1 ampicillin (mFcA) or 4 µg mL-1 tetracycline (mFcT) from cattle fed diets supplemented with 6-7 % of either winery by-products (WB) or water as a control (C). Letters represent statistically similar numbers (95 %), not compared between isolation methods, and numbers in brackets are sample size (n). ! Table A.2. Sequence numbers between samples from cattle fed winery byproduct (WB) and water (C) supplemented feeds over time. ! Sample ID Diet Pen Date Sequences A1 WB A 1 5233 B1 WB B 1 4091 X1 C X 1 4477 Y1 C Y 1 2777 A3 WB A 63 4539 B3 WB B 63 3722 X3 C X 63 3745 Y3 C Y 63 4550 A6 WB A 143 3864 B6 WB B 143 3618 X6 C X 143 4660 Y6 C Y 143 4302 Total 49578 Average 4131.5 Stdev 637.5 .#! ! U$ T$ ! Figure A.1 Principal component analysis of bacterial diversity between fecal samples from cattle fed experimental diets using discrete unweighted UNIFRAC parameters. A. Diversity between cattle fed a diet supplemented with 6-7 % winery by-products (!) and cattle fed a diet supplemented with 6-7 % water (") at d1, 64 and 143 on feed. B. Sample diversity between cattle fed 6-7 % winery by-products (d1", d64# and $d143) and cattle fed 6-7 % water as the control (d1!, d64" and d143%) ! .$! V52/>W/4$O@/B-/2$ ! #&KK! #KKK! "&KK! "KKK! &KK! K! M! K! "KKK! #KKK! $KKK! %KKK! &KKK! O39..?.Y2$S-W/>2-:E$ O/XG/.B/2$@/>$29A@D/$ P! "#! "K! 5! '! %! #! K! K! "KKK! #KKK! $KKK! %KKK! &KKK! C39?%$S-W/>2-:E$ O/XG/.B/2$@/>$29A@D/$ 'KKK! E! %KKK! #KKK! K! K! "KKK! #KKK! $KKK! %KKK! &KKK! O/XG/.B/2$@/>$O9A@D/$ M"! M$! M'! P"! P$! P'! Q"! Q$! Q'! R"! R$! R'! Figure A.2. Bacterial diversity and species richness between samples from cattle fed experimental diets. A) Changes between observed species; B) changes to Shannon’s diversity; and C) Chao1 richness index. ! .%! ! Table A.3 Diversity within bacterial Phyla and Genera populations between cattle fed experimental diets. Numbers in sample ID indicate sampling session: 1 at d1, 3 at d63 and 6 at d143. Letters indicate statistically similar numbers between each Phyla or Genera (P < 0.05). Phylum Genus WB1 C1 WB3 C3 WB6 C6 ANOVA Firmicutes 67.41 65.07 70.03 68.42 70.99 69.60 0.7787 Ruminococcus o_Clostridiales; Other 22.87ab 29.78a 28.11a 19.74b 25.40ab 29.88a 0.0974 3.01c 4.34a 4.29ab 3.35bc 4.69a 3.78abc 0.0309 c_Clostridia; Other 5.14a 3.55ab 3.25ab 4.22ab 3.36ab 2.81b 0.2894 2.42a 1.31b 2.45a 2.25ab 1.25b 2.25ab 0.0872 0.68a 0.42ab 0.36b 0.55ab 0.55ab 0.51ab 0.2907 bc abc ab c c a g_Oscillospira c_Clostridia;g_ unnamed g__Dorea 0.44 0.58 0.64 0.30 0.32 0.78 0.0413 Bacteroidetes 23.80 29.15 21.95 21.56 21.75 23.69 0.4946 Tenericutes 1.40 1.11 1.50 2.59 1.40 1.53 0.7307 g__Sutterella 0.04b 0.09b 0.73a 0.15b 0.11b 0.60a 0.0011 Proteobacteria 1.27 1.09 0.31 0.63 1.34 1.28 0.6356 Spirochaetes 0.69 0.45 1.65 0.44 0.27 0.38 0.5866 a c Cyanobacteria ! 0.71 0.04 abc abc 0.62 abc c 0.34 ab abc 0.41 0.0749 bc 0.25 o__YS2;g_other 0.71 a 0.33 0.40 0.04 0.59 0.25 0.0615 Fibrobacteres 0.84 0.41 0.00 0.31 0.00 0.19 0.6008 Verrucomicrobia 0.48 0.01 0.27 0.40 0.32 0.11 0.5861 Lentisphaerae 0.18ab 0.02c 0.04bc 0.23a 0.08bc 0.13bc 0.0657 Elusimicrobia 0.01 b 0.00 b Planctomycetes 0.09 Fusobacteria ab ab bc 0.04 0.09 a 0.01 b 0.01 b 0.0843 0.00 0.00 0.05 0.00 0.00 0.3864 0.00 0.00 0.00 0.00 0.03 0.00 0.4894 TM7 0.00 0.00 0.01 0.00 0.00 0.01 0.5865 Chloroflexi 0.00 0.00 0.00 0.00 0.00 0.02 0.4894 Actinobacteria 0.00 0.00 0.00 0.00 0.01 0.00 0.4894 Acidobacteria 0.00 0.00 0.00 0.01 0.00 0.00 0.4894 Other 3.11 2.65 3.86 4.64 3.39 2.78 0.3368 Unclassified 0.01 0.00 0.00 0.00 0.00 0.00 0.4894 .&!