SUPPLEMENTATION STRATEGIES TO ENHANCE BEEF CATTLE PRODUCTION EFFICIENCY By MARIANA GARCIA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2016
Â© 2016 Mariana Garcia
To my husband, Ulises, who accompanied me in this wonderful journe y
4 ACKNOWLEDGMENTS I thank God, for His infinite wisdom and mercy, and for letting things happen when they are supposed to happen, exactly when the time is right. I thank my husband, Ulises, for walking this usually complicated and rough path besides me, for being my shelter in the storms, and for always giving immense love and support. I love you to the infinity and beyond. Thanks to my parents, Susana and Camilo, for all their love and care, and for even more. Thanks to my sisters, Macarena, Jimena and Maria Jose, for always being right next to me, in the good and bad times. In add ition , thanks to my nieces and nephew, Victoria, Franco and Ana Lucia, for showing me so much love all the time. I deeply and greatly thank Dr. Nicolas DiLorenzo (and his family, Liza and Lucy), for the immense patience, dedication, and continuous encourag ement throughout these years, and for always letting the doors of his office (and home) open for his students. I would also like to thank my committee members, Dr. Cliff Lamb and Dr. Jose Dubeux, for their contributions and guidance through the development of my program. These two years would have been unbearable without the friendship and guidance of Dr. Martin Ruiz Moreno, whose cheerfulness always enlighten even the darkest moments. I would like to thank Fulbright, for trusting me and giving me the great opportunity to study in the United States. Finally, I greatly give thanks to my colleagues and friends at the North Florida Research and Education Center, whose help and friendship have been invaluable these
5 years, and to all the people that work at the NFREC and make it one of the best research centers in the world.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 2 LITERATURE REVIEW ................................ ................................ .......................... 15 Livestock Production in the Southeast ................................ ................................ .... 15 Nutritional Management in Cow Calf Operations ................................ .................... 15 Frequency of Supplem entation ................................ ................................ ......... 17 Energy Supplementation of Lactating Beef Cows ................................ ............ 20 Nutritional Considerations during Preconditioning and Backgrounding ............ 22 Metabolizable Protein Supplementation ................................ ................................ . 25 Role of Rumen Degradable Protein ................................ ................................ ........ 26 Microbial Protein Synthesis ................................ ................................ ..................... 27 Fermenten as RDP Source ................................ ................................ ..................... 28 3 EF FECT OF FREQUENCY OF SUPPLEMENTATION WITH MEGALAC R ON NON ESTERIFIED FATTY ACIDS AND BLOOD UREA NITROGEN CONCENTRATION IN LACTATING BEEF COWS ................................ ................ 31 Materials and Methods ................................ ................................ ............................ 31 Animals ................................ ................................ ................................ ............. 31 Diets ................................ ................................ ................................ ................. 31 Sampling ................................ ................................ ................................ .......... 32 Blood Analysis ................................ ................................ ................................ .. 32 Statistical Analysis ................................ ................................ ............................ 33 Results and Discussion ................................ ................................ ........................... 33 Conclusions ................................ ................................ ................................ ............ 38 4 EFFECT OF DIFF ERENT INCLUSION RATES OF FERMENTEN ON PERFORMANCE, CARCASS TRAITS, AND TOTAL TRACT DIGESTIBILITY OF GROWING ANGUS CROSSBRED STEERS ................................ ................... 44 Materials and Methods ................................ ................................ ............................ 44 Animals ................................ ................................ ................................ ............. 44 Diets ................................ ................................ ................................ ................. 44
7 Sampling and Laborator y Analysis ................................ ................................ ... 45 Statistical Analysis ................................ ................................ ............................ 47 Results and Discussion ................................ ................................ ........................... 47 Performance and Carcass Traits ................................ ................................ ...... 47 Blood Metabolic Parameters ................................ ................................ ............ 50 Apparent Total Tract Digestibility ................................ ................................ ...... 52 Conclusions ................................ ................................ ................................ ............ 54 5 EFFECT OF FERMENTEN ON NITROGEN METABOLISM AND RUMEN PROFILE OF ANGUS CROSSBRED STEERS ................................ ...................... 6 2 Materials and Methods ................................ ................................ ............................ 62 Animals ................................ ................................ ................................ ............. 62 Experimental Diets ................................ ................................ ........................... 63 Ruminal Profile and Blood Urea Nitrogen ................................ ......................... 63 Apparent Total Tract Digestibility ................................ ................................ ...... 64 Statistical Analysis ................................ ................................ ............................ 66 Results and Discussion ................................ ................................ ........................... 66 Ruminal Fermentation Parameters and Nitrogen Metabolism .......................... 66 Apparent Total Tract Digestibility ................................ ................................ ...... 71 Conclusions ................................ ................................ ................................ ............ 72 LIST OF REFERENCES ................................ ................................ ............................... 78 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 85
8 LIST OF TABLES Table page 3 1 Analyzed composition (DM basis) of pasture (Ryegrass [RG]) and supplements (corn gluten feed [CGF] and Megalac R [M R]). ............................ 39 3 2 Serum concentrations of NEFA and BUN of multiparous lactating beef cows offered energy supplements 3, 5 or 7 d/wk. ................................ ........................ 40 4 1 Ingredients and nutritional composition (DM basis) of experimental diets (Experiment 1). ................................ ................................ ................................ ... 55 4 2 Performance and carcass traits of Angus crossbred steers fed backgrounding diets containing Fermenten at 0, 2 or 4% of dietary DM. ........... 56 4 3 Serum concentrations of BUN, NEFA and glucose of Angus crossbred steers fed backgrounding diets containing Fermenten at 0, 2, or 4% of dietary DM. .... 57 4 4 Intake and apparent total tract digestibility of Angus crossbred steers fed backgrounding diets containing Fermenten at 0, 2 or 4 of dietary DM. ............... 61 5 1 Ingredients and nutritional composition (DM basis) of experimental diets (Experiment 2). ................................ ................................ ................................ ... 73 5 2 Ruminal pH, total VFA concentration, VFA molar proportions, acetate to propionate ratio, NH 3 N and BUN of cannulated Angus crossbred steers fed backgrounding diets containing 0 or 4% inclusion rate of Fermenten in the diet DM. ................................ ................................ ................................ .............. 74 5 3 Intake, apparent total tract digestib ility and BW gain of Angus crossbred steers fed backgrounding diets containing 0 or 4% inclusion rate of Fermenten in the diet DM. ................................ ................................ .................. 77
9 LIST OF FIGURES Figure page 3 1 Serum concentrations of NEFA of multiparous lactating beef cows supplemented 7 (S7), 5 (S5) o r 3 d/wk (S3) during first and second phase s . ..... 41 3 2 Concentrations of BUN of multiparous lactating beef cows supplemented 7 (S7), 5 (S5) or 3 d/wk (S3) on different sampling days . ................................ ...... 42 3 3 Crude protein and TDN content in the DM of samples of ryegrass from paddocks used in the experiment across weeks ................................ ................ 43 4 1 Daily mean values of BUN concentrations of steers receiving a diet with 0 (CTL), 2 (2% FER), or 4% (4% FER) inclusion rate of Fermenten in a backgrounding diet ................................ ................................ ............................. 58 4 2 Daily mean values of serum NEFA concentrations of steers receiving a diet with 0 (CTL), 2 (2% FER), or 4% (4% FER) inclusion rate of Fermenten in a backgrounding diet ................................ ................................ ............................. 59 4 3 Daily mean values of serum glucose concentrations of ste ers receiving a diet with 0 (CTL), 2 (2% FER), or 4% (4% FER) inclusion rate of Fermenten in a backgrounding diet. ................................ ................................ ............................ 60 5 1 Ruminal pH of cannulated Angus crossbred steers receiving a diet with 0 (CTL), or 4% (4% FER) inclusion rate of Fermenten in a backgrounding diet .... 75 5 2 Ruminal concentrations of A mmonia N (NH 3 N) and BUN as affected by hours after feed delivery in cannulated Angus crossbred steers receiving a diet with 0 (CTL), or 4% (4% FER) inclusion rate of Fermenten in a backgrounding diet ................................ ................................ ............................. 76
10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science SUPPLEMENTATION STRATEGIES TO ENHANCE BEEF CATTLE PRODUCTION EFFICIENCY By Mariana Garcia August 2016 Chair: Nicolas DiLorenzo Major: Animal Sciences An experiment evaluated t he effect s of different frequencies of supplementation with CGF and Megalac R ( Church & Dwight, Princeton, NJ) , on the blood profile of suckled beef cows. Cows recei ving equal amounts of an isocaloric and isonitrogenous supplement 3 , 5, or 7 d/wk had similar concentrations of NEFA ( P = 0.42) and BUN ( P = 0.74). R educing the frequency of supplementation did not affect the metabolism o f lactating beef cows and could contribute to reduce co sts. I nclusion rates of Fermenten ( Church & Dwight, Princeton, NJ ) were evaluated on performance a nd metabolism of steers. A first experiment teste d the effects of 0, 2, and 4% inclusion rate of Fermenten in the diet DM on performance of steers . After 56 d under treatment , steers in the 4% treatment had decreased BW ( P = 0.01), ADG ( P < 0.01), DMI ( P = 0.02) and G:F ratio ( P < 0.01). There was no effect of treatment ( P > 0.05) on carcass or blood parameters . Greater inclusion rate of Fermenten resulted in increased ( P < 0.01) DM, OM, NDF, ADF, and CP apparent total tract digestibility. When more mature animals were evaluated, no differences were observed o n fermentation and metabolic parameters, except for concentration s of butyrate , which were greater ( P = 0.01) for steers receiving 4% of F ermenten , than for control . Int ake of all nutrients was reduced ( P < 0.05) for
11 steers receiving Fermenten , but similar nutrient digestibility ( P > 0.05) was observed . In conclusion, Fermenten did not improve performance or metabolism of growing cattle.
12 CHAPTER 1 INTRODUCTION At a national level, Florida is ranked in the 12 th position for inventory of beef cows, and 17 th position when total head of cattle is counted (USDA NASS, 2013) . Most of the cow calf operations base their nutrition programs i n grazing warm season perennial forages, hay or grain byproducts supplementation during the winter, or grazing winter annual pastures during the cool season. This cool season may be coincident with the calving season in some parts of the state. Cooler temp eratures and less forage require to pay additional atte ntion to the care of the cows, for example through supplementation of energy and protein after parturition. Supplementation is directed increase body condition and assure a new pregnancy. In addition, supplementation can also be used to also increase gro wth performance of the calves, during backgrounding or preconditioning to increase their economic value. Supplementation can become a major portion of the activities in a livestock operation and it can significantly impact the cost of production, through an increase in feed purchasing, and labor and time involved in feed delivery. When referring to cow calf operations, the two main objectives of the farm are to produce one calf per cow per year and to spend the least amount of time possible trying to rebreed the cow in the next breeding season. Livestock production, and primarily cow calf operations, is one of principal agricultural economical activities in the state of Florida. The weather and rainfall conditions allows for the production of warm season forages, which provides abundant biomass, even though the nutritional quality is inferior to that of tempe ra te forages, especially crude protein (CP) content, which can be quite low in many warm season
13 grasses grown in Florida. In addition, during the co ol season, due to the lower temperatures and shorter photoperiod, the forages enter a latent state and the p roduction decreases until warmer temperatur es arrive. This situation, typically occurring during the main calving season for many cow calf operations in Florida, forces producers to offer feed supplements to the cows in order to assure an optimal body cond ition score . This is necessary for cows to rebreed successfully and provide sufficient milk to the calf. Once calves are weaned, they may be backgrounded or preconditioned in order to transition them into a finishing phase. Depending on current market sta tus, preconditioning or backgrounding may lead to increased revenues at the time of the sale of calves, especially if commercialized in markets that value those type of animals with a potentially lower health risk, and are willing to pay a bonus for them. Supplementing ca ttle always implies incurring extra costs in the form of feed purchases, labor and hours to deliver sufficient amounts of quality feed to the animals. For that reason, frequency of supplementation and type and quality of nutrien ts should be taken into consideration to avoid unnecessary expenses. For lactating beef cows, energy is the main nutrient required to assure a successful lactation and prompt return to heat and pregnancy. Ruminally protected fats provide long chain fatty acids that ar e not subjected to biohydrogenation in the rumen, potentially provid ing precursors for hormones such as progesterone while providing concentrated energy at the same time. In order to support the growth of newly weaned calves, di ets should contain ~ 14% CP and a relatively low energ etic concentration to prevent early fattening. In addition, at this stage it is critical to develop smooth tissues (like the rumen and the rest
14 of the gastro intestinal tract) and muscle fibers. T he ruminal degradability of the pr otein provides nitrogen for microbial protein synthesis which is of great metabolic value due to its amino acids profile. Additionally , increasing ruminal microbial populations and their activity can also lead to greater digestibility rates . The objective of this study was to evaluate the effect of different frequencies of supplementation in lactating beef cows and the effect of feeding different levels of a rumen degradable prote in (RDP) source, on the performance of post weaned steers.
15 CHAPTER 2 LITERATURE REVIEW Livestock P roduction in the Southeast Livestock production is one of the primary activities in the Southeastern United States. Approximately 28% of the U.S. beef cows and calves are located in this portion of the country, which includes the Southern Plains, mainly in Texas, and the Southeast (CattleFax, 2015) . These regions have the advantage of a longer grazing season, which implies that less supplemental forage is usually required to support beef cattle during the winter, decreasing the costs associated with feed (McBride and Mathews, 2011) . Particularly in the Southeast, c ow calf operations represent the main activity regarding beef production systems. In these operations, the main goal is to achieve successful calving management, with great calf survivability and dam breeding efficiency. Therefore, it is of great interest to increase the efforts directed to enhance reproduction efficiency , which is essential in the beef production process, considering that w ithout reproduction there is no production (Dargatz et al., 2004) . Even though cow calf operations com prise very specific goals, managing these types of operations is often less intensive, when compared to other segments of the beef production industry. For that reason, cow calf production systems are more suitable for producers with limited resources, suc h as time and labor, and particularly for smaller operations (Adkins et al., 2012) . Nutritional Management in Cow Calf O perations In order for cow calf operations to be economically efficient, feed supply should r ely mostly on range and pasture grazing. T h e productivity of the forage resources is related to meteorological conditions, such as average rainfall and temperature in the
16 area. Forage should be harvested by beef cows to fulfill their maintenance and production requirements, to raise a calf, and ide ally with little or no addition of supplement to the diet (McBride and Mathews, 2011) . However, and even when beef cows are kept on pastures for most of the year, few cow calf producers rely exclusively on grazing to supply the amount of feed required to m eet the needs of the cows throughout the year (Short, 2001) . F eed supplements are fed to cattle on forage based diets for: correction of nutrient deficiencies, conservation and improved utilization of forage, enhancement of animal performance and behavior, and greater economic return ( Kunkle et al. , 2000) . Producers located in areas with longer grazing seasons, and milder winters (e.g., the state of Florida) have greater competitive advantages in terms of input costs. Supplemental feed and grain to meet the nutritional requirements of cattle during periods of decreased availability or quality of forage should be considered (Caton and Dhuyvetter, 1997) , even though they greatly impact cow calf production costs (Ramsey et al., 2005; McBride and Mathews, 2011) , and may account for more than half of the total direct cost to cow calf operations (Short, 2001) . Besides influencing the need for additional feed supplements, stocking capacity of range and pasture land (determined by the herbage mass ) may define management practices, such as deciding to market the calves soon after weaning, or keeping them to add weight, and perhaps better selling prices (Hodur et al., 2007) . The main task of nutritionists and cattle farmers is, therefore, to correctl y determine the ingredients, additives, feed processing, and delivery system that will improve forage utilization, performance and efficiency of cattle, at a cost that will positively impact the profitability
17 of the system. As an example, for cattle fed ma inly forage, supplementation should provide enough protein, minerals and vitamins to offset deficiencies in the forage (Kunkle et al., 2000) . When protein is deficient, providing additional energy in the form of supplement may not result in a direct benef it, given that N supply, either to the small intestine or the rumen may be limiting performance. Conversely, increasing the supplementation of protein when energy is deficient, will not result in better performance or increased profitability. Supplementati on should be directed to balance the deficiency, that is, when forages are low in protein, offering supplemental protein will enhance both forage intake and fermentation, therefore improving the protein and energy status of the cow (Mathis, 2003) . When ene rgy is supplemented, and it is balanced with other nutrients, the performance of forage fed cattle, and cattle fed lower quality forage usually show an improvement. However, an energy supplement will probably decrease forage intake and utilization, especia lly when supplements contain an elevated concentration of nonstructural carbohydrates, due to a decrease of the rumen pH and growth inhibition of the fibrolytic bacteria (Kunkle et al., 2000) Frequency of S upplementation According to Drewnoski et al. ( 2014 ) , when there is a reduction in the frequency of supplementation, there is no change in the amount of supplement fed each week, however , there is an increase in the amount fed during each supplementation meal. Restriction to hay access or less frequent sup plementation may be an important management strategy to potentially reduce feed intake while maintaining adequate levels of nutrient supply, without altering performance (Klein et al., 2014) . Reducing
18 frequency of supplementation should help to decrease th e costs of production associated with labor, fuel, equipment, and management (Loy et al., 2007; Cooke et al., 2008; Moriel et al., 2012a; Klein et al., 2014) . However, a reduction in the frequency of supplementation could affect nutrient intake and the con centration of different blood metabolites (Drewnoski et al., 2014) , and therefore some performance parame ters of cattle may be impacted. An experiment performed by Cooke et al. ( 2008) demonstrated that beef heifers grazing a low quality pasture, and receiv ing fibrous energy byproduct supplement daily, had greater ADG, and attained puberty and became pregnant earlier, when compared with heifers supplemented three times per week. Variations in daily intake of protein and energy in less frequently supplemented heifers caused great variations in daily BUN concentration, and decreased IGF I concentrations, reflecting a poorer performance. Conversely, Moriel et al. ( 2012a) observed that heifers receiving a low starch energy supple ment three times per week had sim ilar ADG when compared with cohorts supplemented daily, regardless of the quality of the pasture. Nevertheless, heif ers supplemented daily had better reproductive performance (faster achievement to puberty and pregnancy), and less variation in DMI and bloo d metabolite concentrations (NEFA, glucose, IGF I, and BUN). S upplementing different sources of energy in the form of digestible fiber three times per week to crossbred heifers, resulted in decreased ADG and DMI compared with daily supplementation , withou t affecting feed efficiency, measured as G:F ratio (Loy et al. , 2008) . Reducing the frequency of supplementation of energy products with an elevat ed content of starch impairs rumen function, and decreases forage intake and
19 digestibility (Kunkle et al., 200 0) . Success of less frequent supplementation of energy sources may at least depend in part on the availability of N to favor the synthesis of microbial crude protein (Drewnoski et al., 2014) . W hen studies comparing different frequencies of protein suppleme ntation show no difference in the response of grazing cattle, the ability of the ruminants to recycle N in order to meet the microbial needs may be the cause of this lack of effect (Loy et al. , 2008) . The excess of N in the rumen needs to be metabolize d into urea by the liver, in a process that requires energy . Once converted into urea, N may be recycled and used to supply N to the rumen (Reynolds, 1992) . When providing distillers grains (DG) daily or three times per week to gestating beef cows consumin g grass hay, or alternating the provision of DG and grass hay, Klein et al. ( 2014) reported that total DMI, and therefore total caloric intake (Mcal NE m /d), was lower in this last treatment. However, there was no difference in total BW gain, G:F ratio, bod y condition score, and carcass traits such as intramuscular fat, rib fat, rump fat, and LM area. Even though daily supp lementation produced more steady concentrations of BUN and NEFA, there was no negative effect of reducing frequency of supplementation on cow performance or subsequent calf birth weights. Nutrient utilization differs among animals of different physiological status (i.e., heifers vs. mature dry cows), therefore, effects of different frequencies of supplementation may be best reflected in gro wing animals (Klein et al., 2014) . However, when providing a mixture of 47% soybean hulls, 47% corn gluten feed (CGF), 2% feed grade limestone, and 4% molasses (as fed basis) at 2% of the BW to steers every other day, Drewnoski et al. ( 2014) obtained simil ar ADG, hay DMI, and G:F ratio than when
20 steers were supplemented at 1% BW daily. S upplement s including a mixture of so ybean hulls and CGF have been reported to have great concentration of digestible fiber, low concentration of non structural carbohydrates , and elevated ruminally degradable protein content (Drewnoski and Poore, 2012) . These qualities allow to supplement CGF less frequently, without negatively affect ing digestibility , when cattle is consuming a high forage diet. Energy S upplementation of L actating B eef C ows Energy supplementation may decrease DMI, decrease the ruminal pH, and inhibit digestibility of the diet, especially in high roughage diets. Negative effects may be greater with greater inclusion rates; when concentration s of nonstructura l carbohydrates (NSC) in the diet is great, ruminal pH decreases and the growth of fibrolytic bacteria is depressed. As an alternative, using fibrous by products with low concentrations of NSC may have a lower impact on performance of supplemented cattle ( Kunkle et al., 2000) . Corn gluten feed (CGF) is mainly composed of corn bran and steep liquor, and it is available either in dry or wet forms. The energy value of CGF is almost as high as in corn (75 to 83% TDN), with the difference that most of the energy in CGF is in the form of digestible fiber (bran fraction) . Additionally, CGF also provides fat, which increases the energy density of the diet (Myer and Hersom, 2008) Supplemental fat is often included during postpartum, as an attempt to increase the ener gy density of the diet, and fulfill the energetic demands of the subsequent lactation. This practice is preferred over increasing the content of soluble carbohydrates , such as starch, that can negatively affect digestion, milk composition, and health. Addi tionally, fat supplementation enhances reproductive performance in dairy cows, by improving conception of lactating dairy cows, and stimulating the
21 development of ovarian follicles during the early postpartum period (Staples et al., 1998) . The use of rumen protected fats prevent s the breakdown of fatty acids in the rumen; therefore, little or no biohydrogenation occurs, and unsaturated fatty acids may pass intact to the abomasum, where the acidic conditions dissolve the protection ( i.e. calcium salts), and release the fatty acids to the medium (Goodman, 2005) . S everal hypothesis exist regarding the improved reproductive performance of cows supplemented with fat (Staples et al. , 1998) : 1) fat helps relieve the negative energy status, therefore promot ing an ea rlier return to postpartum estrous or cycles , and improving fertility ; 2) the synthesis of steroids (like cholesterol, a precursor of progesterone) increases fertility ; 3) a manipulation of insulin to stimulate the ovarian follicle development occurs ; and 4) fat may either stimulate or inhibit the production and release of PGF , which influences the persistence of the corpus luteum. However, w hen feeding supplements comprised of either saturated or poly unsaturated fatty acids to Holstein cows in their las t period of gestation, fat supplementation did not increase the concentration of progesterone (P4) and insulin when compared with cows receiving a non fat control supplement . Small amounts of supplement were probably not enough to facilitate increased conc entrations of these metabolites (Moriel et al., 2014). When Brahman crossbred beef cows were supplemented with a source of rumen ine rt PUFA and a control supplement (comprised of a rumen inert indigestible substance), fat supplementation increased pregnancy rates when offered from the beginning of the synchronization protocol unt il 28 d after TAI, attributable in part to the
2 2 beneficial post breeding effects of PUFA on reproductive function ( Lope s et al. , 2009) . Also, c ows undergoing embryo transfer (ET) and supplemented from the end of the synchronization period until 21 d after ET presented greater pregnancy rates, when compared to a control group with no supplementation (Lopes et al., 2009) . Lo pes et al. ( 2011) hypot hesized that strategic post breeding supple mentation with PUFA could have positive effects in cow calf operations as a nutritional tool to improve the reproductive performance of beef Bos indicus cows. Post breeding supplementation of Ca salts of PUFA, esp ecially during the estimated time of luteolysis, increased pregnancy to timed AI, by mechanisms that still need to be further investigated. The authors suggested that supplementation with Ca salts of PUFA for 21 d, beginning at AI, might be an alternative to enhance reproductive efficiency in cow calf operations. Conversely, Moriel et al. ( 2012b ) did not report any difference in conception rate to AI, pregnancy rate to AI, and final pregnancy rate when supplementing a beet pulp based supplement at 1.8 kg/d per cow (control) vs a beet pulp based supplement containing a rumen protected fat (Megalac R) at 1.4 kg/d per cow, either for 30 d after timed AI, or for 10 d before and af ter timed AI. Nutritional C onsiderations during P reconditioning and B ackgrounding After weaning, calves may enter an extensive system where they will grow in a backgrounding program using harvested or grazed forage during the winter, subsequently either entering the feedlot or continuing grazing through the summer before starting the f inishing phase (yearlings). Alternatively, some intensive systems (calf fed steers) include feeding calves with a high concentrate diet immediately after weaning, until they reached the appropriate qualities to be slaughtered (Griffin et al., 2007) .
23 One o f the main reasons for backgrounding cattle is to increase the BW or frame of lightweight cattle before starting a high energy finishing diet. This process allow s smaller calves to deposit muscle and build frame without depositing excess fat . Backgrounding allows the use of low cost feedstuffs such as byproducts, because young, lightweight calves are highly efficient at converting feed into BW gain (Crawford, 2009) . Calves should be grouped according to quality, weight, and sex before introdu cing them into a backgrounding program. Producers in cow calf operations may decide to retain calves after weaning to facilitate the transition to the feedlot phase, as part of a backgrounding system. Part of this transition consist s of adjusting calves to consume grains, byproducts or harvested forages from feed bunks. This practice increases the costs of production, but will result in animals with better health status, better adapted to enter the feedlot yard than a newly weaned calf, and with heavier wei ghts. If backgrounding is coupled with a vaccination program, the system is typically referred to as pre conditioning (Crawford, 2009) . Often, calves are introduced to dry feed during preconditioning; therefore, palatable feedstuff s should be offered to st arting calves. Young growing animals have a decreased DMI capacity, thus nutrient concentrations need to be optimal to meet calf nutrient requirements. Roughage diets and supplemental feeds may account for up to 50 to 70% of the costs associated with the p reconditioning program (Hersom et al., 2014) . According to Lardy ( 2013) , preconditioning prepares calves to enter feedlot in geographical locations different from those they were originally raised in, while adding
24 weight to light steers for a short period of time, usually lasting 30 to 45 d. Even though preconditioning takes place on lightweight calves over a short period of time, small and positive changes in body weight a nd metabolism during this phase may have a positive effect on subsequent calf perfor mance in the feedlot yard (Hersom et al., 2014) . A meta analysis was performed on different p roduction systems to raise weane d calves before finishing (Lancaster et al., 2014) . N ormal weaned calves (calf fed) adapted to a high grain diet after weaning, and fed ad libitum until slaughter were compared to other production systems, including yearling fed steers . The yearling fed systems included grazing on wheat pastures, silage or hay based growing diets, limit fed high concentrate diets, and wintering on low quality forages, followed by summer grazing. All the experiments used similar finishing diets for calf fed and yearling fed steers. Yearling steers entered and finished the feedlot period with greater BW than calf fed steers. They also had greater ADG and DMI, however G:F ratio was greater for calf fed animals, as well as rib fat thickness and kidney, pelvic, and heart fat. There was no difference in hot carcass weight, LM area, yield grade or marbling score. M anagement prior to feedlot entry have an effe ct on animal per formance and carcass quality, when feeding weaned calves either in calf fed systems or as yearlings (Griffin et al., 2007) . When h eavier, large frame calves were placed into the calf feeding system while lighter, smaller framed calves were backgro unded as yearling steers, initial BW was similar betw een treatments at feedlot entry. However, yearling steers had greater final BW and ADG, and stayed less days on feed, but were less feed efficient than calf fed steers. Steers from both treatments were similar in yield grade, marbling
25 score and choice percen tage, but yearling steers had gre ater hot carcass weight and reduced fat thickness. Metabolizable Protein S upplementation In ruminants, dietary protein may be classified in rumen degradable pro tein (RDP) and rumen undegradable protein (RUP). The degradable portion may be comprised of true protein and non protein N. True protein is degraded first to peptides, and then to amino acids by protein and amino acid fermenting bacteria in the rumen, and eventually deaminated into ammonia N or incorporated into microbial protein. Non protein N is present in DNA, RNA, ammonia, amino acids, and small peptides, with the N from peptides, AA, and ammonia being used for microbial growth (Bach et al., 2006) . Prot ein that escapes microbial fermentation and microbial protein, leave the rumen and enter the abomasum and small intestine, and is degraded to amino acids and small peptides, and then absorbed into the portal blood system (Hammond, 1997) . According to the N RC ( 2000) , metabolizable protein is defined as true protein absorbed by the intestine, which may be comprised of microbial protein and RUP. Protein requirement systems in ruminants, separate the N requirements of the ruminant host, from amino acid and N requirements of ruminal microorganisms (NRC, 2000; Dryden, 2008) . The microbia l population in the rumen provide s the ruminants their ability t o increase the quality of the feed, by turning fibrous feeds and low quality protein, even non protein N, into nutritionally valuable animal proteins (Dewhurst et al., 2000) , if energy is not limiting (Hristov et al., 2004) . If N requirements of ruminal mi croorganisms are not adequately met, fermentation of plant cell tissues will decrease, reducing DMI, volatile fatty acid production, and general supply of energy to the animal (Dryden, 2008) .
26 Bacterial crude protein (BCP) may supply most of the metabolizab le protein required by beef cattle, depending on the RUP content of the diet; therefore, the efficiency of microbial crude protein synthesis is of great economic importance (NRC, 2000) . R ole of Rumen D egradable P rotein Efficiency of RDP utilization in the rumen is a major determinant of the economic and environmental impact of production (Hristov et al., 2004) . According to the NRC ( 2000) , the requirement for rumen degradable protein, and non protein N, are equal to microbial crude protein synthesis, assum ing that the ammonia N that escapes from the rumen is equal to the amount of recycled N. Deficiency of ruminal ammonia, favors recycling of N , while excess increases N absorption, thus a balance minimizes both recycling and absorption. Hristov et al. ( 200 4) hypothesized that increasing RDP intake, when energy is not a limiting factor, and maintaining constant RUP and metabolizable protein levels, would derive in excess ammonia directed towards milk protein synthesis in dairy cows. The results, however, sho wed that even though ruminal ammonia N tended to be greater when the RDP supply was high, microbial nitrogen and efficiency of microbial crude protein synthesis were similar to those in cows fed adequate levels of RDP. The authors estimated that levels of ruminally fermentable energy were not sufficient to produce a change in microbial crude proteins synthesis. Different sources of N contribute to the RDP portion. Sannes et al. ( 2002) compared different sources of rumen degradable protein (urea and soybean meal) in the diet of multiparou s Holstein cows, supplying urea as a source of non protein N, and
27 soybean meal, as a source of RDP true protein. There was no difference s in milk yield , milk components for either sources, or microbial crude protein synthesi s. M icrobial Protein S ynthesis Efficiency of microbial crude protein synthesis is maximized when there is an adequate ratio of available energy in the form of fermentable organic matter, to protein. When there is an excess of N relative to energy in the ru men, ruminal ammonia concentration increases, and enters the portal blood through the rumen wall to be transported to the liver, and metabolized to urea (Hammond, 1997) . Increased utilization of N for bacterial synthesis and capture of dietary N into anab olic products, particularly absorbed amino acids, may be influenced by dietary manipulation, mainly by energy levels in the ration . However, there is an upper limit to the overall efficiency of the process : a maximum of 50 to 60% of dietary N, or 70 to 90% of apparent digested N will be converted into amino acids and released into the portal vein (Lapierre and Lobley, 2001) . The liver removes and detoxifies the excess ammonia, primarily by converting it into urea, which is released into the vena cava. The u rea that was not excreted t h rough urine is transferred back into the digestive tract, via saliva or blood urea, may be metabolized by microbial urease and converted to ammonia N, which can reenter the liver or be used for microbial synthesis of amino acids (Reynolds, 1992) . Ruminants should be able to survive and maintain stable rumen conditions when provided with non protein N, however, efficiency of microbial growth is enhanced by addition of amino acids and peptides to their growth medium in vitro (Argyle and Baldwin, 1989 ; Cotta and Russell, 1982 ) . Compared to ammonia, ruminal amino acids and peptides might enhance the rate and extent of BCP synthesis (NRC, 2000) .
28 The balance between N catabolism and anabolism in the rumen is of great importance; if rumen ammonia concentrations are high for long durations, this may result in a low efficiency of N utilization by the animal. Ruminants make a n inefficient use of dietary C P mainly due to the ruminal N metabolism (Broderick et al., 1991) and because microbial protein degradation is not directly coupled to microbial protein synthesis (Sannes et al., 2002) . Ruminal ammonia N is often excessive, and after be ing metabolized to u rea in the liver, if it does not return to the rumen, is lost in the urine (Sannes et al., 2002) . Fermentation rate of feed is positively associated with microbial efficiency (Van Soest, 1994) , allowing an increased growth rate of ruminal microorganisms, b ecause of a dilution in the microbial maintenance requirements. Diff erent carbohydrate types have different fermentation and passage rates, which I turn affect ruminal pH, therefore influencing microbial maintenance requirements (NRC, 2000) . The y ield of microbial mass is related to the amount of available substrate and the energy consumed for maintenance, which is associated with the maintenance requirements and the growth rate, also called dilution rate (Pirt, 1965) . The available energy is used by the m icrobial cells to meet the maintenan ce or growth requirements. M aintenance energy is dependent on the growth rate, with slower growth rates being more energetically expensive than faster growth rates, therefore, cell yields are decreased at slow growth rat es (Dewhurst et al., 2000) Fermenten as RDP S ource Fermenten is a byproduct of lysine production from the microbial fermentation of cereal grains. It is mainly made of dried condensed corn fermentation solubles, processed grain by products, and glutamic acid. Fermenten also contains a readily
29 degradable fraction of NPN in addition to amino acids and peptides bound within an organic complex and combined into a slowly rumen released formulation (Cooke et al., 2009) . When performance of lactati ng dairy cows supplemented with different fermentation byproducts and traditional supplements of non protein nitrogen (urea), and true protein (soybean meal) were compared, r esults showed that overall performance was better when a source of true protein wa s provide d in the diet (Broderick et al., 2000) . Inclusion of Fermenten decreased DMI, BW gain, and milk yield, when compared to soybean meal and urea. However, feed efficiency and N efficiency were similar to urea, but lesser than soybean meal. The use of Fermenten resulted in greater ruminal ammonia N concentration than soybean meal, however, there was no difference in purine derivative excretion, microbial crude protein synthesis, in situ DM digestibility, and acetate to propionate ratio (Broderick et al ., 2000) . A meta analysis was performed to assess the effects of Fermenten and BioChlor (a fermentation by product that decreases the dietary cation anion difference ) on digestibility, microbial protein production and efficiency, and VFA production, in co ntinuous cultures (Lean et al., 2005) . A ddition of Fermenten or Bio C hlor increased DM, OM, and CP digestibility, microbial N production and microbial synthesis efficiency. In addition , there was an interaction between Fermenten and Bio C hlor with inclusion of starch ( particularly sugar ) on CP digestibility and microbial nitrogen production. However, Fermenten decreased propionate concentration, and increased the acetate to propionate ratio, possible due to an increased efficiency of microbial growth (Lean et al., 2005) . Conversely, when comparing ruminal fermentation parameters of lactating dairy
30 cows supplemented with Fermenten in addition to high or low sugar concentrations, Fermenten did not increase digestibility of nutrients, regardless of concentration of sugar (Penner et al., 2009) . Furthermore, i nclusion of Fermenten did not show differences in BUN or glucose concentration in plasma, when compared to other treatments, even though there was a tendency to increase BUN for cows fed Fermenten with no sugar added to the diet. I n vitro experiment s to assess the ruminal degradability of Fermenten were conducted and found that concentration of ammonia N was greater in cultures where Fermenten was added, from the 0 to 2 h of incubation , compared to true protein sources (soybean meal), or non protein nitrogen (urea). After 2 h of incubation, concentrations of ammonia in urea cultures remained greater for the rest of the experiment (Cooke et al., 2009) . Additionally, effects of Fermenten were evalua ted on body and reproductive performan ce of Brahman crossbred heifers; performance of heifers receiving Fermenten was similar to that of heifers receiving urea. There was no difference in ADG, pelvic area, body volume, DMI, or blood metabolites such us BUN , glucose or IGF I concentrations (Cooke et al., 2009) .
31 CHAPTER 3 EFFECT OF FREQUENCY OF SUPPLEMENTATION WITH MEGALAC R ON NON ESTERIFIED FATTY ACIDS AND BLOOD UREA NITROGEN CONCENTRATION IN LACTATING BEEF COWS Materials and Methods The protocols in volving the handling and care of the animals used in this experiment were reviewed and approved by the University of Florida, Institutional Animal Care and Use Committee. The experiment was conducted at the University of Florida IFAS, North Florida Researc h and Education Center, Marian na, from March to April 2014, and the collection period was divided into two sampling phases. Animals Eighteen early lactating beef cows (average BW Â± SD = 499 Â± 21 kg; days in lactation Â± SD = 28 Â± 18 d) were utilized in the experiment. Cows were allocated to a pasture of ryegrass ( Lolium multiflorum L. ), stratified by calf date of birth, and assigned randomly to receive a supplement daily (S7), 5 (S5) or 3 (S3) times weekly. During Phase 1 (d 0 to d 13), cows were supplement ed with corn gluten feed (CGF) at a weekly rate of 4.5 kg as is per cow, and for Phase 2 (d 14 to d 34) Megalac R (MR ; Church & Dwight Co., Princeton, NJ) was added at a weekly rate of 1.6 kg as is per cow, following the same schedule as in the first phase. Cow was considered the experimental unit , for a total of 6 cows/treatment. Diets Hand clipped s ample s were collected at 7 d intervals from the pasture. Hand shears were used to cut the forage. Samples were immediately placed into individual paper bags and transported to the laboratory. Forage and supplements samples were
32 weighted before and after drying at 55Â°C for 48 h , groun d in a Willey mill (Arthur H. Thomas Co., Philadelphia, PA) to pass a 2 mm screen, and composited across weeks. Samples were analyzed for nutrient composition by a commercial laboratory (Dairy One Forage Laboratory, Ithaca, NY). Samples were analyzed by we t chemistry procedures for concentrations of CP, NDF, and ADF, while TDN was calculated using the equation proposed by Weiss et al. ( 1992) . Nutritional values of the pasture and the supplements are described in Table 3 1. Supplement was offered to the cow s at 0700 h daily (S7), on Mondays to Fridays (S5) or on Mondays, Wednesdays, and Fridays (S3). Sampling Blood samples were taken on the last three days of each phase, from d 11 to d 13, and d 32 to d 34, at three times per day; pre supplementation (0 h), and post supplementation (8 and 16 h). The days of collection were Friday, Saturday and Sunday and corresponded with a day when all cows received the supplement (ALL), a day when only S7 cows were supplemented (S7O 1), and a second consecutive day when onl y S7 cows were offered the supplement (S7O 2). Blood samples were used to determine concentrations of blood urea nitrogen (BUN), and non esterified fatty acids (NEFA). Blood A nalysis Blood samples were collected via jugular venipuncture into blood collect ion tubes (10 mL Vacutainer, Becton Dickinson, Franklin Lakes, NJ) with no additives. Blood samples were allowed to clot for 1 h at room temperature, and at least 24 h at 4Â°C, then centrifuged at 2,360 g for 15 min at 4Â°C. Serum was frozen at 20Â°C until analysis of BUN and NEFA.
33 Serum samples were thawed at room temperature before analysis. BUN and NEFA concentrations were determined using quantitative colorimetric kits B7551 (Pointe Scientific Inc., Canton, MI), and the acyl CoA synthetase, acyl CoA oxid ase method (NEFA HR, Wako Pure Chemical Industries, Richmond, VA), respectively. Statistical A nalysis Data were analyzed as a completely randomized design with double repeated measures , using the MIXED procedure (SAS Inst. Inc., Cary, NC). The model state ment included the fixed effects of treatment, day, time(day), and the interactions of treatment Ã— day, and treatment Ã— time(day). Data were analyzed using cow as a random variable. The best fit covariance structure s for both BUN and NEFA were unstructured and compound symmetry for the effects of day and hour(day), respectively. S ignificance was set at P P Results and Discussion There was no effect of frequency of supple mentation on serum concentrations of NEFA ( P = 0.42) and BUN ( P = 0 .74), as seen on Table 3 2. There was a treatment Ã— day interaction ( P < 0.01) for serum NEFA and a day effect for BUN ( P < 0.01). During Phase 1 , serum concentrations of NEFA were similar ( P > 0.05) across treatments on d 11 (CGF ALL) and d 12 (CGF S7O 1). However, on d 13 (CGF S7O 2 ) cows on the S3 schedule had lower serum concentration s of NEFA than S5 cows, and S7 cows had similar concentrations to S3 and S5 (Fig. 3 1). During Phase 1 , c oncentrations of serum NEFA increased ( P < 0.05) from d 11 to 13, for all treatments. Conversely, during the second phase serum NEFA concentrations remained similar w ithin treatment from d 32 to 34 for S3 and S5 cows ( P > 0.05) . However, S7 cows only incre ased serum NEFA concentrations from d 32 to 33 ( P = 0.01); d 34 had similar values to d 32 and 33 ( P >
34 0.05) (Fig. 3 1). Serum NEFA concentrations on d 32 and 33 were lower ( P < 0.05) than concentrations during the first phase, while serum NEFA concentrations on d 34 were similar ( P = 0.72) to that on d 11 (Fig. 3 1). Massive mobilization of NEFA from adipose tissue is the metabolic hallmark of the transition from pregnancy to lactation. The balance between trig lyceride synthesis and lipolysis is represented by the net release of NEFA from adipose tissue, which may be promoted by suppression of the de novo synthesis or uptake, and therefore esterification of fatty acids; by increased lipolysis; decreased intracel lular re esterification of mobilized fatty acid; or by a combination of these metabolic changes (Bell, 1995) . The diffusion of NEFA into the bloodstream provides a source of energy for tissues located throughout the body. However, excessive concentration s of NEFA may become toxic, because the bovine liver has a limited capacity to synthesize tri acyl glycerides ( TAG ) from NEFA. When this limit is reached TAG accumulate in the liver, impairing its normal function, and leading to the development of fatty live r syndrome. Fatty liver hinders gluconeogenesis, which lowers blood glucose and insulin secretion, and in t urn promotes greater fatty acid mobilization, uptake of fatty acids by the liver, and ketogenesis (Adewuyi et al., 2005) . Most of the energy in CGF comes from digestible fiber, which provides a good complement to forage based diets, due to its low starch and elevated fiber energy content. Also, CGF provides fat that will increase the energy density of the diet, and it is a go od source of rumen degradable protein (Myer and Hersom, 2008) . Moriel et al. ( 2012a ) report that when beef hei fers we re supplemented energy in the form of a non fibrous byproduct 3 or 7 d/wk, simila r plasma NEFA concentrations were detected
35 between treatme nts both on days when only heifers supplemented daily receive d the su pplement, and when both groups we re supplemen ted. However, authors also reported that plasma concentration s of NEFA of hei fers supplemented infrequently were more variable on days when al l were supplemented to days when only daily heifers receive supple ment. D aily supplementation reduce d daily variation in nutrient intake, thus maintaining similar levels of metabolites, which result ed in hastened attainment of puberty and pregnancy. Diffe rent frequencies of supplementation (a control group with no supplementation, 3 and 7 d/wk) and the elimination of forage from diets on alternate days while supplementing distillers grains ( DG ) to beef cows during mid to late gestation on a low quality fo rage based diet were tested to evaluate performance and metabolic responses (Klein et al., 2014) . The authors followed a blood collection scheme similar to the one in this experiment and reported a treatment Ã— day interaction for concentrations of serum NEFA; when all treated cows received supplement, serum NEFA concentrations were similar and lower than the control group ( 0.05). However, only cows supplemented daily had stable and lower serum concentration s of NEFA across days, while cows on other su pplementation frequencies had great er day to day variation . After two consecutive days of not receiving supplement, all groups except the daily supplemented, reached the serum concentrations of NEFA of the control group. The au thors indicated that in general, concentrations of NEFA usually increase in ruminants when caloric intake is restricted or decrease d which would explain the increased serum NEFA concentration during Phase 1 of our experiment (Klein et al., 2014) .
36 Conce ntrations of BUN were lower ( P < 0.05) when cows were supplemented CGF onl y (Fig. 3 2). Within phases, concentrations of BUN were similar ( P > 0.05) when all cows were supplemented (ALL) and on the first day when only S7 cows received supplement (S7O 1). H owever, on the second consecutive day of S7 su pplementation only (S7O 2), concentrations of BUN decreased when cows were supplemented with CGF alone, but increased when MR was added to the supplement ( P < 0.05). When N is excessive, relative to energy in the rumen, concentration s of ruminal ammonia increases, nitrogen partially enters the portal blood through the rumen wall and is directed to the liver where it is converted to urea. Urea then circulates in the blood to the kidneys and i s excreted with the urine or re directed to the rumen (Hammond, 1997) . During Phase 1 collection (week 3), the CP concentration of the forage was approximately 14% lower than that of the sample collection period in Phase 2 (week 5), while TDN was similar between the two collection phases (Fig 3 3). There was a decreased CP:TDN ratio in the forage grazed during Phase 1 , which was reflected in decreased BUN concentration (17.42 Â± 0.50 mg/dL) when compared with Phase 2 (20.76 Â± 1.25 mg/ dL ), when the ratio in creased due to increased CP concentration , but similar TDN content in the ryegrass pasture. However, both BUN concentrations were greater than the normal level of 15 mg/dL cited for lactating dairy cows (Hammond, 1997) . When heifers we re offered an energy supplement based on fibrous by prod ucts daily or 3 d/wk, plasma concentrations of BUN were different within days (Cooke et al., 2008) . M ostly, when all heifers receive d supplement, the ones in the daily supplementation schedule had increased concentration s of BUN , but on days when only
37 daily heifers were supplemented, hei fers on the 3 d/wk schedule had increased concentration s of BUN . When applying the same supplementation scheme to mature anima ls, cows receiving daily supplement had greater mean concentrat ions of BUN compared with infrequ ently supplemented cows, and concentrations of BUN increased in a similar pattern for both treatments after supplement consumption. Greater BUN concentrations when no sup plement was offered might be due to N recycling; when there is a deficiency of dietary protein, and not enough N is available, urea is returned to the rumen increasing the ruminal ammonia concentrations; and therefore promoting microbial synthesis and increasing the flow of amino acids to the host animal (Ha mmond, 1997; Lapierre and Lobley, 2001) . When forage fed beef cows in mid to late gestation were supplemented dried di different supplementation schemes (a control group with no supplementation, 3 and 7 d/wk supplementat ion) and eliminating forage from diets on alternate days while supplementing distillers grains ( DG ), resulted in a treatment Ã— time interaction for BUN (Klein et al., 2014) . When all cows were supplemented, concentrations of BUN were greater than that of control. Cows in an alternate feeding scheme and 3 d/wk frequency scheme experienced a great BUN peak, when only cows in the daily scheme were supplemented, but returned to lower levels on the second consecutive days when only cows in the daily supplementa tion sc heme received DG. T he authors indicated that this might be due to excess N originating from DG consumed the previous day. In addition , even though they did not find differences in total BW gain among treatments, infrequent supplementation alt er ed
38 nutrient supply and metabolism more than when cows receive d a supplement in a daily manner (Klein et al., 2014 ) . W hen supplementation frequency was reduced, the patte rn and rate on nutrient intake wa s modified, therefore altering metabolites and hormo ne blood concentrations. Success of less frequent supplementation of energy based supplements may at least partially depend on availability of ruminal N for efficient microbial growth and digestion. (Drewnoski et al., 2014) . Conclusions Decreasing the fre quency of supplementation of energy supplements from daily to 3 or 5 d/wk does not affect serum NEFA nor BUN concentration of lactating Angus crossbreed cows. Decreasing the frequency of supplementation without negatively affecting metabolism and performan ce of lactating cows would decrease t he costs associated with supplementation .
39 Table 3 1 . Analyzed composition (DM basis) of pasture (Ryegrass [RG]) and supplements (corn gluten feed [CGF] and Megalac R [M R]). Component, % RG CGF M R DM 89.96 89.40 97.0 0 CP 29.18 19.70 1.00 a NDF 2 38.88 34.50 0.20 ADF 28.80 10.20 0.10 TDN 67.80 74.00 192.00 Calcium 0.05 9.09 Phosphorus 1.05 0.01 Magnesium 0.38 0.07 Potassium 1.58 0.04 Sodium 0.22 0.12 1 Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY). 2 Amylase used in the NDF analysis process .
40 Table 3 2 . S erum concentrations of NEFA and BUN of multiparous lactating beef cows offered energy supplements 3 (S3), 5 (S5) or 7 (S7) d/wk . 1 S3: Supplement offered 3 d/wk; S5: Supplement offered 5 d/wk; S7: Supplement offered 7 d/wk . 2 SE of treatment means, n = 6 cows /treatment. 3 P value: o bserved significance levels for treatment (TRT) , day (DAY), and hour ( TIME ) effects of frequency of supplementation , and fo r their interaction (TRT x TIME and TRT Ã— TIME(DAY)) . Item S3 S5 S7 SEM 2 TRT 3 DAY 3 TIME(DAY) 3 TRT Ã— DAY 3 TRT Ã— TIME(DAY) 3 NEFA, mEq/L 0.45 0.54 0.53 0.048 0.42 < 0.001 0.006 < 0.001 0.86 BUN, mg/dL 19.30 19.28 18.42 0.907 0.74 < 0.001 < 0.001 0.16 0.39
41 Figure 3 1. Serum concentrations of NEFA of multiparous lactating beef cows supplemented 7 (S7), 5 (S5) or 3 d/wk (S3) during first phase (A, SEM Â± 0.079) and second phase (B, SEM Â± 0.054) of the experiment. 0 0.2 0.4 0.6 0.8 1 1.2 11 12 13 NEFA, mEq/L Day, first phase S3 S5 S7 0 0.1 0.2 0.3 0.4 0.5 0.6 32 33 34 NEFA, mEq/L Day, second phase S3 S5 S7 A B
42 Figure 3 2 . Concentrations of BUN of multiparous lactating beef cows supplemen ted 7 (S7), 5 (S5) or 3 d/wk (S3) on different sampling days. Average of 3 sampling times/d: 0, 8, and 16 h postfeeding. Black bars correspond to corn gluten feed (CGF) feeding. Bars with downward diagonal lines correspond to CGF + Megalac R feeding. Bars without common letters differ ( P < 0.05). b b a c c d 0 5 10 15 20 25 30 11 12 13 32 33 34 BUN, mg/dL Day
43 Figure 3 3 . Crude protein and TDN content in the DM of samples of ryegrass from paddocks used in the experiment across weeks . 65 66 67 68 69 70 22 24 26 28 30 32 34 Wk 1 Wk 2 Wk 3 Wk 4 Wk 5 TDN, %DM CP, %DM CP TDN
44 CHAPTER 4 EFFECT OF DIFFERENT INCLUSION RATES OF FERMENTEN ON PERFORMANCE, CARCASS TRAITS, AND TOTAL TRACT DIGESTIBILITY OF GROWING ANGUS CROSSBRED STEERS Materials and Methods All research procedures were reviewed and approved by the University of Florida, Institutional Animal Care and Use Committee. The experiment was conducted at the University of Florida IFAS, North Florida Research and Education Center, Marianna, from June to October 2015, and was divided into two phases. Animals Eighty one recently weaned Angus crossbred steers (initial BW Â± SD = 189 Â± 22 kg) were utilized in this experiment. For the first phase (d 0 to 56), steers were used in a generalized randomized block design, using initial BW as blocking factor ( L ; light, M ; medium, and H ; heavy steers), and allocated to 9 pens (9 steers/pen) on d 14. Pens were assigned randomly to receive one of three treatments: 0, 2, and 4% inclusion rate of Fermenten (FER ; Church & Dwight Co., Inc., Princeton, NJ) in the diet DM of a ba ckgrounding diet comprised of peanut hulls, corn gluten feed, soybean hulls and soybean meal. For the second phase (d 57 to 112), steers were relocated to a paddock with free access to a basal diet common to all animals, without FER, to assess potential r esidual effects of FER feeding. Diets Sampl es of the diets were taken week ly , dried, ground to pass a 2 mm screen, and composited by treatment. Diet and FER samples were analyzed for nutritional
45 composition by a commercial laboratory (Dairy One Forage Labo ratory, Ithaca, NY). Samples were analyzed by wet chemistry procedures for concentrations of CP, NDF, and ADF, while TDN was estimated using the equation proposed by Weiss et al. (1992). Diets of the first phase were formulated to contain equal amounts of DIP (degradable intake protein) and energy (6.5% DIP, 70.6% TDN, DM basis), using book referenced DIP concentrations of each ingredient. Steers had ad libitum access to diets and water. Composition and nutritional profile of the diets are described in Tabl e 4 1. Sampling and L aboratory A nalysis Initial BW was an average of the unshrunk BW on d 1 and 0. Interim BW were taken at a 14 d intervals in the morning, and ult rasound measures were taken at 28 d interval s on the right flank 12 th rib to determine LM ar ea (LMA), and rib fat thickness (RF). Change in BW, and ultrasound traits were calculated by subtracting the final measurement on d 56 from the measurements taken on d 0, for the first phase, and the final measurement on d 112 from the measurements on d 56 , for the second phase. Final BW was the average of weights taken on d 55 and 56, and d 111 and 112, for the first and second phase s respectively. During the first phase, individual intake data was recorded using a GrowSafe feed intake monitoring system. B lood samples were obtained in the morning, once every 14 d to determine concentrations of glucose, blood urea nitrogen ( BUN ), and non esterified fatty acids ( NEFA ). Blood samples were collected via jugular venipuncture into commercial blood collection tube s (10 mL Vacutainer, Becton Dickinson, Franklin Lakes, NJ) with no additives. Blood samples were allowed to clot for 1 h at room temperature, and at least 24 h at 4Â°C, then centrifuged at 2,360 g for 15 min at 4Â°C. Serum was frozen at 20Â°C
46 until analysis of glucose, BUN, and NEFA. Glucose and BUN concentrations were determined using quantitative colorimetric kits G7521 and B7551, respectively (Pointe Scientific Inc., Canton, MI). Serum NEFA was determined using the acyl CoA synthetase, acyl CoA oxidase me thod (NEFA HR, Wako Pure Chemical Industries, Richmond, VA). Apparent total tract digestibility of DM, OM, CP, NDF, and ADF was measured in a subsample of 27 steers (9 steers/treatment) using indigestible NDF ( iNDF ) as a marker. Feed and fecal grab sample s were collected twice per day, at 0800 and 1700 for a 4 d period, from d 33 to 36, and from d 34 to 37, respectively. After collection, fecal samples were frozen at 20Â°C. At the end of the experiment, feed and fecal samples were thawed, dried at 55Â°C fo r 48 h in a forced air oven, ground in a Willey mill (Arthur H. Thomas Co., Philadelphia, PA) to pass a 2 mm screen, and pooled on an equal weight basis, per steer for determination of nutrient and marker concentration. For determination of feed and fecal DM and OM, approximately 0.5 g of each sample was weighed in duplicate, dried in a forced air oven at 100Â°C for 24 h and ashed at 550Â°C for 6 h. For determination of the fibrous component, samples were weighed in duplicate inside of F57 bags (Ankom Techno logy Corp., Macedon, NY) and analyzed for NDF, using heat amylase and sodium sulfite, and subsequently for ADF as described by Van Soest et al. (1991) in an Ankom 200 Fiber Analyzer (Ankom Technology Corp., Macedon, NY). Concentrations of crude pr otein in the samples were determined by rapid combustion using a macro elemental N analyzer (Vario Max CN, Elementar Americas Inc., Mt. Laurel, NJ) according to the official method 992.15 (AOAC, 1995).
47 For the determination of iNDF, samples of 0.5 g were weighed in duplicate inside of F57 bags (Ankom Technology Corp., Macedon, NY), incubated in the rumen of one cannulated steer for 12 d, then rinsed, and incubated in an Ankom 200 Fiber Analyzer (Ankom Technology Corp., Macedon, NY), following the same protocol of NDF analysis, rinsed again, and finally dried to a constant weight at 60Â°C, as described by AhvenjÃ¤rvi et al. (2000). Apparent total tract digestibility of DM, OM, NDF, ADF, and CP were calcu lated using the following formula: Statistical A nalysis Data were analyzed as a generalized randomized block design, using the MIXED Procedure of SAS (SAS Institute Inc., Cary, NC). Steer was considered the experimental unit, and the model included the random effect of steer , and fixed effect of treatment, block and pen. The best covariance structure was selected for each variable. A repeated measures ANOVA was applied for blood metabolites. For all analysis, significance was set at P 0.05, and tendencies were determined if P > 0.05 and 0.10. Results and Discussion Performance and C arcass T raits As seen on Table 4 2 , steers started the experiment with similar weight among treatments ( P = 0.90). However, by the end of the first phase, 2% FER steers were heavier than 4% FER steers ( P = 0.01), while CTL had similar BW to the other two treatments ( P > 0.05). By the end of the experiment, and likely as a result of
48 compensatory growth , there was no difference among treatments, even though CTL steers had a tendency to be heavier than 4% FER steers ( P = 0.07). Compensatory growth is based on endocrine and metabolic process that accelerate growth after a period of constrained development, usually due to feed restriction (Hornick et al., 2000) . During the f irst phase of the experiment, steer s in the 4% FER treatment had lower ADG than CTL and 2% FER steers ( P < 0.01). Conversely, throughout the second phase of th e experiment, steers ha d similar ADG across treatments ( P > 0.05). Nevertheless, taking both meas urements and finding a general ADG for the whole exper imental period, 4% FER steers had a lower ADG than CTL ( P = 0.02), while 2% FER had similar values to the other two treatments ( P > 0.05). B eef heifers that were fee d restricted for 92 d had greater ADG than heifers fed ad libitum since the beginning of the experiment, when they entered a normal re alimentation period ( Yambayamba et al. , 1996) . In addition, feed restrict ed heifers BW caught up and data showed that their a verage feed consumption was great er during the re alimentation period, even though their overall consumption was lower. T he performance responses mentioned above are possibly explained by DMI differences across treatments . I ntake was individually measured during the first phase o f the exp eriment, and data indicated that CTL steers had greater DMI than 4% FER steers ( P = 0.04), whereas 2% FER steers had similar values to both CTL ( P = 0.99) and 4% FER ( P = 0.05), even though there was a tendency for a difference between 2% FER and 4% FER tr eatments. When considering DMI as percentage of BW, steers in all groups had similar values ( P > 0.05), even though CTL steers tended to have greater intake when compared to 4% FER group ( P = 0.08). Feed efficiency, measured as G:F
49 ratio, was similar between CTL and 2% FER steers ( P = 0.87), and also greater for these groups when compared to 4% FER steers ( P < 0.05). When different sources of rumen available N were supplemented to lactating dairy cows, supplementing Fermenten resulted in a 3.4 and 8.7% lower DMI, relative to urea and soybean meal (SBM) supplementation, respectively, likely due to the low dietary cation anion difference (DCAD) of Fermenten ( Broderick et al. , 2000) . C ows supplemented with Fermenten ex perienced significant BW losses and re duced milk yield compared to urea and SBM, even though feed efficiency was similar for all treatments. Penner et al. ( 2009) fed Fermenten in low and high sugar diets to Holstein cows and reported no difference in DMI, nor found effect s of Fermenten on BW change, even though high sugar diets resulted in greater BW gains. In addition, a sugar Ã— Fermenten interaction for body condition score (BCS), where cows receiving Fermenten in the high sugar diet gained more BCS than cows in the low sugar diet. B eef heif ers grazing bahiagrass pasture and supplemented with an isonitrogenous, iso RDP grain based supplement containing either Fermenten or urea, had no difference in ADG (Cooke et al., 2009) , even though performance of cattle is expected to increase when RDP wa s added to a low forage diet (Kunkle et al., 2000) . Differences in BW and ADG were not reflected in carcass traits (Table 4 2); values for LM area gain and fat thickness gain, measured at the 12 th rib, were similar for all the treatments during both phase s of the experiment ( P > 0.05). Reduced feed intake in 4% FER steers, relative to CTL, may have decreased their basal metabolic rate, by reducing the volume and metabolic activity of the viscera; under normal development conditions, muscle shows the greate st growth rate, however, when growth is
50 constrained, a coordinated decrease of tissue turnover occurs, and weight losses are first c aused by viscera and fat tissue mobilization (Hornick et al., 2000) . As tissues are affected by growth restriction relative to their metabolic activity, the most active tissues will have the greatest weight losses (Drouillard et al., 1991) , and likely the differences in BW obtained in the experiment were due to alterations in the weight of visceral tissues, and not muscle. Yambayamba et al. ( 1996) reported no differences in side muscle weight (obtained from the left side, between the 12 th and 13 th rib, and separated into muscle, bone and fat) relative to side weight between beef heifers feed restricted for 92 days and beef heifers fed ad libitum. Howev er, restricted heifers had greater bone proportion, and less side fat than heifers fed ad libitum. When restricted heifers were also fe d ad libi tum, both groups had similar carcass characteristics. Nevertheless, proportion of liver and spleen relative to empty BW were lower for restricted heifers throughout the experiment (Yambayamba et al. , 1996 ) . Blood M etabolic P arameters There was no effect of treatment on concentrations of BUN ( P = 0.45), NEFA ( P = 0.80), and glucose ( P = 0.12) in blood . However, there was a TRT Ã— DAY interaction ( P < 0.0001) for all the measured blood parameters (Table 4 3). During the first phase of the experiment, only d 28 presented different concentrations of BUN ( P < 0.05) across treatments (Fig. 4 1); 4% FER steers had greater concentration s of BUN than 2% FER and CTL groups ( P = 0.02, P < 0.0, respectively). Conversely, during the second phase, there was more variabilit y in BUN concentrations across treatments and da ys; CTL group generally ha d greater BUN
51 concentrations than 2% FER (d 70, 84, 98; P < 0.01) and 4% FER steers (d 70, 98, 112; P < 0.05). Serum concentrations of NEFA (Fig. 4 2) were generally similar across t reatments for most part of the experiment. However, during the first phase, differences were detecte d on d 0, when CTL steers had lower serum concentrations of NEFA than 2% FER and 4% FER steers ( P < 0.01), and on d 56, when 4% FER steers had greater conce ntrations than CTL and 2% FER steers ( P < 0.01). During the second phase, serum NEFA concentrations tend ed to be greater than the first phase , and on d 70, CTL steers had greater serum concentrations of NEFA than 4% FER steers ( P < 0.01). Serum concentrations of glucose were variable through the experiment (Fig. 4 3). On the first sampling day (d 0), CT L group had greater serum concentrations of glucose than 4% FER and 2% FER steers ( P = 0.04, P = 0.03, respectively). Steers in the 4% FER treatm ent had the g reatest and lowest serum concentration s of glucose ; on d 28, 4% FER steers had greater serum concentration s of glucose than CTL and 2% FER groups ( P < 0.01). Conversely, on d 42, 4% FER steers presented lower serum concentration s of glucose th an the two other groups ( P < 0.01). Other differences were also observed on d 56, when 2% FER steers had greater serum concentration s of glucose than CTL and 4% FER groups ( P < 0.01), and on d 70 and 84, when CTL was greater than 4% FER, and 2% FER, respec tively ( P < 0.05). R eference mean concentrations and standard deviations of serum urea nitrogen, and glucose for Shorthorn calves (mean BW ranging from 166.4 to 290.2 kg) are 12.07 Â± 2.32 mg/dL, and 82.15 Â± 7.38 mg/dL , respectively (Doornenbal et al., 1988 ) .
52 Sources of RDP contribute to microbial protein synthesis by provision of amino acids, peptides, or NH 3 N; when N concentration is greater relative to energy in the rumen, excess ruminal ammonia enters the portal blood, is trans ported to the liver, and i s converted to urea (Hammond, 1997) . Cooke et al. ( 2009) , when supplementing heifers grazing a bahiagrass pasture with a grain based supplement containing different sources of RDP (urea or Fermenten), found greater BUN (average BUN Â± SEM; 11.9 mg/dL Â± 0.39 ) and glucose (average glucose Â± SEM; 80.15 mg/dL Â± 11.7) concentrations than those in this exp eriment, but no difference s between treatments were observed. Concentrations of blood metabolites are correlated to body growth and development. T he similar conc entrations of BUN and glucose in this study were reflected in similar body growth and development of heifers, indicating that Fermenten failed to increase the availability of nutrients to the animal (Cooke et al., 2009) . Other studies indicated n o difference s in plasma concentrations of glucose (65.48 mg/dL), urea N (13.13 mg/dL), and beta hydroxybutyric acid (B HBA, 8.25 mg/dL) of Holstein cows fed low and high sugar diets and supplemented with Fermenten (Penner et al., 2009) . Apparent T otal T ract D igestibility Steers in the 2% FER treatment tended to have a greater intake of DM ( P = 0.09), OM ( P = 0 .09), and CP ( P = 0.08) than the 4% FER steers (Tab le 4 4), while CTL steers had similar values to both 2% FER and 4% FER treatments ( P > 0.05). Intake of NDF and ADF was lower ( P = 0.02, P = 0.01) for 4% FER steers than for the CTL treatm ent, while 2% FER steers had similar values for both groups ( P > 0.05). Because of greater content of CGF, NDF was greater in CTL diet than in the 4% FER treatment.
53 App arent total tract digestibility of DM, OM, NDF, and ADF was greater for 4% FER steers, followed by 2% FER and CTL treatments ( P < 0.01), respectively (Table 4 4). Steers fed 2% FER and 4% FER had similar apparent total tract digestibility of CP valu es betw een them, and both had greater CP digestibility than CTL ( P < 0.01). Because of a tendency to decreased DM and OM intake in 4% FER steers, nutrient digestibility might have been greater due to a longer permanence of the feed in the rumen. A ugmented dietary intake may result in increased ruminal escape of feed nutrients, because of a decreased ruminal digestion of diet components, and increased ruminal fermentation and escape of microbial nitrogen (Robinson et al., 1985) . In addition, apparent digestibility of OM, N, NDF, and ADF decrease d when OM intake levels of dairy cows increase d, which have been due to a greater rate and extent of degradation of feed components soluble in neut ral detergent, when the time spent in the rumen decreased due to increased int ake levels. N o differences in apparent total tract digestibility of the DM, OM, CP, starch, and NDF were obtained for cows on a low and high sugar diet supplemented with Fermenten (Penner et al., 2009) . Conversely, a meta analysis from a data set of continuous culture fermenter trials on studies using Fermenten or BioChlor (a similar product, with differ ent mineral concentration), indicated that Fermenten or BioChlor increased CP digestibility by 11% ( Lean et al., 2 005) . In addition, an interaction with sugar or starch content of the diet was reported , both of which increased C P digestibility when Fermenten wa s included ; furthermore, Fermenten increased DM, and OM digestibility by 3.6 and 7.9%, respectively, compared to control treatment ( Lean et al., 2005) .
54 F iber break down increase when pre formed amino acids are provided in the diet is an effect from the stimulation of the non cellulolytic microbes associated with the fiber, instead of a direct stimulation of the c ellulolytic organisms ( Newbold, 1999) ; non cellulolytic organisms are main contributors to the removal of fiber degradation products, therefore preventing feedbac k inhibition of cellulose lysis (Cheng and McAllister, 1997) . T he Cornell Net Carbohydrate Prot ein System (CNCPS) states t hat non str uctural carbohydrate fermenting bacteria use either ammonia or peptides and amino acids as N source, while structural carbohydrate fermenting bacteria is only able to use and assimilate ammonia, as a source of nitrogen (Russell et al., 1992) . Less NDF content and greater provision of N, in the form of peptides and amino acids, provided in the 4% FER diet could have added a more available source of N and energy, contributing to an increased feed degradation. Conclusions Including Fermenten in a backgrounding diet at 2% of the diet DM did not improve the performance of growing steers. Moreover, including Fermenten at 4% of the diet DM resulted in decreased DMI and poorer performance. Carcass traits were not affected by inc lusion rate of Fermenten. Compensatory growth was exhibited by steers receiving 4% Fermenten, after switching fed to a common basal diet without Fermenten added . Blood metabolite parameters were similar across treatments, even though responses varied among days, and between phases of the experiment. Inclusion of Fermenten resulted in increased apparent total tract digestibility of DM, OM, NDF, ADF, and CP, mainly at the greatest inclusion rate.
55 Table 4 1 . Ingredients and nutrition al composition (DM basis) of experimental diets (Experiment 1) . CTL 1 2% FER 2 4% FER 3 Ingredients, % DM Fiber pellets 4 41.0 41.0 28.0 Soybean hulls pellets 35.0 36.0 38.0 Corn gluten feed pellets 17.0 15.0 12.0 Soybean meal 2.0 1.0 Liquid supplement 5 5.0 5.0 5.0 Fermenten 6 2.0 4.0 Nutritional composition 7 , % DM DM, % 91.6 91.5 91.6 CP 13.7 15.7 16.0 RDP 8 6.8 6.5 6.2 A ndf 54.7 51.4 51.5 ADF 43.0 37.9 39.2 TDN 70 .0 71 .0 71 .0 Calcium 1.03 1.39 1.33 Phosphorus 0.30 0.34 0.31 Magnesium 0.24 0.27 0.26 Potassium 1.32 1.52 1.45 Sodium 0.28 0.45 0.46 1 CTL: no inclusion of Fermenten in the diet 2 2% FER: 2% inclusion of Fermenten in the diet DM 3 4% FER: 4% inclusion of Fermenten in the diet DM 4 Commercial pellets made of cotton and peanut byproducts (AFG Feed LLC, Donalsonville, GA). 5 Molasses based liquid supplement custom formulated to provide vitamins (A, D, E) and minerals and to supply 35 mg of monensin/kg of diet DM (Westway Feed Products Inc., New Orleans, LA). 6 Church & Dwight, Princeton, NJ . 7 Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY). 8 Calculated using book values for rumen degradable protein (RDP).
56 Table 4 2. Performance and carcass traits of Angus crossbred steers fed backgrounding diets containing Fermenten at 0 (C TL), 2 (2% FER) or 4% (4% FER) of dietary DM . Item CTL 2% FER 4% FER SEM 1 P value N 27 27 27 BW, kg d 0 188 189 188 2.7 0.90 d 56 238 ab 242 a 226 b 3.9 0.01 d 112 290 x 288 xy 274 y 5.2 0.05 ADG, kg d 0 56 0.88 a 0.90 a 0.68 b 0.05 0.002 d 56 112 0.94 0.83 0.85 0.04 0.18 d 0 112 0.91 a 0.87 ab 0.77 b 0.04 0.02 DMI d 0 56 6.33 a 6.30 ab 5.62 b 0.20 0.02 % BW 2.96 2.92 2.73 0.08 0.07 G:F 0.14 a 0.14 a 0.12 b 0.005 0.007 LM area gain, cm 2 d 0 56 0.05 0.02 0.01 0.015 0.12 d 56 112 0.09 0.11 0.09 0.017 0.49 12th rib fat thickness, cm d 0 56 0.15 0.14 0.10 0.020 0.17 d 56 112 0.21 0.22 0.19 0.053 0.80 a,b and x,y Means with uncommon superscripts differ at P < 0.05 or P < 0.10, respectively 1 SE of treatment means, n = 27 steers/treatment
57 Table 4 3. Serum concentrations of BUN, NEFA and glucose of Angus crossbred steers fed backgrounding diets containing Fermenten at 0 (CTL), 2 (2% FER), or 4% (4% FER) of dietary DM . 1 SE of treatment means, n = 27 steers/treatment. 2 P value: o bserved significance levels for the effects of treatment (TRT), DAY , and their interaction (TRT x DAY). Item CTL 2% FER 4% FER SEM 1 TRT 2 DAY 2 TRT Ã— DAY 2 BUN, mg/dL 10.91 10.58 10.76 0.19 0.45 <0.0 01 <0.0 01 NEFA, mEq/L 0.28 0.29 0.29 0.01 0.80 <0.0 01 <0.0 01 Glucose, mg/dL 58.15 56.76 55.29 0.98 0.12 <0. 001 <0.0 01
58 Figure 4 1. Daily mean values of concentrations of BUN of steers receiving a diet with 0 (CTL), 2 (2% FER), or 4% (4% FER) inclusion rate of Fermenten in a backgrounding diet; SEM at each day averaged 0.37 mg/dL . 8 9 10 11 12 13 14 15 0 14 28 42 56 70 84 98 112 BUN, mg/dL Day CTL 2% FER 4% FER
59 Figure 4 2. Daily mean values of serum concentrations of NEFA of steers receiving a diet with 0 (CTL), 2 (2% FER), or 4% (4% FER) inclusion rate of Fermenten in a backgrounding diet; SEM at each day averaged 0.02 mEq/L. 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0 14 28 42 56 70 84 98 112 NEFA, mEq/L Day CTL 2% FER 4% FER
60 Figure 4 3. Daily mean values of serum concentrations of glucose of steers receiving a diet with 0 (CTL), 2 (2% FER), or 4% (4% FER) inclusion rate of Fermenten in a backgrounding diet; SEM at each day averaged 1.82 mg/dL. 35 40 45 50 55 60 65 70 0 14 28 42 56 70 84 98 112 Glucose, mg/dL Day CTL 2% FER 4% FER
61 Table 4 4. Intake and apparent total tract digestibility of Angus crossbred steers fed backgrounding diets containing Fermenten at 0 (CTL), 2 (2% FER) or 4 (4% FER) of dietary DM. Item CTL 2% FER 4% FER SEM 1 P value N 9 9 9 Intake, kg/d DM 6.56 xy 6.75 y 5.51 x 0.392 0.07 OM 6.08 xy 6.13 y 5.03 x 0.361 0.07 NDF 3.87 b 3.62 ab 2.98 a 0.224 0.02 ADF 2.93 b 2.64 ab 2.18 a 0.168 0.01 CP 0.81 xy 0.96 y 0.79 x 0.051 0.06 Digestibility, % DM 44.07 a 55.52 b 61.90 c 1.349 < 0.01 OM 44.70 a 55.88 b 61.84 c 1.368 < 0.01 NDF 30.36 a 39.72 b 47.64 c 1.795 < 0.01 ADF 25.56 a 32.66 b 40.52 c 1.831 < 0.01 CP 37.82 a 58.49 b 65.19 b 2.205 < 0.01 a, b and x,y Means with uncommon superscripts differ at P < 0.05 or P < 0.10, respectively 1 SE of treatment means, n = 9 steers/treatment
62 CHAPTER 5 EFFECT OF FERMENTEN O N NITROGEN METABOLISM AND RUMEN PROFILE OF ANGUS CROSSBRED STEERS Materials and Methods An experiment was conducted at the University of Florida IFAS, North Florida Research and Education Center, Marianna, from January to February 2016. All research procedures were reviewed and approved by the University of Florida, Institutional Animal Care and Use Committee. Animals Eight Angus crossbred steers (BW Â± SD = 568 Â± 123 kg) fitted with flexible ruminal cannulas (Bar Diamond, Inc., Parma, ID) were assigned randomly to a switchback design to allow for 8 replications per treatment. The experiment c onsisted of two periods of 24 d. Treatments were selected in correspondence w ith the two diets that had significant differences in performance of steers in p revious experiments (Chapter 4); 0, and 4% inclusion rate of Fermenten ( FER ; Church & Dwight Co., Inc., Princeton, NJ) in the diet DM of a backgrounding diet comprised of peanut hulls, corn gluten feed, soybean hulls and soybean meal. Steers were housed in individual pens, and within each period, from d 0 to 13, were adapted to the diets. On d 0 of e ac h period, steers were weighed to calculate the offering of feed at a rate of 2.8% of the BW (DM basis). Diets were offered daily at 0900 h. Bunks were monitored daily to adjust feed offering. From d 14, orts were recorded daily, removed from the bunks and dried in a forced air oven until constant weight, to estimate the DM of the refusals. Water was available for ad libitum consumption.
63 Experimental D iets Diets were offered in a constant ratio of 92% of a basal TMR (common to both treatments), and 8% of a top dressed premix, containing corn gluten feed pellets ( CGF ), a molasses based vitamin and mineral supplement ( Westway Feed Products Inc.,New Orleans, LA) , and FER and soybean hulls, for the 4% FER inclusion, or soybean meal, for the 0% FER inclusion. Di ets were formulated to contain equal amounts of DIP (degradable intake protein) and energy (6.5% DIP, 70.6% TDN, DM basis), using book referenced DIP concentrations of each ingredient (Table 5 1). Ruminal P rofile and B lood U rea N itrogen Samples of ruminal fluid and blood were collected on d 18 of each period, before feeding (0 h), and every 3 h post feeding, for 24 h. Content of the rumen was collected from different sections of the ruminal cavity, and approximately 100 mL of ruminal flu id was obtained after filtration through four layers of cheese cloth. Immediately after collection, ruminal fluid was analyzed for pH using a pH meter (Corning Pinnacle M530, Corning Inc., Corning, NY), and two 10 mL aliquots per time point per steer were acidified adding 0.1 mL of 20% H 2 SO 4 solution, to stop fermentation. Before storage, one aliquot of each time point and steer, was transferred into polypropylene vials (12 mm Ã— 75 mm; Fisherbrand; Thermo Fisher Scientific Inc., Waltham, MA) in duplicates. Samples were stored frozen at 20Â°C for determination of concentrations of volatile fatty acids ( VFA ), and ammonia nitrogen ( NH 3 N ). Concentrations of VFA were determined in a water based solution using ethyl acetate extraction. For analysis, samples were thawed at room temperature, and centrifuged for 10 min at 10,000 Ã— g (Avanti J E, Beckman Coulter Inc., Palo Alto, CA). After centrifugation, the pellet was reserved for NH 3 N analysis, while the ruminal fluid
64 supernatant was mixed with a meta phosphoric a cid:crotonic acid (internal standard) solution at a 5:1 ratio, frozen overnight, thawed and centrifuged for 10 min at 10,000 Ã— g . Supernatant was transferred into glass tubes and mixed with ethyl acetate in a 2:1 ratio of ethyl acetate to supernatant. Tube s were agitated, and the ethyl acetate fraction, located on the top, was transferred to vials. Samples were analyzed by gas chromatography (Agilent 7820A GC, Agilent Technologies, Palo Alto, CA) using a flame ionization detector and a capillary column (CP WAX 58 FFAP 25 m 0.53 mm, Varian CP7767, Varian Analytical Instruments, Walnut Creek, CA). Concentrations of NH 3 N were measured after centrifugation of the remnant pellet at 10,000 Ã— g for 15 min at 4Â°C following the phenol hypochlo r ite technique describ ed by Broderick and Kang (1980) with the following modification: absorbance was read at 620 nm in flat bottom 96 well plates using a plate reader (DU 500 Beckman Coulter Inc., Fullerton, CA ). Blood samples were obtained at the time points mentioned before, to determine concentrations of blood urea nitrogen ( BUN ). Samples were collected via jugular venipuncture into commercial blood collection tubes (10 mL Vacutainer, Becton Dickinson, Franklin Lakes, NJ) with no additives. Blood samples were allowed to clot for 1 h at room temperature, and at least 24 h at 4Â°C, then centrifuged at 2,360 g for 15 min at 4Â°C. Serum was frozen at 20Â°C until analysis. BUN concentrations were determined using quantitative colorimetric kit B7551 (Pointe Scientific Inc., Canton, MI). Apparent T otal T ract D igestibility Apparent total tract digestibility of DM, OM, CP, NDF, and ADF were determined using indigestible NDF ( iNDF ) as a marker . Feed and fecal grab samples were collected
65 twice per day, at 0800 and 1700 for a 4 d period, from d 20 to 23, and from d 21 to 24, respectively. After collection , fecal samples were frozen at 20Â°C. At the end of the experiment, feed and fecal samples were thawed, dried at 55Â°C for 48 h in a forced air oven, ground in a Willey mill (Arthur H. Thom as Co., Philadelphia, PA) to pass a 2 mm screen, and pooled on an equal weight basis, by steer and period, for determination of nutrient and marker concentration. For determination of feed and fecal DM and OM, approximately 0.5 g of each sample was weighe d in duplicate, dried in a forced air oven at 100Â°C for 24 h and ashed at 550Â°C for 6 h. For determination of the fibrous component, samples were weighed in duplicate inside of F57 bags (Ankom Technology Corp., Macedon, NY) and analyzed for NDF, using heat amylase and sodium sulfite, and subsequently for ADF as described by Van Soest et al. (1991) in an Ankom 200 Fiber Analyzer (Ankom Technology Corp., Macedon, NY). Concentrations of crude protein in the samples were determined by rapid combustion using a macro elemental N analyzer (Vario Max CN, Elementar Americas Inc., Mt. Laurel, NJ) according to the official method 992.15 (AOAC, 1995). For the determination of iNDF, samples of 0.5 g were weighed in duplicate inside of F57 bags (Ankom Technology Corp., Macedon, NY), incubated in the rumen of one cannulated steer for 12 d, then rinsed, and incubated in an Ankom 200 Fiber Analyzer (Ankom Technology Corp., Macedon, NY), following the same protocol of NDF analysis, rinsed again, and finally dried to a constant weight at 60Â°C, as described by AhvenjÃ¤rvi et al. (2000).
66 Apparent total tract digestibility of DM, OM, NDF, ADF, and CP were calculated using the following formula: Statistical A nalysis Data were analyzed as a switchback design, using the MIXED Procedure of SAS (SAS Institute Inc., Cary, NC ). Steer was considered the experimental unit, and for ruminal pH, VFA concentrations, NH 3 N, and BUN the model included the random effect of steer, and fixed effect of treatment, period, order(period), time, and treatment Ã— time interaction . For apparent total tract digestibility, the model included the fixed effect of steer, and the random effect of treatment, period, and order(period) . Ruminal pH, VFA concentrations, N H 3 N, and BUN were analyzed as repeated measures using the MIXED procedure of SAS, selecting the covariance structure with the smallest Akaike Information Criterion ( AIC ) values. For all analysis, significance was set at P 0.05, and tendencies were determined if P > 0.05 and 0.10. Results and D iscussion Ruminal F ermentation P arameters and N itrogen M etabolism Steers in both treatments had similar ruminal pH ( P = 0.32; Table 5 2 ) , al though there was a significant effect of time ( P < 0.01), and a tendency ( P = 0.09) for a TRT Ã— TIME interaction (Fig. 5 1). There was no effect of treatment on total VFA concentration ( P = 0.28), or molar proportion of the main VFA ( P > 0.05). However, there was a tendency for a treatment effec t on acetate ( P = 0.06) and greater molar proportion of butyrate ( P = 0.01) in steers receiving the 4% FER diet. The ratio between acetate to propionate (A:P) did not differ with the addition of Fermenten to the diet.
67 Nitrogen metabolism parameters were s imilar between treatments; there was no effect of treatment on ruminal NH 3 N ( P = 0.97) or BUN ( P = 0.95) concentration (Table 5 2). However, there was a significant effect of time on both parameters ( P Fig. 5 2 ). F or a RDP concentration of approxi mately 6.5% of diet DM, NH 3 N expresses its peak 3 h after feeding, gradually decreases until 12 h post feeding, a nd rises again at 21 h, likely due to N recycling (Fig. 5 2). C oncentrations of BUN, show a peak 3 h after NH 3 N, at 6 h after feeding, and re mains fairly similar across time. Rumen pH is dependent on several factors, one of them being microbial cell yield, which is variable and affects the production of fermentation acids from available organic matter truly digestible in the rumen (Allen, 1997 ) . E fficiency of microbial protein synthesis may affect rumen pH, as ruminal degradable OM is directed to synthesis of microbial protein instead of being available for fermentation acid production (Penner et al., 2009) . Therefore, if microbial protein synthesis was stimulated, and resulted in greater yield of microbial cells per unit of ruminal degradable OM, an increase in ruminal pH should be expected. However, inclusion of Fermenten in the diet DM of steers, did not result in greater pH than that of steers fed a control diet. Similar results were reported by Penner et al. ( 2009) , who in cluded Fermenten in the diet DM of lactating Holstein cows fed 2 dietary sugar concentrations, and found no effect of Fermenten in rum en pH. Inclusion of Fermenten did not affect rumen pH or total VFA production of continuous culture fermenters (Lean et al., 2005) . However, propionate production was significantly lower by effect of Fermenten, which in turn resulted in greater acetate to propionate r atio. L ower production of propionate would be expected due to an improved
68 efficiency in the microbial growth of non fibrous carbohydrate fermenting bacteria, which would have been able to incorporate the peptides provided by Fermenten into bact erial protein (Lean et al., 2005) . Conversely, Penner et al. ( 2009) reported no effect of Fermenten on ruminal VFA total production or individual molar proportion, of lactating Holstein cows fed 2 diets with different sugar concentrations. When sources of rumen degradable N for lactating dairy cows were compared, supplementing Fermenten resulted in less VFA total production than that of urea supplementation, but similar to that of soybean meal (Broderick et al., 2000) . Molar proportions of the main VFA were similar among Fermenten, soybean meal, and urea supplementation (Broderick et al., 2000) . However, in our experiment a shift in fermentation occurred, and butyrate molar proportions were greater for 4% FER steers. Greater butyrate molar proportion and a t endency to lower acetate molar proportion in 4% FER steers could be explain ed by short chain fatty acid interconversions and use by rumen anaerobic bacteria, which provide intermediary metabolites that promote the completion of microbial metabolic cycles ( Hackmann and Firkins, 2015) . S ome strains of Butyrivibrio fibrisolvens are acetate stimulated and their production of butyrate is facilitated by a butyryl CoA/acetate CoA transferase, rather than by a butyrate kinase enzyme; butyryl CoA/acetate CoA transfe rase converts butyryl CoA directly to butyrate, using acetate as a coenzyme A acceptor (Diez Gonzalez et al., 1999) . As acetate is converted to acetyl coA, further phosphotrans acetylase and acetate kinase action promotes additional ATP formation, and fina lly regenerates acetate, which is available to enter a new cycle (Russell, 2002). Bacteria expressing butyryl CoA/acetate CoA transferase yield greater quantities of butyrate and
69 consume more acetate (Hackmann and Firkins, 2015). Additionally, different gr oups of Butyrivibrio have variable effect on lipid ruminal biohydrogenation, in relation to the main pathway used for butyrate synthesis ( Pa illard et al., 2007) ; groups with elevated butyrate kinase activity are able to produce sterate from linoleic acid, while those expressing butyryl CoA/acetate CoA transferase are not. A study reported that supplementation with Fermenten promoted a greater concentration of linoleic and linolenic acids in milk fat of lactating Holstein cows fed low su gar diets, which indicates a decreased lipid biohydrogenation of fatty acids in the rumen, since mammals are unable to synthesize those (Penner et al., 2009) . These results would correspond with the current study, and it could be speculated that Fermenten stimulates Butyrivibrio strains that utilize the butyryl CoA/acetate CoA transferase pathway, thus shifting ruminal fermentation, by increasing butyrate and decreasing acetate production, and promoting the escape of lipids from biohydrogenation. Butyrate i s the preferred energy source for mature ruminal papillae (Baldwin and Jesse, 1992) , most of it being converted to ketone bodies or CO 2 by the epithelial cells (Bergman, 1990) , and is related to induced rumen epithelial cell proliferation, when infused rap idly in adult sheep (Sakata and Tamate, 1978) . Even though RDP concentrations were similar in both diets, a lower NH 3 N concentration was expected for 4% FER steers, due to a supposedly greater N assimilation by the rumen microbial population when peptides and amino acids are supplemented (Blake et al., 1983) . U se of amino acids by rumen bacteria would be directed for microbial protein synthesis and fermentation, which would turn amino acids in to a source of energy and would also release ammonia (Cotta and Russell, 1982) .
70 Rumen NH 3 N concentration is also related to the solubility of the N sources and how rapidly they are hydrolyzed in the rumen; in a study performed by Hume ( 1970) the rate and extent of NH 3 N accumulation was similar among casein and gelati n, both sources of protein N and urea, which is rapidly degrade d in the rumen. An in vitro study indicated that for the first two hours of incubation, substrate s containing Fermenten had greater NH 3 N concentration than cultures containing urea and soybean meal (Cooke et al., 2009) . However, after this period, concentration of NH 3 N in urea containing cultures surpassed that of Fermenten, and remained greater until the end of the incubation period. Fermenten supplementation increases the ruminal NH 3 N concentration, compared to soybean meal, a source of true protein N (Broderick et al., 2000) . U sing NH 3 N as a sole parameter for estimate microbial protein synthesis may be inconvenient , since low concentrations could indicate a highly efficient usage ra te, as well as deficient concentrations for microbial protein synthesis (Firkins et al., 2007) . Yet, prev ious continuous culture studies refer that there is a r elation between NH 3 N concentrations and ENU (efficiency of N utilization) by rumen microbes, si nce as ENU increases, a related decrease in NH 3 N accumulation in the medium occurs (Bach et al., 2005) . However, a meta analysis performed by Lean et al. ( 2005) indicated that addition of Fermenten to the substrate of continuous culture fermenters resulte d in increased NH 3 N concentration as well as microbial protein nitrogen. A study reported that l actating cows re ceiving Fermenten tended to have greater NH 3 N concentration, effect that was not fully attribute d to the addition of Fementen, but also to a g reater CP concentration in the diet of cows receiving Fermenten (Penner et al., 2009) . Additionally, cows fed Fermenten in a low sugar concentration diet tended to have
71 greater plasma urea N (Penner et al., 2009) . Similar BUN concentrations in the current study indicate similar degradation rates of the different sources of RDP, as unused ruminal NH 3 N is converted to urea in the liver, after entering the portal blood through the rumen wall (Hammond, 1997) . The lack of a TRT x TIME interaction for both NH 3 N and BUN concentrations may be due to the similar provision s and degradation rates of RDP in both diets. Additionally, return of rumen NH 3 N concentrations to values similar to those expressed at the beginning of the collection, even after experiences depr essed concentrations, could be explain by provision of N to the rumen by mechanisms of N recycling (Lapierre and Lobley, 2001) . Apparent T otal T ract D igestibility There was an effect of treatment on DM ( P = 0.03), OM ( P = 0.02), NDF ( P < 0.01), and ADF ( P < 0.01) intake; inclusion rate of 4% of Fermenten in the diet DM resulted in decreased nutrient intake (Table 5 3). Despite these differences, digestibility values for DM ( P = 0.45), OM ( P = 0.24), NDF ( P = 0.13), and ADF ( P = 0.13) remained similar betwee n treatments, even though 4% FER steers numerically had greater digestibility values. Dry matter (DM) intake was also lower for 4% FER ( P = 0.03) steers, when tabulated as percentage of the BW (Table 5 3), even though BW gain was similar between the two gr oups. Usually, results are divergent whe n Fermenten is used in vivo than when it is included in the substrate of in vitro or batch culture studies. Lean et al. ( 2005) constru cted meta analysis models from data of 15 continuous culture fermenter trials with Fermenten or BioChlor ( Church & Dwight Co., Inc. Princeton, NJ , a similar product with greater DCAD [dietary cation anion difference] ) and reported that inclusion of Fermenten incr eased DM, OM, CP, and nonstructural carbohydrate (NSC) digestibility,
72 but n ot affecting NDF or total carbohydrate digestibility. A study indicated that DMI was similar to that of control diet when Fermenten was added to low or high sugar diets, and reported no effect of treatment or interaction between Fermenten and concentration of sugar on DM, OM, CP, starch, or NDF apparent total tract digestibility (Penner et al., 2009) . S upplements containing Fermenten significantly reduced DMI when compared to urea or soybean meal containing supplements likely due to low DCAD values of Fermenten ( 16, 285, 303 m E q/kg of DM for Fermenten, urea, and soybean meal, respectively) (Broderick et al., 2000) . Reduced feed intake when DCAD is low, mainly when it presents negat ive values, may be associated with palatability of the anionic salts so urce, or with a negative effect of metabolic acidosis on intake, which occurs when dietary anions are increased in the diet (Hu and Murphy, 2004) . Even though Broderick et al. ( 2000) reported differences in DMI, in situ DM digestibility of alfalfa hay was similar across treatments. Conclusions Inclusion of Fermenten resulted in similar rumen fermentation parameters and N metabolism than the control diet, except for a significant increase in the synthesis of butyrate and lower acetate production in steers re ceiving Fermenten. S teers consuming Fermenten had decreased DMI, may be due to a lower DCAD, however this did not result in differen ces in nutrient digestibility or BW gains.
73 Table 5 1. Ingredients and nutritional c omposition (DM basis) of experimental diets (Experiment 2) . CTL 1 4% FER 2 Ingredients , % diet DM Fiber pellets 3 4 1.0 41.0 Soybean hull s pellets 35.0 38.0 Corn gluten feed pellets 17.0 12.0 Liquid supplement 4 5.0 5.0 Soybean meal 2.0 Fermenten 5 4.0 Nutritiona l composition 6 , % DM DM 88.3 87.9 CP 14.6 14.3 RDP 7 6.8 6.2 Andf 58.3 59.1 ADF 44.6 45.4 TDN 69.0 69 .0 Calcium 0.87 0.91 Phosphorus 0.41 0.35 Magnesium 0.29 0.27 Potassium 1.62 1.56 Sodium 0.233 0.246 1 CTL: no incl usion of Fermenten in the diet 2 4 % FER: 4% inclusion of Fermenten in the diet DM. 3 Commercial pellets made of cotton and peanut byproducts (AFG Feed LLC, Donalsonville, GA). 4 Molasses based liquid supplement custom formulated to provide vitamins (A, D, E) and minerals and to supply 35 mg of monensin/kg of diet DM (Westway Feed Products Inc., New Orleans, LA). 5 Church & Dwight, Princeton, NJ . 6 Analyzed by a commercial laboratory using a wet chemistry package (Dairy One, Ithaca, NY). 7 Rumen Degradable Protein (RDP) calculated using book values.
74 Table 5 2. Ruminal pH, total VFA concentration, VFA molar proportion s , acetate to propionate ratio, NH 3 N and BUN of cannulated Angus crossbred steers fed backgrounding diets containing 0 (CTL) or 4% (4% FER) inclusion rate of Fermenten in the diet DM . Item CTL 4% FER SEM 1 TRT 2 TIME 2 TRT x TIME 2 N 8 8 Ruminal pH 6.12 5.98 0.096 0.32 < 0.01 0.09 Total VFA , m M 139.83 148.34 5.2 88 0.28 < 0.01 0.54 VFA m olar proportion, mol/100 mol Acetate 68.92 67.67 0.42 0.06 < 0.01 0.29 Propionate 18.57 19.21 0.33 0.19 < 0.01 0.22 Isobutyrate 0.75 0.65 0.106 0.51 0.19 0.29 Butyrate 8.90 9.58 0.16 0.01 < 0.01 0.54 Isovalerate + 2 MB 3 1.19 1.16 0.05 4 0.68 < 0.01 0.10 Valerate 1.26 1.32 0.04 3 0.37 < 0.01 0.87 Caproate 0.41 0.41 0.045 0.92 < 0. 01 0.60 A:P 4.31 4.10 0.52 1 0.78 0.65 0.32 NH 3 N, m M 5.68 5.65 0.797 0.97 < 0.01 0.62 BUN, mg/dL 11.69 11.75 0.629 0.95 0.01 0.29 a,b,c Within a row, means with different superscripts differ, P < 0.05. 1 SE of treatment means, n = 8 steers/treatment . 2 P value: o bserved significance levels for treatment (TRT) and time effects of inclusion rate of Fermenten in the diet DM and for their interaction (TRT x TIME). 3 MB = Methylbutyrate .
75 Figure 5 1. Ruminal pH of cannulated Angus crossbred steers receiving a diet with 0 (CTL), or 4% (4% FER) inclusion rate of Fermenten in a backgrounding diet; SEM = 0.11. 5.5 5.7 5.9 6.1 6.3 6.5 6.7 6.9 0 3 6 9 12 15 18 21 24 pH Time postfeeding, h CTL 4% FER
76 Figure 5 2. Ruminal concentrations of Ammonia N (NH 3 N) and BUN as affected by hours after feed delivery in cannulated Angus crossbred steers receiving a diet with 0 (CTL), or 4% (4% FER) inclusion rate of Fermenten in a backgrounding diet; SEM at each time point averaged 0.76 m M , and 0.70 mg/dL for NH 3 N a nd BUN, respectively. 8 9 10 11 12 13 14 0 2 4 6 8 10 12 14 0 3 6 9 12 15 18 21 24 BUN, mg/dL NH 3 N, m M Time post feeding, h NH3-N BUN
77 Table 5 3. Intake, app arent total tract digestibility and BW gain of Angus crossbred steers fed backgrounding diets containing 0 (CTL) or 4% (4% FER) inclusion rate of Fermenten in the diet DM . a,b,c Within a row, means with different superscripts differ, P < 0.05. 1 SE of treatment means, n = 8 steers/treatment. Item CTL 4% FER SEM 1 P value N 8 8 Intake, kg/d DM 15.8 14.5 1.18 0.03 OM 14.5 13.3 1.08 0.02 NDF 8.2 7.4 0.58 0.003 ADF 6.0 5.4 0.42 0.002 CP 2.4 2.8 0.24 0.058 Digestibility, % DM 61.7 64.0 2.24 0.45 OM 63.5 66.1 1.52 0.24 NDF 50.0 53.9 1.60 0.13 ADF 44.6 48.4 1.53 0.13 CP 63.4 74.0 2.64 0.012 DMI, % BW 2.7 2.5 0.06 0.03 BW gain, kg 611.1 609.1 47.97 0.58
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85 BIOGRAPHICAL SKETCH Mariana Garcia was born in Asuncion, Paraguay. In 2011 she received a degree in Agricultural Engineer with a major in a nimal s ciences from Universidad Nacional de Asuncion, Paraguay. On 2013, she was awarded a Fulbright Scholarship to pursue graduate studies in United States, and on 2014, she was accepted as student by the Department of Animal Science at University of Florida. She developed her research under the supervision of Dr. Nicolas DiLorenzo, at the North Florida Research and Education Center, in Marianna, Florida. Mariana focuses on the use of feed additives to enhance the performance of beef cattle, and decrease the costs of production associated with feed and management in cow calf operations.