Cows and on Animal Performance THESIS. Presented in Partial Fulfillment of the Requirements for the degree Master of Science in

Transcription

1 Effect of Bovamine on Ruminal, Post-Ruminal, and Total Tract Digestibilities in Dairy Cows and on Animal Performance THESIS Presented in Partial Fulfillment of the Requirements for the degree Master of Science in the Graduate School of The Ohio State University By Catherine E. Dickey, B.S. Graduate Program in Animal Sciences The Ohio State University 2016 Thesis Committee: Dr. Maurice Eastridge, Advisor Dr. Jeffrey Firkins Dr. William Weiss

2 Copyright by Catherine Eileen Dickey 2016

3 ABSTRACT Increasing feed efficiency in dairy cows is one of the primary goals of the dairy industry. The use of direct-fed microbials (DFM) is now commonly being evaluated to improve efficiency and reduce the use of antibiotics. This 2-part study evaluated the effects of feeding Bovamine to dairy cows. Bovamine is a DFM consisting of Lactobacillus acidophilus and Propionibacterium freudenreichii. A total of 30 lactating, non-cannulated Jersey cows were used for 12 weeks to evaluate the effects of Bovamine on animal performance. There were 15 control cows that were not fed Bovamine and 15 treatment cows that received Bovamine. The effects of the DFM on digestibility were investigated with 8 lactating, cannulated Jersey cows for 12 weeks. The Bovamine was mixed with ground corn and given daily as a topdress. For the production experiment, there were no differences in dry matter intake (DMI), milk fat and protein percentages, and body condition score (BCS) between the treatments. However, milk yield, fat-corrected milk yield, and energy-corrected milk yield were greater in cows that received Bovamine. Additionally, the milk urea nitrogen was greater in the treated cows. Also, there was a trend for increased efficiency (milk/dmi) in Bovamine -fed cows. For the digestibility experiment, there were no differences between treatments for ph, ammonia concentration, total volatile fatty acids (VFA), and molar proportion of ii

4 VFA in the rumen. Additionally, there were no differences observed in total tract digestibilities between the treated and control cows. Based on this study, feeding Bovamine to dairy cows increased milk production, but it did not appear to have an effect on the rumen environment or total tract digestibility. iii

5 ACKNOWLEDGEMENTS First I would like to thank my advisory committee. I am thankful for the opportunity that my advisor, Dr. Eastridge, provided that allowed me to further my education. I am also very grateful for his abundant patience and support throughout my graduate work. My committee members, Drs. Firkins and Weiss, have also been very helpful throughout this process. I appreciate all their help and the knowledge they have shared. Secondly, I want to thank the faculty, staff and fellow graduate students in the Department of Animal Sciences. I am very appreciative of the assistance, encouragement, and support from Josie Plank, the ruminant nutrition laboratory manager. Her willingness to help and positive outlook are very admirable. I am also very thankful for everything Yairy Roman Garcia has done. Not only has she been an exceptional friend, she has always been willing to help with my work in the lab, on the farm, and in class. I am glad we had the opportunity to meet and become good friends. I would additionally like to thank Paula Chen, Brooklyn Wagner, and Jerad Jaborek for their friendship, support, and assistance. Other graduate students that must also be mentioned for their help at the farm include Garth Ruff, Alex Tebbe, and Logan Morris. I could not have accomplished everything without the help of many undergraduate students. Their assistance on the farm and in the lab kept me sane and I am glad they were able to put up with me at times when I was stressed out and exhausted. iv

6 The willingness of some of them to work in the early morning hours is very commendable. Although there were several, they were all able to contribute to the success of this project at one point or another; thank you Rachel Nelson, Priscila Bernhard, Doug Liebe, Kendal Searer, Kaylin Krueger, Rachel Patton, Jaime Uren, Hannah Gaitan, and Marisa Joldrichsen. Additionally, I would like to thank the managers and crew at Waterman Dairy Farm, in particular, Rebekah Meller and John Lemmermen. Their cooperation at the farm made this project possible. I appreciate their reliability and assistance. All the workers at the farm made my time there much more enjoyable. Finally, I cannot go without giving thanks to my parents. Their continual support, unconditional love, and faith in me are things that I will always cherish. I would not be who I am today without them. Also, I must thank my best friend Renee and my friend Sarah for always listening, giving advice, and offering help. I would not have accomplished all that I have without them. v

7 VITA 2009 Covington High School 2014 B.S. Animal Sciences and Biology, University of Findlay 2014-Present...Graduate Research Associate, Department of Animal Sciences, The Ohio State University Major Field: Animal Sciences FIELDS OF STUDY vi

8 TABLE OF CONTENTS ABSTRACT... ii ACKNOWLEDGEMENTS... iv VITA... vi LIST OF TABLES... x LIST OF FIGURES... xi LIST OF ABBREVIATIONS... xii CHAPTER 1: INTRODUCTION... 1 CHAPTER 2: LITERATURE REVIEW... 3 I. Improving Efficiency of Dairy Cows... 3 II. Direct-Fed Microbials... 4 III. Bovamine... 6 A. Mode of Action... 6 B. Beef Cattle Studies... 7 C. Dairy Cattle Studies... 9 IV. Estimating Digestibility A. Digesta Markers vii

9 B. Sampling Sites V. Summary CHAPTER 3: EFFECT OF BOVAMINE ON RUMINAL, POST-RUMINAL, AND TOTAL TRACT DIGESTIBILITIES IN DAIRY COWS AND ON ANIMAL PERFORMANCE MATERIALS AND METHODS I. Experimental Design II. Housing and Diets III. Digesta Markers IV. Sample Collection and Laboratory Analyses V. Statistical Analyses RESULTS AND DISCUSSION I. Animals and Treatments II. Feed Composition III. Dry Matter Intake IV. Milk Production and Composition V. Body Weight and Body Condition Score VI. Efficiency VII. Rumen Fermentation VIII. Ruminal, Post-Ruminal, and Total Tract Digestibilities viii

10 CHAPTER 4: CONCLUSIONS REFERENCES ix

11 LIST OF TABLES Table 1. Ingredient composition of TMR for all cows..39 Table 2. Chemical composition of the experimental TMR...40 Table 3. Effect of Bovamine on performance of cows in the production experiment.41 Table 4. Effect of Bovamine on performance of cows in the digestibility experiment...42 Table 5. Least squares means for ruminal fermentation characteristics for control cows and cows fed Bovamine...43 Table 6. Effect of Bovamine on ruminal, post-ruminal, and total tract digestibilities...44 x

12 LIST OF FIGURES Figure 1. Milk yield of control and treated cows in the production experiment.45 Figure 2. Dry matter intake of control and treated cows in the production experiment.45 xi

13 LIST OF ABBREVIATIONS ADG BCS BW Co-EDTA CP Cr-EDTA DFM DIM DM DMI ECM FCM FP iadf ICP indf LP MUN average daily gain body condition score body weight cobalt-ethylenediaminetetraacetic acid crude protein chromium-ethylenediaminetetraacetic acid direct-fed microbial days in milk dry matter dry matter intake energy corrected milk fat corrected milk fluid phase of reticular fluid indigestible acid detergent fiber inductively coupled plasma spectrophotometry indigestible neutral detergent fiber large particles of reticular fluid milk urea nitrogen xii

14 NDF NEL OM PEG RFI SP TMR VFA neutral detergent fiber net energy for lactation organic matter polyethylene glycol residual feed intake small particles of reticular fluid total mixed ration volatile fatty acids xiii

15 CHAPTER 1: INTRODUCTION Cost of feed for dairy cows makes up the largest cost of producing milk. Therefore, the efficiency of feed utilization by dairy cows must be a major point for improving the profitability of dairy operations. Direct-fed microbials (DFM) are live bacterial and fungal microorganisms. These can be supplemented in the feed to potentially improve efficiency, therefore reducing the amount of feed per unit of milk produced. Bovamine (Nutrition Physiology Company, LLC) is a DFM that consists of Lactobacillus acidophilus and Propionibacterium freudenreichii. These bacteria should beneficially work together because L. acidophilus produces lactate and P. freudenreichii utilizes lactate and produces propionate. As a precursor for gluconeogenesis, the production of propionate should result in more energy availability for cows, consequently causing greater milk production and therefore possibly an overall improved efficiency. This two-part study was conducted to evaluate the effect of feeding Bovamine to lactating Jersey dairy cows. The effect on animal performance was investigated by analyzing BW, BCS, and milk yield and composition. The hypothesis was that cows given Bovamine will have greater yield of milk and milk components and improved feed efficiency. 1

16 Additionally, the effects of Bovamine on rumen fermentation, and ruminal and total tract digestibilities were explored. The hypothesis was that cows given Bovamine would have increased ruminal, post-ruminal, and total tract digestibilities. 2

17 CHAPTER 2: LITERATURE REVIEW I. Improving Efficiency of Dairy Cows The world s human population is continuously increasing. In addition to this increase, urbanization is on the rise. This means that fewer people are living in rural areas and maintaining their own livestock. These two factors plus the growth of income are increasing demand for livestock products (Thornton, 2010). Dairy cattle not only provide dairy products, but also meat, both of which are necessary to feed this growing population. To combat this issue, either a substantial increase in the number of cattle and other livestock will be needed or the efficiency of feed utilization will have to improve. Being that available land is limited, the solution appears to be the improvement of feed efficiency. By making cows more efficient, other problems also are solved. Dairy cattle are often blamed for polluting the environment with nitrogen and phosphorus that is excreted in their manure (Spears et al., 2003a, b). An improvement in the utilization of these nutrients in animals will decrease the excretion of these elements into the environment. In addition to utilizing these nutrients more efficiently, the cost of feed can be decreased for producers. Feed is easily the greatest expense for a dairy operation. The more efficiently a cow can utilize feed, using a greater percentage of the feed for 3

18 production and less for maintenance so that more milk is produced per unit of feed, the greater the profit margin will be for the farmer. II. Direct-Fed Microbials There are many ways to improve feed efficiency in dairy cattle. One of these is through the use of direct-fed microbials (DFM) to potentially improve rumen fermentation or intestinal health to improve digestion and reduce the need for antibiotics. Without question, concern has increased about the use of antibiotics in animal production. Consequently, the shift away from antibiotics also has led to increased usage of DFM (Krehbiel et al., 2003; Raeth-Knight et al., 2007). The term probiotic was defined by Fuller (1989) as a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance. However, because the addition of microorganisms to a ruminant diet often have their effect in the rumen rather than in the intestines, the FDA began requiring manufacturers to use the term DFM instead of probiotic and defined a DFM as a source of live (viable) naturally-occurring microorganisms (Yoon and Stern, 1995). A DFM may be used as an additive or preservative for silage or hay, replacement for antibiotics in stressed cattle, to increase milk production in dairy cows or BW in beef cattle, or to increase feed efficiency (Yoon and Stern, 1995; Krehbiel et al., 2003). The mode of action for DFM is still uncertain; however, suggested mechanisms depend on the type, amount, and frequency of DFM fed (Elghandour et al., 2015). Brown and Nagaraja (2009) stated that bacterial DFM typically have an effect on the 4

19 gastrointestinal tract, whereas fungal DFM generally have more of an impact in the rumen. There are, however, some studies that have shown that bacterial DFM improve the rumen environment (Ghorbani et al., 2002; Nocek et al., 2006). Some suggestions for the mechanism of action of DFM include the modification of gut microbial population, change of rumen fermentation, increased nutrient flow to the small intestine, improved digestibility, and enhanced immune response (Yoon and Stern, 1995; Krehbiel et al., 2003). For bacterial DFM, Brown and Nagaraja (2009) proposed a few mechanisms that include production of antimicrobial substances, competition for nutrients and attachment site in the intestines, and immunostimulation. Buntyn et al. (2016) also suggested indirect mechanisms including competitive exclusion, improved digestion and nutrient utilization, immunostimulation, and improved gut microbiota. Although there are multiple suggested modes of action for DFM, there is agreement on a few possible mechanisms. Further research of specific DFM would need to be done to determine the exact mechanisms. A DFM can either be fungi or bacteria. Common fungi that are used as DFM include yeast, typically Saccharomyces cerevisiae, and molds, usually Aspergillus niger or Aspergillus oryzae. According to Brown and Nagaraja (2009), feeding fungal DFM can increase the number of bacteria in the rumen, change fermentation products, and stimulate fiber-digesting and lactate-utilizing bacteria. The use of bacteria as a DFM usually involves lactic acid-producing bacteria or lactic acid-utilizing bacteria (Elghandour et al., 2015). Bacteria such as Bacillus, Bifidobacterium, Enterococcus, Lactobacillus, Propionibacterium, and more specific species like Megasphaera elsdenii are commonly used as DFM (Elghandour et al., 2015; Buntyn et al., 2016). 5

20 III. Bovamine Bovamine is a product of Nutrition Physiology Company, LLC. It is a DFM consisting of two bacteria: Lactobacillus acidophilus strain NP51 and Propionibacterium freudenreichii strain NP24. Bovamine was first introduced in 1994 for beef cattle. The company later expanded product options so that there is now Bovamine Defend for beef cattle, Bovamine Dairy for dairy cattle, and Poultrimax for poultry (Nutrition Physiology Company, 2016). There have been more studies to determine the effects of Bovamine in beef cattle compared to dairy cattle. Fortunately, recent interest has led to an increasing number of studies in dairy cattle. A. Mode of Action 1. Lactobacillus acidophilus The bacterium L. acidophilus is a gram-positive facultative anaerobe that is rodshaped. It is a lactic acid-producing bacterium and is obligate homolactic, meaning that it converts glucose only to lactic acid. This process occurs via glycolysis and because of the lack of oxygen, pyruvate is reduced to lactic acid by NAD + -dependent lactate dehydrogenase (Axelsson, 2004). The production of lactic acid results in a decrease in ph. This drop in ph may be the reason L. acidophilus has been shown to be antagonistic toward pathogenic bacteria (Krehbiel et al., 2003). Bacteriocins, which are bacterial proteins, produced by L. acidophilus also are thought to help protect against pathogenic bacteria (Krehbiel et al., 2003). 6

21 2. Propionibacterium freudenreichii This bacterium is a gram-positive obligate anaerobe that utilizes lactic acid. It utilizes lactic acid by converting 3 moles of lactate to 2 moles of propionate, 1 mole of acetate, 1 mole of CO2, and 1 mole of H2O (Piveteau, 1999). Propionate is a primary precursor for hepatic gluconeogenesis, so the production of propionate by P. freudenreichii could increase metabolizable energy. B. Beef Cattle Studies Nutrition Physiology Company, LLC claims that when Bovamine is given to beef cattle, it supports the immune system and reduces pathogens, improves feed efficiency and rate of gain, and it maximizes energy capture (Nutrition Physiology Company, 2016). Several studies have evaluated the effects of Bovamine on the reduction of pathogens, particularly Escherichia coli O157:H7. Cull et al. (2012) examined the efficacy of a vaccine and Bovamine used separately and together in commercial feedlot cattle against fecal shedding of E. coli and found that the DFM did not affect fecal shedding. On the contrary, Younts-Dahl et al. (2005) reported that, when finishing steers were given three different levels of Bovamine, there was a reduction in fecal shedding of E. coli. Fecal shedding of E. coli O157 was linearly decreased when treating steers with different levels of the DFM (Elam et al., 2003). A meta-analysis was conducted to evaluate the efficacy of DFM, primarily Bovamine, on the reduction of E. 7

22 coli O157 shedding in beef cattle, and it revealed that there was a significant reduction (Wisener et al., 2015). Direct-fed microbials may also be fed to beef cattle as a way of preventing or minimizing acidosis. Wilson and Krehbiel (2012) stated that Lactobacillus acidophilus can help prepare the rumen for an acidic environment, allowing the microbes to adjust to a lower ph. Ghorbani et al. (2002) claimed that Propionibacterium should also help in the prevention of acidosis because of its ability to convert lactate to propionate, therefore lessening the amount of lactate in the rumen. There is, however, a lack of studies using Bovamine as a preventative method for acidosis in beef cattle. The use of Bovamine to improve performance and efficiency in beef cattle has been commonly studied. Cull et al. (2012) noted that feedlot cattle treated with Bovamine had a greater gain-to-feed ratio than cattle that were not given the DFM but didn t observe a difference in average daily gain (ADG). Although there was no difference in ADG or DMI, finishing steers fed different levels of Bovamine had a greater gain-to-feed ratio than control cattle (Vasconcelos et al., 2008). Similarly, Elam et al. (2003) did not detect any significant differences in DMI, ADG, or gain-to-feed ratio when comparing feedlot cattle given different levels of Bovamine to control cattle. These results suggest that Bovamine may increase the gain-to-feed ratio in beef cattle, but the exact mechanism is unknown because the DMI and ADG often appear unaffected. 8

23 C. Dairy Cattle Studies Bovamine in dairy cattle has not been as extensively studied as it has been in beef cattle. Boyd et al. (2011) observed no differences in DMI, milk yield, or feed efficiency when lactating cows were treated with Bovamine. Because Bovamine should increase blood glucose concentration due to the production of propionate by P. freudenreichii, Boyd et al. (2011) took blood samples from dairy cows, but reported that serum glucose concentrations in cows fed this DFM were not increased. Ferraretto and Shaver (2015) observed no differences either in milk yield or feed efficiency; however, there was a trend for DMI to be lower in cows treated with Bovamine than control cows. O Neil et al. (2014) reported that lactating dairy cows had lower DMI when fed Bovamine, which increased efficiencies of actual milk and ECM, but they did not observe a difference in yields of milk or ECM. Similar to Boyd et al. (2011), West and Bernard (2011) detected no difference in DMI between control cows and cows treated with Bovamine. There was a trend for greater milk yield in the treated cows, and ECM yield and feed efficiency were greater in the treated cows (West and Bernard 2011). The effect these bacteria have on digestibility has also been studied. Boyd et al. (2011) noted that lactating dairy cows treated with Bovamine had improved apparent total tract digestibility of CP and NDF. Ferraretto and Shaver (2015) evaluated total tract starch digestibility but detected no differences in cows treated with Bovamine and control cows. Raeth-Knight et al. (2007) assessed apparent total tract digestibilities of DM, NDF, CP, and starch when cows were treated with Bovamine and detected no differences between treated and control cows. However, there was a trend for an 9

24 elevation in ruminal propionate concentration. Lastly, Osman et al. (2012) observed an increase in total volatile fatty acids (VFA) in the rumen of dairy cows treated with Bovamine compared to control cows. IV. Estimating Digestibility Digestibility essentially refers to the extent to which feed that is consumed is absorbed or consumed by microbes before passing through the animal. Simply collecting fecal samples and comparing them to the feed that was consumed would give an estimate of total tract digestibility. The issue with this is that a total collection of the feces would be necessary, which is laborious and stressful to the animal (Huhtanen et al., 1994). Additionally, in ruminants, it is often important to know the digestibility that occurs in the rumen and not just the total tract. To measure digestibility at different points throughout the digestive tract, samples need to be taken from different areas of the digestive tract. The use of digesta markers allows subsamples of digesta or feces to be taken to calculate digestibility. A. Digesta Markers Owens and Hanson (1992) define a marker as a reference compound used to evaluate chemical and physical aspects of digestion. Several markers have been tested in the past, including the use of internal and external markers. The compound used as a marker cannot be absorbed or affected by the digestive tract, it must associate and flow with the material it is marking, and it must be measurable (Owens and Hanson, 1992). 10

25 Because of the difficulty in collecting a representative sample, the use of more than one marker is often employed so that the different particle sizes and the fluid in digesta can be more accurately measured. France and Siddons (1986) compared the use of a single marker, double marker, and triple marker method and concluded that the single and double marker methods result in under- or overestimated flows. Using the triple marker method, markers are then available for each component: small particles, large particles, and the fluid portion of the digesta. A few of the markers that have been previously investigated for large particles of digesta include lignin, indigestible acid detergent fiber (iadf), and indigestible neutral detergent fiber (indf). According to Titgemeyer (1997), the use of lignin as a marker is not suitable because of high variability in its recovery. Also, Titgemeyer (1997) stated that some diets contain insufficient amounts of iadf for it to be measured accurately. Huhtanen et al. (1994) evaluated multiple markers, including indf and iadf, and concluded that indf was a more suitable marker because of a higher recovery. Additionally, Ahvenjärvi et al. (2003) provided evidence that indf primarily associates with large particles. Rare earth elements and chromic oxide are commonly used as markers. However, there are issues using chromic oxide because it flows with the fluid phase sometimes and does not associate exclusively with a particular phase (Firkins et al., 1986; Titgemeyer, 1997). Ytterbium can be analyzed by atomic absorption spectrometry rather than neutron activation like many other rare earth elements (Siddons et al., 1985). Ahvenjärvi et al. (2003) claimed that Yb primarily associates with small particles. Beauchemin and 11

26 Buchanan-Smith (1989) also concluded that Yb strongly bonds to feed, but acknowledged that migration can occur. There are typically three markers considered for the fluid phase: polyethylene glycol (PEG), cobalt-ethylenediaminetetraacetic acid (Co-EDTA), and chromiumethylenediaminetetraacetic acid (Cr-EDTA). There have been issues using PEG because some may be absorbed or precipitated and there is a lack of an accurate method of analysis (Downes and McDonald, 1964; Udén et al., 1980). Both Co-EDTA and Cr- EDTA were considered suitable markers by Udén et al. (1980). Downes and McDonald (1964) also concluded that Cr-EDTA is a satisfactory marker. The use of Co-EDTA in dairy cows reduces the concentration of milk fatty acids and should therefore be avoided (Shingfield et al., 2008; Taugbøl et al., 2008). There are two ways to dose external markers: pulse-dosing or continuous. The advantage of a pulse-dose is that it disturbs the animal less than continuous dosing, but it only provides a snapshot and flow of the marker may be less consistent (Owens and Hanson, 1992). With continuous infusion, concentration of the marker should be more uniform in the digesta and after a steady state has been reached, the concentration should remain constant (Owens and Hanson, 1992). In both types of dosing, multiple markers can be used. B. Sampling Sites To estimate ruminal and post-ruminal digestibilities, samples must be obtained post-ruminally. Traditionally, samples have been taken from the duodenum by using 12

27 duodenally cannulated animals. However, secretions from the abomasum can increase endogenous N supply (Huhtanen et al., 1997). Additionally, animals require surgery for placement of the duodenal cannula, intense management of the animals is required, and duodenally cannulated animals tend to have a shortened lifespan (Hristov, 2007). Previous researchers trying to estimate digesta flow have compared sampling sites and have had variable results with duodenal sampling, and there are more difficulties with using duodenal cannulas than with omasal or reticular sampling (Hristov, 2007; Fatehi et al., 2015). As an alternative to duodenal sampling, omasal sampling has been investigated. Huhtanen et al. (1997) proposed an omasal sampling technique in which a device was placed in the omasum with a tube connected to it that went through the ruminal cannula, which was connected to a compressor/vacuum pump that would pull out fluid from the omasum into a collecting vessel. Ahvenjärvi et al. (2000) used this technique to compare duodenal and omasal sampling and concluded that omasal sampling is a promising alternative to duodenal sampling. Additionally, a meta-analysis indirectly validated the accuracy of omasal sampling measurements, indicating it could replace duodenal sampling (Huhtanen et al., 2010). Ahvenjärvi et al. (2001) modified the omasal sampling technique proposed by Huhtanen et al. (1997) by making the sampling tube larger, using a different type of valve, and inserting a weight into the abomasum. Several studies have since used these techniques, emphasizing the successful replacement of duodenal sampling with omasal sampling (Reynal and Broderick, 2005; Brito et al., 2007; Reveneau et al., 2012). 13

28 More recently, researchers have been investigating the use of reticular sampling as an alternative to omasal sampling. Ahvenjärvi et al. (2000) compared omasal and duodenal sampling and reported that omasal samples deviated more from calculated true digesta. Additionally, the technique used for omasal sampling might reduce dry matter intake (DMI; Krizsan et al., 2010). Hristov (2007) compared duodenal and reticular sampling, using a 250-mL bottle with a lid to collect the reticular fluid. Results from this study showed that there are differences in the composition and nutrient flow of duodenal and reticular fluid; however, the author concluded that sampling from the reticulum can provide reliable estimates for ruminal degradability of DM (Hrsitov, 2007). Krizsan et al. (2009) compared omasal and reticular sampling, with the reticular fluid being sieved through a 1-mm sieve, and concluded that digesta collected from the reticulum is representative of digesta collected from the omasum. In a later study, Krizsan et al. (2010) also compared reticular and omasal sampling, again filtering the reticular fluid through a 1-mm sieve, and also concluded that reticular sampling is a promising alternative to omasal sampling. In the latter study, there were no differences in nutrient flow between the two methods of sampling. Additionally, the digesta composition was not significantly different, with the exception that the neutral detergent fiber (NDF) was higher in the omasal fluid, which the authors concluded could have been due to the size of sieve used on the reticular fluid (Krizsan et al., 2010). Fatehi et al. (2015) compared omasal sampling to ruminal and reticular sampling. In this study, samples from the 3 sites were not sieved, filtered through a 5.6-mm sieve, 14

29 and filtered through an 11.6-mm sieve. They concluded that the samples sieved through an 11.6-mm sieve did not differ from the particle size distribution of feces from the same cows. They also found that flow estimates were more similar between the reticular and omasal samples than between the ruminal and omasal samples and concluded that sampling from the rumen is less promising than reticular sampling as an alternative to omasal sampling. V. Summary Because Bovamine consists of lactate-producing bacterium, L. acidophilus, and lactate-utilizing bacterium that produces propionate, P. freudenreichii, the two bacteria should work together to increase concentration of blood glucose, which could have beneficial effects in cattle. The use of Bovamine in dairy cattle has had varying results on performance; however, some studies have observed improved feed efficiency (West and Bernard, 2011; O Neil et al., 2014). Few studies have evaluated the effects of feeding Bovamine on nutrient digestibility. The triple marker procedure is recommended (France and Siddons, 1986) to accurately measure digestibility from the omasum and rumen, from which samples likely underrepresent long particles. By sampling throughout the digestive tract, ruminal, post-ruminal, and total tract digestibilities can be evaluated. Recently, the use of reticular sampling has been investigated. The objective of the study was to determine the effects of feeding Bovamine to lactating dairy cows on ruminal, post-ruminal, and total tract digestibilities, and on animal performance. The hypotheses were that cows given 15

30 Bovamine would have increased ruminal, post-ruminal, and total tract digestibilities, and that Bovamine -fed cows would have greater yields of milk and milk components and improve feed efficiency. 16

31 CHAPTER 3: EFFECT OF BOVAMINE ON RUMINAL, POST-RUMINAL, AND TOTAL TRACT DIGESTIBILITIES IN DAIRY COWS AND ON ANIMAL PERFORMANCE MATERIALS AND METHODS I. Experimental Design For the production experiment, 30 (10 primiparous and 20 multiparous) lactating, non-cannulated Jersey cows began the trial on June 8, 2015 and ended on August 31, All procedures were approved by The Ohio State University Institutional Animal Care and Use Committee (Protocol # 2015A ). The study was conducted as a randomized complete block design with a 2-week covariate period and a 10-week experimental period. Cows were blocked by parity (5 blocks of primiparous cows and 10 blocks of multiparous cows), calving date, and milk yield and averaged 147 ± 49 DIM at the start of the study. Within each block, cows were randomly assigned to either control (n=15) or treated with Bovamine (n=15). The digestibility experiment consisted of 8 (4 primiparous and 4 multiparous) lactating, ruminally cannulated Jersey cows that were used in a cross-over design for 12 weeks with a 2-week adjustment period, 4-week experimental period, 2-week washout period, and another 4-week experimental period. All of the cows were not at similar DIM and pregnancy status, so 4 of the cows were in the experiment from June 8, 2015 to August 31, 2015 and the other 4 were in the experiment from December 14, 2015 to 17

32 March 7, The cows averaged 167 ± 103 DIM at the start of the experiment. In both parts of the experiment, treatments were assigned with 2 cows (1 primiparous and 1 multiparous) randomly selected as the control cows and the other 2 cows (1 primparous and 1 multiparous) treated with Bovamine. Because it was a cross-over design, after the first 6 weeks, the treatment for each cow was switched. II. Housing and Diets All cows were housed in a tie-stall barn at the Waterman Dairy Farm at The Ohio State University. During the summer experiments, which included the entire production experiment and half of the digestibility experiment, there was an aisle between the control and treated cows. In the winter, the control and treated cows were on opposite ends of a row of stalls. This housing arrangement was used to avoid contamination by control cows. In addition, there were separate shovels, brooms, and feed scrapers for each treatment that were color coded and kept on the appropriate side of the barn, depending on the treatment designation. There was a feed barrel for each cow that was also color coded and labeled with the cow s number. All cows had ad libitum access to water and were fed the same TMR (Table 1) twice daily with feed being mixed at 1700 h. Dry matter intake (DMI) was recorded daily for each cow. Using the feed barrels, feed for the control cows was weighed first, and then the treated cows feed was weighed. Half of the feed was dumped in the evening and the remaining half was dumped from the barrels the next morning. The feed was pushed up in the middle of the day and once at night. The cows were fed for 4 to 5% orts to 18

33 minimize sorting and ensure that the top dress was consumed. Each evening after the cows were let out for milking, the refusals were swept up, put in the corresponding cow s barrel, and then weighed to obtain the daily intake. Dry matter intake data were averaged by week for each cow for statistical analyses. The control cows were individually fed a topdress consisting of 454 g/d of ground corn. Treated cows were also individually fed a topdress daily; however, it consisted of 453 g of ground corn and 1 g of Bovamine, which was labeled to contain 1 x 10 9 CFU/g of Lactobacillus acidophilus strain NP51 and 2 x 10 9 CFU/g of Propionibacterium freudenreichii strain NP24. Both topdresses came from the Ohio Agricultural Research and Development Center (OARDC) feed mill with the control topdress bagged before the Bovamine topdress was mixed and bagged. All cows were given the control topdress during the covariate period, then the appropriate topdress was given during the experimental period. For the digestibility experiment, the control topdress was also given to all cows during the adjustment and washout periods. III. Digesta Markers To determine the effect of Bovamine on ruminal, post-ruminal, and total tract digestibilities, the use of digesta markers was necessary. The triple marker procedure was used in this trial with indigestible neutral detergent fiber (indf) for the large particles, ytterbium (Yb) acetate tetrahydrate for the small particles, and chromiumethylenediaminetetraacetic acid (Cr-EDTA) for the fluid phase (France and Siddons, 1986). The Yb acetate tetrahydrate and Cr-EDTA were continuously infused through the 19

34 rumen cannula of the cows during d 20 to 28 of both experimental periods. A peristaltic pump was used to pump the mixed solution of Yb acetate tetrahydrate and Cr-EDTA from a flask through tubing that hung above the cows then went down and through the cannula. The cows were infused at all times of the day except when they were let out to be milked, at which time the pumps were paused and the cannula plugs were replaced with the usual plugs without a hole for the tubing. The Yb acetate tetrahydrate was infused at a rate of 2.8-g Yb/cow/d, and the Cr- EDTA was infused at a rate of 2.8-g Cr/cow/d (Fatehi et al., 2015). The Yb acetate tetrahydrate and Cr-EDTA were made so that each cow received 1-L of each solution every day they were infused. To make the Yb acetate tetrahydrate solution, 6.8 g of Yb acetate tetrahydrate was weighed, dissolved in deionized water, and brought to volume in a 1-L volumetric flask. The Cr-EDTA was made according to Binnerts et al. (1968) with the exception that more water was added to make the solution 1 L rather than 500 ml. IV. Sample Collection and Laboratory Analyses A. Feed Samples Weekly samples of the corn silage, alfalfa baleage, and total mixed ration (TMR) were taken and dried in the oven at 55 C for at least 2 days. The DM percentages of the corn silage and alfalfa baleage were used to adjust the amount that was weighed when mixing the daily TMR. The DM percentage of the TMR was used to calculate the DMI of each cow. Any refusals that were wetted were sampled to adjust the DMI of the 20

35 individual cow. The refusals that were not wetted were assumed to be representative of the TMR, so the DM percentage used to calculate DMI was the same as the TMR. Additionally, on the days that reticular fluid and fecal samples were taken in the digestibility experiment, samples of the feed offered and refused were also taken. This included background feed offered and refusal samples from each cow the day before the cows were infused with the digesta markers, 3 feed offered, and the 3 refusal samples taken on the last 3 days of each experimental period. The 3 feed offered samples from each cow were composited by weighing the same amount of each sample after they had been dried at 55 C. The 3 refusal samples from each cow were composited based on the total weight of the refusals the day each sample was taken. Samples of feed offered and refused were mistakenly not taken during the last 3 days of the first experimental period of the first part of the digestibility experiment. A sample of the TMR from the following week and the background refusal samples were used to represent the missed samples. The dried TMR samples were ground to 1-mm in a Wiley Mill (Thomas Scientific, Swedesboro, NJ). They were then analyzed for total DM in the oven at 105 C overnight, ash in a muffle oven at 550 C overnight (AOAC, 2000), NDF with sodium sulfite and heat-stable α-amylase using an ANKOM 200 Fiber Analyzer (ANKOM Technology, Macedon, NY; Van Soest et al., 1991), CP by Kjeldahl determination using a Foss Tecator (Foss, Eden Praire, MN; Bremmer and Mulvaney, 1982), and starch (Karkalas, 1985; Knudsen, 1997; Hall, 2009). The dried, ground composited feed samples from the digestibility experiment were sent to Service Testing and Research (STAR) Laboratory (OARDC, Wooster, OH) 21

36 to be analyzed for concentrations of Cr, Yb, and other macro and micro minerals. There were a total of 12 composited feed offered samples, 1 TMR sample that represented the missing feed samples from the last week of the first experimental period of the first part of the digestibility experiment, and 16 refusal samples. The samples were digested in perchloric acid and then analyzed via inductively coupled plasma spectrophotometry (ICP). B. Milk Samples The cows were milked twice daily during the trial. Milk yield was recorded at every milking using Afimilk (Afimilk USA, Inc., Fitchburg, WI). Milk samples were taken at 4 consecutive milkings each week. These samples were sent to DHI Cooperative, Inc. (Columbus, OH) where they were analyzed for the concentrations of fat and true protein using infrared spectroscopy (B2000 Infrared Analyzer, Bentley Instruments, Chaska, MN) and milk urea nitrogen (MUN) using a Skalar SAN Plus segmented flow analyzer (Skalar, Inc., Norcross, GA). Milk component percentages for each cow were weighted for the amount of milk per milking. Milk yield and milk composition data were averaged weekly for each cow for statistical analyses. C. Body Weight and Body Condition Score All cows were weighed at the beginning and end of the trial and each week during the experimental period. The cows were scored on their body condition at the beginning 22

37 of the covariate and experimental periods, every 3 wk during the experimental periods, and at the end of the trial. A 1 to 5 scale was used to score the cows, with 1 representing a thin cow and 5 signifying a fat cow. D. Rumen Fluid Samples Grab samples of rumen fluid were taken from various areas of the rumen through a rumen cannula from each cow on 2 consecutive days during wk 4 of each experimental period at approximately 4 h post-feeding. At this time, a ph meter was used to immediately record the ph of the rumen fluid. A 50-mL sample was taken each time and 3 ml of 6N HCl added to stop fermentation. The samples were then frozen at -20 C until analyzed. After all rumen fluid samples had been collected they were thawed and prepared for volatile fatty acid (VFA) and ammonia analyses by centrifuging at 15,000 x g for 15 min and filtering the supernatant through Whatman number 1 filter paper (Whatman International, Maidstone, UK; Harvatine et al, 2002). VFA was analyzed using a gas chromatographer (GC) with 2-ethyl butyric acid used as an internal standard. Ammonia concentration was analyzed with a standard colorimetric assay (Chaney and Marbach, 1962). E. Reticular Fluid Samples Samples of reticular fluid were taken from each cow at the beginning of wk 4 of each experimental period prior to infusing the digesta markers, which was considered a 23

38 background sample, and samples were taken the last 3 d of each experimental period and composited. A 250-mL bottle with a stopper attached was used to obtain the samples by going through the rumen cannula and reaching the reticulum (Hristov, 2007). The stopper remained in the bottle until placed in the reticulum where the stopper was removed, the bottle filled with reticular fluid, and then the stopper was put back in the bottle before the bottle was removed from the reticulum and pulled out of the cow. At each sampling time, 500 ml of fluid was collected. This was measured in a graduated cylinder, and the fluid was poured through a 9.53-mm sieve. The composited samples were taken 4 times per day every 2 h over the 3 d, shifting the sample times each day so that a 24-hour schedule was covered and a total of 12 samples was collected from each cow during each experimental period. These 12 samples were then frozen at -20 C. Prior to analysis, each sample was separated into 3 phases: large particles (LP), small particles (SP), and fluid phase (FP). To do this, each sample was first thawed and then gently inverted multiple times to mix thoroughly. For the background samples, 250 ml was measured. For the composited samples, 3-L was measured to be separated. The fluid was poured through a piece of 100-µm fabric (03-100/32 Nitex Nylon fabric from Sefar Filtration, Inc.). The particles that remained on the fabric, which were the LP, were squeezed so that the majority of the fluid went through the fabric. All the fluid collected after sieving through the fabric was then centrifuged at 1,000 x g for 10 min to separate the SP from the FP (Krizsan et al., 2010). The 3 phases of each sample were dried at 55 C. The large and small particles were ground through a 1-mm screen in a Wiley Mill (Thomas Scientific, Swedesboro, 24

39 NJ). As with the feed samples, the large and small particles were also analyzed for total DM, ash, NDF, CP, and starch as described earlier. The FP samples were analyzed for all of these components except for NDF. All dried phases of all composited reticular fluid samples were sent to the STAR Laboratory (Wooster, OH) to be analyzed for Cr and Yb concentrations. The samples were digested in perchloric acid and then analyzed via ICP. F. Fecal Samples Fecal grab samples were taken from each cow prior to infusing, which was a background sample, and samples were taken the last 3 d of each experimental period. The samples were taken 2 times per day every 4 h over the 3 d, shifting the sample times each day so that a 24-hour schedule was represented and a total of 6 samples were collected from each cow during each experimental period. The samples were then frozen at -20 C until they were dried in the oven at 55 C. The dried samplers were composited per cow for each period using equal portions from each day. The dried background and composited samples were ground through a 1-mm screen with a Wiley Mill (Thomas Scientific, Swedesboro, NJ) and then analyzed for total DM, ash, NDF, CP, and starch as described previously. All dried, ground, and composited fecal samples were sent to the STAR Laboratory (Wooster, OH). The samples were digested in perchloric acid and then analyzed via ICP for Cr and Yb. Fecal DM excretion was calculated from Yb infused and its concentration in feces. 25

40 G. In Situ Samples All background and composited dried, ground feed offered and refusal samples and the 3 dried phases of composited reticular fluid, and the dried, ground background and composited fecal samples were analyzed for indigestible NDF via in situ analysis. Samples were analyzed in duplicate within a cow, and 2 cows were used for each sample, so that there were 4 replicates of each sample. There were also 2 empty bags put in each cow to be used as blanks. In the bags with pore size of 50-µm (ANKOM Technology, Macedon, NY) 3 g were weighed for each sample. The bags were put into mesh bags with a chain attached, which were put into the rumen of cannulated cows and were left there for 288 h (Huhtanen et al., 1994). Once all mesh bags were removed, they were opened and the bags were taken out and rinsed thoroughly in cold water. The bags were then dried in an oven at 55 C and weighed. The remaining sample in each bag was then analyzed for NDF with sodium sulfite and heat-stable α-amylase using an ANKOM 200 Fiber Analyzer (ANKOM Technology, Macedon, NY; Van Soest et al., 1991). V. Statistical Analyses All performance data from both experiments were analyzed using the PROC MIXED procedures of SAS (version 9.4, SAS Institute, Cary, NC). The variables treatment, week of the experimental period, and the interaction treatment x week were used as fixed effects, and cow was a random effect. Block was also used as a random effect for the production experiment analyses. The averages from the 2-wk covariate period were used as a covariate adjustment. The DMI, milk yield, FCM yield, ECM 26

41 yield, milk fat percentage and yield, milk protein percentage and yield, MUN, body weight (BW), change in BW, DMI as a percentage of BW, and body condition score (BCS) were all analyzed as repeated measures. Additionally, efficiency was calculated multiple ways (milk/dmi, FCM/DMI, ECM/DMI, and energy efficiency; discussed in results section below) and analyzed as repeated measures. An autoregressive order 1 covariate structure and the LSMEANS statement were used with each analysis. Treatments were considered significantly different when P 0.05 and a trend was declared when 0.05 < P The data for ph, ammonia, and VFA in the rumen fluid samples, and the nutrient flow and digestibility data were analyzed using the PROC MIXED procedures of SAS (version 9.4, SAS Institute, Cary, NC). Treatment was a fixed effect, but cow was regarded as a random effect. The LSMEANS statement was used with all analyses. Again, treatments were considered significantly different when P 0.05, and a trend was declared when 0.05 < P RESULTS AND DISCUSSION For the production experiment, the covariate data for each variable accounted for a significant amount of variation (P<0.05). Weeks of the experimental period were significantly different (P<0.05) for all data analyzed, with the exception of energy efficiency, which was as expected as cows advanced in lactation during the study. Also, interactions of treatment and week of the experimental period were not observed (P>0.10) for any of the data analyzed. The effects of the treatments are discussed below. 27

42 I. Animals and Treatments During the production experiment, there were 2 cows, cows 11 and 497, that became rather ill, having watery diarrhea and no appetite. Cow 11 was removed from the experiment, causing unequal cell size. Cow 497 was sick during wk 7 and 8 of the experimental period; however, all the data was still used in the statistical analyses. Both cows were Bovamine -fed cows; all control cows remained healthy throughout the trial. The cows that were part of the digestibility experiment during the summer all remained healthy, with the exception of one cow, 492, that also had diarrhea; however, she was only sick for a couple days at the end of week 1 and beginning of week 2 of experimental period 2. The intake data for those 2 days was not used in the statistical analysis. This cow was being fed Bovamine at the time. All control cows during the summer, and all cows that were part of the experiment during the winter remained healthy. II. Feed Composition The chemical composition of the TMR can be seen in Table 2. There were differences between these values and the values of the formulated ration. The expected CP was 18.9% and the actual values were 17.4% and 18.1% for the production and digestibility experiments, respectively. NDF was formulated at 30.1% and the actual values were 28.2% and 29.4%, respectively. Differences could be due to sampling error, errors in mixing, changes in feed chemical composition, or variation in laboratory analyses (Weiss et al., 2016). Additionally, a different TMR mixer was used in the winter 28

43 part of the digestibility experiment than what was used for the summer feeding, which could have affected the variation in chemical composition of the samples taken. III. Dry Matter Intake DMI was similar (P>0.10) between control cows and cows that were fed Bovamine (Tables 3 and 4). Others (Boyd et al., 2011; West and Bernard, 2011) that treated dairy cows with Bovamine also did not see a difference in DMI. O Neil et al. (2014) reported that cows fed Bovamine consumed less DM per day than control cows; however, composition of the TMR was not provided, so in comparison to this study, the effect on DMI may have been due to different diets fed. The results from the digestibility experiment (discussed below) revealed that there were no differences in total tract digestibility of OM or ruminal VFA profiles. Oba and Allen (2003) observed ruminal propionate infusions increased the proportion of propionate in the rumen and increased concentrations of propionate and glucose in the blood, while also observing a decrease in DMI with level of propionate infused. Thus, the similar total tract digestibilities and VFA profiles in our study may have attributed to the similar DMI. In the production experiment, DMI as a percentage of BW was greater (P=0.05) in Bovamine -fed cows (Table 3), which was not expected given that DMI (kg/d) was similar between treatments. 29