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1 EXOGENOUS FIBROLYTIC ENZYME OR ANHYDROUS AMMONIA EFFECTS ON THE NUTRITIVE VALUE, INTAKE, AND DIGESTION KINETICS OF BERMUDAGRASS AND THE GROWTH OF BEEF CATTLE By JUAN JOSE ROMERO GOMEZ A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009
2 2009 Juan Jose Romero Gomez
3 To my dear parents, Luz and Marino
4 ACKNOWLEDGMENTS I would like to thank my committee super visor, Dr. Adesogan for his invaluable support and dedication during my M.S. program He has not only been my professor, but he has been a mentor and guide for my professional development. I would also lik e to thank Dr. C.R. Staples and Dr. L.E. Sollenberger for their priceless advice and guidance as members of my committee. Special thanks to Dr. W.F. Brown for his initial involvement in my committee and for givi ng me the opportunity to start my M.S. program at UF. I am indebted to Miguel Zarate, Osca r Queiroz, and Junghun Han for their involvement in my first experiment. For almost ten months dur ing and after this experiment, Miguel became my friend as well as my brother. I will always treasure his invaluable help and friendship. In addition, I would like to thank Oscar for his extraordinary help, advice, and friendship; he was like an older brother to Miguel and I. I am grateful to Junghun Hun for his constant support. I will always remember his great work ethic and willingness to help. The four of us formed a formidable team and spent good and bad times together for almost a year. I greatly appreciate the efforts of Ka thy Arriola and Dr. Max Huisden during the times of intensive sampling for my first experiment. Kathys expert advice and help influenced my development as a professional and I will always be thankful to her for the time she spent training me in the Ruminant Nutrition laboratory. Dr. Maxs experience and support while he worked as Dr. Adesogans laboratory technician were particularly useful to me during my experiments. I would like to thank Nicholas Londono, Be rt Faircloth and t he Santa Fe Beef Ranch staff for their involvement in my second experiment. Their many contributions
5 allowed the experiment to proceed in a timely manner. I am particularl y grateful to Bert for transporting animals and feeds needed for the experiments on numerous occasions. Special thanks are also due to the staff of the Sheep Physiology Unit and the Beef Teaching Unit, particularly Ken Clyatt and Jesse Savell for their assistance with logistics. I would also like to thank Jerry Wasdin for his useful advice and help during the planning and implementation st ages of the experiments. I am very grateful to Jill Bobel and Jan Ki vipelto for their invaluable help during the laboratory analyses, to Jae-Hyeong Shin fo r his help with VFA analysis, to Eduardo Alava and Miriam Garcia for their advice and assistance with various measurements, to Julio Schlaefli for helping to chop the hay, to Shirley Levi for her help with various tasks, to James Colee for his advice with the statistical analysis, and to Chaevien Clendinen for her help with some laboratory analysis. Lastly, I would to thank Melazero, Barbon, Miedoso, Bravito, Cachn and Burga for being such a nice steers and for letting us wo rk with them. I will always treasure them in my heart; they became like my dogs her e in the US. I cannot finish this acknowledgments without thanking my roomma te Jorge Camejo for being such a good friend to Miguel and me. Thanks for not complain ing about all the hay and odors that we brought to the house. I am grateful that God involved these great people in my life.
6 TABLE OF CONTENTS Page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES ............................................................................................................ 8LIST OF FIGURES .......................................................................................................... 9LIST OF ABBR EVIATIONS ........................................................................................... 11 CHA PTER 1 INTRODUC TION .................................................................................................... 152 LITERATURE REVIEW .......................................................................................... 17Warm-Season Grasses ........................................................................................... 17Definition and Importance ................................................................................. 17Characteristics of C4 Photosynt hesis ................................................................ 17Subtypes of C4 Photosyn thesis ........................................................................ 18Practical Implications of C4 Photosyn thesis ...................................................... 19Factors Affecting Digesti bility of Forages ................................................................ 20Impact of C4 Grass Anatomy on Forage Digesti bility ........................................ 20Influence of organs and t heir tissue pr oportions ........................................ 21Influence of tissue type on di gestion .......................................................... 23Impact of the Forage Cell Walls on Di gestibility ............................................... 30Chemical composition of forage ce ll walls ................................................. 30Cell wall development and its impact on diges tion ..................................... 34Cross-linking mediated by hydrox ycinnamic acids and its impact on digestion ................................................................................................. 35Methods to Improve Forage Nu tritive Value and Inta ke .......................................... 40Anhydrous Ammonia ........................................................................................ 41Exogenous Fibrolyt ic Enzymes ........................................................................ 43Types of enzymatic activi ties ..................................................................... 44Synergy betw een enzym es ........................................................................ 47Attachment of enzymes ............................................................................. 48Product inhi bition ....................................................................................... 49Cellulose degradati on in nat ure ................................................................. 49Exogenous fibrolytic enzymes in rumi nant diets ........................................ 523 EXOGENOUS FIBROLYTIC ENZYME OR ANHY DROUS AMMONIA EFFECTS ON THE NUTRITIVE VALUE, INTAKE, AND DIGESTION KINETICS OF BERMUDAGRASS AND THE GROW TH OF BEEF CATTLE ................................ 61Introduc tion ............................................................................................................. 61Materials and Methods ............................................................................................ 62
7 Forage Treat ments ........................................................................................... 62Experiment 1 .................................................................................................... 63Cattle and diets .......................................................................................... 63Sampling and analysis ............................................................................... 64In situ ruminal degradabi lity ....................................................................... 65Rumen fermentati on paramet ers ............................................................... 66Ruminal fluid volume and liquid rate of pa ssage ........................................ 67Experiment 2 .................................................................................................... 67Statistical Analysis ............................................................................................ 68Results and Discussion ........................................................................................... 70Nutritional Co mposition .................................................................................... 70Voluntary Intake ............................................................................................... 71In vivo apparent digestibi lity ............................................................................. 72In Situ Rumen Di gestion Kine tics ..................................................................... 73Rumen Fermentati on Parame ters .................................................................... 75Ruminal Fluid Volume and Li quid Rate of Passage ......................................... 76Growth Perform ance of Steers ......................................................................... 77Conclusi ons ............................................................................................................ 784 GENERAL SUMMARY AND RECOMMENDA TIONS ............................................. 90LIST OF RE FERENCES ............................................................................................... 94BIOGRAPHICAL SKETCH .......................................................................................... 111
8 LIST OF TABLES Table Page 2-1 Tissue proportions in organs of diffe rent fo rage types (From Wilson, 1993). ..... 222-2 Percentage of tissue types in cro ss-sections (CS) of warm-and cool-season grass leaf blades from 4 to 8-wk-o ld plants. (Modified from Akin, 1989). Number of sample s not stated. ........................................................................... 243-1 Chemical composition of untreat ed (CON), enzyme-treated (ENZ) and ammoniated (AMN) hay harvested at two regrowth interval s (RI) (n=6) ............. 793-2 Effect of treating bermudagrass hay har vested at two regrowth intervals with a fibrolytic enzyme or ammonia on the voluntary inta ke of steers ...................... 803-3 Effect of treating bermudagrass hay har vested at two regrowth intervals with a fibrolytic enzyme or ammonia on in vivo apparent digestibility by steers ......... 813-4 Effect of treating bermudagrass hay har vested at two regrowth intervals with a fibrolytic enzyme or amm onia on kinetics of in situ ruminal DM digestion. ...... 823-5 Effect of treating bermudagrass hay har vested at two regrowth intervals with a fibrolytic enzyme or ammonia on in situ ruminal DM digestion after different incubation periods. ............................................................................................. 833-6 Effect of treating bermudagrass hay har vested at two regrowth intervals with a fibrolytic enzyme or ammonia on rumen fermentat ion parameters .................. 843-7 Effect of treating bermudagrass hay har vested at two regrowth intervals with a fibrolytic enzyme or ammonia on rumi nal fluid volume and fluid kinetics ......... 853-8 Effect of treating bermudagrass hay har vested at two regrowth intervals with a fibrolytic enzyme or ammonia on growth performance of steers ...................... 86
9 LIST OF FIGURES Figure Page 2-1 The C3 and C4 anatomy and photosynthetic pathw ay. (Volenec and Nelson, 2007). ................................................................................................................. 182-2 Cross sections of young (a, head emergence) and mature (b, grain maturity) stems of Sorghum bicolor .. ................................................................................. 232-3 Scanning electron micr ograph of bermudagrass (I eft) and orchardgrass (right) leaf blades incubated fo r 48 h in ru men fluid.. .......................................... 242-4 Chemical structural characteristics of typical lignin precursors (coniferyl, sinapyl and p -coumaryl alcohols) and hydro xycinnamic acids (ferulate, p coumarate and sinapate) found in fo rage cell walls (H atfield, 1999). ................. 332-5 Diferulate cross-linking of arabinoxylan chains and incorporation into lignin via active me chanisms. ...................................................................................... 332-6 Schematic representat ion of a plant cell and wall development (Jung and Allen, 1995). ....................................................................................................... 352-7 Impact of diferulate formation in nonlignified walls upon wall structural polysaccharide degradation by fungal hydrolases (Grabber 1998b) ................... 372-8 Attachment of ferulates to lignin by two distinct mechanisms: Passive mechanism (LFP Complex A) and Activ e radical-coupling mechanism (LFP Complex B; Ralph, 1996). .................................................................................. 392-9 Schematic representat ion of the major enzymes involved in cellulose hydrolysis (Beauchemin et al., 2004). ................................................................. 452-10 Schematic representati on of the hemicellulose enzymes involved in the degradation of arabinoxylan (B eauchemin et al., 2004). .................................... 462-11 Synergism between Penicillium pinophilum cellobiohydrolase (I and II) and endoglucanases (EI to EV) in solubilizi ng cotton fiber (Bhat and Hazlewood, 2001). ................................................................................................................. 482 -12. Hydrolysis of amorphous and microcr ystalline cellulose by non-complexed (A) and complexed (B) cellulase syst ems. ............................................................... 523-1. Temperature (C), rainfall (mm) and solar radiation (watts/m2) during the growing period of the hays (Florida automated weather network, 2009). ........... 873-2. In situ DM digestibili ty of 5and 13-wk regrowth of bermudagrass hay that were untreated (C) or treated with en zymes (E) or a mmonia (A). ............................... 88
10 3-3. Effect of treating be rmudagrass hay harvested at two regrowth intervals with a fibrolytic enzyme or ammonia on ru minal-N ammonia concentrations of steers. ................................................................................................................. 89
11 LIST OF ABBREVIATIONS ADF Acid detergent fiber ADG Average daily gain ADICP Acid detergent insoluble crude protein ADL Acid detergent lignin AMN Ammonia-treated bermudagrass hay BW Body weight CBM Carbohydrate binding molecule CON Untreated bermudagrass hay CP Crude protein CSA Cross sectional area DM Dry matter DMI Dry matter intake EFE Exogenous fibrolytic enzymes ENZ Enzyme-treated bermudagrass hay EPI Epidermis IVDMD In vitro dry matter digestibility MES Mesophyll NAD-ME Nicotinamide adenine dinucleotide malic enzyme NADP-ME Nicotinamide adenine dinuc leotide phosphate malic enzyme NCPAR Non-chlorenchymatous parenchyma NDF Neutral detergent fiber NDFD Neutral detergent fiber digestibility NDS Neutral detergent solubles NMR Nuclear magnetic resonance
12 OM Organic matter PBS Parenchyma bundle sheath PEP-CK Phosphoenolpyruvate carboxykinase RI Regrowth interval RUBISCO Ribulose biphosphate carboxylase/oxygenase SCL Sclerenchyma SE Standard error VFA Volatile fatty acid VT Vascular tissue Wt weight
13 Abstract of Thesis Pres ented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Master of Science EXOGENOUS FIBROLYTIC ENZYME OR ANHYDROUS AMMONIA EFFECTS ON THE NUTRITIVE VALUE, INTAKE, AND DIGESTION KINETICS OF BERMUDAGRASS AND THE GROWTH OF BEEF CATTLE By Juan Jose Romero Gomez December 2009 Chair: Adegbola T. Adesogan Major: Animal Sciences The objectives were to compare the effect of exogenous fibrolytic enzyme (Biocellulase A20) or anhydrous ammonia (4% DM) treatment on the nutritive value, voluntary intake, and digestion kinetics of Coastal bermudagrass [ Cynodon dactylon (L.) Pers.] hay harvested at two regrowth interval s and to determine if feeding the treated hays improves the growth of beef cattle. In Experiment 1, 6 individually housed, ruminally-cannulated Brangus steers (BW 216 6 kg) were used in an experiment with a 6 x 6 Latin square design with a 3 (additives) x 2 (regrowth intervals) factorial arrangement of treatments. Ea ch period consisted of 14 days of adaptation, 7 days of digestibility measurements, 4 days of in situ degradability, 1 day of rumen rest and 1 day of rumen fluid fermentation measurem ents. Steers were fed hay ad libitum supplemented with sugar cane molasses and distillers grain at a rate that met maintenance energy requirements. Differences in prevailing weather conditions (e.g. temperature) during growth of the 5and 13wk hays were associated with greater fiber concentrations in the 5-wk hay. Ammonia treatment decreased most fiber fractions and increased the crude protein (CP) concentration particularly for the ma ture lignified 13-wk
14 hay. Enzyme treatment did not affect most nutritional components but slightly increased CP concentration. Enzyme application did not affect intake measures but ammoniation decreased intake. Ammoniation increased diges tibility of DM, OM, NDF, hemicellulose ADF and cellulose across regrowth interval s, but reduced CP digestibility across regrowth intervals. Enzyme application increa sed NDF and hemicellulose digestibility of the 5-wk hay. Ammoniation increased the ruminal in situ DM degradation of the hay and ruminal ammonia concentration but enzyme treatment did not. In Experiment 2, 90 Angus and Brangus steers (308 37 kg) were stratified by weight and randomly allocated to 18 1.01-ha pens containing bahiagrass pasture ( Paspalum notatum Fl gge). Three pens were assigned randomly to each treatment. The same hays and supplement fed in Experiment 1 were fed in Experiment 2. The treated hays from both regrowth intervals were fed in round-bale feeders to cattle in respective pens in quantities sufficient to ensure ad libitum access for 56 d. Steers were adapted to diets for 6 d, and full body weights were obtained on two consecutive days at the beginning (d 7 and 8) and end (d 50 and 51) of the me asurement period. W eekly supplement allocations were fed in open troughs in equal amounts three times per week. Refused hay was weighed on d 30 and 49. Ammoniation increased hay DMI and tended to increase final BW and ADG. Enzyme treatment increased DMI of the 5-wk hay but had no effect on growth performance. In conclu sion, ammoniation improved the nutritional composition and digestibility of the hays and resulted in a trend for increased growth. Enzyme treatment improved hay CP concen tration, and improved the intake and NDF and hemicellulosedigestibilit y of the 5-wk hay but di d not improve growth.
15 CHAPTER 1 INTRODUCTION Forages are the major feed source fo r ruminant animals and they represent approximately 61% and 83% of the ration of dairy and beef cattle in the U.S., respectively (Barnes and Nelson, 2003). In tropical areas of the world, warm-season grasses make up around 85% of the feed suppl y for meat, milk, and fiber production (Coleman et al., 2004). In the southeastern of the U.S., warm-season grasses are the basis of livestock production (Pitman, 2007) and a ll beef cattle operations in Florida rely on forages as the primary s ource of nutrients (FASS, 2000). During the growing season, plant maturity and environmental conditions such as elevated temperatures and prolonged drought stress, re duce the quality of warmseason grasses (Pitman and Holt, 1982). For instance, the higher temperatures at which warm-season grasses grow are associated with increased lignification and reduced tissue and cell wall degradability (Colem an et al., 2004). Because of these and other complex factors, warm-season grasse s often have lower quality than cool-season grasses (Minson, 198 0; Pitman, 2007). Several methods have been developed to improve forage quality (Fahey et al., 1993; Berger et al., 1995) and anhydrous a mmonia application is one of the most effective methods (Berger et al., 1995). Ammoniation has improved the forage quality of cool(Wanapat et al., 1985; Flachowsky et al., 1996; Wang et al., 2004) and warmseason forages (Brown, 1988; Brown and Kunkle 2003; Krueger et al., 2008) but it is not used widely because it is costly, potent ially toxic and caustic (Rotz and Shinners, 2007). Recent research has focused on using exogenous fibrolytic enzymes (EFE) as a potential alternative to im prove forage quality and animal performance (Beauchemin et
16 al., 2004; Adesogan, 2005). Supplementing dair y cow and feedlot cattle diets with EFE has improved cell wall digestion and anima l performance (McAllister et al., 1999; Beauchemin et al., 2003; Arriola et al., 2007) and a few EFE have been used successfully to enhance the digestibility of warm-season grasses (Dean, 2005; Krueger et al., 2008). However, the results of EF E application on forage quality and animal performance have been equivocal (McAllister et al., 2001; Wang and McAllister, 2002; Adesogan, 2005). Thus, more research is needed to develop consistently effective enzyme products and improved application strategies. This research is needed particularly for intrinsically recalcitrant warm-season forages, since more attention has been focused on using EFE on cool-season forages Krueger et al. (2 008) reported that an EFE was more effective at improving the in take and digestibility of a 5-wk regrowth of bermudagrass hay when applied at cutting instead of at hay baling or feeding,. Limited information is availabl e on whether EFE-induced increases in forage intake and digestion depend on forage regrowth interval If EFE efficacy is independent of regrowth interval, application to higher-yield ing mature forages may be advisable. The objectives of this study were to ev aluate the effect of applying an EFE enzyme or anhydrous ammonia to bermudagrass hay [ Cynodon dactylon (L.) Pers.] on its nutritive value, voluntary intake, digesti on kinetics and the growth of beef cattle.
17 CHAPTER 2 LITERATURE REVIEW Warm-Season Grasses Definition and Importance Warm-seas on grasses are the backbone of live stock systems in much of the world and make up perhaps 85% of the feed supply for meat, milk and fiber production in warm-climate areas (Coleman et al., 2004). In t he southeast of the Un ited States and in the tropics, they are the basis of most livestock systems (Pitman, 2007). The optimum growth temperature of warm-season grasses is 35 to 38C (Long, 1999) and they are characterized by the C4 photosynthetic system (Moser et al., 2004). Characteristics of C4 Photosynthesis In order to comprehend C4 photosynthesis, it is necessary to understand the C3 pathway, because the former is an adaptation of the latter (Moser et al., 2004). In the C3 pathway, ribulose biphosphate carboxylase/ oxygenase (rubisco) in mesophyll cells (MES) fixes CO2 to the 5-carbon sugar, ribulose-1,5-biphosphate generating an unstable six-carbon compound t hat immediately forms two 3-carbon molecules of 3phosphoglycerate, which are subsequently c onverted to glucose (Nelson and Cox, 2008) (Figure 2-1). However, rubisco can also fix O2 and cause photorespiration because of its oxygenase activity (McA dam and Nelson, 2003). Photorespiration reduces the efficiency of C fixation during photosynthesis by 10 to 50% or more, depending on the prevailing te mperature (McAdam and Nelson, 2003). Warm-season grasses possess unique anatomical features t hat reduce photorespiration and make the C4 photosynthetic pathway more efficient. T he most notable feature is the Kranz anatomy (Rudall, 2007), which has m any vascular bundles surrounded by a
18 specialized, large, chloroplast-containing parenchyma bundle sheath (PBS; Rudall, 2007), which in turn is surrounded by a conc entric layer of MES cells (Moore et al., 2004). The Kranz structure acts to inhibit photorespiratory CO2 loss by maintaining high CO2 concentrations in the bundle sheath cells, or by promoting refixation of evolved CO2 as it diffuses outwardly thr ough the MES cells (Kennedy, 1976). In the C4 pathway, CO2 is fixed initially in the MES as four-carbon intermediates by PEP carboxylase, which has a greater affinity for CO2 than rubisco. The four-carbon intermediates are then transported to the PBS where they are decarboxylated, releasing CO2. This process prevents photorespiration and increases the concentration of CO2 in the PBS, where the rest of the photosynthetic reactions occur (Moore et al., 2004). Consequently, photosynthesis is more efficient in C4 grasses. Figure 2-1. The C3 and C4 anatomy and photosynthetic pathw ay. (Volenec and Nelson, 2007) CA= Carbonic anhydrase, PEPc= Phosphoenolpyruvate carboxylase. Subtypes of C4 Photosynthesis There are three subtypes of C4 photosynthesis depending on the decarboxylating enzyme in the PBS and chloroplast structur e and arrangement (Moser et al., 2004). The enzymes and associated subtypes include phosphoenolpyruvate carboxykinase
19 (PEP-CK), nicotinamide adenine dinucleotide malic enzyme (NAD-ME) and nicotinamide adenine dinucleotide phosphate malic enzyme (NADP-ME). Decarboxylation occurs in the cytoplasm in PEP-CK types and in the mitochondria in NAD-ME and NADP-ME types (Moor e et al., 2004). Subtypes of C4 grasses also vary in PBS anatomy and function, which influences their photosynthetic efficiency (Moore et al., 2004). From a photosynthetic and cell wall anatomy perspective, NADP-ME types seem to be more efficient, because their bundle sheath cells have a suberized lamella between the border of thei r secondary and primary wall (Hattersley and Browning, 1981). This suberized layer keeps the CO2 inside the cell reducing its leakage, thus greatly reducing the risk of photorespira tion, and consequently increasing efficiency (Moore et al., 2004). This group includes some of the mo st productive C4 grasses such as corn ( Zea mays), sorghum ( Sorghum bicolor ) and bahiagrass ( Paspalum notatum ). In contrast, NAD-ME types are more sensitive to O2 and may be less efficient because their bundle sheath cells lack the s uberized layer and leak more O2 (Ehleringer and Pearcy, 1983). Examples of NAD-ME type are broomcorn ( Panicum miliaceum ) and bermudagrass ( Cynodon dactylon ). The PEP-CK subtype will not be discussed further because very few species in this group are used in agriculture. Practical Implications of C4 Photosynthesis Waterand N-use efficiency. Warm-season grasses use water efficiently, requiring about one-third to one-half as much wa ter to produce a unit of dry matter (DM) compared to C3 grasses (Moser et al., 2004). This is because C4 plants maintain high photosynthetic rates at lower rates of stomatal conductance than most C3 plants, resulting in higher rates of carbon fixation per unit of wa ter transpired (Ehleringer and Monson, 1993). Furthermore, C4 grasses have a higher N-use efficiency than C3
20 grasses (Wedin, 2004). High rates of leaf photosynthesis can occur in C4 grasses with one-half or less of the leaf N required fo r the same photosynthetic rate by C3 species (Brown, 1978). This is because C4 plants use less rubisco than C3 plants for the same photosynthetic rate due to their Kranz Anatomy and their special pathway (Wedin, 2004). Long (1991) estimated that at 30C with normal atmospheric CO2 concentrations, a C4 leaf will need 13 20% of the rubisco found in C3 leaves to maintain the same photosynthetic rate. Nutritional quality. Warm-season grasses vary tremendously in forage quality depending on maturity, leaf:stem ra tio, inherent nutritive value of leaves and stems and environmental factors during their growth (Moser et al., 2004). Nevertheless, they generally are of poor er quality than C3 grasses. The C4 grasses have lower protein concentrations compared to C3 grasses because they have less rubisco and associated photosynthetic enzymes that can a ccount for 50% of leaf N in C3 grasses (Wedin, 2004). Minson (1980) also noted that CP c oncentrations of a large number of C4 grasses averaged 4 6% less than that of C3 species and concluded that occurrence of CP deficiency among livestock fed C4 grasses is much greater. Moreover, C4 grasses possess special anatomical feat ures and cell wall components t hat limit their intake and digestibility. Factors Affecting Digestibility of Forages Impact of C4 Grass Anatomy on Forage Digestibility The anatomy of plant organs and their constit uent tissues is important in ruminant nutrition because they affect cr itically the intake and digestib ility of forages (Coleman et al., 2004). Firstly, they affect intake by affecting chewing and rumination time (Coleman et al., 2004), particle size reduction (Wils on and Kennedy, 1996), and passage rate
21 (Kennedy and Doyle, 1993). Secondly, the ex tent of lignification of plants largely determines their digesti bility (Akin, 1989). In most of the ruminant nutri tion literature, nutritional valu e of forages is estimated from chemical composition. However, this review will focus on chemical and anatomical features because both influence forage quality. Anatomical features are particularly critical in C4 grasses because the Kranz structure of their leaves has a detrimental impact on plant degradati on (Akin et al., 2007). Influence of organs and their tissue proportions The digestibility of blades, sheaths and stems is associated with the relative proportion of tissue types in each organ with diges tible or indigestible cell walls (Wilson, 1993; Wils on and Hatfield, 1997). However, focusing on tissue proportions alone ignores the impact on digestibility of interce llular air spaces or the thickness or degree of lignification of cell walls in the various tissues; consequently, tissue proportions may not explain smaller differences in vitro DM digestibility (IVDMD) between species or cultivars (Wilson, 1993). Leaf Blade. Due to the anatomical stru ctures associated with the C4 photosynthetic pathway, C4 grasses generally have significantly more vascular bundles in the leaf than C3 grasses (Dengler et al., 1994). Consequently, there is a higher proportion of the less digestible, thick-wall ed, lignified tissues (PBS, sclerenchyma, and vascular tissue) in C4 than C3 grasses (Table 2-1). The NADP-ME type of C4 grasses have lower proportions of PBS than PEP-CK and NAD-ME types (Dengler et al., 1994). Studies conducted to evaluate the impact of these anatomical differences between C4 grass subtypes on digestibility have not been conclusive (Akin et al., 1983a; Wilson and Hattersley, 1989).
22 Table 2-1. Tissue proportions in organs of different forage types (From Wilson, 1993). Proportion of tissue in cross sectional area (%) Cell Type C4 grass ( Panicum maximum ) C3 grass ( Lolium multiflorum ) Blade SheathStem Blade SheathStem Epidermis 22 4 2 23 NM 2 Mesophyll 31 7 2 66 86 2 PBS 1 24 7 0 5 0 0 Sclerenchyma 2 6 8 1 10 12 NCPAR 2 14 66 75 2 NM 75 Vascular tissue (without phloem) 6 9 12 3 4 9 Phloem <1 1 1 <1 <1 <1 1PBS= parenchyma bundle sheath, 2NCPAR= non-chlorenchymatous parenchyma. NM= not mentioned Leaf Sheath. The anatomy of leaf sheaths is in termediate between those of the blade and stem, but more like t hat of the stem. Sheaths also have lignin concentrations between those of blades and stems and consequently their IVDMD falls between those of blades and stems (Wilman and Altimimi 1982). Sheath tissue proportions do not appear to change with maturity (Cherney and Marten, 1982), but there are marked increases in wall thickness of lignified cells (Wilson, 1976) and a marked decrease in sheath IVDMD with maturity (37.2% for yo ung vs. 55.8% for mature bermudagrass tissue, Akin et aI., 1977). Stem. Stems differ from leaf blades in that their tissue characteristics change greatly with maturity (Cherney and Marten, 1982) such that stem IVDMD can be similar to or greater than that of l eaves when young, but lower than that of leaves when mature due to a faster rate of dec line in IVDMD (Hacker and Mins on, 1981). When the stem is young (Fig. 2-2) the vascular tissue (VT) is in isolated bundles but as it matures, the bundles link together through lignification of the interfascicular nonchlorenchymatous parenchyma cells (NCPAR), which form a strong, indigestible tissue (Wilson, 1993)
23 Eventually, this tissue forms a ring that embraces the entire cortical region of sclerenchyma (SCL) and epidermis in most grasses and constitutes a powerful barrier to digestion (Wilson, 1993). For instance, th e neutral detergent fiber (NDF) digestibility (NDFD) of Sorghum bicolor stem was 55% at the 12-leaf stage but it deceased to 30% at the mature grain maturity stage (Wilson and Hatfield, 1997). Figure 2-2. Cross sections of young (a, head emergence) and mature (b, grain maturity) stems of Sorghum bicolor Solid black areas indicate lignification. e=epidermis, s=sclerenchyma, m=mesophy ll, vz= vascular lignified zone. p= pith parenchyma. Modified from Wilson (1993). Influence of tissue type on digestion Epidermis (EPI). The epidermis is the outermost (der mal) cell layer in plants. It covers the entire plant surface (Rudall, 2007 ) and constitutes approximately 26% of the cross sectional area (CSA) of bermudagrass leaf blades (Table 2-2). The outer tangential walls of the EPI become thickened, lignified, and completely covered with cuticle with increased maturity (Akin, 1989; Wilson, 1993). The cuticle consists of complex waxes, cutin and phenolic com pounds (Gevens and Nicholson, 2000); therefore, it is not usually degraded by ru minal microbes (Akin, 1989). Thus, the outer surface of the EPI is impervious to micr obial digestion and penetration (Monson et al. 1972) except through stomata or breaks caused by prehens ion or chewing (Wilson,
24 1993). After 48 h of digestion in rumen fluid, the cuticle of bermudagrass and orchardgrass leaves were intact (Fig. 2-3). Other tissues were lost from orchardgrass leaves except SCL and lignified VT; w hereas changes in bermudagrass leaves only included loss of MES and parti al degradation of EPI and PBS. Table 2-2. Percentage of tissue types in cross-sections (CS) of warm-and cool-season grass leaf blades from 4 to 8-wk-o ld plants. (Modified from Akin, 1989). Number of samples not stated. Tissue type Grass Total vascular tissue Lignified vascular tissue PBS1 Phloem EPI2 SCL3 MES4 Cynodon dactylon 37 5 28 4 26 10 27 C4 grass mean+ 22 4 15 2 35 6 38 C3 grass Mean+ 15 7 6 2 23 6 57 1PBS= parenchyma bundle sheath, 2EPI= epidermis, 3SCL= sclerenchyma, 4MES= mesophyll +Average value for several species. Figure 2-3. Scanning electron micrograph of bermudagrass (Ieft) and orchardgrass (right) leaf blades incubated for 48 h in rumen fluid. M= mesophyll, E= epidermis, B= parenchyma bundle sheath, C= cuticle, S= sclerenchyma and V= vascular tissue. Modi fied from Akin (1989). In most C3 grasses, the EPI is lost from the leaf quickly during chewing and digestion because it is attached to MES cells, whereas in C4 grasses the EPI is not lost readily (Akin et al., 1983a), because it is attached to the major and intermediate-sized vascular bundles through thick-walled SCL girder cells (Wilson, 1993). The epidermis is
25 not easily broken down in C4 grasses because the walls of the adjacent long cells in paradermal view are linked together with str ong dove tailed joints (Coleman et al., 2004). Consequently breakages must occur by splitting across walls rather than separation at the middle lamella (Wilson et al., 1989). The comparable joints in most C3 grasses are straight sided, which appears to facilitate easy splitting along the middle lamella (Wilson et al., 1989). The EPI in C4 grasses can be a strong barrier to digestion depending on maturity and environment (Akin, 1989). Nevertheless, c hemical treatments like sodium hydroxide and anhydrous ammonia can dissolve or crack the cuticle, respectively and potentially increase microbial colonization of cell walls a nd the rate and extent of digestion. Certain plant aerobic pathogenic fungi produce cu tinases (Kolattukudy, 1985), which may degrade the cuticle and EPI, but this hypothesis is yet to be validated by research. Mesophyll (MES). The MES comprises the chlorenchymatous tissue internal to the EPI in leaves (Rudall, 2007) and represents around 27% of leaf blade CSA tissue in bermudagrass (Akin, 1989). It is composed of thin-walled cells that form the main volume of tissue in all leaves but only a small part of the volu me of stems (Wilson, 1993). In C3 leaves, MES cells are arranged more loosely than in the C4 grasses because significantly more inte rcellular spaces exist in C3 (30%) than C4 grasses (22%) (Dengler et al., 1994). This creates a larger surface for bacteria to attach and helps detachment of individual cells, which can aid rate of passage (Wilson, 1993). Mesophyll cell walls never lignify; therefore, they ar e one of the most rapidly digested cell types (Akin, 1989). For such cells, digestion is essentially completed in less than 12 h (Chesson et al., 1986). However, MES cells of C4 grasses like bermudagrass are
26 digested more slowly than those of C3 grasses partly because of the reasons mentioned previously but also because phenolic compounds are more abundant in C4 grass MES cells of C4 grasses (Akin, 1989). Parenchyma Bundle Sheath (PBS). The PBS is a highly specialized group of chlorenchymatous cells surrounding the VT in leaves (PBS in C3 grasses do not have chloroplasts). It is commonly called the Kranz sheath in C4 grasses, along with the radial distribution of MES cells around the PBS (Moore et al., 2004). The PBS cells contain a high proportion of prot ein, because of their large chloroplasts, and starch and hence are a significant source of eas ily digestible substrates in C4 grasses (Wilson, 1993). They represent around 28% of the leaf blade CSA ti ssue in bermudagrass (Akin, 1989). Secondary thickening occurs in PBS cell wa lls with increasing maturity, resulting in about five times the thickness of MES walls (Wilson, 1990). The degree of lignification of the PBS can vary with m any factors (Akin, 1989) including stressful growth conditions. Akin et al. (1 983b) reported that irrigated C4 grasses had significantly lower digestibility than their non-irrigated counterparts, partially because of increased PBS lignification. They also reported that PBS digestion in Panicum spp. can be incomplete and is always slow, taking 48 to 72 h or longer. The NADP-ME sub-types of C4 grasses have a suberized lamella in their PBS but NAD-ME types do not. It was hypothesized that this suberized lamella acts as a barrier to the digestible nutrient-rich cytosol contents of PBS since it cannot be digested by rumen microbes (Wilson and Hattersley, 1983). Akin et al (1983a) evaluated the impact of the suberized layer on digestibility of the PBS among different C4 grasses by visual estimation of tissue loss in cross-sections after incubation in rumen fluid. Interestingl y, they reported that for a
27 group of Panicum spp. the PBS walls of the NAD-ME grasses were more slowly digested than those of NADP-ME. However, subsequent comparison with a wider range of genera did not substantiate this conclu sion (Wilson and Hattersley, 1989). Akin and Burdick (1977) highlighted the pot ential benefits of improving th e rate of digestion of the PBS of bermudagrass. They stated that in or der for bacteria to access the PBS cytosol, they would first have to degrade the PBS cell wall fast enough before it leaves the rumen. It takes 48 -72 h to digest the PBS (Akin et al., 1983a) which implies that they could be usually incompletely digested in hi gh-producing cattle that have high passage rates. Chemical treatments have been explored to improve digestion of the PBS in bermudagrass. After treatment with NaOH and incubation in rumen fluid, almost complete degradation of PBS was report ed (Akin and Hartley, 1992). The main component of the suberized layer is suberin, which is similar to cutin in composition (Kolattukudy, 1985); therefore, cutinases (and suberinases) may also aid PBS degradation by degrading suberin. Nonchlorenchymatous Parenchyma (NCPAR). Nonchlorenchymatous parenchyma cells are typically thin-walled and often polyhedral or variously shaped (Rudall, 2007). They can be quite large in size (40-140 m) and variable in function (Rudall, 2007). Leaf NCPAR represents about 14% of the leaf blade tissue CSA in C4 grasses but only 2% in C3 grasses (Wilson, 1993). Nevertheless, they are thin-walled in leaves and usually are rapidly and almost ent irely digested (Wilson et al., 1991). In contrast, leaf sheath and stem NCPAR can be large contributors to the low digestibility of leaf sheath and stem, respectively. This pr oblem is exacerbated by their
28 extensive presence in such tissues (66 and 75% of CSA tissue, respectively; Wilson, 1993), which reflects their large volume and their capacity to develop a thick secondary wall that can undergo lignification. The NCPAR cells form a solid tissue joined by a middle lamella with little intercellular space (Wilson, 1993). When stems are young, the NCPAR cells are easily digested and lost fr om stem sections; however, as stems mature, the NCPAR tissue becomes more li gnified from the outer stem inwards, particularly for the cells between the vascular bundles in the main vascular ring (Akin, 1989; Wilson, 1993). Clearly, NCPAR have a pivotal role in limiting sheath and stem digestion. The stem is usually a key fraction that influences overall plant digestibility, because it comprises much of the weight and decreases rapidly in quality with maturity (Cherney and Marten, 1982). Since NCPAR comp rises 75% of the tissue CSA in the stem, improving its digestion should be central in any attempt to improve forage digestibility. Therefore, more research should be conducted on improving the accessibility of enzymes and microbes to NCPAR. Sclerenchyma (SCL). The sclerenchyma is the supporting or protective tissue categorized as fibers (long and narrow) and scl ereids (variously shaped), which develop a thickened lignified wall with maturity (R udall, 2007). The SCL represents10, 6 and 8% of blade, sheath and stem CSA tissue respectively, in C4 grasses (Akin 1989). In grass blades and sheaths, SCL are more or less universally present as discrete patches above and below the vascular bundles, and at t he leaf margins (Wils on, 1993). In the grass stem, they are similarly associat ed with vascular bundles as SCL caps, and may form a complete ring of tissue around the outside of the stem between the EPI and vascular tissue (Wilson, 1993). This ring could act as another protective sheath that
29 hinders access of enzymes and microbes to t he inner NCPAR in t he stem. Therefore, enhancing the degradation of the SC L would likely improve the in take and digestibility of the stem. The SCL also can have a critical impact on intake by slowing down particle size reduction because of their structur al role in the plant (Rudall, 2007). Vascular Tissue (VT). In this document, xylem and phloem tissue will be jointly referred as the VT. Phloem tissue consists of thin-walled cells and because they do not lignify, they are quickly digested (Akin, 1989). Ho wever, they form a small fraction of the tissue volume in leaves, sheaths, and stem s (approximately 4% of leaf blade CSA tissue in bermudagrass; Akin, 1989). Xylem tissue consists of thick-wa lled cells that are heavily lignified whether in blades, sheaths, or stems. In thes e plant organs, xylem accounts for 6, 9 and 12% of CSA tissue of C4 grasses, respectively and it is considered indigestible (Wilson, 1993). Most rumination ac tivity is directed towards destruction of the VT structure and most of the undigested fiber particles appearing in feces are parts of the VT with attached or isolated SCL strands (Wilson, 1993). Thus, degradation of this structure is crucial to improve intake, digestion and passage of C4 grasses. Middle lamella: Intercellular component. The middle lamella is the layer between walls of neighboring cells (Rudall, 2007). The middle lamella-primary wall region is where lignification starts and is more concentrated (Jung and Allen, 1995); therefore, the middle lamella is the most powerful barrier to microbial degradation in recalcitrant tissues (Coleman et al., 2004). It cements cells together reducing exposure of the cell outer surface (Wilson, 1993). Consequently, cell wall digestion can only start from the less lignified lumen (Jung and Alle n, 1995) after cells have been physically ruptured (Wilson, 1993).
30 Impact of the Forage Cell Walls on Digestibility Chemical composition of fora ge cell walls The major chemical components of ce ll walls are various structural polysaccharides and lignin (Theander and West erlund, 1993). Protein, minerals, and lipids are minor components. Grasses also contain small but im portant amounts of hydroxycinnamic acids (ferul ates and p-coumarates) (Hatfi eld et al., 2007). Structural polysaccharides can have complex molecular st ructures, yet if removed from the cell wall they are degraded readily to their component monosaccharides by microbial enzymes (Hatfield et al., 1999a). This sect ion briefly describes such structural polysaccharides and then details their in teraction in the cell wall matrix. Cellulose. Cellulose is the most abundant struct ural polysaccharide in forage cell walls. It is a linear polymer of glucose linked by 1,4 glycosidic bonds. It has a simple primary structure and a complex tertiary st ructure and the repeating unit is cellobiose (Bhat and Hazlewood, 2001). Indivi dual cellulose molecules ar e extremely large and are arranged into bundles known as microfibrils (Nelson and Cox, 2008). Hydrogen bonds between cellulose molecules hold the microfibrils together (Nelson and Cox, 2008). In some regions the cellulose chains ar e highly ordered and strong hydrogen bonds hold them together in structures called cryst allites, whereas loosely-arranged cellulose molecules form the amorphous regions (Bhat and Hazlewood, 2001). Pure cellulose is quickly and completely degraded ruminally as is cellulose cross linked to hemicelluloses alone (Hatfield et al., 1999a). Hemicellulose. Hemicellulose is the second most abundant plant structural polysaccharide. It is present in association with cellulose in the walls of most plant species and can be extracted with alkalis fr om delignified walls (Bhat and Hazlewood,
31 2001). It is composed of a range of cell wall heteropolysaccharides different in component sugars and linkages (Hatfield et al., 2007). The most abundant of the heteropolysaccharides are the xy lans, which are composed of a -1,4-linked xylose backbone with branch substitutions (Hatfield et al., 2007). The type and frequency of branch substitutions varies with species and stage of development (Hatfield et al., 2007). Grass xylans have complex structures and contain substitutions of arabinose or glucuronic acid or both (Hatfield, 2007). In addition, some of the arabinose residues contain ferulic acid and to a much lesser ext ent p-coumaric acid ester linked to the C-5 hydroxyl group (Hartley, 1972). The ferulates can form linkages to one another, creating diferulate structures that crosslink arabinoxylan chains in grass cell walls (Ralph et al., 1994a). Several complementary enzymes are needed for hemicellulose degradation (Bhat and Hazlewood, 2001) and these are discu ssed later in the enzyme section. In the absence of lignin, intimate association of xylan and cellulose does not inhibit the rate of digestion of either polysacchari de in rumen fluid (Weimer et al., 2000). Pectins. Pectins account for a very small fr action of grasses ( 10 g/g or less of DM; Hatfield et al., 1999a) and they are r apidly and extensively degraded from cell wall matrices (Hatfield et al., 1999a). Due to thei r minor role in grass cell walls, they will not be discussed further. Proteins. Proteins generally make up less than 5 g/g of the grass cell wall depending on tissue type and maturity (Hatfield et al., 1999a). As with polysaccharides, proteins outside the wall matrix are suscept ible to degradation, but those within the wall may be completely resistant to degradation and pass intact through t he digestive tract.
32 Structural proteins like extensin, appear to play critical roles in cross-linking wall components, particularly in primar y walls (Hatfield et al., 1999a). Lignin. Lignin is defined as a group of polymer ic natural products arising from an enzyme-initiated dehydrogenative pol ymerization of its primar y precursors, coniferyl, sinapyl, and p-coumaryl alcohol (Sarkanen and Ludwig, 1971). Coniferyl and sinapyl alcohols are the most import ant in grasses (Hatfield et al., 2007). Peroxidases and/or oxidases react with lignin precursor alcohols to form lignin (Fig. 2-4). These reactions result in one-electron oxidized products t hat undergo radical coupling reactions to produce a growing polymer of lignin (Hatfield et al., 1999a). However, the strict chemical definition of lignin ma y not be compatible with the i dea of lignin from a nutritional perspective, because other components can become incorporated into the "lignin" polymer (Hatfield et al. 1999a). Consequent ly, Hatfield et al. (1999a) proposed that lignin should be defined as a phenolic-derived macromolecule that interacts with other wall polymers to provide structural integr ity, resistance to degradation, and water impermeability. Deleterious effects of lignin on digestibility result from its interaction with cell wall polymers (Hatfield et al., 1999a). Hydroxycinnamic acids. The difunctional hydroxyci nnamates (ferulic acid, p -coumaric acid and sinapic acid) are structurally related to lignin precursors and they may attach to lignin, playing critical roles in regulating wa ll matrix organization (Hatfield et al. 1999a). Hydroxycinnamic acids can cross-link polysaccharides wit h other polysaccharides or lignin (Fig 2-5; Ralph, 1996), and both of these result in decr eased digestibility (Hatfield, 1993).
33 Figure 2-4. Chemical structural characterist ics of typical lignin precursors (coniferyl, sinapyl and p -coumaryl alcohols) and hydro xycinnamic acids (ferulate, p coumarate and sinapate) found in fo rage cell walls (Hatfield, 1999). Figure 2-5. Diferulate cross-lin king of arabinoxylan chains and incorporation into lignin via active mechanisms. Schematically s hown are incorporati on of a)the 8-O-4 diferulate and b) the 5-5 diferulate, which can produce a very highly crosslinked matrix. Lignin potential attachm ent points are signaled by arrows. Me= methyl group (Modified from Ralph, 1996)
34 Cell wall development and its impact on digestion According to Terashima et al. (1993), t he growth and development of the cell wall in plants can be divided into two phases: Primar y Wall Phase of Development. Primary wall growth is the phase when the plant cell is increasing in size through wa ll elongation (Jung and Allen, 1995). After plant cells have reached mature size, additional development of the primary wall or deposition of a secondary wall st ructure can occur (Hatfield et al., 2007). However, any additional material is deposited on the cell lumen side of the wall; thus, decreasing cytoplasmic space (Terashima, 1993) (Figure 2-6). Primary cell wa lls are composed of cellulose, hemicellulose (primarily xylans), pectin and small amounts of proteins at this stage (Hatfield, 1993). In grasse s, hydroxycinnnamic acids are also present particularly ferulates and small amounts of p-coumaric acid; both are esterified to arabinoxylan polymers (He and Terashima, 1989; 1990). There is no depositi on of lignin during this phase (Jung and Allen, 1995). For the few tiss ues that develop thickened primary cell walls, the additional wall ma terial appears to be similar in composition to previously deposited primary cell wall (J ung and Engels, 2002). However, during the secondary stage of growth, the primary wall becomes the most lignified portion of the cell wall (Jung and Allen, 1995). Secondary Wall Phase of Development. The process of secondary wall thickening starts after ma ture cell size has been attained. Polysaccharides added are richer in cellulose than xylans and pecti n is no longer aggr egated (Jung and Allen, 1995). Also, ferulates (Jung, 2003) and p-coumaric acids (Lam et al., 1992) are being continuously added. Lignin depositi on starts at this phase in the middle lamella primary wall region
35 Figure 2-6. Schematic repres entation of a plant cell and wall development (Jung and Allen, 1995). (Jung and Allen, 1995) and continues on the lumen side of the wall (Terashima et al., 1993). Because lignin deposition lags behind polysaccharide aggregat ion, the most recently deposited polysaccharides are not lignifi ed; therefore, they are more digestible (Jung and Allen, 1995). In contra st, the middle lamella primar y wall region is the most intensely lignified and least digestible (Jung and Allen, 1995) region. Th is partly explains why microbes digest recalcitrant cells from the inside out (Grant, 2009). Cross-linking mediated by hydroxycinna mic acids and its im pact on digestion Conclusive evidence of the cross-link ing function of hydroxycinnamic acids was initially provided by Lam et al. (1 991; 1992), who developed a technique to quantitatively distinguish hydroxycinammic acid s that are ester linked, ether linked and both ester and ether linked. Hy droxycinnamic acids can form ester or ether bonds with lignin but only form ester bonds with polysacc harides (Ralph and Helm, 1993). Lam et al. (1991; 1992) reported that p -coumaric acid often is esterified to lignin, but also quite frequently involved in ether bonding; however it did not appear to form cross-linked
36 structures with both ester and ether linkages. Whereas, ferulic acid was mostly both esterified and etherified and thus it was invo lved in cross-linkages between lignin and arabinose, though it occurred also in the esterified only form. The fact that no ferulic acid was found in the etherified only form su ggests that only the pre-esterified form is incorporated to lignin via ether bonds to form the cross-link (Lam et al., 1992). In another study, Ralph et al. (1994b) showed that ferulate and p -coumarate molecules are esterified to arabinoxylan in gr asses; however, the majority of p -coumarates are ester linked to lignin. In grasses, the m ode of attachment seems to be similar across species, with the acid group esterified to the primary hydroxyl at the C-5 position when arabinose is in the furanose form. Mueller-Har vey et al. (1986) estimated that ferulic acid was substituted on 1 in 15 arabinose units in barley (Hordeum vulgare L.) straw. However, Ralph et al. (1995) mentioned that this level of substitution is based on extractable ferulates and it probably underestimates total feruloylation within the grass. Cross-linking of cell wall co mponents has a marked influence on numerous wall properties such as accessibility, extensibilit y, plasticity, digestibility, and adherence (Hatfield et al., 1999b). The formation of diferulates from ferulic acid monomers ester linked to arabinose in a polysaccharide chain is all that is required to covalently couple two polysaccharides, and this can occur in two wa ys (Hatfield et al., 1999b). The first one is a photochemical process, which produces cyclodimers and the second is by radical mediated dimerisation, which produces a range of dehydrodimers (Hatfield et al., 1999b). In the photochemical process, the plant has no control over the process but the radical-mediated dimerisation process involves sufficient control by the plant to optimize
37 wall cross linking (Hatfield et al., 1999b). Diferulate concentrations have been underestimated for many years due to limitat ions of analytical techniques and they could account for up to 70% of total ferula tes present in grasses (Ralph, 1996). Crosslinks mediated by diferulates between polysacchar ides have a structural role in plants and depress the rate and possibly the ext ent of polysaccharide degradation (Grabber 1998b) (Figure 2-7). Figure 2-7. Impact of diferulate formation in nonlignified walls upon wall structural polysaccharide degradation by fungal hydrolases (Grabber 1998b) Using nuclear magnetic resonance (NMR) sp ectrometry on ryegrass, Ralph et al. (1995) demonstrated that ferulates attached to C-5 of arabinose units do form covalent linkages to coniferyl and sinapyl alcohol resi dues in lignin. Mor eover, they confirmed that ferulates bound to arabinoxylans could bec ome covalently linked to monolignols by radical coupling reactions (active mechanism ) besides the commonly accepted ligninferulate -ethers (passive mechanism; Figure 2-8). This means that ferulates act as nucleation or initiation sites for the lignification process (Ralph et al., 1995) because
38 radical coupling to lignin can only occur if fe rulates react with monolignols. Hatfield et al. (1999) suggested that the positioning of feru lates within the wall might regulate lignin formation patterns and control cross-linking within wall matrices. Consequently, controlling the level of total feruloylation shoul d affect directly extent of cross-linking. Except for -ether, linkages arising from radi cal coupling reactions cannot be released by common solvents used for ferulate quantification (Ralph et al., 1995). This is because high-temperature alkaline hydrolysis only works for and -ether structures, but many other st ructures are involved when r adical coupling occurs (Ralph et al., 1995). Consequently, about 50% of total fe rulates are not released from lignified walls during quantification, resulting in underes timation of cross linking (Grabber et al., 1995). Ralph et al. (1995) suggested that NMR is the only accurate method for their total quantification. Grabber (2005) mentioned that each unit of lignin depressed cell wall degradability by two units in his model based on maize ( Zea mays L.) cell walls. Lignin appears to act as a physical barrier to the microbial enzymes that degrade cell-wall polysaccharides (Jung and Deetz, 1993; Hatfiel d et al., 2007). Therefore, lignified forage tissues are only partially digestible, whereas non-lignified tissues of mature forages remain completely digestible (J ung and Engels, 2002). Rumen microbes cannot overcome this barrier to cell wall polysa ccharide digestion because lignin cannot be degraded in the anaerobic rumen environm ent (Hatfield et al., 2007). In the past, the composition of ligni n was hypothesized to impact the degree to which cell wall digestion is limited by li gnin (Jung and Deetz, 1993). However, recent reports indicate that lignin composition does not play a direct role in cell wall digestibility
39 (Grabber et al., 1998a; Jung et al., 1999). Appar ently, the decrease in cell wall digestion associated with increased syri ngylto guaiacyl-type lignin observed previously (Jung and Deetz, 1993) reflects accumulation of poorly digested, lignified, secondary cell walls that are intrinsically hig her in syringyl lignin concentration (Jung and Engels, 2002; Grabber, 2005). Figure 2-8. Attachment of ferulates to li gnin by two distinct mechanisms: Passive mechanism (LFP Complex A) and Activ e radical-coupling mechanism (LFP Complex B; Ralph, 1996). Jung and Allen (1995) suggested that ferulic acid esters of arabinoxylan alone (excluding the ether cross-linkage with ligni n) could interfere with digestion of the polysaccharides they are esterified to by hindering the alignment of xylanase with its substrate, which is necessary for hy drolysis (Gorbacheva and Rodionova, 1977; Mitsuishi et al., 1988). Furthermore, Jung et al. (1991) demonstrated that the presence of hydroxycinnamic esters reduced polysacc haride digestion. When ferulates cross-link arabinoxylans and lignin via ester and ether linkages, res pectively, the extent of digestion is reduced dramatically (Jung and Deetz, 1993; Grabber et al., 1998 a,b). Jung and Allen (1995) hypothesiz ed that this happens because the ester portion of the
40 ferulate bridge is no longer available to be cleaved by enzymes since the lignin polymer is in such close proximity that ferulate este rase can no longer attach appropriately to its substrate. In addition, the authors mentioned that anaerobic cleavage of ether linkages is not known to occur. Hence, ferulate ether s accumulate in the residues remaining after forage digestion (D.R. Mertens and H .G. Jung, unpublished data cited by Jung and Allen, 1995). Grabber et al. (1998 a,b) demonstrated that when lignin was added and crosslinked to a cell wall model based on maize cells, the rate and extent of cell wall degradation was depressed with a more pro nounced impact on the arabinoxylans than on any other wall polysaccharide. This was cl ear proof of the impact of ferulate crosslinking of hemicelluloses to lignin on diges tion. Casler and Jung (1999) reported that higher levels of ferulate cross-linking at the same lignin concentration reduced NDF digestibility of smooth bromegrass. In summary, forages are very complex substrat es for ruminal digest ion. So far, this review has described impacts of different anatomical and chemical features on forage digestion. It is clear that lignification of the EPI of the whole pl ant, the PBS of the leaf, and the SCL ring of the stem impedes microbial degradation of the plant cell wall. In addition, though to a lesser exte nt, diferulate crosslinking of polysaccharides hinders cell wall degradation (Hatfield et al., 1999a). The rest of the review focuses on methods that have been used to attempt to improve cell wall digestion in order to enhance animal performance. Methods to Improve Forage Nutritive Value and Intake Many treatments have been appli ed to low quality forages to improve their nutritive value, voluntary intake and ra te and extent of digestion in order to improve nutrient
41 availability to the ruminant animal (Berger et al., 1995). De tailed descriptions of such physical, chemical, and biological treatment s were outlined by Fahe y et al. (1993) and Berger et al. (1995). Am ong the existing methods, ammonia application is a proven technology to improve forage quality and is the most widely used chemical method in the US (Berger et al., 1995). Application of EFE is a more recent technology that holds promise for improving for age quality but avoids some problems associated with ammoniation. The rest of this review will summarize ke y effects of ammoniation on forage nutritive value and animal performance and discuss the underlyi ng principles and efficacy of EFE application to forages. Anhydrous Ammonia Several studies have c ompared the efficacy of improving forage digestibility with ammonia and other alkaline compounds like NaOH, CaOH and urea. In a classical study, Wanapat et al. (1985) demonstrated that NaOH was the most effective treatment at improving digestibility of barley straw ( Hordeum vulgare), followed by anhydrous ammonia. Similar results were obtained by Haddad et al. (1995) and Flachowsky et al. (1996) with wheat straw ( Triticum spp. ). Nevertheless, NaOH is less widely used than ammonia (Berger et al., 1995) because it exac erbates the N deficiency of low quality forages (Moss, 1990), places a high sodium load on the animal (Haddad et al., 1995), and is very dangerous to handle d ue to its caustic nature. Anhydrous ammonia acts as an alkali afte r it dissolves in and deprotonates water and is transformed into ammonium hydr oxide (Solomons and Fryhle, 2004). The hydroxide functional group saponifies este r bonds (Solomons and Fryhle, 2004) that bind lignin-crosslinked hydroxycinnamic acid s to sugars on a hemicellulose chain. Consequently, ammoniation increases hemicellulose solubilization in forages (Berger et
42 al., 1995), leading to increases in the rate and extent of forage digestion (Brown, 1988; Wang et al., 2004). These fact ors culminate in increased intake, energy supply to and performance of animals fed ammoniated fo rages (Brown, 1988; Fahey et al. 1993; Flachowsky et al., 1996), parti cularly when such forages are mature and highly lignified (Fahey et al., 1993; Brown and Kunkle, 2003). An additional benefit of ammoniation that is lacking with other forage treatment methods is the increased N concentration of the ammoniated forage (Berger et al., 1995; Sollenberger et al., 2004). This attribute is particula rly important for low quality forages like warm-season grasses that have characteristically low protein concentrations (Brown and Kunkle, 2003). Ho wever, applying excessive levels of ammonia can depress intake due to the st rong odor, and more ammonia than desired may be trapped in forages with moisture c oncentrations exceeding 25-30%, even at the 4% (DM basis) recommended rate (Brown and Kunkle, 2003). Consequently, ammonia should be applied only at 4% to forages with moisture conc entrations at or below 25 30%. Ammoniation improves the quality of lignified forages, but animal and human safety concerns have limited its use (Rotz and Shinners, 2007). It can be toxic to cattle when applied to forages with high soluble sugar concentrations, due to formation of 4methyl imidazole. In addition, direct expos ure of ammonia to humans can cause severe burns, blindness, and even death (Rotz and Sh inners, 2007). For these reasons and others like the cost and availability of am monia, other forage treatment methods are required (Adesogan, 2003).
43 Exogenous Fibrolytic Enzymes Recent reductions in enzyme manufacturi ng costs along with more active and better defined enzyme preparations have stimulated researchers to re-examine the role of EFE in ruminant diets (McAllister et al., 2001). Some studies reported that adding EFE to diets of dairy and feedlot cattle has considerable potential to improve milk production and weight gain (Sanc hez et al., 1996; Yang et al. 1999; McAllister et al., 1999; Krueger et al. 2008; Arriola et al., 2007); however, experimental results have been inconsistent (Luchini et al., 1997; Dhim an et al. 2002; Beauchemin et al. 2003; Yescas-Yescas et al., 2004). Wang and McAlli ster (2002) argued that much of the EFE research has been focused on animal responses to different commercial enzyme products, yet little attention has been paid to how they differ and the mechanisms of enzyme action. An additional, related problem is that commercial EFE products are marketed based on specified levels of activi ty of one or two key enzymes (typically xylanase and cellulase) without reference to others that could be present (McAllister et al., 2001). This implies that enzyme products with similar activities of main enzymes may have other enzyme activities that comp lement or hinder acti vity of the main enzymes. This presents a serious problem for interpreting and comparing results from enzyme studies in the literature (Wang and McAllister, 2002). In most cases, enzyme activities suppl ied by commercial EFE products are not novel to the rumen. Therefore, EFE act on the same cell wall targets as endogenous enzymes (Wang and McAllister, 2002). This mi ght explain why many EFE treatments have improved the rate but not the extent of plant cell wall digestion (Feng et al., 1996; Wang et al., 2004) and had equivocal effects on forage nutritive value and animal performance. The remainder of this review focuses on th e science underlying fibrolytic
44 enzyme action and effects of EFE application on cell wall concentration, digestibility and animal performance. Types of enzymatic activities Cellulases. The term cellulase refers to a broad group consisting of many fibrolytic enzymes. Fig. 2-9 gives an overview of the su ccessive action of some of such enzymes. a) Endoglucanases (1,4--D-glucan-4-glucanohydr olases (EC 188.8.131.52)) Endoglucanases specifically cleave the internal -1,4-glycosidic bonds of amorphous and swollen celluloses as well as cello-oligosaccharides but they are generally inactive towards crystalline cellulose and cellobiose (Bhat and Hazlewood, 2001). Microorganisms secrete mu ltiple endoglucanases (I, II III, IV, V) with a wide range of substrate specificit ies and thereby cause efficient hydrolysis of complex substrates (Bhat et al., 1990). End pr oducts of endoglucanase hydrolysis include oligosaccharides of various lengths (Lynd et al., 2002). b) Exoglucanases (4--D-glucan glucanohydrolases (cellodextrinases, EC 184.108.40.206) and 1,4-D-glucan cellobiohydrolases (cellobiohydrolases, EC 220.127.116.11) Exoglucanases act on the reducing or non-reducing ends of cellulose fibrils, liberating either glucose (glu canohydrolases) or cellobiose (cellobiohydrolase) as major products (Lynd et al., 2002). Cellobiohydrol ases are highly active on amorphous and swollen cellulose, but degrade poorly crysta lline cellulose and cello-oligosaccharides (Wood and Bhat, 1988). These enzymes are specific for -1,4-glycosidic bonds, but are inactive on cellobiose. There are two classes of cellobiohydrolases. Cellobiohydrolase I hydrolyses the cellulose chain prefer entially from the reducing end whereas Cellobiohydrolase II attacks the chain from the non-reducing end (Bhat and Hazlewood,
45 2002). Glucanhydrolases only release glucose from the non-reducing end (Wood and McRae, 1982). c) -glucosidases or -glucoside glucohydrolases (EC 18.104.22.168). -glucosidases can be classified as either aryl -D-glucosidases (hydrolyzing aryl-D-glycosidases exclusively), cellob iases (hydrolyzing diglucosides and cellooligosaccharides) or -glucosidases with broad substrate specificities (Bhat and Hazlewood, 2001). -glucosidase sequentially removes one glucose unit from either the reducing end, the non-reducing end or both ends (Bhat and Hazlewood, 2001). Figure 2-9. Schematic representation of the major enzymes involved in cellulose hydrolysis (Beauchemin et al., 2004). Bl ack circles indicate glucose units. Hemicellulases. Hemicellulases are composed of a myriad of enzymatic activities. A broad schematic representation of their action is shown in Fig. 2-10. Xylanases are specific for the internal -1,4 linkages of polymeric xylan and are designated as endoxylanases (EC 3.2.1. 8), which yield xylooligomers, and -1,4
46 xylosidase (EC 22.214.171.124), which yields xylose (Bhat and Ha zlewood, 2001). Most endoxylanases are specific for unsubstituted (not branched with acetic acid, glucuronic acid or arabinose) xylosidic linkages of xylans and release both substituted and unsubstituted xylo-oligosaccharides. In cont rast, some endoxylanases are specific for xylosidic linkages adjacent to substituted groups in the main xylan chain (Bhat and Hazlewood, 2001). Other hemicellulase enz ymes which degrade the side chains are mannosidase (E.C. 126.96.36.199), -L-arabinofuranosidase (E.C. 188.8.131.52), -Dglucuronidase (EC 184.108.40.206), -D-galactosidase (EC 220.127.116.11) acetyl-xylan esterases (18.104.22.168) and ferulic acid esterase (EC 22.214.171.124) (Beauchemin et al., 2004). Figure 2-10. Schematic representation of the hemicellulose enzymes involved in the degradation of arabinoxylan (Beauchemin et al., 2004). PA= phenolic acid, Ac= acetic acid, triangle= glucoronic acid, square= arabinose.
47 Synergy between enzymes Synergism between cellulases. Cellulase enzyme systems exhibit higher collective activity than the sum of the ac tivities of individual enzymes, a phenomenon known as synergism. Five forms of synergism have been r eported (Lynd et al., 2002): Endo-exo synergy between endoglu canases and exoglucanases, Exo-exo synergy between exoglucanases processing from the reducing and non-reducing ends of cellulose chains, Synergy between exoglucanases and -glucosidases that remove cellobiose (and cellodextrins) as end products of exoglucanases, and Endo-endo synergy between different types of endoglucanases (Klyosov, 1990) Intramolecular synergy between catalytic domains and carbohydrate -binding molecules Bhat and Hazlewood (2001) speculated that the exo-exo synergism was due to their ability to expose new hydrolysis sites to each other, as well as their ability to act from reducing and non-reduci ng ends (Barr et al., 1996). The presence of endoglucanases with different subs trate specificities (I, II, I II, IV, V) would increase the synergistic efficiency further, as shown in Fig. 2-11. Wood (1992) suggested that the five endoglucanases of T. reesei are primarily responsible for decreasing the degree of polymerization by internally cleaving cellulose chains at relatively amorphous regions, thereby generating new cellu lose chain ends susceptible to the action of cellobiohydrolases (Teeri et al., 1998). Synergy between hemicellulases. Efficient and complete hydrolysis of xylan requires the synergistic action of main and side-chain enzymes with different
48 specificities (Coughlan et al., 1993). Two ty pes of synergy between such enzymes have been described in vivo (Coughlan et al., 1993): Homeosynergy: This occurs between tw o or more differ ent types of sidechain-cleaving enzymes or between tw o or more types of main-chain cleaving enzymes. (e.g., feru lic acid esterase and -L-arabinofuranosidase; endoxylanases and -xylosidases) Heterosynergy: This occurs between side-chain and main chain cleaving enzymes (e.g., ferulic acid esterases and endoxylanases). Figure 2-11. Synergism between Penicillium pinophilum cellobiohydrolase (I and II) and endoglucanases (EI to EV) in solubilizi ng cotton fiber (Bhat and Hazlewood, 2001). Attachment of enzymes Carbohydr ate-binding molecules (CBMs) ar e non-catalytic structures that have been found in most cellulases. The CBM a ffects binding to the cellulose surface, presumably to facilitate cellulo se hydrolysis by bringing th e catalytic domain in close proximity to the substrate (Lynd et al., 2002). The presence of CBMs is particularly
49 important for the initiation and activity of exoglucanases (Teeri et al., 1998). Cellulases that adsorbed to a greater extent increased t he rate and extent of crystalline cellulose hydrolysis when compared with cellulases that adsorbed less strongly (Klyosov, 1990). Product inhibition As with most enzymes, high concentrations of the hydrolysis products of cellulases and xy lanases often inhibit their action. E ndoglucanases (Bhat et al., 1989) and most cellobiohydrolases are inhibited by cellobiose (Wood and McCrae, 1986). However, concentrations of glucose up to 100 mM had little effect on many cellobiohydrolases and endoglucanases (Bhat et al., 1989). Like wise, xylanases and endoxylanases are believed to be inhibited by high concentrations of xylobiose, but not by xylose (Bhat and Hazleewood, 2001). In contrast, -glucosidases are inhibited by glucose and other monoand disaccharides (Bhat et al., 1993). These inhibition s suggest strongly that different enzymes, each with sufficiently hi gh activity levels are needed to ensure unimpeded and thorough degradation of plant cell walls. The challenge for researchers is to formulate products with ideal proporti ons of each type of enzyme to overcome product inhibition and ensure enzyme efficacy. Cellulose degradation in nature An understanding of mechanism s of cellulose degradation in nature is essential to appreciate differences between degradation c apabilities of EFE and ruminal fibrolytic enzymes. There is a distinct difference in the strategy for degrading cellulose among cellulolytic microorganisms, depending on w hether they are aerobes or anaerobes (Lynd et al., 2002). Most EFE are produced by aerobes w hereas, ruminally produced fibrolytic enzymes are from anaerobes (Lynd et al., 2002).
50 Complexed cellulase systems (Cellulose degradation by anaerobes ). In this system, the majority of ce llulolytic anaerobes (bacteria and fungi) do not release extracellular cellulase, instead their complexed cellulases are localized directly on the surface of the cell or the cell-glycocalyx matrix, which is called the cellulosome (McAllister et al., 1994; Fig. 2-12). Most ce llulolytic anaerobes including those in the rumen grow optimally on cellulose when atta ched to the substrate, and this contact appears to be necessary for catalysis (W ang and McAllister, 2002). Due to their anaerobic metabolism, such microbes have low yields of enzymes compared to aerobes; consequently, production of EFE from aerobes is preferred in the industry (Bhat and Hazlewood, 2001). In addition, such low yields partly explain why supplemental EFE complement the activity of ruminal fibrolytic enzymes despite similarities in the types of enzymes involved. Lynd et al. (2002) hypothes ized that anaerobic bacteria develop cellulosomes to degrade cellulosic material because they cannot effectively penetrate cellulosic material and also because their anaerobic metabolism requires them to be as efficient as possible in enzyme synthesis. The cellulo some seems to allo w cooperative enzyme activity in close proximity to the bacterial cell, enabling optimum synergism between the cellulases presented on the cellu losome (Lynd et al., 2002) At the same time, it minimizes the distance over which hydrolysi s products must diffuse, allowing efficient uptake of oligosaccharide products by the mi crobes (Bayer et al., 1994). According to Lynn et al. (2002), cellulosomes are efficient at hydrolyzing microcrystalline cellulose because they have adequate ratios of enzymatic activities that optimize synergism and their structure spaces enzymes and CBMs in a manner that favors synergism.
51 Many rumen bacteria including Ruminococcus species (Ding et al., 2001) and Fibrobacter succinogenes (Miron and Forsberg, 1999) seem to produce cellulosomes. There is evidence indicating that anaerobic fungi also utiliz e cellulosomes for hydrolysis of crystalline cellulose (Hazlewood and G ilbert, 1998). Inhibiti on of adhesion of microbes to cellulose or detachment of microbes already adherent can reduce or completely prevent cellulose utilization by ruminal microbes (Wei mer et al., 1993). Non-complexed cellulase systems (cellulose degradation by aerobes). Aerobic cellulolytic bacteria and fungi produce high cell yields characteristic of aerobic respiratory growth (Bhat and Hazlewood, 2001). Aerobic cellulose degraders utilize cellulose through production of substantial amounts of extracellula r cellulase enzymes that can be recovered from cult ure supernatants (Schwarz, 2001). While many aerobic bacteria adhere to cellulose, physical contact between microbes and cellulose does not appear to be necessary for their cellulolytic action (Lynd et al., 2002). Cellulolytic filamentous fungi and actinomycete bacteria can penetrate plant cell walls through hyphal extensions, thus pres enting their cellulase systems in close proximity to the substrate in confined caviti es, even if they are not strictly adhering (Eriksson et al., 1990). The production of free standing cellulases may therefore suffice for efficient hydrol ysis of cellulose under thes e conditions because loss of enzymes and hydrolytic products due to diffu sion is likely to be limited (Lynd et al., 2002). In contrast, when enzymes from aer obic organisms are sprayed on forages or added to diets, they may be less effectiv e because they cannot cross anatomical barriers to the digestible cell wall components. More research is needed in this area to understand how to optimize the interaction of EFE with di etary substrates.
52 Figure 2 -12. Hydrolysis of amorphous and mi crocrystalline cellulose by non-complexed (A) and complexed (B) cellulase system s. The solid squares on the right extremes of cellulose chains repr esent reducing ends, and the open squares on the left extremes represent nonr educing ends. Amorphous and crystalline regions are indicated. Cellulose, en zymes, and hydrolytic products are not shown to scale. From Lynd et al. (2002) Exogenous fibrolytic enzymes in ruminant diets This section initially describes the mode of action of EFE in ruminant diets, then discusses the non-enz ymatic factors affectin g the response to EFE addition to forages and diets fed to ruminants. Mode of action of EFE in ruminant diets. McAllister et al. (2001) divides the mode of action of EFE in ru minant diets as follows:
53 a) Pre-consumption effects Application of EFE to ruminant diets has caused pre-consumptive effects, particularly the release of soluble carbohydrat es (Hristov et al., 1996; Nsereko et al., 2000) due to partial solubilization of NDF and ADF (Gwayumba and Christensen, 1997; Krause et al., 1998; Nsereko et al., 2000; Krueger, 2007) but this effect is not always observed (Krueger et al., 2008). This resp onse most likely depends on the degree of lignification of the forage and the type of enzyme used. Nser eko et al. (2000) demonstrated compelling evidence that applying enzymes to feed causes structural changes that make feed more amenable to fu rther degradation. In their trial, EFE application (Multifect Xylanas e, experimental preparation and Sumizyme X, Monsanto Co., St. Louis, MO) improved alfalfa hay ( Medicago sativa ) digestibility in rumen fluid even when substrates were autoclaved and washed to remove enzyme residues and hydrolysis products, leaving on ly structural changes as the possible explanation for the digestibility improvement. McAllister et al (2001) reported that EFE application caused appearance of digestive pits in cell walls of barley straw though the application rates tested were greater than those recommended by the manufacturer. Collectively, these studies demonstrate that EFE exert pre-ingestive effects that enhance cell wall utilization. b) Ruminal effects Most of the improvements in forage qual ity resulting from EFE application are attributable to ruminal e ffects (Beauchemin et al., 2003) In the rumen, EFE are generally more stable than previ ously thought (Hristov et al., 1998; Morgavi et al., 2000a, 2001). Application of enzymes to feeds prior to ingestion enhances the
54 adhesion of the enzyme to t he substrate, which increa ses the resistance of the enzymes to proteolysis and prolongs their re sidence time within the rumen (McAllister et al., 2001).There are two mechanisms by which EFE influence ruminal fiber utilization: a.1) Direct hydrolysis The fact that EFE remain active in the rumen raises the possibility that they may improve digestion through direct hydrolysis of ingested feed withi n the rumen (Rode et al., 2001). Dong (1998 cited by McAllister et al., 2001) noted that applying an EFE to a grass hay diet for sheep increased endoglucanas e activity in ruminal fluid, but this activity accounted for 0.5% of the total endoglucanase activity in the rumen. This suggests that addition of EFE hardly affected the hydrolytic capacity conferred by endoglucanase in the rumen but the author acknowledged problems with his sampling procedure for particle-associated activity. More work is needed to substantiate the latter report and to quantify improvements in digestion that are entirely due to direct cell wall hydrolysis in the rumen by added EFE. a.2) Synergism with rumen microbes Synergism between EFE and rumen microbes has enhanced ruminal fiber digestion (Morgavi et al., 2000c). Adding ex ogenous enzymes to the diet increases the hydrolytic capacity within the rumen mainly due to increased bacterial attachment (Yang et al., 1999; Morgavi et al., 2000b; Wang et al., 2001) and stimulation of rumen microbial populations (Wang et al., 2001; Ns ereko et al., 2002). These responses may have occurred because nutrients released by init ial enzyme action attracted bacteria to the digestion site, stimulating further micr obial degradation (McAllister et al., 1994).
55 The model described above is widely acc epted. However, Krueger et al. (2008) reported that EFE improved forage digesti on even when applied to the harvested hay several months before it was f ed. In that study, the activi ty of EFE on the stored hay was not evaluated. Therefore, it is not clear if the impr ovement was caused before or during ingestion and ruminal digestion of the hay. Perplexingly, applying the same EFE to the hay at feeding did not improve digestibility. Nevertheless, the success of the harvest time application holds promise for EFE use in production systems where adding EFE at feeding is impractical. Non-enzymatic factors affecting efficiency of EFE. Many factors influence EFE effects on ruminant diets, and the most important of these will be discussed in this section. a) Manufacturing process Exogenous fibrolytic enzymes are produced by a batch fermentation process, mostly with aerobic fungi or bacteria (Cowan 1994). Once the fermentation is over, EFE are separated from ferm entation residues and the source organism (Beauchemin et al., 2004) by chemical and physical processes (Gashe, 1992). The types and activity of EFE produced depend on the strain select ed, the growth substrate and culture conditions (Considine and Coughlan, 1989). Batch to batch variations in enzyme activity may arise from changes in factors that influence the fermentation process including substrate moisture c ontent, culture depth, O2 and CO2 concentrations, and prevailing temperature and pH (Cons idine and Coughlan, 1989). Morgavi et al. (2000a; 2001) reported that in some cases carriers and stabilizers added during manufacturing increased the survival of EFE in the rumen. van de Vyver
56 et al. (2004) and Adesogan (2005) also ment ioned that co-factors and natural or artificially-induced enzyme glycolysation are im portant for ensuring ruminal stability and function of EFE. These factors also may be important determinants of how long EFE can survive outdoors, especia lly when applied to feeds stored for long periods before they are fed. b) Influence of pH and temperature Most commercial EFE products that hav e been evaluated in ruminant diets were originally designed for non-feed applications (Bhat and Hazlewood, 2001). This partially explains why the pH and temperature optima of most such enzyme products are 4 5 and 60C, respectively (Beauchemin et al., 2004). In general, fungal cellulases are optimally active between pH 4.0 6.0 (Wood, 1985). Endoglucanases, cellobiohydrolases, xylanases, and glucosidas es from mesophilic fungi are optimally active between 40-55C (Bhat et al., 1989; Coughlan et al., 1993). In addition, most xylanases have low acidic pH optima (Wong et al., 1988). Ruminal conditions are often very different from these opt ima, with a relatively const ant temperature of 39C and a pH approximating 6.0 (Van Soest, 1994). Th erefore, many commercial EFE have suboptimal enzymatic activities when analyz ed under ruminal conditions or added to ruminant diets (Kung et al., 2002; Vicini et al., 2003). Attempts have been made to standardize methods of determining enzym e activity (Colombatto and Beauchemin, 2003); however, they are based on using pure substrates. Therefore, they do not accurately indicate how much of plant substrate will be degraded by the EFE (Beauchemin et al., 2004). More research is needed to develop realistic cell wall models to analyze activities of EFE that will be added to ruminant feeds.
57 c) Specificity to the substrate Enzyme-substrate specificity is a wid ely-accepted phenomenon in the literature (Nelson and Cox, 2008). On a fundamental level, the comp lexity of plant cell walls complicates targeting EFE to such substr ates. Ideally, each com ponent of the cell wall (e.g.cellulose, hemicelluloses, etc.) should be targeted by one or more enzymes but this is difficult because the concentration of each cell wall component varies with tissue type; moreover, the proportions of each ti ssue vary with plant species and growth conditions. For instance an EFE treatment (Xylanase B, Biovance Tech. Inc., Omaha, NE, and Spezyme CP, Genencor, Rochester, NY combination) improved ADG of alfalfa and timothy hay, but had no similar effect on barley silage (Beauchemin et al., 1995). Also, EFE that were most effective at im proving the 18-h DMD of alfalfa hay differed from those that were most ef fective on corn silage (Colom batto et al., 2003). These and other studies indicate str ong enzyme-feed specificity. Lignin and its crosslinking to hemicelluloses can conceivably obscure the specificity of EFE to plant cell walls because it prevents enzyme accessibility to digestible polysaccharides(Wang et al., 2004). Pretreatments with en zymes from aerobes like -etherases, ligninases, laccases, glucose oxidase or chemicals may be required for proper hydrolysis of such substrates. d) Influence of the animal Animal factors like low ruminal pH and high passage rates that compromise ruminal fiber digestion are common in high -producing early-lactation dairy cows. Such cows are typically in negative energy bal ance and therefore need all the energy that they can assimilate to cope with milk production. In these critical situations where fiber digestion is depressed and energy requirement is greatest, response to added EFE is
58 usually greatest (Beauchemin et al., 2004). Gr eater intake, body-weight gain and milk yield responses to EFE treatments have been reported in early-lactation cows compared to those from their midto late-l actation counterparts (S chingoethe et al., 1999; Knowlton et al., 2002). Consequently, Zheng et al. (2000) recommended dosing with EFE soon after parturition. In summary, it seems that current ly available EFE are only capable of improving digestibility and animal performance in situations when normal fiber digestion is compromised. Theref ore, research on more effective EFE is needed to facilitate their use in production si tuations where digestion of fiber is not compromised by animal factors, but by forage factors like lignification. Method of providing EFE. The effects of EFE will be influenced by the different forms of providing EFE as described next. a) Time of application Little or no time is required for EFE to atta ch to substrates (Beauchemin et al., 2004). Lewis et al. (1996) reported no difference in response to applying EFE to diets immediately before feeding or 24-h earlier. Some in vitr o studies support this report (Colombatto, 2000), but others revealed that a 24-h EFE-pretreatment period was advantageous for improving forage digestibility (Krueger, 2007). Therefore, more research is needed in this area. b) Method of EFE application Spraying in liquid form on the feed is a more effective way of applying EFE than adding the powder form (Beauchemin et al., 2004). Infusing EFE in the rumen was not effective compared to spraying EFE on the f eed (Lewis et al., 1996). Beauchemin et al. (2004) stated that close association between the substrate and EFE is needed for EFE
59 to bind strongly enough and to prevent its removal and destruction by proteolytic activity in the rumen. Consequently, spraying is the most widely used method of applying EFE. A related question is which fraction of the diet should be treated with the EFE. Based on their higher cell wall concentration, the forage component should be the target; however, contradictory reports exist in the literature. Digestibility and milk production were improved when EF E was applied to the concent rate fraction instead of the TMR (Yang et al., 2000). Others found no difference between applying EFE to dry forage or dry forage plus concentrate (Yang et al., 1999; Dean, 2005). Such counterintuitive results may reflect the presence of non-fibrolytic activities in the EFE. c) Rate of enzyme supplementation An enzyme product that has the ideal enzymat ic activities for the target substrate can be ineffective if applied in excessive or insufficient amounts (Sanchez et al., 1996; Beauchemin et al., 2004). Kung et al. (2000) reported that cows fed a low level of enzyme tended ( P < 0.10) to produce more milk t han those fed a high level of enzyme. Others have mentioned that in vivo responses to increasing levels of dietary EFE are typically non-linear (Beauchemin, 2004). A possible explanati on was given by Nsereko et al. (2002), who documented a quadratic re sponse in total bacterial numbers in ruminal fluid with increasing levels of enzyme application. These authors speculated that application of a moderate level of EF E to ruminant feeds caused some beneficial disruption of the surface stru cture of the feed either before or after ingestion. Whereas the high EFE rate decreased this benefit because excess EFE attached to the feed and thereby restricted microbial adhesion and digestion. Adesogan (2005) proposed an
60 alternative explanation that negative feedback inhibition of enzymes by hydrolysis products may explain poorer effica cy at high application rates. Summary. This first half of this review discussed the C4 photosynthetic pathway of warm-season grasses, and the chemical and anatomical features that limit the digestibility of such forages by ruminants. The second half summarized the benefits and challenges of improving the quality of such forages wit h ammonia and subsequently discussed the mode of action of EFE and factors that have led to inconsistent effects of EFE on forage quality and animal performance. Mo st of the studies on EFE application to feeds involved addition of EFE to diets f ed to dairy cows or feedlot cattle. Little attention has been paid to using EFE to improve the performance of beef cattle fed warm-season grasses in cow-calf producti on systems that are common in the Southeast. Therefore the objective of this study was to evaluate the effect of applying an exogenous fibrolytic enzyme or anhy drous ammonia to bermudagrass hay [ Cynodon dactylon (L.) Pers.] on its nutritive value, vo luntary intake, digestion kinetics and the growth of beef cattle.
61 CHAPTER 3 EXOGENOUS FIBROLYTIC ENZYME OR ANHY DROUS AMMONIA EFFECTS ON THE NUTRITIVE VALUE, INTAKE, AND DIGESTION KINETICS OF BERMUDAGRASS AND THE GROWTH OF BEEF CATTLE Introduction Forages are the major feed source fo r ruminant animals and they represent approximately 61 and 83% of the ration of dairy and beef cattle in the US, respectively (Barnes and Nelson, 2003). In the southeaste rn US warm-season grasses are the basis of livestock production (Pitman, 2007). For Flor ida in particular, a ll beef cattle operations rely on forages as the primary source of nutrients (FASS, 2000). Warm-season grasses have lower nutritional value than cool-season grasses because they are less digestible and contain less CP (Minson, 1980). Several methods have been proposed to improve forage qualit y (Fahey et al., 1993) and anhydrous ammonia application is one of the most effect ive (Berger et al., 1995). Ammoniation has improved the nutritive value of cool(Wanapat et al., 1985; Flachowsky et al., 1996; Wang et al., 2004) and warm-season forages (Brown, 1988; Brown and Kunkle, 2003; Krueger et al., 2008) but it is not widely used because of it s cost, potential toxicity and caustic nature (Rotz and Shinners, 2007). Recent research has focused on using exogenous fibrolytic enzymes (EFE) to improve forage nutritive value and animal performance. Supplementing dairy cow and feedl ot cattle diets with EFE has improved cell wall digestion and animal performance (McA llister et al., 1999; Beauchemin et al., 2003; Arriola et al., 2007). Some EFE also have been used to enhance the digestibility of warm-season grasses (Dean, 2005; Krueger et al., 2008). However, results of EFE on forage nutritive value and animal performa nce have been equivocal (McAllister et al., 2001; Wang and McAllister, 2002; Adesogan, 2005) Thus, more research is needed to
62 develop consistently effective enzyme pr oducts and improved application strategies, particularly for warm-season forages. Krueger et al. (2008) reported that when applied at cutting instead of at hay bal ing or feeding, an EFE was mo re effective at improving the intake and digestibility of a 5-wk regrow th of bermudagrass hay. Limited information is available on whether EFE-induced incr eases in forage intake and digestion depend on forage regrowth interval. If EFE effi cacy is independent of regrowth interval, application to higher-yielding mature forages would be advisable. The first objective of this study was to evaluate the effect of applying an EFE or anhydrous ammonia on the nutritive value, voluntary intake, and digestion kinetics of bermudagrass [ Cynodon dactylon (L.) Pers.) hay harvested at two regrowth intervals. A second objective was to determine the effect of the treatments on the growth of beef cattle. The first hypothesis was that applic ation of anhydrous ammonia and EFE would improve the nutritive value and voluntary in take of bermudagrass hay and the growth of beef cattle. The second hypothesis was that ammoniation would be more effective in the more mature bermudagrass, whereas the EFE would be more effective in the less mature bermudagrass. Materials and Methods Forage Treatments An establis hed stand of Coastal bermudagrass ( Cynodon dactylon [L.] Pers) owned by a local hay producer in Alachua County, Florida was staged in August, 2007 by mowing to a 4-cm stubble and removing the residue. The field subsequently was fertilized with N (69 kg / ha) and the grass in each half of the field was allowed to regrow for 5 or 13 wk concurrently such that the respective harvest dates were 28 September and 17 November,, 2007. On eac h harvest date, the grass wa s mowed in 1 d to a 4-cm
63 stubble with a New Holland 617 mower conditioner (New Holland North America, New Holland, PA). Average precipitation (mm), temperature (C) and solar radiation (watts/m2) in Alachua during the growth periods we re obtained from University of Florida Forage Research Unit weather database (FAW N, 2007) and are shown in Figure 3-1. Mowed forage in two of every three wi ndrows was untreated (CON) and the third windrow was sprayed (10 g/ton) with an exogenous fibrolytic enzyme product (ENZ; Biocellulase A20, Loders Croklaan, Channa hon, IL) using a tractor-mounted 57-L continuous flow sprayer (FIMCO, North Siou x, SD) fitted with a th ree-nozzle boom. The enzyme had previously increased the in vi tro digestibility of bermudagrass (Dean, 2005) and intake and in vivo digestibi lity of bermudagrass in beef cattle (Krueger et al., 2008),. Endoglucanase and xylanase activities of the enzyme were determined under ruminal conditions (pH 6 and 39C), according to Colombatto and Beauchemin (2003), were 71.3 nmol of glucose released/min per mg a nd 206.5 nmol of xylose released/min per mg of enzyme powder, respectively. Hays were stored in round bales in a covered shed. For ammoniation, untreated round bales we re stacked on each other, covered with 0.15-mm plastic, and treated with anhydrous a mmonia (4% of DM) as described by Brown (1988) and Brown and Kunk le (2003). The forage was allowed to react with the ammonia for 6 wk and then vented to release ammonia gas. Experiment 1 Cattle and diets The Institutional Anim al Care and Use Committee of the University of Florida approved the animal protocol for this exper iment. Six yearling, ruminally cannulated Brangus steers (216 6 kg of BW) were de wormed with Ivomec (Merial, Duluth, GA)
64 and assigned to six treatments arranged in a 3 (additives) x 2 (regrowth intervals) factorial arrangement in a exper iment with a 6 x 6 Latin square design. Each of the six, 27-d periods consisted of a 14-d adaptation period, followed by 7-d of voluntary intake and digestibility measurements (total fece s collection method), 4-d of in situ degradability, 1-d without measurements and 1-d of rumen parameters. Steers were housed in individual 4 x 12 m pens in an open-sided barn equipped with continuous lighting. After storage for 4 mo, each hay bale wa s chopped to 15-cm lengths using a tub hay grinder (Roto Grind, model 760, Burrows enterprises, Greely, CO ). Steers were fed 110% of the previous days intake to a llow ad libitum intake at 1000 and 1600 h. A supplement formulated to meet the main tenance energy needs of a 250-kg steer gaining 0.58 kg/d (NRC, 2000) was fed in a separate container at 0900 h. The supplement consisted of 2 kg of sugar cane molasses (DM = 83%; CP = 11.7%), 0.8 kg of dried distillers grains with solubles (NDF= 37.3%; CP = 30%) and 0.06 kg of a mineral vitamin mix (Lakeland Animal Nutr ition, Lakeland, FL) containing 21% NaCl, 13% Ca, 6% P, 1% Mg, 0.95% Zn, 0.8% K, 0.4% S, 0.4% Fe, 0.22% Mn, 0.2% Cu, 800 mg/kg of Fe, 200 mg/kg of Co, 175 mg/kg of I, 48 mg/kg of Se, 45, 454 IU /kg of vitamin A and 9,091 IU /kg of vitamin D3 (DM basis). Feed grade ur ea (Southeastern Minerals Inc., Bainbridge, GA) containing 46% N was added and mixed (0.03 kg/hd/d) with the supplements of steers fed CON and ENZ hays to minimize the risk of N deficiency in the rumen. Sampling and analysis Feces were collected using fecal bags twice a day (0900 and 1700h) during the 7d digestibility trial. A subsample was taken after weighing and stored. Daily s amples of
65 hay, supplements, refusals, and feces taken dur ing the 7-d digestibili ty trial collection period were analyzed for DM ( 48 h at 60C). Dried samples were composited by steer within each period, ground to pass a 1-mm scr een using a Wiley mill (Arthur H. Thomas Company, Philadelphia, PA) and analyzed for ash (600C at 2 h, AOAC, 2000), NDF and ADF (Van Soest, 1991) using an ANKOM 200 Fiber Analyzer (ANKOM, Macedon, NY). Amylase was used for NDF analysis but sodium sulfite was not used. Hays also were analyzed for ADL according to Van Soest (1991) and NDF, NDS (neutral detergent solubles, 100-NDF), and ADL results were expressed on an organic matter basis. Cellulose (ADF minus ADL) and hemicel lulose concentrations (NDF minus ADF) and apparent digestibilities of DM, OM, NDF hemicellulose, ADF and cellulose were calculated. Crude protein concentration of ha ys and distillers grain were calculated as N x 6.25 after N concentration was determined using a Vario MAX CN Macro Elementar Analyzer (Elementar Analysensysteme GmbH, Hanau, Germany) by the Dumas combustion method (AOAC, 2000). Hay ADF re sidues were also analyzed for CP to estimate ADICP concentration. In situ ruminal degradability The in situ ruminal DM disappear ance of each hay was determined in triplicate on d 22-25 within steers consuming the same hay in that period. Hay samples taken representatively at the beginn ing of each period were dried for 48 h at 60C, ground to pass a 4-mm screen using a Wiley mill and weighed (5 g DM) into 10 x 20 cm ANKOM R1020 in situ bags (ANKOM, Macedon, NY), which were tied with rubber bands. The bag pore size was 50 m and the sample size to free bag surface area ratio was 12.5 mg/cm2. Bags were attached to a rope with clips, placed in the ventral sac of the rumen for 0, 3, 6, 9, 12, 18, 24, 48, 72, a nd 96 h and removed simultaneously. Upon removal
66 from the rumen, bags were washed with cold water to re move adherent particles and bacteria and then washed in a commercial washing machine (Kenmore, Benton Harbor, MI) using a cool-wash cycle without soap. Wa shed bags were dried for 48 h at 60C and weighed. The model of Me rtens (1977) was fitted to the DM degradation data using the NLIN procedure of SAS, version 9.1 (2009, SAS Inst., Inc., Cary, NC). The model is of the form: R(t) = Di x (e-kd(t-L))+Io where R(t) = Total indigested residue at any time t, Di = potentially digestible fraction, kd = fractional rate of digestion of Di, t = time incubated in the rumen in h, L = discrete lag time in h, and Io = fraction not digested after 96h of incubation. Effective degradability was calculated using the Orskov and McDonald (1979) formula: Effective degradability = A + D x [kd / (kd + kp)] Where A = washout fraction, D = potentially digestible fraction, kd = fractional rate of digestion, kp = assumed ruminal passage rates of 0.046,.according to Pond et al. (1989) Rumen fermentation parameters Samples (100 ml) of ruminal fluid were taken by aspiration at 0800 h (1 h before feeding) and hourly thereafter until 2000 h on d 27. The samples were immediately filtered with 2 layers of cheese cloth, and analyzed for pH using a calibrated electrode (Accumet, model HP71, Fisher Scientific, Pittsburgh, PA). Subsequently, the ruminal fluid was acidified to pH < 2 with 50% H2SO4 and then frozen at -40C for further analysis. Thawed samples were centrif uged at 8000 x g for 20 min at 4C and the supernatant was analyzed for VFA and lactate (Muck and Dickerson, 1988) using a High Performance Liquid Chromatograph (Hitac hi, FL 7485, Tokyo, Japan) coupled to a
67 UV detector. Ammonia-N c oncentration was measured usi ng an adaptation of the Noel and Hambleton (1976) procedure that involved colorimetric N quantification on a Technicon Auto Analyzer (Technicon, Tarrytown, NY). Ruminal fluid volume and liquid rate of passage Ruminal fluid volume and dilution rate were measured on d 27 using samples taken at the same time as those anal yzed for VFA. Cobalt EDTA was prepared according to Uden et al. (1980). Twenty eight grams of a cobal t chelate (4 g of Co) were diluted in 1 L of distilled water and dosed into the rumen. Rumen contents were hand thoroughly mixed to aid equilibrat ion of the marker. Ruminal fluid samples were taken 3 min later and processed in the same way as those reserved for VFA analysis. The cobalt concentration of the ruminal fluid supernatant was measured with a Perkin Elmer 5000 (Wellesley, MA) atomic absorption spectrom eter. Ruminal fluid di lution rates were calculated as the slope of the natural logarithm of cobalt concentration on time postdosing and are expressed as percentage of volume/hour. Ruminal fluid volumes were calculated by dividing the Co dose by the extrapolated Co concentration at time 0 (dosing). Experiment 2 The hays and supplements used in Experiment 1 also were used in Experiment 2. Ninety Angus and Brangus steers (308 37 kg ) were stratified by weight, dewormed with Ivomec and randomly allocated to 18 1.01-ha pens (5 steers per pen) containing dormant bahiagrass ( Paspalum notatum Fl gge) pasture. Forage mass was not measured in the pens because the bahiagrass was minimal and dormant during the experiment. Three pens were assigned rando mly to each treatment. The CON, ENZ, and AMN hays from both regrowth intervals were weighed and fed in round-bale
68 feeders to cattle in respective pen s in quantities sufficient to ensure ad libitum access for 55 d. Steers were adapted to diets for 6 d, and full body weights were obtained on two consecutive days at the beginning (d 7 and 8) and end (d 54 and 55) of the measurement period. Weekly supplement a llocations were fed in one open trough in each pen in equal amounts three times per week and they were consumed completely. Refused hay was collected and weighed on d 30 and 55. Statistical Analysis Data from Experiment 1 were analyzed usin g the MIXED procedure of SAS v9.1 (2009, SAS Inst., Inc., Cary, NC). The model used to analyze intake and digestibility data was: Yijkl = + i + j + ij + rk + cl + ijkl Where: = general mean i = effect of additive i j = effect of regrowth interval j ij = effect of additive i regr owth interval j interaction rk = effect of period k cl = random effect of steer l ijkl = experimental error A similar model that excluded the steer effect was used to analyze hay composition data. Rumen fe rmentation data was analyzed wit h the GLIMMIX procedure of SAS and a repeated measures statement that used the autoregressive [ar(1)] covariate structure. The model was similar to the intake model but included effects of time and interactions with time.
69 Experiment 2 had a completely randomized, split-plot design. The main and subplots were the additive regrowth interval interaction and breed, respectively, and pen was the experimental unit. The initial and fi nal weight and ADG were analyzed using the MIXED procedure of SAS v9.1 and the model was: Yijkl: + i + j + ij + k + ik + jk + ijk + ijk Where: = general mean i = effect of additive i j = effect of regrowth interval j ij = effect of additive i regr owth interval j interaction k = effect of breed k ik = effect of additive i breed k interaction jk = effect of regrowth inte rval j breed k interaction ijk = three way interaction effect of additi ve i, regrowth interval j, and breed k ijkl = error effects Main plot (additive regrowth interval) error was additive regrowth interval and sub-plot error was the residual. A simila r model was used for estimating treatment effects on intake and gain to feed ratio (G:F ) but the breed effect and its interactions were omitted because steers were group-fed in pens. Contrast statements were used to determi ne the effects of en zyme treatment (ENZ vs. CON), ammoniation (AMN vs. CON), and regrowth interval (5 vs. 13 wk) and the interactions of regrowth interval with the respective additives. Significance was declared at P < 0.05, and only significant interactions effects are discussed.
70 Results and Discussion Nutritional Composition The 5-wk hay had greater ( P < 0.001) concentrations (% DM) of NDF (77.7 vs. 72.7), hemi cellulose (36.8 vs. 33.4), ADF (40.9 vs. 39.4), ce llulose (34.4 vs. 32.4) and CP (14.3 vs. 11.5) and less ( P < 0.001) NDS (Neutral deter gent solubles) (22.3 vs. 27.3), ADL (6.42 vs. 6.98) and ADICP ( P = 0.03; 9.82 vs. 10.37 % of total CP) than the 13-wk hay (Table 3-1). Although fiber concentr ations often increase with plant maturity, higher fiber concentrations in bermudagrass wit h shorter regrowth intervals compared to longer regrowth intervals have been reported in the literature on some occasions, as is the case in this study (Joliff et al., 1979; Mandebvu et al., 1999 and Scarbrough et al., 2006). Chronological age effects on bermudagr ass nutritional value can be confounded with environmental conditions influence on plant growth and development changes during growing period (Joliff et al., 1979). In this experiment, differences between the average temperatures during the growth of the 5and 13-wk hays (25.2 vs. 21.7oC; Figure 3-1) partly explain the greater fiber concentrations of the 5-wk hay. As reported in other studies on tropica l grasses (Krueger et al. 2008), enzyme application had no effect on fiber fractions or lignin. Ho wever, enzyme application increased CP concentration of the 5-wk but not the 13-wk hay (enzyme x regrowth interval interaction, P = 0.03) but had contrasting effe cts on ADICP of 5and 13-wk hays (enzyme regrowth interval interaction, P = 0.004). The enzyme used in this study had increased the CP concentration of guineagrass hay (Panicum maximum Jacq.; Tous et al., 2006) but had no effect on CP of bermudagrass hay (Krueger et al., 2008). The mechanism by which enzyme application increases CP concentration is unclear particularly because quantities added in the form of the enzyme were minute.
71 Ammoniation resulted in reduced NDF, hemicellulose, ADICP, and ADL concentration and greater, NDS, cellulose, a nd CP concentrations and these effects were more pronounced in the 13-wk hay (regr owth interval ammonia interaction, P < 0.05). Others have reported t hat the efficacy of ammoniat ion increased with maturity (Fahey et al., 1993; Brown and Kunkle, 2003; Sollenberger et al., 2004). The reduction in NDF by ammoniation reflects solubilizat ion of hemicellulose (Saenger et al., 1983; Haddad et al., 1995) and hydroxycinnamic co mpounds (Fahey et al., 1993; Chesson, 1993; Wang et al., 2004) in the alkali due to breakage of ester bonds. As reported previously (Haddad et al., 1995; Krueger et al., 2008), ammoni ation did not affect ADF concentration yet it reduced ADL concentration particularly for the 13-wk hay (regrowth interval ammonia interaction P = 0.04). Similar effects of ammoniation on lignin have been attributed to solubilization of -aryl ether linkages within the lignin macromolecule (Chesson, 1993). The increase in CP caused by ammoniation agrees with other studies (Wanapat et al., 1985; Flachowsky et al., 1996; Krueger et al., 2008). The reason for the greater CP response to am moniation in the more mature forage is unclear but the outcome is particularly beneficial for rumi nants fed warm-season grasses, which have greater yields and lower CP concentrations as they mature (Mos er et al., 2004). Voluntary Intake Intakes (kg/d) of total diet DM, OM, CP, NDF, hemicellulose, ADF and cellulose ( P 0.008) were greater by steer s fed the 5-wk than those fed the 13-wk hay (8.0 vs. 7.5; 7.3 vs. 6.9; 1.3 vs. 1.1, 4.3 vs 3.7; 2.1 vs. 1.8, 2.2 vs. 1.9 and 1.8 vs. 1.6, respectively; Table 3-2). Intakes of hay DM, OM, CP NDF, hemicellulose, ADF and cellulose ( P 0.008) were greater in the 5-wk than the 13-wk hay (5.1 vs. 4.6; 4.9 vs. 4.4; 0.7 vs. 0.5, 4.0 vs. 3.4, 1.9 vs. 1.6, 2.1 vs. 1.8, 1.8 vs. 1.5 respectively). Enzyme application did not
72 affect these intake measures but ammoniation decreased ( P 0.05) total intakes of DM, OM, NDF and hemicellulose (7.6 vs. 8.0, 6. 9 vs. 7.3, 3.7 vs. 4.2 and 1.7 vs. 2.1, respectively). Ammoniation also decreased ha y NDF and hemicellulose (3.4 vs. 3.9 and 1.5 vs. 1.9) and tended ( P < 0.08) to decrease intake of total ADF (2.0 vs. 2.1) and hay DM, OM and ADF (4.7 vs. 5.1, 4.5 vs. 4.9 and 1.9 vs. 2.0). Ammoniation increased total and hay CP intake ( P < 0.001; 1.3 vs. 1.1 and 0.8 vs. 0.5). In other studies, ammoniation of hay increased (Flachowsky, 1996; Krueger et al., 2008) or did not affect (Ward and Ward, 1987; Wang et al., 2004) DMI. However, am moniation has decreased hay intake particularly when applied at rate s of 3% or greater to forages with 30% moisture or more due to the strong smell and reduced acceptability of the ammoniated hay (Streeter et al., 1981; Grovum and Chapman, 1988; Brown and Kunkle, 2003). The use of partially enclosed feedbunks that conc entrated the odor of the ammoniated hay may have caused the adverse effect s of ammoniation on intake. In vivo apparent digestibility Digestibility (%) of CP, NDF, hemicellu lose, ADF and cellulose were greater ( P 0.002) in the 5-wk hay compared to the 13-wk hay (67.5 vs. 64.6, 58.1 vs. 51.2, 69.8 vs. 63.2, 46.8 vs. 40.2 and 58.3 vs. 50.9; Table 3-3). Howeve r, there was no difference between 5wk and 13-wk DM and OM digestibilities. Enzyme application did not affect digestibility (%) of DM, OM, CP, ADF and cellulose but increased NDF and hemicellulose digestibility of the 5-wk hay (57.8 vs. 53.2 and 69.3 vs. 62.3, respectively) and decreas ed that of the 13-wk hay (46.7 vs. 49.6 and 56.7 vs. 61.1; enzyme regrow th interval interaction, P 0.009). When the same enzyme was applied to a 5-wk regrowth of Coastal bermudagrass hay, digestibility of NDF by steers also was increased (Krueger et al., 2008). The improvement in NDF-
73 digestibility may have only occurred in t he 5-wk hay because it contained less lignin than the 13-wk hay. The response probably reflects a pre-ingestive hydrolytic effect of the enzyme on the forage. Ammoniation increased digestibility ( P 0.007) of DM (66.9 vs. 63.2%), OM (66.4 vs. 62.5%), NDF (60.3 vs. 51. 4%), hemicellulose (74.8 vs 61.7), ADF (47.7 vs. 41.2%) and cellulose (61.0 vs. 50.5). Ammoniatio n cleaves ester linkages and solubilizes hydroxycinnamic acids, hemicellulose, and ac etyl groups in the cell wall (Wang et al., 2004), thereby disrupting the lignin-hemicellulose matrix and facilitating cell wall degradation by microbial enzymes (Jung and Allen, 1995). Greater ADF and cellulose digestibility with ammoniation probably reflect reduced encrustat ion of cellulose by lignin and increased exposure of t he cellulose polymer to mi crobes (Fahey et al., 1993). However, ammoniation decreased ( P < 0.001) CP digestibility (62.2 vs. 67.5), which contradicts the results of Kr ueger et al. (2008) on a 5 wk r egrowth of bermudagrass hay. These different responses may be due to use of a lower ammoniation rate (3% of hay DM) in the latter study. In Situ Rumen Digestion Kinetics Discrete lag times were unaffected by additiv es but were longer ( P < 0.001) for the 5-wk (2.8 h) compared to the 13-wk hay (1 .5 h; Table 3-4). Lag time is influenced by factors such as the rate of hydration of the substrate, microbial attachment, and nutrient limitations (Lopez, 2005). The lower concentration of the digestible, nutrient-rich NDS fraction in the 5-wk hay likely reduced the rate of substrate colonization causing a longer lag phase. Compared to values for the 13 wk hay, in situ DM digestion of the 5 wk hay was less ( P < 0.05) during the first 6 h of digestion, sim ilar at 12, 18, and 24 h, and greater
74 ( P < 0.05) between 36 and 96 h (Figure 3-2; Table 3-5). Enzyme application did not affect in situ DM digestion but am moniation consistently increased ( P < 0.05) in situ digestion and the response was greater in the 13-wk hay during the first 48 h of incubation (ammonia regrowth interval interaction, P < 0.05). The 5-wk hay contained less (P < 0.05) soluble (12.7 vs. 13.6%) and indigestible (40.9 vs. 43.5%) fractions and more ( P < 0.001) of the potentiall y digestible fraction (46.4 vs 42.9%) than the 13-wk hay. These results reflect the greater NDF and ADF concentrations and lower lignin concentration of the 5-wk hay (Table 3-1). As in the study of Krueger et al. (2008) enzyme application to bermudagrass hay, had no effect on in situ digestibility of DM. Mc Allister (2001) stated t hat in most studies, enzymes have not increased the potentially diges tible fraction, but they have increased the rate of digestion (Feng et al., 1996). Such increases in the digestion rate occurred mainly in reports on cool-season or temper ate forages, which have considerably lower NDF concentrations than warm-season grasses. Ammoniation increased the soluble fracti on and effective degradability of the hays to a greater extent in the 13-wk versus 5-wk hay (ammonia regrowth interval interaction, P = 0.002). Ammoniation also increased ( P < 0.001) the potentially digestible (50.3 vs. 42.7%) fraction and decreased ( P < 0.001) the indigestible fraction (34.6 vs. 45.3%) of 5 and 13-wk hays, whereas it only increased the degradation rate of the 13-wk hay (ammonia regrow th interval interaction, P = 0.04). Ammoniation may have increased the soluble fraction by w eakening the physical structure and properties of the cell wall (Harbers, 1982) thereby increasing its fr agility (Selim, 2002, 2004). Effects on the potential and indigestible fractions likely reflect hydrolysis of ester and
75 ether linkages and solubilization of cell wall components (Chesson, 1993). The greater degradation rate and effective degradability responses to ammoniation of the 13-wk hay agree with reports that the alkali is more effective on more mature, lignified forages (Fahey et al., 1993; Brown and Kunkle, 2003; Sollenberger et al., 2004). Rumen Fermentation Parameters Ruminal pH was unaf fected by regrowth in terval or enzyme or ammonia treatment (Table 3-6). When applied to forages, enzymes have not affected ruminal pH (Lewis et al., 1996; Yescas-Yescas et al., 2004; Dean, 2005). Also, ammoniation of hay has not affected ruminal pH in previous studies (Z orrila-Rios, 1985; V agnoni et al., 1995). Ruminal ammonia concentration was no t affected by maturity or enzyme application. In contrast, ammoniation increased ( P = 0.004) ruminal ammonia-N concentration (15.1 vs. 11.4 mg/dl) when com pared to control, reflecting increased supply of ruminally available N from the ammoniated hay (Fahey et al., 1993). In agreement with Vagnoni et al. (1995), ammoniation reduced the initial rate of release and subsequent rate of decrease in ruminal ammonia-N concentration (Figure 3-3; treatment time interaction, P < 0.001). A more gradual release of ruminal ammonia may benefit rumen microbes due improved stability of N supply. Mean ruminal ammonia-N concentrations of all diets exc eeded the level (5 mg /dl; Satter and Slyter, 1974) that limits microbial protein synthes is. However, those of control and enzyme diets were slightly below the threshold prio r to feeding the concentrate in the morning and after feeding the hay in the evening due to the rapid solu bilization of the supplemented urea.. Enzyme application and regrowth interval did not affect total VFA concentration or VFA molar proportions except t hat the 5-wk hay had more ( P = 0.002) acetate (58.3 vs.
76 56.5 mol / 100 mol) and less ( P = 0.001) butyrate (16.1 vs. 17 .8) than the 13-wk hay, reflecting the greater NDF concentration and digestibility of t he 5-wk hay. Higher acetate proportions and lower butyrate proportions also were reported when less mature tall fescue hay was fed to steers in stead of mature hay (Fieser and Vanzant, 2004). Relatively high proportion of butyrate in ruminal fluid in this study reflect the fermentation of the supplementary molasses (Wing et al., 1988) and distillers grains (Leupp et al., 2009) in the steer diets. Ammoniation increased ( P = 0.02) total VFA concentration (155.4 vs. 144.3 mM) as repor ted previously in beef cattle fed ammoniated bermudagrass hay (Wyatt et al., 1989) or tall fescue (Chestnut et al., 1987). Such results are probably a consequenc e of greater digestion and fermentation of ammoniated hay. Ammoniation also decreased ( P = 0.006) the molar proportion of propionate (17.9 vs. 19.1) and increased ( P = 0.02) the acetate to propionate ratio (3.4 vs. 3.2). Ruminal Fluid Volume and Liquid Rate of Passage Ruminal fluid volume, dilution rate, turnover time and passage rate were unaffected by additive treatment or regrowth i nterval (Table 3-7). Judkins and Stobart (1988) noted that enzyme treatment of an alfalfa hay based diet did not affect rate of dilution or ruminal fluid volume in lambs. In addition, Beauchemin et al. (1999) reported that enzyme treatment of a barley silage ba sed TMR did not affect liquid passage rate in dairy cows. However, Zorrilla-Rios (1985) reported that ammoniat ion of wheat straw increased liquid passage rate in steers. Ammoniation may have not increased liquid passage rate in this study because it reduced DMI and tended ( P = 0.13) to reduce ruminal fluid volume. Rate of fluid passage val ues (%/h) observed in this trial (14 15.7) were relatively high but similar to those of beef steers fed old world bluestem
77 ( Bothriochla inermedium ) (14.7 %/h; Coleman et al., 1984) or tall fescue ( Festuca arundinacea ) plus soy hulls (13 %/h; Richards et al., 2006) for ad libitum intake. Growth Performance of Steers Enzyme treatment only increased hay and total DMI when the 5-wk hay was fed (enzyme regrowth interval interaction, P < 0.007; Table 3-8). In contrast, ammoniation increased hay and total DMI at both regrowth intervals but the increase was greater by steers fed the 5-wk hay (ammonia x regrowth interval interaction, P < 0.05). Ammoniation also tended ( P = 0.11) to increase final BW (347 vs. 342 kg) and ADG (0.79 vs. 0.70 kg/d), but enzyme treatment had no effect on measures of growth. Ammoniation has improved the ADG of beef cows and steers fed low quality forages (Ward and Ward, 1987; Brown,1988; Flachowsky et al., 1996) by improving intake and digestion (Flachowsky et al., 1996), w hereas, enzyme application has had equivocal effects on the intake, digestion, and ADG of ruminants fed low quality forages (Beauchemin et al., 1995; Robison et al., 2001; Krueger et al., 2008). Ammoniation did not affect gain to f eed ratio, but enzyme application tended to reduce the ratio in the 5-wk hay and increase it in the 13-wk hay. As in this study, Krueger et al. (2008) report ed greater intake of enzym e-treated 5-wk hay without a corresponding ADG increase. These results suggest that the increased nutrient supply resulting from enzyme treatment was insufficient for improving gain or was used inefficiently. Therefore, future studies on enzyme treatment of forages or diets should estimate nutrient partiti oning and retention. Differences in intake responses to treatments between Experiments 1 and 2 reflect the different feeding strategies used. Contrasting intake responses to ammoniation reflect use in Experiment 1 of feedbunks that concentrated the ammonia odor to a much
78 greater extent than the hay ring feeders used in Experiment 2. Th at enzyme treatment increased intake of the 5-wk hay in Experiment 2, but not in Experiment 1, suggests that chopping hays before they were fed in Exper iment 1 may have reduced benefits of enzyme treatment because choppi ng forages increases DMI (F ahey et al., 1993; Berger et al., 1995). Conclusions The 5-wk hay had more NDF, hemicellulose, ADF, cellulose, and CP and les s lignin than the 13-wk hay, but the NDF, hem icellulose, ADF, cellulose and CP digestibility of the 5-wk hay was greater. Ammonia treatment improved the nutritional composition of bermudagrass hay by reducing NDF, hemicellulose, and lignin concentrations and increasing cellulose and CP concentrations particularly in the 13-wk hay. Ammoniation increased DMI in the pastu re-based experiment but reduced hay DMI in the confined feeding experiment likely du e to concentration of the ammonia odor. Ammoniation improved the extent of total tract apparent digestibility and extent and rate of DM digestion of the hays in the rum en and tended to increase the ADG and final BW of steers. Except for slightly increasing CP concent ration, enzyme treatment did not improve the nutritive value or in situ rumen diges tion of bermudagrass hay. However, enzyme treatment increased the NDF and hemicellulose digestib ility of the 5-wk hay and increased the DMI of this hay wh en offered at pasture, but this did not result in improved growth performance. As hy pothesized, ammoniation ha d more pronounced effects on the more mature (13 wk) hay, whereas enzym e application only resulted in beneficial effects when applied to the 5-wk hay.
79 Table 3-1. Chemical composition of untreated (CON), enzyme-treated (ENZ) and ammoniated (AMN) hay harvested at two regrowth intervals (RI) (n=6) Component, % of DM 5-wk regrowth 13-wk regrowth SE P values CON ENZ AMN CON ENZ AMN RI ENZAMN ENZRI AMNRI OM 95.6 94.7 95.6 95.5 95.7 95.6 0.1 0.006 0.010.9 <0.001 0.7 NDS 21.4 20.9 24.7 25.0 25.3 31.4 0.5 <0.0010.84<0.0010.40 0.004 NDF 78.6 79.2 75.2 75.0 74.7 68.6 0.5 <0.0010.84<0.0010.40 0.004 Hemicellulose 38.1 38.4 34. 1 35.7 35.3 29.1 0.5 <0. 0010.95<0.0010.47 0.01 ADF 40.6 40.8 41.2 39.3 39.4 39.5 0.3 <0.0010. 660.17 0.85 0.43 Cellulose 34.1 34.3 34.9 32.0 32.1 33. 1 0.2 <0.0010.43<0.0010.98 0.47 ADL 6.47 6.49 6.29 7.32 7.23 6.40 0.2 <0.0010.820.003 0.73 0.04 CP 12.2 12.9 17.8 9.1 9.2 16.3 0. 2 <0.0010.01<0.0010.03 <0.001 ADICP (% of CP) 10.1 9.9 9.4 10.9 11.9 8.3 0. 3 0.03 0.20<0.0010.04 0.004 SE values represent the variability of sa mples collected daily for 7d from hay offe red to each steer and composited by period.
80 Table 3-2. Effect of treating bermudagrass hay harvested at two regrowth intervals with a fibrolytic enzyme or ammonia on the voluntary intake of steers Item 5-wk regrowth 13-wk regrowth SE P values CON ENZ AMN CON ENZAMN RI ENZ AMN ENZx RI AMNx RI Total Net intake, kg/d DM 8.12 8.10 7.81 7. 80 7.457.29 0.24 0.007 0. 34 0.05 0.43 0.62 OM 7.47 7.40 7.16 7.16 6.836.67 0.23 0.008 0.29 0.05 0.50 0.63 CP 1.19 1.22 1.35 1.00 0.971.20 0.03 <0.0010.90 <0.0010.20 0.28 NDF 4.42 4.45 4.03 3. 98 3.713.34 0.18 <0.001 0.39 0.002 0.31 0.40 Hemicellulose 2.21 2.24 1. 90 1.96 1.831.52 0.09 <0. 0010.48 <0.0010.26 0.34 ADF 2.21 2.20 2.13 2.02 1.881.83 0.09 <0.0010. 33 0.08 0.37 0.48 Cellulose 1.86 1.85 1.80 1.64 1.531. 54 0.08 <0.0010.38 0.23 0.45 0.72 Hay intake, kg/d DM 5.25 5.22 4.96 4. 92 4.574.44 0.24 0.007 0. 35 0.06 0.43 0.62 OM 5.01 4.94 4.74 4.70 4.374.24 0.23 0.008 0.29 0.06 0.50 0.63 CP 0.64 0.67 0.89 0.45 0.420.74 0.28 <0.0010.90 <0.0010.20 0.28 NDF 4.12 4.15 3.73 3. 68 3.413.04 0.19 <0.001 0.39 0.002 0.31 0.40 Hemicellulose 2.00 2.03 1. 68 1.74 1.621.30 0.09 <0. 0010.48 <0.0010.26 0.34 ADF 2.13 2.12 2.04 1.94 1.791.75 0.10 <0.0010. 33 0.08 0.37 0.48 Cellulose 1.79 1.78 1.73 1.57 1.461. 47 0.08 <0.0010.38 0.23 0.45 0.72
81 Table 3-3. Effect of treating bermudagrass hay harvested at two regrowth intervals with a fibrolytic enzyme or ammonia on in vivo apparent digestibility by steers Digestibility, % 5-wk regrowth 13-wk regrowth SE P values CON ENZ AMN CON ENZ AMN RI ENZ AMN ENZxRI AMNxRI DM 63.0 64.9 67.4 63.3 62.9 66.4 0. 860.14 0.31 <0.0010.14 0.40 OM 62.4 64.3 67.0 62.7 62.4 65.8 0. 880.16 0.31 <0.0010.15 0.32 CP 68.1 70.2 64.0 66.9 66.6 60.3 0. 80<0.0010.30 <0.0010.15 0.14 NDF 53.2 57.8 63.2 49.6 46.7 57.4 1. 43<0.0010.46 <0.0010.004 0.35 Hemicellulose 62.3 69.3 77.9 61.1 56.7 71. 6 2.54<0.0010.50 <0.0010.009 0.20 ADF 44.1 46.0 50.1 38.3 36.9 45.3 2.430.002 0.91 0.007 0.45 0.82 Cellulose 54.8 57.6 62.4 46.2 47.0 59.6 1.80<0.0010.29 <0.0010.55 0.09
82 Table 3-4. Effect of treating bermudagrass hay harvested at two regrowth intervals with a fibrolytic enzyme or ammonia on kinetics of in situ ruminal DM digestion. Measure 5-wk regrowth 13-wk regrowth SE P values CON ENZ AMN CON ENZAMN RI ENZ AMN ENZxRI AMNxRI Lag phase, h 2.7 2.7 3.0 1.2 1.3 2.0 0.4 <0.001 0.84 0.12 0.91 0.41 Soluble fraction, % of DM 12.1 12.3 13.8 11.8 12.616.4 0.4 0.02 0.23 <0.001 0.48 0.002 Potentially digestible fraction, % of DM 43.9 43.3 52.1 41.5 38.848.4 1.2 <0 .001 0.12 <0.001 0.32 0.52 Fractional digestion rate, %/h 6.12 6.74 6.08 5.56 5.897.43 0.48 0.96 0.28 0.05 0.74 0.04 Indigestible fraction, % of DM 44.1 44.5 34.1 46.6 48.635.2 1.0 <0 .001 0.18 <0.001 0.38 0.39 Effectivea degradability 37.1 37.9 43.3 34.2 34.446.1 0.9 0.07 0.53 <0.001 0.65 0.002 aEffective degradability calculated assu ming a passage rate of 0.046.
83 Table 3-5. Effect of treating bermudagra ss hay harvested at two regrowth interval s with a fibrolytic enzyme or ammonia on in situ ruminal DM digestion a fter different incubation periods. Hour 5-wk regrowth 13-wk regrowth SE P values CON ENZ AMN CON ENZAMN RI ENZ AMN ENZxRI AMNxRI 0 12.1 12.3 13.8 11.8 12.616.4 0. 4 0.02 0.23 <0.001 0.48 0.002 3 14.2 14.3 16.0 15.4 16.320.0 0.5 <0.001 0.19 <0.001 0.36 0.003 6 19.9 20.7 22.4 21.3 22.028.0 0. 9 0.001 0.30 <0.001 0.99 0.007 12 31.0 32.2 35.6 30.3 30.741.2 1. 0 0.12 0.32 <0.001 0.65 0.002 18 38.6 39.9 44.8 36.6 36.949.6 1. 2 0.91 0.44 <0.001 0.58 0.003 24 43.9 45.1 51.2 41.2 41.254.9 1. 2 0.24 0.56 <0.001 0.54 0.003 36 50.1 50.8 58.6 46.8 46.360.5 1. 1 0.01 0.90 <0.001 0.50 0.006 48 53.1 53.4 62.3 49.7 48.962.9 1.0 0.001 0.70 <0.001 0.46 0.02 72 55.3 55.1 65.0 52.2 50.864.4 1.0 <0.001 0.32 <0.001 0.44 0.13 96 55.8 55.5 65.7 53.0 51.364.7 1.0 0.001 0.23 <0.001 0.41 0.27
84 Table 3-6. Effect of treating bermudagrass hay harvested at two regrowth intervals with a fibrolytic enzyme or ammonia on rumen fermentation parameters Parameter 5-wk regrowth 13-wk regrowth SE P values CON ENZ AMN CON ENZ AMN RI ENZ AMN ENZxRI AMNxRI pH 6.54 6.53 6.44 6.50 6.53 6.52 0. 060.70 0.87 0.36 0.65 0.19 Ammonia (mg/dl) 11.4 12.2 16.3 11.5 10.9 13.9 1.5 0.26 0.91 0.004 0.55 0.33 Total VFA (mM) 142.7 147.0 161.7 145.9141.3149.1 5.9 0.20 0.98 0.02 0.33 0.10 VFA (mol / 100 mol) Acetate 58.3 58.3 58.3 56. 0 56.3 57.3 0.8 0.002 0. 85 0.39 0.81 0.35 Propionate 19.0 19.1 17.7 19.1 19.8 18.1 0.5 0.24 0.38 0.006 0.53 0.69 Butyrate 15.7 15.4 17.3 18.1 17.2 18. 2 0.7 0.001 0.34 0.19 0.64 0.23 Isobutyrate 3.1 3.5 3.0 3.0 2.9 3.2 0.2 0.25 0.41 0.93 0.26 0.36 Isovalerate 3.8 3.7 3.8 3.8 3.7 3. 3 0.2 0.43 0.65 0.24 0.89 0.22 Acetate : Propionate 3.2 3.2 3.4 3.1 3.0 3.3 0.1 0.17 0.55 0.02 0.91 0.80
85 Table 3-7. Effect of treating bermudagrass hay harvested at two regrowth intervals with a fibrolytic enzyme or ammonia on ruminal fluid volume and fluid kinetics Measure 5-wk regrowth 13-wk regrowth SE P values CON ENZAMN CON ENZAMN RI ENZ AMN ENZxRI AMNxRI Ruminal fluid volume, L 45.1 48.1 38.1 45.2 48.342.9 3. 90.48 0.30 0.13 0.99 0.42 Dilution rate, %/h 15 14.4 15.7 14 14. 314.6 1.20.37 0.87 0.51 0.64 0.97 Rate of fluid passage, L/hr 6.6 6.4 5.9 6.3 6.5 6.1 0. 50.96 0.94 0.21 0.51 0.42 Turnover time, h 6.8 7.4 6.5 7.4 7.4 7.0 0.70.41 0.60 0.51 0.59 0.93
86 Table 3-8. Effect of treating bermudagrass hay harvested at two regrowth intervals with a fibrolytic enzyme or ammonia on growth performance of steers Parameter 5-wk regrowth 13-wk regrowth SE P values CON ENZ AMN CONENZAMN RI ENZ AMN ENZxRI AMNxRI Total DMI 6.1 6.8 7.1 6.9 6.7 7.2 0.150.040.10 <0.0010.007 0.02 Hay DMI 3.5 4.2 4.3 4.2 4.0 4. 5 0.120.020.05 <0.0010.003 0.03 Initial wt, kg 305 306 306 309 306 311 3.3 0.27 0.73 0.70 0.56 0.79 Final wt, kg 343 341 346 340 341 348 3.9 0.98 0.84 0.11 0.60 0.42 ADG, kg/d 0.77 0.70 0.82 0.64 0. 730.77 0.060.230.84 0.11 0.18 0.50 Gain:feed ratio 0.12 0.10 0.12 0.09 0.110.11 0.010.12 0.82 0.49 0.10 0.27
87 Figure 3-1. Temperature (C), rain fall (mm) and solar radiation (watts/m2) during the growing period of the hays (Florida automated weather network, 2009). 0.0 10.0 20.0 30.0 AugustSeptemberOctoberNovemberTemperature (C) 0 50 100 150 AugustSeptemberOctoberNovemberRainfall (mm) 2800 2850 2900 2950 3000 AugustSeptemberOctoberNovemberSolar Radiation (watts/m2)
88 Figure 3-2. In situ DM digestibility of 5and 13-wk regrowth of bermudagrass hay that were untreated (C) or treated wit h enzymes (E) or ammonia (A).* and + indicate significant effects of maturity and ammoniat ion at that incubation time.
89 Figure 3-3. Effect of treating bermudagrass ha y harvested at two regrowth intervals with a fibrolytic enzyme or ammonia on ru minal-N ammonia concentrations of steers.. Error bars indicate the 95% c onfidence interval of the means; treatment time interaction, P < 0.001. Arrows labeled C and H indicate times when concentrate and hay were fed respectively.
90 CHAPTER 4 GENERAL SUMMARY AND RECOMMENDATIONS In the southeastern U.S., warm-season gras ses are the basis of livestock production (Pitman, 2007), but t hey have intrinsically low nutritional value. Several methods have been proposed to improve their quality, and ammonia application is one of the most effective. Howe ver, ammoniation is costly, potentially toxic and caustic, therefore it is not used widel y. Recent research has focused on enzyme treatments to improve forage quality but this effort has had inconsistent results and has been used primarily on cool-season grasses. Consequent ly, two experiments were initiated to evaluate the effect of applyi ng an exogenous fibrolytic enzyme or anhydrous ammonia to Coastal bermudagrass hay harvested as 5or 13-wk regrowths on forage nutritive value and growth of beef cattle. In Experiment 1, 6 ruminally-cannulat ed Brangus steers (BW 216 6 kg) were used in an experiment with a 6 x 6 Latin squar e design with a 3 (additives) x 2 (regrowth intervals) factorial arrangement of treatm ents. Each period c onsisted of 14 d of adaptation, 7 d of digestibilit y measurements, 4 d of in si tu degradability, 1 d of rumen rest and 1 d of ruminal fluid ferment ation measurements. Steers were housed individually and fed hay ad libitum. A supplem ent consisting of sugar cane molasses, distillers grain and a mineral and vitamin prem ix was fed at a rate that met maintenance energy requirements (NRC, 2000). Urea (0.03 kg/hd/d) was mixed with the concentrate and offered to steers fed control and enzyme-treated hays to ensure that N deficiency did not limit rumen function. Ammonia treatment improved the nutritional composition of the hay and this effect was more pronounced in the more mature and lignified 13-wk hay. Enzyme did not
91 affect most nutritional components but slightly increased CP concentration of 5-wk hay. Enzyme application did not affect intake m easures, but ammoniation decreased intake probably because the enclosed feedbunks concentrated the strong ammonia odor. Ammoniation increased digestibi lity of DM, OM, NDF, hemicel lulose, ADF and cellulose across regrowth intervals, but enzyme application only increased NDF and hemicellulose digestibility of the 5-wk hay. Ammoniat ion also improved the DM degradation in the rumen but enzyme treatment did not. In Experiment 2, 90 Angus and Brangus steers (308 37 kg) were stratified by weight and randomly allocated to 18, 1. 01-ha pens located on dormant bahiagrass pasture. Three pens were assigned randomly to each treatment. The same hays and supplement fed in Experiment 1 were fed in Experiment 2. The treated hays from both regrowth intervals were weighed and fed in round-bale feeders to cattle in respective pens in quantities sufficient to ensure ad libitum access for 55 d. Steers were adapted to diets for 6 d, and full body weights were obtained on two consecutive days at the beginning (d 7 and 8) and end (d 54 and 55) of the measurement period. Weekly supplement allocations were fed in open troughs in equal amounts three times per week and they were completely consumed. Refused hay was weighed on d 30 and 55. Ammoniation increased hay DMI and tended to increase final BW and ADG. Enzyme treatment increased DMI of the 5-wk hay but had no effect on growth performance. In summary, ammoniation improved the forage quality of the hays and enhanced the growth of steers. En zyme treatment had few beneficial effects on the nutritional value of the hays and did not improve the growth performance of the steers.
92 Ammoniation had more pronounced effects on t he more mature (13 wk) hay, whereas enzyme application only resulted in beneficia l effects when applied to the 5-wk hay. Like others in the literature, this study indicates that much work needs to be done to improve the consistency and efficacy of enzyme treatment of forages and diets before it can be recommended for routine use by cattle producers. Enzyme treatment of forages differs from chemical treatment bec ause the enzymes involved are not a simple chemical entity, instead they are a myriad of complex proteins that only work when certain conditions are met (Nelson and Cox, 2008). As mentioned by Wang and McAllister (2002), measuring animal responses to commercial enzyme products without a proper understanding of the mechanisms underlying their ac tion will only confuse and delay the practical adoption of this technology Therefore, additional research on animal responses to enzyme addition to diet s should be delayed until a mechanistic investigative approach is impl emented that describes the fu ll array of enzymes needed for cell wall hydrolysis and outlines the conditi ons required for synergistic effects that result in through degradation of cell walls. Also, presence in appropriate proportions of subtypes of xylanase and cellulase enzymes that are critical for thorough cell wall hydrolysis should be verified in addition to measuring cellulase and xylanase activities. Since enzymes are expected to release oligosaccharides and monosaccharides as a consequence of their catalytic actions, the Van Soest detergent system (Van Soest et al., 1967, 1991) should be considered inadequate for evaluating improvements in enzyme potency at cell wall hydrolysis because it does not measure sugars. Rather, techniques like the Uppsala di etary fiber system should be used (Theander et al., 1995). This system uses a gas chromatographiccolorimetric-gravimetric method for
93 determination of total dietary fiber as neut ral sugar residues, uronic acid residues, and Klason lignin. This approach is more labor intensive and costly than the Van Soest system but it has been used by the lignocellu losic degradation industry, partly because it reflects enzyme effects on cell wall co mponents more accurately. Hydroxycinnamic compounds in forages should also be measured routinely when evaluating the impact of enzyme treatments because they impede en zymatic cell wall degradation. Finally, the impacts of anat omic structures and tiss ue arrangement of forages on cell wall digestibility shoul d be considered in order to develop improved enzyme cocktails that hydrolyze cell walls consist ently and thoroughly. In particular, future research should evaluate effects of adding cutinases, suberinases, esterases, and etherases to xylanase cellulase enzyme cocktails in order to enhance the removal of specific anatomical and c hemical features that im pede plant cell wall digestion.
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111 BIOGRAPHICAL SKETCH Juan Jose Romero was born in Lima, Peru in 1984. He graduated from San Agustin High School in 2001 and his favorite courses were biology, chemistry and history. He was raised in a family deeply in volved in Agricultural Sciences research; therefore, he was exposed to the agricultural c hallenges in Peru at a very early age. He realized that there was a big gap between t he modern agriculture industry on the coast of Peru and the agriculture of subsistence in the Andes and he promised himself that he would endeavor to improve agriculture in the Andes. His parents ta ckled the challenges of farming in the Andes from a Crop Science perspective, but he always liked cows and llamas more, so he decided to enroll in the Animal Science program at Universidad Nacional Agraria La Molina (UNALM). During his studies he came across a paper titled Effect of alkali pretreatment of wheat st raw on the efficacy of exogenous fibrolytic enzymes. Prior to that time, had no knowle dge about improving the quality of straw but knew that cereal straw was an abundant re source in the Andes. Consequently, he presented a seminar on the subject to his cl assmates at the University and conducted his undergraduate thesis research on the subjec t. He submitted his Literature Review on the subject to the 2007 Alltech Young Animal Scientist Contest for Latin America and was awarded the prize for the second best report in the region. Afterwards, he received his B.S. from UNALM in 2007 and finished at t he top of his Animal Science class. Soon after his graduation, he was admitted to UF to pursue an M.S. in Animal Science under the supervision of Dr. Bill Brown. The fa ct that he was going to work on improving forage quality was particularly exciting to him. After Dr. Brown moved to the University of Tennessee, he was supervised by Dr. Adesogan until he completed his M.S. program. During his time at UF, his underst anding of constraints of, and methods to
112 improve forage digestibility was greatly enhanced and he presented his research findings at the Annual Meeting of the American Society of Anim al Science in Montreal in 2009. He plans to continue working on the subject so that he can develop a practical solution for improving digestibility of low-quality forage in the Andes.