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1 ESTIMATION OF APPARENT DIGESTIBILITY OF SIX FORAGES USING TWO DIFFERENT DIGESTIBILITY MARKERS By CHUNALA ALEXICO NJOMBWA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012
2 2012 C hunala A lexico N jombwa
3 To my family
4 ACKNOWLEDGMENTS My profound thanks should go to almighty God for clearing the way for me to reach this far in academi c circles. I do not take this for granted but I only see this as a wonderful blessing upon me. I also thank my family for encouragement and support during the time I was thousands of miles away from home. I would like to express my deepest gratitude to Dr. Cliff Lamb for his endless support during my academic programme. He is wonderful, objective and solution oriented such that in him I found every reason to smile. His sociable altitude and a great family made US my home. And because of his mentorship, I ha ve acquired a variety of invaluable skills which I will utilize for the development of livestock sector of Malawi. In addition, I would like to convey my sincere gratitude to Dr. Nicolas DiLorenzo, a committee member and a friend. The skills he has imparte d in me shall stay forever and will be beneficial for millions of poor people in Malawi and world over. Again, his open door policy is incredible. Many thanks should also go to United States Agency for International Development (USAID) for financial suppor t which enabled me to take this challenge. I am also extremely grateful for the wonderful times I have shared with Vitor, Guilherme, Kayln, Tera, and Francin e Your social and academic support is something I will never forget. You made me feel great amon g all Malawians at UF. I wish to thank the NFREC beef crew, Harvey, Butch, David, and Don for all their assistance with my trial. Cutting grasses every morning with a chopper, collecting fecal samples everyday are among the most wonderful experiences I wi ll never forget. Words cannot describe my appreciation for the tireless hours they put in to make my research trial a success. In addition, I am grateful for their understanding, guidance, and patience throughout the research period.
5 TABLE OF CONTENTS P age ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 2 LITERATURE REVIEW ................................ ................................ .......................... 14 Forage Quality and Adaptation ................................ ................................ ............... 14 Cool Season Grasses ................................ ................................ ...................... 14 Warm Season Grasses ................................ ................................ .................... 16 Variation of Forage Digestibility ................................ ................................ ........ 19 Methods of Estimating Digestibility ................................ ................................ ......... 24 In Vitro Methods ................................ ................................ ............................... 24 In Vivo Methods ................................ ................................ ................................ 24 Concepts of Marker Techniques ................................ ................................ ............. 26 Categories and Analysis of Digestibility Markers ................................ .............. 26 Characteristics of Good Digestibility Markers ................................ ................... 28 Utilization of Digestibility Markers ................................ ................................ ..... 32 Advantages of Marker Techniques ................................ ................................ ... 34 Challenges of Using Digestibility Markers ................................ ........................ 34 Chromic oxide and Titanium dioxide ................................ ................................ 35 3 ESTIMATION OF APPARENT DIGESTIBILITY OF SIX FORAGES USING TWO DIFFERENT DIGESTIBILITY MARKERS ................................ ..................... 37 Materials and Methods ................................ ................................ ............................ 37 Annual Winter Forages (Experiment 1) ................................ ............................ 37 Annual Summer Forages (Experiment 2) ................................ ......................... 38 Animals and Management (Experiment 1) ................................ ....................... 39 Animals and Management (Experiment 2) ................................ ....................... 40 Sample Collection and Preparati on (Experiment 1 and 2) ................................ 41 Sample Analysis (Experiment 1 and 2) ................................ ............................ 42 4 RESULTS AND DISCUSSION ................................ ................................ ............... 48 Results ................................ ................................ ................................ .................... 48 Experiment 1 ................................ ................................ ................................ .... 48 Experiment 2 ................................ ................................ ................................ .... 49 Discussion ................................ ................................ ................................ .............. 51
6 Nutrient Digestibility of Forages ................................ ................................ ........ 51 Effectiveness of TiO 2 and Cr 2 O 3 as Digestibility Markers ................................ 51 Conclusion ................................ ................................ ................................ .............. 55 5 COMMERCIAL BEEF PRODUCTION IN MALAWI ................................ ................ 62 Introductory Re marks ................................ ................................ .............................. 62 Results and Discussion ................................ ................................ ........................... 63 Farm Ownership and Objectives ................................ ................................ ...... 63 Breeds, Breeding Methods and Animal Performance ................................ ....... 64 Nutritional Management ................................ ................................ ................... 65 Parasites and Diseases Management ................................ .............................. 66 Animal Housing and Handling Facility ................................ .............................. 67 Conclusion and Recommendation ................................ ................................ .... 67 L IST OF REFERENCES ................................ ................................ ............................... 74 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 79
7 LIST OF TABLES T able p age 3 1 Composition, yield and analyzed nutrient content (DM basis) of the winter annual pastures used in Exp. 1. ................................ ................................ ......... 46 3 2 Composition, yield and analyzed nutrient content (DM basis) of the summer annual pastures used in Exp. 2. ................................ ................................ ......... 47 4 1 Nutrient intake and digestibility by heifers fed winter forages, using Cr 2 O 3 or TiO 2 as indigestible marker and under two fecal sampling protocols in Exp. 1. .. 56 4 2 Apparent total tract digestibility of nutrients measured using Cr 2 O 3 or TiO 2 as indigestible markers in heifers fed three winter forages in Exp. 1. ...................... 57 4 3 Nutrient intake and digestibility by heifers fed summer forages, using Cr 2 O 3 or TiO 2 as indigestible mark er and under two fecal sampling protocols in Exp. 2. .. 58 4 4 Interaction between indigestible marker used and fecal collection protocol on digestibility of nutrients in heifers fed three su mmer forages in Exp. 2. .............. 59 4 5 Apparent total tract digestibility of nutrients measured using Cr 2 O 3 or TiO 2 as indigestible markers in heifers fed three summer forages in Exp. 2. .................. 60 4 6 Interaction between type of forage consumed and indigestible marker used on digestibility of nutrients in heifers fed three summer forages in Exp. 2. ......... 61 5 1 Farm ownership, objectives and cattle breeds presented in percentages of the nine visited beef producing farms. ................................ ................................ 68 5 2 Cattle population, breeding seaso n, breeding methods, pregnancy rate and birth weight expressed as percentage of 9 beef producing farms. ...................... 69 5 3 Mortality rates, weaning and mature weight and number of animals sold per ann um expressed as percentage of 9 visited beef producing farms of Malawi ... 70 5 4 Animal feeding and feed production expressed as percentage of 9 visited beef producing farms of Malawi. ................................ ................................ ......... 71 5 5 Forage preservation and feed supplementation practices expressed as percentage of 9 beef producing farms of Malawi. ................................ ............... 72 5 6 Animal housing, disease and parasite management expressed as percentage of 9 beef producing farms of Malawi. ................................ ................................ .. 73
8 LIST OF ABBREVIATIONS ADF Acid detergent fiber ADL Acid detergent lignin AIA Acid insoluble ash APL A cid peroxide lignin CIAT International centre for tropical agriculture CP Crude protein D Day DM Dry matter GIT Gastro intestinal tract H hour NAA Neutron activation analysis NDF Neutral detergent fiber OM Organic matter OMD Organic matter digestibilit y VFA Volatile fatty acids
9 A bstract of T hesis P resented to the G raduate S chool of the University of Florida in P artial F ulfillme nt of the Requirements for the D egree of Master of Science ESTIMATION OF APPARENT DIGESTIBILITY OF SIX FO RAGES USING TWO DIFFERENT DIGESTIBILITY MARKERS By Chunala Alexico Njombwa December 2012 Chair: G.C. Lamb Major: Animal Science s T he objectives of the two experiments were to compare apparent total tract digestibility of nutrients of summer and winter annual forages ; and to compare the efficacy of TiO 2 and Cr 2 O 3 as digestibility mark ers for fresh forages fed ad libitum and to determ ine effects of performing 2 vs. 3 per d ay f ecal sample collection to measure digestibility. Ryegrass ryegrass + oat, a nd ryegrass + triticale Mulato II, millet and sorghum we re fed to 12 Angus and Angus crossbred heifers Heifers were dose d at 1200 h with 10 g of Cr 2 O 3 and 10 g of TiO 2 via gelatin capsule s F eed and fecal samples were collected within 5 days In experime nt 1, no effect of forage, marker, sampling schedule, or marker x sampling schedule i nteraction was found for the digestibility variables measured (P > 0.05). In experiment 2, sampling effect, sampling protocol x marker and forage x marker were observed ( P < 0.05) on digestibility of forages Both Cr 2 O 3 and TiO 2 may be used indistinctively to estimate digestibility of winter forages while only TiO 2 may be used for summer forages Increasing sampling frequency to 3x a day may yield more desirable results whe n Cr 2 O 3 is used for fresh summer forages. In addition, a survey was conducted to determine status of commercial beef production in Malawi. Lack of proper strategic breeding and animal performance
10 monitoring, inadequate nutritional management and insufficie nt farm mechanization in majority beef farms indicate that beef production is at infant stage in Malawi. Research and dissemination of technologies to farmers should be intensified to enhance beef production.
11 CHAPTER 1 INTRODUCTION One of the most fundam ental measurements used to determine the nutritive value of a feed is digestibility. Digestibility of a feed determines the amount of nutrients that are actually absorbed by an animal and, therefore, the availability of these nutrients for maintenance, gro wth, reproduction, and production of other desirable products such as meat and milk (Ibrahim and Olaloku, 2000). Feedstuffs of high digestibility are often associated with high nutritive values and accelerated animal performance. Consequently, feedstuffs o f low digestibility are associated with low nutritive quality and may not provide sufficient nutrients for successful animal performance (Kham et al., 2003). Therefore, knowledge of the digestibility of feed should be assessed in livestock production syste ms to ensure that livestock producers optimize efficiency of production within their operation. This would allow livestock producers the opportunity to choose, produce, and provide feeds of high quality that offer more nutrients for animal growth and produ ction, resulting in higher yields. Classically, several techniques have been used to estimate digestibility. Among various techniques in use, markers have been widely accepted applications to estimate digestibility and organic matter intake. Markers are indicator substances which are inert (non digestible) in the gastrointestinal tract (GIT) of an animal. Characteristics of a good marker are: 1) they are strictly non absorbable; 2) they do not affect or are affected by the GIT or microbial population; 3) they are physically similar to or closely associated with feed material; and 4) methods of estimation in digesta samples must be specific, sensitive, and not interfere with other analyses (Kham et al., 2003).
12 Although markers are widely used to estimate fe ed digestibility, erratic results have been reported when administered across a wide range of feedstuffs. It has been reported that some markers yield accurate estimates of digestibility of specific feeds while overestimating or underestimating digestibili ty of other feeds (Sunvold and Cochran, 1991). Thus, there may be an inconsistency in feed evaluation which may jeopardize the process of evaluating and allocating the feed to meet maintenance and achieve desired growth and performance of the animal. This also makes it difficult to determine the most efficient feeding strategies to maximize animal productivity and economic returns. Therefore, research aimed at evaluating accuracy of markers in estimating digestibility of various feeds is of extreme importan ce. This ensures more precise and accurate estimates of digestibility which can enhance production of high valued feeds and the allocation of feeds to appropriate groups of animals; thereby, enhancing animal performance and returns. Chromic oxide (Cr 2 O 3 ) has been the most commonly used digestibility marker. However, there are reports that Cr 2 O 3 is associated with health risks such as carcinogenic effects (Myers et al., 2004). As a consequence, titanium dioxide (TiO 2 ) has been explored as a potential altern ative digestibility marker with no reported health risks that may be legally added to feeds as color additive at amounts that do not exceed 1% of finished product (Titgemeyer et al., 2001). Several studies have indicated the feasibility of using TiO 2 as a viable total tract digestibility marker in rats, chicken, pigs, dairy cows (Myers et al., 2004), and beef cattle (Titgemeyer et al., 2001). However, little research has been completed to test its efficacy in estimating digestibility of some forms of feedst uffs and in various feeding conditions.
13 Currently, there is no available data to assess the viability of using TiO 2 as a digestibility marker in beef cattle fed fresh forages on an ad libitum basis, or to quantify the effect of collecting fecal samples 2 or 3 per day. Therefore, the efficacy of TiO 2 as a marker for these conditions is unknown, limiting its application as a digestibility marker in experiments. Therefore, two studies were conducted with objectives to: 1) compare total tract digestibility of nutrients for three cool season and three warm season forages and 2) to determine the efficacy of two digestibility markers titanium dioxide (TiO 2 ) and chromic oxide (Cr 2 O 3 ) sampled 2 or 3 per day on estimating digestibility. Ryegrass ( Lolium multiflo rum Lam.), ryegrass combined with oat ( Avena sativa ), and ryegrass combined with triticale ( Triticosecale rimpau ) were the cool season forages evaluated, whereas sorghum ( Sorghum bicolor ), pearl millet ( Pennisetum glaucum ), and Mulato II ( Brachiaria hybrid ) were the warm season grasses evaluated.
14 CHAPTER 2 LITERATURE REVIEW Animal productivity and profitability of livestock enterprises are directly linked to nutrition which is mostly determined by amount of feed consumed, nutritive quality, and digestibil ity of the diet consumed by livestock (Marais, 2000). Digestibility provides an estimate of the quantity of consumed feed, or specific components of the feed available for an animal to digest and absorb. These components are used for growth, maintenance, r eproduction, and production of meat or milk of livestock species. However, digestibility is useful only if it is accurately estimated (Morais et al., 2010). Therefore, accurate knowledge of the digestibility of feedstuffs is essential for the establishment of effective feeding strategies to optimize the profitability of livestock production systems. Forage Quality and A daptation Forages form a larger proportion of ruminant diets; however, producers should be aware of the existence of variations in seasonal response of forages if they are to maximize forage production and utilization. Some forages are well adapted and more productive during the cool periods of the year (i.e., winter), whereas others are more productive during the warm periods of the year (i. e., summer), hence the terms cool season and warm season forages. Cool S eason G rasses Annual ryegrass ( Lolium multiflorum ) is one of the most widely grown cool season grass in southeastern United States (US) covering greater than one million hectares, ann ually. Ryegrass is considered the best quality winter forage for the southeastern US due to its high dry matter (DM) digestibility (typically > 65%), excellent animal
15 performance, low seed costs, seed availability, and adaptation to a wide variety of envir onments. Ryegrass also has a crude protein (CP) content that exceeds the requirements for most classes of livestock (Blount et al., 2009; Blount et al., 2010). Annual ryegrass can be seeded alone but a more common production practice is to seed it in mixtu res with other cool season forages such as oats and triticale. Triticale ( Triticosecale rimpau ) is a hybrid between wheat ( Triticum ) and rye ( Secale ). It take its name from the first five letters of Triticum and last four letters of Secale. Triticale is w ell adapted to the southern parts of US and peninsular Florida. It has the forage quality of wheat and the excellent disease resistance of rye. Dry matter digestibility of triticale ranges from 60% to 79% while CP content ranges from 11% to 22%, depending on stage of maturity (Keuren and Underwood, 1990). Triticale is best utilized as ensiled haylage or silage because it does not respond well to intense grazing; however, when used in grazing systems, it is important to consider blending it with ryegrass to promote a longer growing season (Blount et al., 2010). Oat ( Avena sativa ) is a palatable grass with a DM digestibility range of 56% to 77% and CP content ranging from 11% to 20% depending on the stage of maturity (Keuren and Underwood, 1990). Peak season of forage production for ryegrass is later than that of oat, rye or triticale. Therefore, when grown in a mixture of oat with rye or triticale provides a faster growing component of the blend, earlier grazing, and as the oat declines in late winter, ryegra ss production peaks. The result is an extension of cool season forage production with high quality ryegrass forage (Blount et al., 2009: Blount et al., 2010).
16 Warm S eason G rasses Warm season grasses are the dominant forage crops used for livestock product ion in tropical regions of the world. The majority of these warm season grasses have seasonal growth, with most of forage production occurring during the spring, summer, and early fall months (Vendramini et al., 2010). Sorghum ( Sorghum bicolor ), pearl mill et ( Pennisetum glaucum ), and Mulato II ( Brachiaria hybrid) are among the most widely used warm season grasses in the US. As with other warm season grasses, these grasses are characterized by high DM content, and relatively low CP and DM digestibility compa red to cool season grasses. Banks (1998) reported DM yield of 11 tons per ha, 12% CP and DM digestibility of 67% for sorghum. Pearl millet has slightly greater feeding value as compared with sorghum, with DM yield of 12 ton per ha, CP content of 18% a nd DM digestibility 69%. Both sorghum and pearl millet may be used as fresh fodder, pasture, silage, or hay (Lang, 2001). However, stage of maturity at harvest, soil fertility, and management impact the quality of sorghum and pearl millet. Although sorg hum is widely used as forage, there are concerns about the potential toxic effects to animals. Leaves of sorghum plants may be toxic as a result of high concentrations of hydrogen cyanide (prussic acid), especially in young dark blue colored regrowth after experiencing drought conditions. Unlike sorghum, pearl millet does not produce prussic acid (Banks, 1998; Cook et al., 2005). However, there have been incidences of nitrate poisoning from pearl millet. Nitrate poisoning results from the ingestion of forag e containing high concentrations of nitrates (NO 3 ; Lang, 2001). Normally, safe levels of nitrates in forages vary depending on the physiological state of
17 the cattle. Nitrate nitrogen (NO 3 ) concentrations > 0.88% are considered unsafe for pregnant females w hereas for non pregnant females, NO 3 concentrations > 1.76% are considered toxic. In addition, forages with NO 3 concentrations < 1.76% may be limit fed to non pregnant animals at specific inclusion percentages. For example, NO 3 concentrations between 1.54 and 1.76% in feedstuffs should be supplied at the rate of 25% of total DM of the ration whereas concentrations of 0.88 to 1.54% and 0.66 to 0.88% may be limit fed at inclusion rates of 35 to 40% and 50%, respectively. As for pregnant females, limited inclu sion rates of 50% must be observed when concentrations of NO 3 are between 0.44 and 0.66% (Lang, 2001). Accumulation of NO 3 occurs in plants that are fertilized with N at high rates, but grow at slow rates. Slow growth usually occurs because of insufficien t soil moisture, but may also be the result of heavy cloud cover, shading, cool temperatures, or frost. Controlling access to toxic forage and dilution with other feeds are methods that may be used to control toxicity (Hannaway and Larson, 2004). In additi on, application of N fertilizer in pearl millet should remain below 250 kg per ha in order to reduce concentrations of NO 3 (Banks, 2002). Furthermore, Banks (2002) indicated that application of N fertilizer for Pearl Millet should be applied at 70% of the application rates used for corn fertilization with the first 50% applied at planting and the remaining 50% after first cutting, to avoid risk of toxicity. As technology in agriculture advances, forage breeding programs have been established to create be tter quality forages that can enhance livestock production to feed the growing human population. One of the products of cross breeding and selection is the development of the cultivar Mulato II ( Brachiaria hybrido ). Mulato II is a
18 Brachiaria hybrid develop ed and released in 2004 by the Tropical Forage Programme, International Centre for Tropical Agriculture (CIAT), Cali, Colombia. Mulato II is a cross between Brachiaria ruziziensis Brachiaria brizantha and Brachiaria decumbens Mulato II is adapted to many soil types ranging from sand to clay ( pH range of 5.5 6.0) and is superior in tolerating drought (up to 6 months), burning, and pests (such as spittle bugs; Vendr amini et al., 2011; Argel et al., 2007). In addition, it also demonstrates high plant vigor and fast recovery after grazing (Argel et al., 2007). Argel et al. (2007) also reported Mulato II to have DM yields of 3 ton/ha/cutting, CP of 11.4%, and DM diges tibility of 66% during periods of rain. However, CP (8.4%) and IVDMD (61%) declined during the dry season. Generally, Mulato II has a range of 11 to 16% for CP and 55 to 60% for TDN (Vendramini et al., 2011). Mulato II does not tolerate water logging but has demonstrated good compatibility with other forages, particularly legumes. In most regions of Sub Saharan Africa and Malawi (located 13 0 55S and 33 0 42E) in particular, napier grass ( Pennisetum purpureum ) is commonly used in livestock production syst ems. Unlike other grasses used in other parts of the world, napier grass is favored because it is a multipurpose grass. Apart from being used as livestock feed, napier grass may also be planted along contours during cultivation to reduce soil erosion. Ther efore, napier grass is a good fit in the Malawi agricultural production system, which is characterized by high land pressure and competition for land between human food production and livestock feed production. napier grass produces DM ranging from 2 to10 ton/ha when unfertilized, 10 to 30 ton/ha/yr and may increase to 85 ton/ha/yr when fertilized. These production rates appear to be significantly greater than
19 those reported for mulato II, sorghum, and millet (Cook et al., 2005). However, napier grass requ ires deep well drained loam soils with pH ranging from 4.5 to 8.2 and requires 150 to 300 kg/ha/yr of N to achieve high nutrient and DM production. These requirements increase the cost of production of napier grass. Unlike the temperate grasses such as an nual ryegrass, oat, and triticale, CP content in Napier grass drops more rapidly with maturity. At 6 wk regrowth, 10% CP of Napier grass was reported, whereas at 10 wk of regrowth, the CP content decreased to 7.6% (Cook et al., 2005). This is attributed to efficiency of N utilization of Carbon 4 (C4) grasses as stage of maturity increases. Napier grass belongs to a group of C4 grasses. The C4 grasses are efficient in utilization of N, thus there is a rapid decrease in N content with advanced maturity result ing in reduced CP. In general, CP content in leaves ranges from 9.5 to 19% whereas DM digestibility ranges from 68 to 74%. Napier grass is regarded as palatable, with high quality, and tolerant to drought conditions (Cook et al., 2005), but quality is aff ected by soil fertility, harvesting frequencies, and management. Variation of F orage D igestibility Forage plants form a great proportion of diets of ruminant animals. Just like other plants, forage plant cells have walls which form the structural framewor k that provides mechanical support for the plant, involved in water balance, ion exchange and also enclose and hold together all organelles (Moore and Jung, 2001). The cell walls constitute cellulose, hemicellulose, and lignin. During primary growth, cells experience an increase in size (Jung and Allen, 1995; Moore and Jung, 2001). However, after increasing in size and elongation, secondary growth is initiated in which cells undergo extensive thickening of walls (Jung and Allen, 1995). During this developme nt,
20 cellulose, and hemicellulose composition of cell wall increases (Buxton and Redfearn, 1997). In addition to this, there is increased deposition of phenolic acids and lignin starting from outside the cell wall progressing to the inside part of the cell wall (Jung and Allen, 1995). This is done to enhance the structure and strength of the cell wall, facilitate water transportation, create a major line of defense against pathogens, insects and other herbivores and also to impede degradation of cell wall po lysaccharides (Hatfield and Vermervis, 2001). When ruminants consume forages, digestion of a great proportion of these forages is aided by bacteria and fungi that exist in the rumen. The rumen microbes release enzymes which hydrolyze cellulose and hemicel lulose to produce volatile fatty acids (VFA) such as propionate (substrate for gluconeogenesis), acetate (precursor for milk fat and a source of energy for muscles) and butyrate (source of energy for rumen cells). These VFAs are absorbed in the rumen and u sed in metabolic processes that support life and performance of ruminants. The rate and efficiency at which microbes digest the cell wall components is what determines digestibility of forages (Jung and Allen, 1995). However, microbial efficiency in diges tion of the cell wall components is affected by plant development. At maturity, the plant cell walls accumulate high cellulose and hemicellulose, both of which are encrusted with lignin. The cellulose and hemicellulose are compacted together and this reduc es surface area for microbial attachment, thereby increasing the duration of hydrolysis and reducing forage digestibility. In addition to this, lignin provides a physical barrier and also shields microbial enzymes from accessing cellulose and hemicellulose Through phenolic acids, lignin develops linkages with cell wall polysaccharides and these linkages change the orientation of cellulose, reducing the opportunity for
21 hydrolysis of cellulose to occur. The reduced rate and efficiency of rumen microbes in di gestion of the cell wall result in a reduction in overall digestibility of forages (Jung and Allen, 1995). Many studies (McMillan et al., 2006; Firdous and Gilan, 1999; Cochran et al., 1986; Sunvold et al., 1991) have reported variations in total tract dig estibility of different forages. In general, these differences are attributed to variation in levels of accumulation of cell materials which is affected by nature of the forage, stage of maturity, species, and proportion of different components of the fora ge (Buxton et al., 1997; Firdous and Gilan, 1999). Class of the forage Forages can be classified into Carbon 3 (C3) and Carbon 4 (C4) plants depending on the way in which they assimilate carbon dioxide into their system. The first products of photosynthesi s in C3 plants are compounds with three carbon atoms, whereas in C4 plants the first compounds have four carbon atoms (Ehleringer et al., 1997). Many studies have reported differences in digestibility between C3 and C4 plants. In most situations, C3 plants have greater digestibility than C4 plants due to differences in structural composition. In general, C4 plants contain a greater proportion of vascular bundles and also have tightly packed mesophyll cells, compared to C3 plants that have a larger proportio n of leaf mesophyll cells which are loosely packed (with large intercellular spaces), which provides enough surface area for microbial attachment in the rumen thereby increasing the rate of digestion (McMillan et al., 2006; Akin, 1986; Buxton and Redfearn, 1997). The higher proportion of vascular bundles in C4 plants increases the proportion of forage requiring microbial digestion, reducing the rate and efficiency of digestion because microbes require additional time to digest forage. Tightly
22 packed mesophy ll cells in C4 forages reduces surface area for microbial attachment in the rumen, decreasing the rate of digestion (McMillan et al., 2006). Temperate grasses and legumes are generally regarded as C3 plants, whereas tropical grasses are regarded as C4 plan ts (Akin, 1986; Sollenberger, 2011). Therefore, these differences between C3 and C4 describe why temperate or winter grasses, such as ryegrass, oat, and triticale have greater digestibility than tropical or warm season grasses such as sorghum, millet, and Mulato II. In addition, variations in digestibility have been reported between legumes and grasses. In general, legumes are more digestible than grasses, since legumes tend to have less fiber and greater CP content (Buxton and Redfearn, 1997). This provide s readily available nitrogen for protein synthesis by rumen microbes thereby enhancing degradation of legumes in the rumen. Whereas grasses have low CP content and increased levels of cellulose and hemicellulose, which requires a longer duration for microb es to digest, hence decreasing digestibility (Sollenberger, 2011). Forage m aturity Within the same species of forage, digestibility varies at different stages of maturity. Under typical conditions, young forages have greater digestibility than mature fora ges (McMillan et al., 2006). Ammar et al. (2010) reported a decline in digestibility of Avena sativa (oat), Trifolium alexandrium and Vicia sativa forages as stages of maturity increased. As the forages mature, the compaction and quantity of cell wall co ntents, such as cellulose and hemicellulose increase. Accumulation of these components reduces digestibility of the forage because rumen microbes require additional time to digest these structural carbohydrates to become available for animal use. In additi on, as the forage plant cell develops, phenolic acids and lignin are deposited in the maturing
23 cell wall in specific structural conformations, and in a strict developmental sequence to enhance the strength of the cell wall (Buxton and Redfearn, 1997). Lig nin is the key element that limits cell wall digestibility. The phenolic acids facilitate linkages between lignin and cell wall polysaccharides resulting into changes in orientation of these polysaccharides thereby physically shielding them from enzymatic hydrolysis (Jung and Allen., 1995). As the cell wall matures, lignin composition changes from guaiacyl type to syringyl type lignin. Unlike p hydroxyphenol and guaiacyl type lignin, the syringyl type lignin protects a greater proportion of cell wall from d igestion because it is more linear in structure and extends further into the secondary wall of the cell, thereby linking with more polysaccharides and reducing cell wall digestibility (Jung and Allen., 1995). Therefore, increased concentrations and composi tion of lignin reduces the percentage of the digestible portion of the forage resulting in reduced digestibility of the forages as stage of maturity increases. Within the vertical orientation of forage Moving up the plant, there is variation in digestibili ty. Studies have demonstrated that digestibility is increased towards the upper portion of the plant compared to the lower portions of the plant. Normally, lower portions of the plant have a low leaf to stem ratio (Ball et. al., 2001). Stem material of all forages has vascular tissues which have more sclerenchyma cells. The sclerenchyma cells are greater in cell wall concentration than cell content due to extensive secondary thickening and they also contain high concentrations of lignin, reducing digestibil ity. In contrast, forage leaves tend to have more mesophyll cells that undergo little secondary wall thickening and deposit virtually no lignin, thus fewer materials is present to prohibit digestion resulting in increased digestibility (Jung and Allen, 199 5).
24 Methods of E stimating D igestibility Several methods of estimating digestibility have been developed for experimental use. Digestibility estimations have been developed for both in vitro and in vivo experimental methods. In V itro M ethods In vitro meth ods involve assimilating conditions of the rumen in a laboratory and estimate the breakdown of forage or feed. Forage or feed is combined with rumen fluid and allowed to undergo anaerobic fermentation for 24 h (to represent similar conditions in the rumen (Kham et al., 2003). After incubation for 24 h, the mixture is exposed to hydrochloric acid and pepsin for 48 h, to represent similar conditions to the ruminant abomasum (Kham et al., 2003). Although this method is less costly, less labor intensive, and c an provide good estimates of feed digestibility, the major challenge has been to create similar conditions to the rumen and abomasum (Judkins et al., 1990). Characteristics such as gastric motility, interaction of microflora in the rumen environment, phys iological changes in the animal that may affect digestibility, and interactions between feed constituents in the rumen environment have been difficult to duplicate. In some cases, these conditions have resulted in digestibility values that may not be achie ved with in vivo methods (Judkins et al., 1990; Cochran et al., 1986). In V ivo M ethod s Unlike the in vitro method, in vivo methods of estimating digestibility are performed in the gastrointestinal tract. This method involves measuring feed intake and fecal output, which are used for calculating feed digestibility. Three methods for in vivo estimates of digestibility have been developed: 1) total fecal collection, 2) in sacco, and 3) indigestible markers.
25 Total fecal collection The total fecal collection me thod involves collection and weights of all feed intake and fecal material, estimating the difference between the two. This method is an excellent estimate of digestibility; however, this technique also is labor intensive, expensive, and frequently impract ical for larger animals, because of the number of times animals need to be handled (Rymer, 2000). In sacco technique The in sacco technique, feed samples are placed in nylon bags and are mechanically suspended in the rumen of ruminally fistulated animals for a specific period of time to allow microbial digestion to take place. The microbes (especially protozoa) and digestive agents found in the rumen enter the bags through the pores and digest the feed; therefore, the ideal pore size should range from 40 t o 60 m (Vanzant et al., 1998). Larger pore sizes are discouraged because they result in loss of feed from the bag, whereas smaller pore sizes frequently become blocked, restricting circulation across the bag resulting in reduced rate of degradation of the feed (Vanzant et al., 1998). This technique helps to estimate lag extent (delay in digestion attributed to time required for wetting of feed and attachment of bacteria to the feed) and rate of DM and nutrient disappearance (Udn and Van Soest, 1984). How ever, the in sacco technique fails to mimic some processes of digestion, such as mastication, rumination, and passage which affect digestion of feed in the rumen (rskov et al., 1980; Vanzant et al., 1998).
26 Marker technique s Marker techniques have been wid ely applied in animal nutrition experiments. They are used to estimate digestibility of DM and nutrients, determine ruminal passage of digesta and fluids, and also DM intake of grazing ruminants (Cochran et al., 1986; Marais, 2000). The marker techniques i nvolve the use of markers which serve as indicator substances which are inert in the GIT (Rymer, 2000). Concentrations of markers in the feed and in the feces are determined and these concentrations are used to calculate estimated digestibility of the feed Concepts of Marker Techniques Categories and Analysis of Digestibility Markers There are two categories of digestibility markers; internal markers and external markers. Internal markers are indigestible materials occurring naturally in forages or feeds such as silica, Acid Insoluble Ash (AIA), and lignin. These digestibility markers form an integral part of feedstuffs (Marais, 2000). In contrast, external markers are materials that are not part of the normal diet and are added to the diet. Common externa l markers are metal oxides such as chromic oxide, rare earth metals such as dysprosium chloride, and chromium mordanted fiber (Kham et al., 2002). Compared to internal digestibility markers, these external digestibility markers are expensive. Several anal ytical methods for external digestibility markers have been developed (Short et al., 1996; Titgemeyer et al., 2001; Myers et al., 2004; Fenton and Fenton, 1979; Hill and Anderson, 1958; Czarnocki et al., 1961). However, the common trait among these methods is the use of light absorbance properties of some digestibility markers. Reactions of some external digestibility markers such as TiO 2, Fe 2 O 3 Cr 2 O 3 Ytterbium oxide and Dysprosium chloride with reagents, result into a solution changing
27 color. When TiO 2 r eacts with H 2 O 2 the end result is production of an orange or yellow color (Titgemeyer et al., 2001). The color produced by the external marker has a distinct absorbance at specific wavelengths of light and this property is used to quantify concentrations o f the marker in fecal samples. Of the inert digestibility markers, research has more recently focused on TiO 2 (Short et al., 1996; Titgemeyer et al., 2001; and Myers et al., 2004). In separate studies, Myers et al. (2004) used light wavelength of 406 nm w hereas Titgemeyer et al. (2001) used 410 nm to determine absorbance and concentration of TiO 2 in fecal samples The six step procedure for calculating TiO 2 concentrations in feed and feces is as follows: 1) preparation of duplicate 0.5 g samples into 250 mL macro Kjeldahl digestion tubes including a baseline sample of feces (or duodenal, ileal digesta,or forage) devoid of TiO 2 for background correction (Myer et al., 2004); 2) addition of a reaction catalyst containing 3.5g K 2 SO 4 and 0.4g CuSO 4 to each vial ; 3) addition of 13 mL of concentrated H 2 SO 4 to each vial and digest samples at 420C for 2 h; 4) remove heat and allow cooling for a minimum of 30 min; 5) addition of 10 mL 30% H 2 O 2 to each vial and allow cooling for 30 minutes; and 6) allow total liquid weight to increase to 100 g with distilled water and filtering through Whatman No. 541 filter paper to remove precipitate and finally, followed by absorbance measurement at 410 nm. The spectrophotometer is calibrated with working standards, prepared by add ing 0, 2, 4, 6, 8, and 10 mg of TiO 2 to blank tubes. The analysis of Cr 2 O 3 usually requires an initial oxidation of the organic matter by dry (Fenton and Fenton, 1979) or wet (Hill and Anderson, 1958; Czarnocki et al., 1961) ashing, followed with a more se vere oxidation of Cr 2 O 3 into the predominantly soluble
28 dichromate form using combinations of either sulfuric acid, nitric acid, perchloric acid, hydrogen peroxide or sodium molybdate. The concentration of dichromate ion may be determined spectrophotometric ally at 440 nm wavelength or by atomic absorption spectroscopy. These procedures generally require sample sizes of 0.5 to 1.0 g (Suzuki and Early, 1991). Some rare earth external digestibility markers like Lanthanum oxide and Samarium may be analyzed by Ne utron Activation Analysis (NAA). This involves bombarding the sample with neutrons, causing elements (markers) to form radioactive isotopes. The radioactive emissions and radioactive decay paths for each element are known. Using this information, it is pos sible to study the spectra of emissions of radioactive samples, and determine the concentration of the elements within it. This analytical procedure does not destroy the sample (Glascock, 2004). Internal digestibility markers such as lignin, NDF and ADF ca n be analyzed using the cell wall analytical procedures by Van Soest (Van Soest et al., 1991; Goering and Van Soest, 1970). These techniques involve boiling samples in detergent solution and this removes all other portions of the cell except the targeted i nternal marker (Goering and Van Soest, 1970). The concentration of the internal marker AIA can be determined by drying and ashing samples in 2 M hydrochloric acid for 5 minutes. The ash content is then determined gravimetrically after filtering, washing th e hydrolysate to remove the acid and reashing (Van Keulen and Young, 1977). After calculating the concentration of the marker in the feed and feces, digestibility is estimated. Characteristics of G ood D igestibility M arkers A good digestibility marker canno t be digestible or absorbable in the GIT (Marais, 2000) and should pass through the GIT unaffected. Publications have established that internal digestibility markers are more susceptible to digestion and absorption compared
29 to external markers, because int ernal markers are natural components of the feedstuffs. When estimating digestibility of feedstuffs using markers, the concentration of marker in the feed and fecal material are critical parameters. When the marker is digested or absorbed in the GIT, there is reduced recovery and concentrations in the feces resulting in overestimation of digestibility (Owens et al., 1992; Rymer, 2000; Judkins et al., 1990). Therefore, an excellent marker must be indigestible and non absorbable to ensure full recovery in the fecal material, thereby providing an accurate estimation of the digestibility of the feed. In addition, an excellent marker must not alter the function of the GIT (Sunvold and Cochran, 1991). A ruminant GIT is a complex system involving complex relation ships among microbes, the endocrine system, and enzymes which are coordinated under specified ranges of pH and temperature (Kham et al., 2003). Interference of this environment will result in malfunctioning of the GIT, reducing its ability to digest feeds. Some external markers, such as rare earth metals, are believed to influence gut fill, thereby reducing DM intake. Therefore, markers that affect the normal function of the GIT should be avoided by scientists to ensure accurate estimation of digestibility. A good marker must be physically similar to or closely associate with feed material, which ensures even distribution of the marker in the feed and fecal material (Marais, 2000). This reduces chances of inaccurate estimation of the digestibility of the fe ed due to varying concentrations of the marker in fecal material. An ideal digestibility marker must have a specific method of analysis and the method must not interfere with other analyses (Marais, 2000). A marker that fulfills all of these conditions is considered
30 ideal for use in digestibility experiments because they are likely to have a high recovery rate, enhancing accurate estimation of digestibility. In general, most external markers (metal oxides and rare earths) have been found superior in estima ting digestibility compared to internal digestibility markers. In most cases, they may also be applied across a range of feeds and feedstuffs in different feeding conditions but provide estimations of digestibility and fecal output not significantly differ ent from the total fecal collection method. Average estimates of daily fecal output obtained from analyzing both Cobalt and Ytterbium concentrations (2.39 and 2.59 kg/d, respectively) did not differ from total collection value (2.48 kg/d) (Brandyberry et a l., 1991). In addition, Myers et al. (2004) observed a reduction in the diurnal effect on the excretion pattern of both TiO 2 and Cr 2 O 3 in sheep. However, use of TiO 2 as a marker underestimated (from 1.6 to 4.3 %) digestibility compared to total fecal coll ection (Titgemeyer et al., 2001). In contrast, TiO 2 was equally effective as Cr 2 O 3 in estimating rate of passage of digesta in sheep (Myers et al., 2004). Among external digestibility markers, some are less effective than others. Pond et al. (1985) indica ted the possibility of reduced recovery in feces and overestimation of digestibility by Fe 2 O 3, which was attributed to Fe 2 O 3 being heavy and not mixing well with digesta. In addition, rare earth metals are more expensive than some metal oxides. Furthermore detection of some of these rare earth elements requires specialized analysis such as neutron activation which is expensive (Prigge et al., 1981). Therefore, Cr 2 O 3 has been the most favored marker in digestibility trials because it is cheap, rarely found in feedstuffs, easy to analyze, and in most cases, it provides similar digestion coefficients as total fecal collection (Mroz et al., 1996; Brisson, 1956). More recently,
31 TiO 2 has become a useful marker which is easy to analyze, has no reported cases of ca rcinogenic effects, and can be legally added to feeds as a color additive (Titgemeyer et. al., 2001). For internal digestibility markers, Sunvold and Cochran (1991) recommended acid detergent lignin (ADL), acid peroxide lignin (APL), and AIA as sound marke rs for estimating organic matter digestibility (OMD) of grass hay diets. Results indicated OMD estimates derived by ADL ratio, APL ratio and AIA ratio (63.5%, 62.0% and 63.5% respectively) for brome grass hay, which was similar to total fecal collection (6 1.2%) measurement. Likewise, OMD for prairie hay by ADL, APL, and AIA ratio (60.1%, 48.2% and 54.9% respectively) did not differ from total fecal collection (54.4%) measurements (Sunvold and Cochran, 1991). However, these markers yielded OMD estimates of a lfalfa that differed from those derived by total fecal collection, which was attributed to relatively low concentrations of these marker (less than 6% in Alfalfa) and possibility of contamination, especially with AIA (Sunvold and Cochran, 1991). High var iations in digestibility estimates by AIA and silica have been associated to contamination of feedstuffs with dirt (Van Dyne and Lofgreen, 1964; Van Keulen and Young, 1977). Furthermore, Fahey and Jung (1983) explained the possibility of losing lignin duri ng analysis as a result of destruction by reagents, pseudo digestion, true digestion, and metabolization in the GIT. Therefore, the application of internal digestibility markers is limited since there is currently no single marker that is effective in esti mating digestibility of all feeds and feedstuffs. Therefore, it is prudent to utilize markers that have been identified as accurate to assess digestibility to avoid erratic results (Judkins et. al., 1990).
32 Utilization of D igestibility M arkers External di gestibility markers may be administered orally by mixing with the diet before feeding or dosed using gelatin capsules. Digestibility markers may also be placed directly into the rumen in fistulated animals. Several studies have reported variations in diges tibility or feed intake estimation with different methods of administering markers (Brandberry et al., 1991; Prigge et al., 1981; Langland et al., 1963; Brisson et al., 1957). Directly incorporating the marker with the feed may resulted in estimations of d igestibility which could not be accurate and this in other cases, could be attributed to the fact that animals may not consume all of the feed provided to them, and it is difficult to account for the marker remaining in the orts, hence erratic estimation o f digestibility (Brandyberry et al., 1991). Direct placement of marker in the rumen is mostly recommended when the objective is to estimate the passage rate of the feed (Myers et al., 2005). After completion of an adaptation period, fecal samples should be collected, composited, and concentration of the marker in both feces and feed measured. Fecal sampling frequency depends on the frequency of external marker administration and the objective of the experiment. More frequent marker administration (6 per day) may require less frequent fecal sample collection, whereas less frequent marker administration (1 or 2 per day) may require more frequent fecal sampling (Brisson et al., 1957; Prigge et al., 1981). No significant differences were reported in fecal o utput obtained by Co and Yb 2 O 3 ( 2.39 and 2.60 kg/d, respectively) using fecal samples collected in the morning or afternoon only compared to estimates ( 2.48 kg/d) by total fecal collection (Brandyberry et al.,1991).
33 In addition, 4 and 8 per day fecal sa mpling were used to determine interval required for marker equilibrium and to evaluate diurnal effects on marker recovery, respectively (Brandyberry et al., 1991). Fecal outputs in beef cows were evaluated using YbCl 3 as digestibility marker (Prigge et al. 1981). Fecal output estimates of 2.74 and 3.00 kg/d from samples collected 1 per d at 0800 h and 1600 h, respectively, did not differ from the 2.87 kg/d obtained from 2 per d sampling at 0800 and 1600 and these did not further differ from 2.83 kg/d fe cal output obtained by total collection (Prigge et al., 1981). In addition, it was determined that samples should be taken at 4 h intervals for 48 hours to estimate diurnal variation of Cr 2 O 3 and YbCl 3 excretion (Prigge et al., 1981). Prior to that (Briss on, 1956), it was determined that fecal samples should be collected 4 per day to successfully establish excretion patterns of Cr 2 O 3 in cattle dosed 1 2 or 6 per day. Again, Brisson et al. (1957) recommended 1 per day fecal sampling to estimate fec al output when cows were dosed 6 per day at equal intervals. In addition, Myers et al. (2005) indicated the possibility of reduced frequency of sampling in sheep dosed 2 without markedly affecting mean concentrations of Cr 2 O 3 and TiO 2 Although variation s in frequency of fecal sample collection exist, it is important that fecal samples are collected in such a way that represents multiple time periods to overcome diurnal variations associated with external marker excretion (Myers et al., 2005; Prigge et al ., 1981; Brisson et al., 1957; Hardison et al., 1955; Langland et al., 1963). Once concentrations of marker in feed and fecal samples are determined, digestibility of forage or feed is calculated using the formula: DM digestibility = 100 (100 % marker in feed / % ma rker in feces).
34 Advantages of M arker T echniques Unlike total fecal collection techniques (where all feces are collected and weighed), with marker techniques only small samples of feces are utilized, which reduces collection of all feces (Rym er, 2000). In certain situations, particularly with cattle, total fecal collection is especially challenging. Thus, markers provide an alternative by using fecal samples from the rectum and using the marker to estimate total fecal output. Challenges of Us ing Digestibility M arkers Although markers are widely used to estimate feed intake, passage rate, and digestibility, speculation still remains regarding the accuracy of specific markers used in estimating these responses. Erratic results have been reported when markers were administered across a wide range of feeds under different feeding conditions (Sunvold et al., 1991). These variations have been attributed to a range of factors such as metabolism and absorption of markers in the GIT, diurnal effects on marker recovery, uneven distribution of markers in the feces, sampling errors, and analytical errors which affect measurement of marker concentrations in feed and feces (Rymer, 2000). In addition, the mechanism of administration of external markers, also a ffects recovery, as a result of residual markers that are not accounted for when the marker is incorporated into the diet (Brandyberry et al., 1991). These factors negatively affect recovery and concentration of markers in feces, thereby influencing variat ions in estimates of DM digestibility, passage rate, and DM intake in nutrition experiments (Marais, 2000). Therefore, it is important to note that markers are selected for specific diets, feeding conditions, and that these selected markers should be valid ated before use in different
35 feeds and feeding conditions in order to achieve accurate estimates of digestibility (Judkins et al., 1990). Chromic oxide and Titanium dioxide Both Cr 2 O 3 and TiO 2 are metal oxides. Chromic oxide is the most commonly used diges marker, Cr 2 O 3 was used in 72% of studies published by the Journal of Animal Science between 1986 and 1995 (Titgemeyer, 1997). In addition, chromic oxide also is the most extensively studied external digestibility marker and has been found to be effective in ruminants, monogastrics, birds and in multiple feedstuffs such as grass hay, legume hay, and concentrates (Hill and Anderson, 1958; Brandberry et. al., 1991; and Titgem eyer et. al., 2001). Additional studies (Hardison et al., 1955; Brisson et al., 1957; Langland et al., 1963; and Prigge et al., 1981; Fenton and Fenton, 1979; Czarnock et. al., 1961; Suzuki et. al., 1991) have developed analytical procedures, evaluated and derived techniques based on dosing, recommended fecal collection frequencies on different diets and feeding conditions when Cr 2 O 3 is used in digestibility experiments However, although Cr 2 O 3 has been the most frequently used marker, there are reports ass ociating it with health risks (specifically those that may be carcinogenic; Myers et al., 2004). In contrast, TiO 2 has been recently explored as a potential alternative digestibility marker, with less health risks, that may be legally added to feeds (Tit gemeyer et al., 2001). Several studies have indicated the feasibility of using TiO 2 as a viable total tract digestibility marker in rats, chicken, pigs, dairy cows (Myers et al., 2004) and beef steers (Titgemeyer et al., 2001). However little has been done to test its efficacy in estimating digestibility in a variety of forms of feed or feeding conditions. Currently, there is no
36 available data to explain the viability of using TiO 2 as a digestibility marker in beef animals fed fresh forages on an ad libitum basis. In addition, no data is available to either quantify the effect of differing sampling frequencies. Therefore, additional data is necessary to further evaluate the use of TiO 2 to broaden its application as a digestibility marker in digestibility exp eriments.
37 CHAPTER 3 ESTIMATION OF APPARE NT DIGESTIBILITY OF SIX FORAGES USING TW O DIFFERENT DIGESTIBIL ITY MARKERS Materials and Methods Two experiments were carried out with the objectives of comparing summer and winter annual forages in terms of apparen t total tract digestibility of nutrients. Secondary objectives were to compare the efficacy of titanium dioxide (TiO 2 ) and chromic oxide (Cr 2 O 3 ) as digestibility markers for fresh forages fed on an ad libitum basis, and to determine the effects of performi ng a 2 vs. 3 per day fecal sample collection protocol to measure digestibility. Annual W inter F orages (Experiment 1) The study was conducted at the University of Florida Feed Efficiency Facility (FEF) in Marianna, FL. Ryegrass ( Lolium multiflorum Lam cu ltivar Prine), blend of Ryegrass ( Lolium multiflorum Lam cultivar Prine) and Oat ( Avena sativa cultivar Horizon 201), and the blend of ryegrass ( Lolium multiflorum Lam cultivar Prine) and triticale ( Triticosecale rimpau cultivar Trical 342) were the forage s used in this study. In November 2010 these forages were sown on prepared seedbeds in 0.7 ha paddocks at the following rates: 35 kg ha 1 of ryegrass; 67 kg ha 1 of oat combined with ryegrass sown at the rate of 30 kg ha 1 ; and triticale was sown at the ra te of 274 kg ha 1 in combination with ryegrass at 17 kg ha 1 All pastures were fertilized twice by the Altha, FL Farmers Coop. The first fertilization was done 28 d after planting at the rate of 57 kg ha 1 of N (NH 4 NO 3 ) and 22 kg ha 1 of S while the seco nd was done after another 28 d from the first fertilization at the rate of 57 kg ha 1 of N (NH 4 NO 3 ) and 11 kg ha 1 of S. All grasses were cut fresh using a chopper at 15 cm stubble height. Forages were cut every morning starting at d 98 from planting and c ontinued until the end of the study.
38 Herbage mass of each forage treatment was estimated at the beginning of the experiment. Ryegrass, Oat + Ryegrass, and Triticale + Ryegrass treatments had herbage mass estimated at 2,258 kg ha 1 3,463 kg ha 1 and 5,198 kg ha 1 respectively (Table 3 1 ). The botanical composition of each treatment was described in order to estimate DM contribution of each forage to the total herbag e mass of the treatment (Table 3 1 ). For Ryegrass treatment, 100% of the total herbage mass w as from Ryegrass alone. Ryegrass contributed 91.9%, whereas Oat and weeds contributed 4.1% each to the total herbage mass of Oat + Ryegrass treatment. Triticale made 61.1% while Ryegrass was 38.9% of the total herbage mass of Triticale + Ryegrass treatment Furthermore, Oat + Ryegrass, and Triticale + Ryegrass treatments had DM contents of 16.34% and 16.48% respectively whereas the Ryegrass treatment had 15.81% DM. The CP content for Ryegrass treatment was 17.73%, NDF and ADF estimates were 41.67% and 22.67 % respectively. For Oat + Ryegrass treatment, CP was at 18.27%, while NDF was 44.46% and ADF was 24.13%. As for Triticale + Ryegrass treatment, the CP, NDF and ADF were estimated at 16.32%, 49.56%, and 27.56% respectively (Table 3 1). Annual S ummer F orages (Experiment 2) In experiment 2, three summer forages, Mulato II (hybrid Brachiaria Pearl Millet ( Pennisetum glaucum Sorghum bicolor ) were separately planted in three pens (0.7 ha each) in June 2011. Mulato II w as planted on June 2, 2011 on a prepared seedbed at a seeding rate of 1 kg ha 1 On June 27, remaining two pens on a prepared seedbeds at a seeding rates of 33 kg ha 1 both. All grasses were fertilized by Altha, FL Farmers Coop. with 57 kg ha 1 of N (NH 4 NO 3 ) and
39 11 kg ha 1 of S on d 44 for Mulato II, whereas for Pearl Millet and Sorghum sudan was done on d 19 from planting. These grasses were cut fresh every morning using a chopp er at 15 cm stable height starting from d 91 after planting and this continued until the end of the study. All three summer forages were characterized in terms of botanical composition, yield and nutrient content on DM basis (Table 3 2). The herbage masses for Mulato II, Pearl Millet and Sorghum sudan were 3,132 kg DM ha 1 1,519 kg DM ha 1 and 1,644 kg DM ha 1 respectively. Millet contributed 100% to the total herbage mass of Pearl Millet treatment, while Sorghum sudan made 91.5% of total herbage mass of So rghum sudan treatment with the remaining 3.5% being a contribution from weeds. Mulato II had a significant proportion of weeds, such that Mulato II contributed 51.8% to the total herbage mass of the Mulato II treatment while the remaining 48.2% was a contr ibution from weeds. Furthermore, DM compositions for Mulato II, Pearl Millet and Sorghum sudan were 32.55%, 22.30% and 20.73% respectively. In addition, Pearl Millet had the highest CP (23.18%) followed by Sorghum sudan (18.29%) and lastly, Mulato II at 17 .83%. The NDF for Sorghum sudan was 54.99% followed by Mulato II at 54.33% and Pearl Millet at 51.74%. The ADF estimates indicate that Mullato II had lowest value 27.28% while Pearl Millet and Sorghum sudan had 30.93% and 30.78% respectively (Table 3 2) A nimals and M anagement (Experiment 1) Angus and Angus crossbred heifers (n=12) were used in this study. On d 0, heifers were randomly assigned to one of the 3 forage treatments and allowed to graze for 28 d. These heifers were weighed on d 29 (364 52 kg o f BW) stratified by weight, and then randomly assigned to pens (2 heifers per pen) in the University of Florida Feed
40 Efficiency Facility (FEF) located in Marianna, FL. From d 29 to 44 heifers were offered daily fresh cuts of the same forage treatments the y were grazing and were also provided with fresh water both on ad libitum basis throughout the study period. Individual intake was monitored in the FEF using a GrowSafe system (GrowSafe Systems Ltd., Alberta, Canada). Every heifer became the experimental u nit once entering the FEF. Again, from d 29 to d 44, heifers were bolus dosed with 2 gelatin capsules: one containing 10 g of TiO 2 and the other containing 10 g of Cr 2 O 3 using a balling gun. These gelatin capsules were fed once per day at 1200 h. Feed sam ples were collected from d 39 to d 43 and fecal samples were collected by rectal grabs from d 40 to d 44 of the study allowing 12 d stabilization of the digestibility markers and also for heifers to adapt to the marker and feeding in the FEF. Animals and M anagement (Experiment 2) A total of 12 Angus and Angus crossbred heifers were selected for enrollment in the study and were moved into the Feed Efficiency Facility and penned in groups of 6 per pen. Heifers were offered water and bahia grass hay on ad lib itum basis for 1 wk to adapt them to eating from the FEF bunks before commencement of the study. On d 0, Heifers were weighed (194 11 kg of BW) stratified by weight, and randomly assigned to pens (2 heifers per pen) in the FEF. Heifers were fed grass f resh cuttings from their respective treatments and were also provided with fresh water both on ad libitum basis throughout the study period. Individual intake was monitored in the FEF using a GrowSafe system (GrowSafe Systems Ltd., Alberta, Canada). Every heifer became the experimental unit once entering the FEF, as feed intake was recorded individually. Beginning on d 7, heifers were bolus dosed with 2 gelatin capsules: one containing 10 g of TiO 2 and the other containing 10 g of Cr 2 O 3 using a balling gun. These gelatin
41 capsules were fed only once per d at 1200 h from d 7 until d 19. Feed samples were collected from d 14 to d 18 and fecal samples were collected by rectal grabs from d 15 to d 19 of the study. This was to allow 8 days of marker stabilization and adaptation of animals to the feed. Sample Collection and P reparation (Experiment 1 and 2) Feed samples for daily DM determination were collected in paper bags 2 daily at 0800 h and 1600 h, and the average DM value of the two samples was used to dete rmine daily DM intake. Feed samples were dried at 100C for 24 h in order to determine DM. Feed samples for nutritive value analyses were collected once daily after feeding and were stored in plastic bags and frozen at 20C for posterior freeze drying to avoid loss of nutrients through continued activities of enzymes in the forages soon after cutting. These samples were then freeze dried at 50 o C in order to reduce loss of volatile elements in the forage through heat destruction. Thereafter, the samples we re ground through a 2 mm screen before analysis. Forage samples from the cutting pastures were collected from 3 areas of 0.25 m 2 within each pasture to determine the botanical composition of the forages. These samples were stored in paper bags and transpor ted to the lab. All 3 samples per pasture were combined into 1 sample and weighed. For experiment 1, while still fresh, each forage sample was sorted into the different species present in each pasture as follows: RG treatment: sorted into ryegrass wer e sorted into different species present in each pasture as follows: Mulato and other species for Mulato II treatment, Pearl millet and other species for Pearl Millet treatment,
42 and finally, Sorghum sudan and other species for Sorghum sudan treatment. After sorting, each subsample was weighed fresh and then dried at 100C for 24 h to determine percentage DM contribution of each species to the total herbage mass expressed as kg of DM ha 1 Fecal samples were collected three times a d at 0800 h, 1200 h and 1 600 h by grabbing from the rectum. After every collection, fecal samples were stored in plastic bags and frozen at 20C for posterior freeze drying at 50 o C and subsequent grinding through a 2 mm screen. Fecal samples for the 5 collection days were compos ited within heifer. Two separate pools of composited samples were created to test the effect of adding a noon fecal collection: one containing equal amounts of 0800 h, 1200 h, and 1600 h fecal samples while the other part containing equal amounts of 0800 h and 1600 h fecal samples. These two pools of composited samples were analyzed separately. Sample A nalysis (Experiment 1 and 2) TiO 2 analysis In both experiment 1 and 2, TiO 2 was analyzed following the procedure of Myers et al. (2004). A 1.0 g sample of d ried feces was weighed into weight papers in duplicate and each sample wrapped in a weight paper was placed into 250 mL macro Kjeldahl digestion tubes. In each run, a blank sample was included (fecal sample devoid of Titanium dioxide and treated as the res t of the samples) to account for any absorbance due to the components of the fecal matter. Again, included in each run, were standards prepared by adding 0, 2, 4, 6, 8, and 10 mg of TiO 2 each one placed in digestion tubes without fecal samples. As with oth er samples, weight paper was included in each of the 0 standards. These standards were used to develop a calibration curve. One CT 37 FisherTab tablet (containing 3.5g K 2 SO 4 + 0.4g CuSO 4 ) was added to each digestion
43 tube as a reaction catalyst and subsequ ently, 3 mL of concentrated sulfuric acid was added to each digestion tube. With the manifold on top of the tubes, samples were digested for 2 h at 420 o C. After turning off the digestor, cooling was allowed for at least 30 min. then, 10 mL of 30% H 2 O 2 was added to each digestion tube and further cooling allowed for another 30 min. Later, total liquid weight of each tube was brought to 100 g using distilled water. Thereafter, the liquid was filtered through Fisherbrand P8 Grade filter paper to remove any pre cipitate. A 96 well plate map was created and from each sample 200 L was transferred into the plate in duplicate wells using a pipette. At least two wells of the plate were left unfilled to be used as a correction for empty well absorbance. The absorbanc e was read at 405 nm wavelength using a Bechman DU 500 Spectrophotometer. Cr 2 O 3 analysis For Cr 2 O 3 concentration in feces, approximately 0.5 g 0.05 g of ground samples were dried at 105C for 24 h to determine DM, after which the samples were ashed at 550C for 3 h to determine OM. The method of Williams et al. (1962) was used to digest Cr 2 O 3 in the samples. Briefly, 3 mL of acid manganese sulfate and 4 mL of potassium bromate were added to the ashed samples and heated in a hot plate for approximately 7 minutes after which, 12.5 mL of calcium chloride were added and samples were brought to volume in a 100 mL volumetric flask. After digestion, Cr 2 O 3 concentration was determined by atomic absorption spectrophotometry (358 nm with an air plus acetylene fl ame; AA 6300; Shimadzu Corp., Kyoto, Japan). Analysis of NDF, ADF, and DM Determination of NDF in samples was conducted using an Ankom 200 Fiber Analyzer (Ankom Technology, Fairport, NY) according to procedures of Van Soest et al.
44 (1991; as modified by An kom Technology). Approximately 0.5 g 0.05 g of the ground composite fecal sample for each animal and each diet were placed into individual F57 filter bags (Ankom Technology) and heat sealed. During the NDF procedure, heat amylase (Ankom Technology) and sodium sulfite were added to both feed and fecal samples (dried at 55C for 2 d). Crude p rotein To determine CP (N 6.25) concentrations in feed and feces, approximately 0.250 g 0.005 g of ground feed or fecal s amples were placed into a crucible for total N analysis by rapid combustion using a macro elemental N analyzer (Vario Max CN, Elementar Americas Inc., Mt. Laurel, NJ) following official method 992.15 (AOAC, 1995). Statistical A nalysis (experiment 1 and 2) Both experiment 1 and 2 were split split plot design in which the whole plot tested the forage treatment effect, the split plot tested the fecal collection schedule (2 vs. 3) and the split split plot tested the marker effect (Cr 2 O 3 vs. TiO 2 ) using heife r as the experimental unit. The data was analyzed using the MIXED procedure of SAS. All values reported are least square means and significance was declared if P < 0.05 The model used to analyze the results was: (BC)jk +(TBC)ijk + Eijk Where; Yijk = digestibility of forage Ti = forage effect (i = 1, 2, and 3) Bj = time of collection effect (j = 2 and 3)
45 (TB)ij = forage time of collection Ck = marker effect (k = TiO2 and Cr2O3) (TC)ij = f orage marker effect (BC)jk = time of collection marker effect (TBC)ijk = forage time of collection marker Eijk = experimental error.
46 Table 3 1. Composition, yield and analyzed nutrient content (DM basis) of the winter annual p astures used in Exp. 1. Treatment a Item Ryegrass b O at + R yegrass c T riticale + R yegrass d Herbage mass at the beginning of digestibility phase, kg DM ha 1 2,258 3,463 5,198 Botanical composition, % of total herbage mass DM e Triticale 61.1 Ryegrass 100 91.9 38.9 Oat 4.1 Weeds 0 4.1 0 Analyzed composition, % of DM DM 15.81 16.34 16.48 CP 17.73 18.27 16.32 NDF 41.67 44.46 49.56 ADF 22.67 24.13 27.56 a Three 0.7 ha pastures were planted on a prepared seedbed for each treatmen t. Pastures were planted on November of 2010 and fertilized 28 d after planting with 57 kg ha 1 of N (NH 4 NO 3 ) and 22 kg ha 1 of S. A second fertilization with 57 kg ha 1 of N (NH 4 NO 3 ) and 11 kg ha 1 of S took place 56 d after planting. b Seeding rate = 3 5 kg ha 1 of Lolium multiflorum Lam cv. Prine. c Seeding rate = 67 kg ha 1 of oat ( Avena sativa cv. Horizon 201) plus 30 kg ha 1 of ryegrass ( Lolium multiflorum Lam cv. Prine). d Seeding rate = 274 kg ha 1 of triticale ( Triticosecale rimpau cv. Trical 324) p lus 17 kg ha 1 of ryegrass ( Lolium multiflorum Lam cv. Prine). e Average of three 0.25 m 2 samples taken from each 0.7 ha pasture.
47 Table 3 2 Composition, yield and analyzed nutrient content (DM basis) of the summer annual pastures used in Exp 2. Treatment a Item Mulato II b Millet c Sorghum d Herbage mass at the beginning of digestibility phase, kg DM ha 1 3,132 1,519 1,644 Botanical composition, % of total herbage mass DM e Mulato Brachiaria 51.8 Sorghum Sudan 96.5 Millet 100 Weeds 48.2 0 3.5 Analyzed composition, % of DM DM 32.55 22.30 20.73 CP 17.83 23.18 18.29 NDF 54.33 51.74 54.99 ADF 27.28 30.93 30.78 a Three 0.7 Ha pastures were planted on a prepared seedbed for each treatment. Pastures were planted o n June 2, 2011 (Mulato) and on June 27, 2011 (Sorghum Sudan and Millet). All pastures were fertilized on July 15, 2011 with 57 kg ha 1 of N (NH 4 NO 3 ) and 11 kg ha 1 of S. b Seeding rate = 1 kg ha 1 of Brachiaria hybrid Mulato II. c Seeding rate = 33 kg h a 1 Pennisetum glaucum ). d Seeding rate = 33 kg ha 1 Sorghum bicolor ). e Average of three 0.25 m 2 samples taken from each 0.7 ha pasture.
48 CHAPTER 4 RESULTS AND DISCUSSI ON Results Experiment 1 Daily nutrient intake by the heifers during the study period was estimated. No forage treatment effect was observed ( P > 0.05) on DM, OM CP NDF and ADF intake (Table 4 1 ). Intakes for DM, OM, CP, NDF and ADF across the forages averaged 4.54 kg/d, 4.05 k g/d, 0.79 kg/d, 2.05 kg/d and 1.0 kg/d, respectively. No significant forage treatment effect was observed ( P > 0.05) on the total tract digestib ility of winter forages (Table 4 1 ). Across forage treatments, average apparent nutrient digestibility were 59%, 61%, 60%, 49%, and 35% for DM, OM, CP NDF and ADF respectively. Furthermore, neither fecal sample collection protocol (2 vs. 3 per day) nor markers affected apparent total tract digestibility ( P > 0.05) of DM, OM, CP NDF and ADF from the three winter f orages (Table 4 1 ). In addition, no interactions were found ( P > 0.05) between sample collection protocol and marker for apparent nutrients digestibility of DM, OM, CP, NDF, and ADF across all winter for ages used in this study (Table 4 1 ). It is worth no ting that within TiO 2 as a marker, there were no significant differences ( P > 0.05) in apparent total tract digestibility of nutrients across sampling schedules (Table 4 2 ). Averaged across 2 and 3 sampling schedules, the apparent total tract nutrient di gestibility measured using TiO 2, were 62.1%, 64.2%, 62.7%, 52.1%, and 37.8% for DM, OM, CP NDF and ADF respectively. Similarly to TiO 2 no significant effects ( P > 0.05) were observed in apparent total tract digestibility of nutrients across 2 and 3 samp ling schedules when Cr 2 O 3 was used as a marker (Table 4 2 ). The averages across sampling schedules were 56.3% for DM, 58.6% for OM, 57.5% for CP,
49 46.2% for NDF, and 31.4% for ADF. In addition, no interactions were observed ( P > 0.05) between the markers an d forages (Table 4 2 ). Experiment 2 During the study period, daily intake of DM, OM, CP, NDF and ADF was not affected ( P > 0. 05) by forage treatment (Table 4 3 ). Across the forages, intake of DM, OM, CP, NDF and ADF was averaged 3.6 kg/d, 2.98 kg/d, 0.7 kg /d, 1.93 kg/d and 0.87 kg/d respectively. Similarly to nutrient intake, apparent total tract digestibility by heifers was not affected ( P > 0.05) by forage treatment. The values of apparent total tract digestibility across the forage treatments averaged 56 .2%, 65.9%, 68.4%, 49.7% and 32.7% for DM, OM, CP, NDF and ADF respectively. There was no effect of marker ( P > 0.05) on apparent total tract digestibility of DM, OM, CP NDF and ADF from the three summer forages. Furthermore, there was no interaction ( P > 0.05) between sample collection protocol and marker for apparent digestibility of ADF. However, unlike in experiment 1, interactions were found ( P < 0.05) between sample collection protocol and marker for apparent nutrients digestibility of DM, OM, CP and NDF across all summer for ages used in this study (Table 4 3 ). Under a 2 sampling protocol, apparent total tract digestibility of DM, OM, CP and NDF measured using Cr 2 O 3 were significantly decreased ( P < 0.05) compared with those obtained using TiO 2 (Tabl e 4 4 ). Total tract apparent digestibility of DM, OM, CP and NDF were 7.7%, 5.7%, 5.4% and 8.8% lower with Cr 2 O 3 than when using TiO 2 under a 2 sampling protocol, respectively. Similarly, using Cr 2 O 3 under a 2 sampling protocol yielded digestibility valu es of DM, OM, CP and NDF that were decreased ( P < 0.05) compared with estimates obtained with either Cr 2 O 3 or TiO 2 under a 3 sampling protocol. However, no differences ( P > 0.05) were observed in total tract nutrient
50 digestibility of summer forages when a 3 fecal sampling protocol was implemented (Table 4 4 ). However, ADF digestibility calculated with reference to Cr 2 O 3 under a 2 sampling protocol did not differ ( P > 0.05) from ADF digestibility measured with TiO 2 under a 2 sampling protocol and Cr 2 O 3 under 3 sampling protocol. Under 2 sampling protocol, ADF digestibility estimated by Cr 2 O 3 (23.8%) was 16.3% less than the values estimated with reference to TiO 2 (39.9%) under 3 sampling protocol. Apparent total tract digestibility of DM, OM, CP, NDF a nd ADF measured using TiO 2 under 2 sampling protocol were not different ( P > 0.05) from those measured with Cr 2 O 3 or TiO 2 under 3 sampling protocol Table 4 4 ). There was a marker effect ( P < 0.05) on apparent total tract digestibility of DM, OM, CP and NDF across sampling schedul es (Table 4 5 ). Averaged across 2 and 3 sampling schedules, the apparent total tract nutrient digestibility measured using TiO 2, were 58.3%, 67.6%, 69.8%, and 52.0% for DM, OM, CP and NDF respectively. Whereas using Cr 2 O 3, appa rent total tract digestibility of DM, OM, CP and NDF across 2 and 3 sampling protocol were 53.7%, 64.3%, 66.6%, and 46% respectively. However, no marker effect ( P > 0.05) was found on apparent digestibility of ADF. In addition, an interaction was observe d between marker and forage ( P > 0.05) on the apparent total tract digestibility of all nutrients measured (Table 4 5 ). When using TiO 2 no differences were observed in nutrient digestibility across forages (Table 4 6 ). However, when using Cr 2 O 3 the total tract digestibility of DM and NDF were decreased ( P < 0.05) in Pearl millet compared with Mulato II (Table 4 6 ).
51 Discussion Nutrient Digestibility of Forages The lack of differences in nutrient digestibility in forages may result from the relatively high quality of the forages tested. Mean DM apparent total tract digestibility of winter grasses (combinations of Oat + Ryegrass, and Triticale + Ryegrass) obtained in this study were within the ranges reported by Keuren and Underwood (1990) of the grasses sep arately. Dry Matter digestibility ranges of 60% to 79% and 56% to 77% for Triticale and Oat respectively were reported by Keuren and Underwood (1990). Similarly, mean DM digestibility for Mulato II and Sorghum sudan reported in this study fell within the r anges of previous reports(Vendramini et al., 2011; Lang, 2001) with DM digestibility for Mulato II ranging from 55 to 60%, while a range of 55 to 70% for DM digestibility of Sorghum sudan. However, DM digestibility for Ryegrass and Millet were slightly low er than the ranges by other reports (Blount et al., 2009; Keuren and Underwood, 1990) and this may be a factor of differences in forage management and increased rate of passage in the GIT due to low DM content since harvesting in this study was done at an early stage of growth. Effectiveness of TiO 2 and Cr 2 O 3 as Digestibility Markers The effectiveness of TiO 2 in estimating digestibility of winter forages was similar to that of Cr 2 O 3 in our study. Furthermore, collecting fecal samples by rectal grab 2 a day (at 0800 h and 1600 h) was as sufficient as obtaining samples 3 a day (0800 h, 1200 h and 1600 h) in estimating digestibility of winter grasses. Contrary to our findings, Titgemeyer et al. (2001) reported that total tract DM digestibility measured using TiO 2 was underestimated ( P < 0.01) by 1.1 to 5.5 percentage units while total tract DM digestibility calculated using Cr 2 O 3 was overestimated ( P < 0.01) by 2.0 percentage
52 units in study 2 except in study 3 where estimates calculated using Cr 2 O 3 were not di fferent from ( P = 0.31) those obtained by total fecal collection. These observations were collected from 2 steers limit fed corn based diets in study 2 and 8 steers fed corn based diets on ad libitum basis in study 3. This contrast may be as a result of di fference in the diets and method of administering the marker. Digestibility markers behave differently when used across different diets (Sunvold and Cochran, 1991; Judkins et al., 1990). In our experiments, forage diets were used whereas Titgemeyer et al. (2001) used corn based diets and this may have attributed to Cr 2 O 3 and TiO 2 behaving differently. In addition, in our experiment, the markers were packed in gelatin capsules and administered orally using a balling gun and this ensured that the entire marke r was taken by the animal. In contrast, markers were mixed with feed or dietary supplements in feeders in the study by Titgemeyer et al. (2001) and this may have resulted in some proportions of the markers not being consumed by steers thereby affecting mar ker recovery and digestibility calculations. Unlike in the winter forage study, the presence of an interaction between sampling protocol and markers in summer forage study indicates differences in the effectiveness of Cr 2 O 3 and TiO 2 to estimate digestibili ty of forages with varying characteristics. As indicated by Judkins et al. (1990) different digestibility markers behave differently when used across different diets, this may be applicable in our study as total tract digestibility estimates of summer for ages calculated with reference to Cr 2 O 3 were affected by sampling frequency while those calculated with reference to TiO 2 were not affected. This may have to do with issues concerning marker and digesta association. Similar studies (Titgemeyer, 1997; Owen and Hardson, 1992; Preggie et al., 1981) have
53 described Cr 2 O 3 as a marker that does not mix completely with digesta in the GIT. Chromic oxide often is criticized because it does not associate specifically with either the particulate or fluid phase (Titgem eyer, 1997). Again, it was reviewed by Titgemeyer (1997) that Cr 2 O 3 does not seem to mix completely with ruminal contents, particularly when supplied in gelatin boluses and that collection of representative fecal sample can alleviate the effect. Currently, it has not been clearly established as to how specific feed types affect the behavior of the Cr 2 O 3 However, Titgemeyer (1997) suggested that the variable fecal recoveries and diurnal pattern of excretion for Cr 2 O 3 presumably are a result of temporal sequ estration of the Cr 2 O 3 in the rumen that results from poor mixing with digesta. All summer forages in this study had DM almost 2 that of individual winter forage which means their retention time in the rumen may be higher than that of winter forages and t his may have allowed more time for temporal sequestration of Cr 2 O 3 in the rumen hence differences in marker concentration in the fecal sample thereby affecting digestibility values calculated with reference to Cr 2 O 3 when fecal samples were collected 2 a d ay (at 0800 h and 1600 h). The variation between total tract digestibility of summer forages calculated with reference to Cr 2 O 3 and those calculated with reference to TiO2 under 2x sampling, may be as a result of differences in the rate of recovery, excre tion pattern and ability of marker to mix with digesta when used in different forages. Many reports (Titgemeyer, 1997; and Priggie et al., 1981) have indicated diurnal variations in the flow of Cr 2 O 3 and these variations have been reported to be more prono unced when Cr 2 O 3 is dosed once. Although not much has been reported on the behavior of TiO 2 in comparison with Cr 2 O 3 Myers et al. (2005) reported consistently higher mean concentrations ( P < 0.05) of TiO 2
54 in fecal samples than Cr 2 O 3 at every sampling time in all three experiments. These observations were obtained from eight ewes fed 100% forage diet (brome hay), 50% forage diet and 25% forage diets in experiments 1, 2, and 3 respectively. Markers were dosed intraruminally twice a day (0600 and 1800 h) and samples were collected at 6 h interval for six days in all experiments. Again, Myer et al. (2005) observed a more erratic pattern of the flow of TiO 2 excretion and further described this as a reflection of the normal pattern of digesta flow and thus provid ing a more accurate representation of marker excretion pattern than Cr 2 O 3 This implies that TiO 2 associated well with the digesta than Cr 2 O 3 in diets used and that TiO 2 recovery was good compared to Cr 2 O 3 In our study, collecting fecal samples 2 a day (0800 h and 1600 h) may have been insufficient to accurately estimate total tract digestibility of the summer forages using Cr 2 O 3 However, increasing the sampling frequency to 3 a day may have reduced effects of diurnal variations and recovery and incomp lete mixing with digesta thereby resulting in collection of representative fecal samples hence total tract digestibility of nutrient calculated with reference to Cr 2 O 3 did not differ from those calculated with reference to TiO 2 when fecal samples were coll ected 2 a day (0800 h and 1600 h) and 3 a day (0800 h, 1200 h and 1600 h). When using TiO 2 no differences were observed in nutrient digestibility across forages. However, when using Cr 2 O 3 the total tract digestibility of DM and NDF were decreased in Pe arl millet compared with Mulato II. Although it is hard to find a factor influencing this variation, but differences in DM and NDF content between Pearl millet (22.30%) and Mulato II (32.55%) may have increased the rate of passage for Pearl
55 millet thereby affecting Cr 2 O 3 recovery and subsequent estimates of DM and NDF digestibility. Conclusion Titanium dioxide and chromic oxide are indigestible markers that can be used to calculate total tract digestibility of fresh winter and summer forages fed to rumina nts. Collecting fecal samples at 0800 and 1600 h was sufficient for measuring digestibility in winter forages, eliminating the need for a 1200 h fecal sampling. Both Cr 2 O 3 and TiO 2 may be used indistinctively to estimate digestibility of winter forages whi le only TiO 2 may be used to estimate total tract digestibility for summer forages if fecal samples are collected 2 a day at 0800 h and 1600 h. Caution should be observed when using Cr 2 O 3 in estimating total tract digestibility of fresh summer forages as 2 sampling frequency (0800 h and 1600 h) may result in underestimations of nutrient digestibility. Increasing sampling frequency to 3 a day (0800 h, 1200 h and 1600 h) may yield more desirable results when Cr 2 O 3 is used as a digestibility marker for fresh summer forages. However standard errors of the mean reported in this study, especially for digestibility of fiber fractions may be of concern. The use of digestibility markers that associate more intimately with the forage (e.g. internal markers) and com paring our results with total fecal collection method should be tested in future studies.
56 Table 4 1. Nutrient intake and digestibility by heifers fed winter forages, using Cr2O3 or TiO2 as indigestible marker and under two fecal sampling protocols in Exp 1. Treatment a P value Item Ryegrass O at + R yegrass b T riticale + R yegrass c SEM d Forage tr ea t ment S ampling p rotocol e Marker f Samp ling Marker g Intake h kg/d DM 4.34 4.88 4.4 0.72 0.85 OM 3.87 4.31 3.97 0.64 0.88 CP 0.77 0.89 0.71 0.12 0.6 NDF 1.81 2.17 2.18 0.33 0.68 ADF 0.88 1.04 1.1 0.17 0.64 Digestibility, % DM 50.3 62 65.4 5.9 0.21 0.31 0.1 0.87 OM 51.8 64.8 67.5 5.8 0.18 0.25 0.1 0.91 CP 55.8 58.2 66.1 5 0.36 0.57 0.13 0.85 NDF 3 0.8 57.3 59.4 8.7 0.08 0.33 0.14 0.96 ADF 13.9 42.9 46.8 9.7 0.07 0.14 0.18 0.62 a Winter forages were cut fresh every day from 0.7 ha pastures at 0800 h and offered ad libitum. b C ombination of Oat and Ryegrass. c C ombination of Triticale and Ryegrass. d P ooled standard error of treatment means, n = 4 heifers/treatment. e Effect of fecal sample collection protocol: 2 samples per day (0800 and 1600 h) vs.3 samples/d (0800, 1200 and 1600 h). f Effect of indigestible marker used to calculate apparent total tract digestibility: 10 g/d of each Cr 2 O 3 and TiO 2 were dosed once daily at 1200 h in two separate gelatin capsules. g Sampling protocol indigestible marker interaction. h Intake during the 5 d digestibility measurement period of the experiment.
57 Table 4 2 Ap parent total tract digestibility of nutrients measured using Cr2O3 or TiO2 as indigestible markers in heifers fed three winter forages in Exp. 1. Marker a P value Item Cr 2 O 3 TiO 2 SEM b Marker effect Marker forage c Digestibility, % DM 56.3 62.1 3.82 0.13 0.82 OM 58.6 64.2 3.75 0.13 0.81 CP 57.5 62.7 3.38 0.16 0.89 NDF 46.2 52.1 5.44 0.18 0.85 ADF 31.4 37.8 6.07 0.21 0.77 a Heifers were dosed once daily with 10 g/d of both Cr 2 O 3 and TiO 2 once daily at 1200 h in two separate gelatin capsules. b Pooled standard error of treatment means, n = 4 heifers/treatment. c Effect of interaction between forage consumed (ryegrass, oat + ryegrass, or triticale + ryegrass) and marker used.
58 Table 4 3 Nutrient intake and digestibility by heifers fed sum mer forages, using Cr2O3 or TiO2 as indigestible marker and under two fecal sampling protocols in Exp. 2. Treatment a P value Item Mulato II Millet Sorghum SEM b Forage tr eatment Sampling p rotocol c Marker d Samp ling M arker e Intake f kg/d DM 3.79 3.55 3.46 0.66 0.94 OM 3.12 2.96 2.86 0.56 0.95 CP 0.72 0.73 0.65 0.10 0.85 NDF 2.05 1.88 1.87 0.39 0.93 ADF 0.92 0.84 0.84 0.16 0.92 Digestibility, % DM 57.0 51.6 60.1 5.39 0.55 0.05 0.07 0.02 OM 6 4.1 63.6 70.1 4.32 0.47 0.04 0.09 0.02 CP 67.8 65.9 71.4 4.09 0.63 0.05 0.08 0.03 NDF 50.7 44.5 53.9 6.40 0.58 0.06 0.07 0.02 ADF 30.8 27.6 39.6 8.92 0.63 0.08 0.17 0.20 a Summer forages were cut fresh every day from 0.7 ha pastures at 0800 h and offer ed ad libitum. b Pooled standard error of treatment means, n = 4 heifers/treatment. c Effect of fecal sample collection protocol: 2 samples per day (0800 and 1600 h) vs. 3 samples/d (0800, 1200 and 1600 h). d Effect of indigestible marker used to calculate ap parent total tract digestibility: 10 g/d of each Cr 2 O 3 and TiO 2 were dosed once daily at 1200 h in two separate gelatin capsules. e Sampling protocol indigestible marker interaction. f Intake during the 5 d digestibility measurement period of the experime nt.
59 Table 4 4 Interaction between indigestible marker used and fecal collection protocol on digestibility of nutrients in heifers fed three summer forages in Exp. 2. 2 samples/d k 3 samples/d l Item Cr 2 O 3 TiO 2 Cr 2 O 3 TiO 2 SEM m Digestibility, % DM 48.2 a 55.9 b 59.2 b 61.6 b 3.9 OM 60.2 a 65.9 b 68.4 b 70.0 b 3.03 CP 62.6 a 68.0 b 70.6 b 72.2 b 2.89 NDF 40.5 a 49.3 b 53.0 b 55.9 b 4.41 ADF 23.8 a 30.6 ab 36.3 ab 39.9 b 6.15 a,b Within a row, means without a common superscript differ ( P < 0.05). k Fecal samples collected by rectal grab at 0800 and 1600 h. l Fecal samples collected by rectal grab at 0800, 1200, and 1600 h. m Pooled standard error of treatment means, n = 4 heifers/treatment.
60 Table 4 5 Apparent total tract digestibility of nutr ients measured using Cr2O3 or TiO2 as indigestible markers in heifers fed three summer forages in Exp. 2. Marker a P value Item Cr 2 O 3 TiO 2 SEM b Marker effect Marker forage c Digestibility, % DM 53.7 58.3 3.25 0.02 0.005 OM 64.3 67.6 2.61 0.03 0.006 CP 66.6 69.8 2.47 0.03 0.006 NDF 46.8 52 3.82 0.02 0.005 ADF 30.1 34.9 5.29 0.06 0.004 a Heifers were dosed once daily with 10 g/d of both Cr 2 O 3 and TiO 2 once daily at 1200 h in two separate gelatin capsules. b Pooled standard error of treatment m eans, n = 4 heifers/treatment. c Effect of interaction between forage consumed (Mulato Brachiaria Sorghum Sudan, or Millet) and marker used.
61 Table 4 6 Interaction between type of forage consumed and indigestible marker used on digestibility of nutrients in heifers fed three summer forages in Exp. 2. Mulato II k Millet l Sorghum m Item Cr 2 O 3 TiO 2 Cr 2 O 3 TiO 2 Cr 2 O 3 TiO 2 SEM n Digestibility o % DM 59.0 b 53.5 ab 46.0 a 57.2 ab 56.1 ab 64.1 b 4.47 OM 65.9 ab 63.9 ab 59.3 a 67.9 ab 67.8 ab 73.6 b 3.43 CP 69.3 ab 67.8 ab 61.9 a 69.8 ab 68.6 ab 74.3 b 3.29 NDF 53.1 b 49.9 ab 38.1 a 50.9 ab 49.2 ab 58.5 b 5.24 ADF 34.8 ab 30.5 ab 20.7 a 34.6 ab 34.7 ab 44.5 b 6.93 a,b Within a row, means without a common superscript differ ( P < 0 .05). k Brachiaria hybrid Mulato II planted in three 0.7 ha pastures at a seeding rate of 1 kg ha 1 l Pennisetum glaucum ) planted in three 0.7 ha pastures at a seeding rate of 33 kg ha 1 m Sorghum bicolor ) planted in three 0.7 ha pastures at a seeding rate of 33 kg ha 1 n Pooled standard error of treatment means, n = 4 heifers/treatment. o To measure digestibility, 10 g/d of each Cr 2 O 3 and TiO 2 were dosed once daily at 1200 h in two separate gelatin capsule s to use and indigestible marker and fecal samples collected by rectal grab at 0800, 1200, and 1600 h during 5 consecutive days.
62 CHAPTER 5 COMMERCIAL BEEF PROD UCTION IN MALAWI Introduct ory Remarks Malawi, located in sub Saharan Africa, has a huge potential for beef production. This is attributed to extensive availability of grasses during rainy season and abundant crop residues ( groundnuts haulms, maize stover ) which can be used as feed for beef cattle. Currently, the beef cattle populat ion is approximately 1,060,000 (Department of Animal Health and Livestock Development, 2011). Most of these cattle are kept by small scale farmers in herds of grazing on communal range lands without any supplementation (Chintsanya et al., 2004). Apart from generating income, small scale farmers use beef cattle as source of power for farming activities, pa ying dowries (wedding gifts) and as a symbol of wealth in the society (Chintsanya et al., 2004). In 1957, the Malawi government and development partners started promoting stall feeding programs with the aim of improving beef cattle productivity and qualit y (Spurling et al., 1972). This led to commencement of what is known as commercial beef production. Commercial beef production is a high input and business oriented production system characterized by intensive feeding of beef cattle up to the time they are ready for sale or slaughter (Chintsanya et al., 2004). Therefore, commercial beef producing farmers regard beef cattle raring as a business from which they can generate income on a regular basis. Currently, there is no clear documentation about general ma nagement (breeds, breeding methods, feeding, housing, parasites, and diseases management) of beef cattle on these farms hence limited chances of improving productivity and profitability of beef enterprise. Therefore, our objectives were to
63 conduct a survey to ascertain 1) breeds and breeding methods, 2) nutritional management, 3) parasites and diseases management, and 4) to determine animal housing, farm equipment and animal handling facilities available in commercial beef cattle farms of Malawi. This infor mation is important for determining research needs that can lead to improvement of beef production in Malawi. This survey took place on nine commercial beef producing farms of Malawi and this represented 100% of recorded commercial beef farms. During the i nterviews, farm managers were asked to provide information on animal reproduction, statistics, nutrition management, animal health and farm mechanization. In order to capture this information, both closed and open ended questions were used and in the surve y and responses for each question are pres ented as percentages in Tables 5 1 5 2, 5 3, 5 4, 5 5 and 5 6. Results and Discussion Farm O wnership and O bjectives Out of 9 commercial beef farms in Malawi, 78% are privately owned while 22% belong to the governm ent. The majority (67%) of the farms are exclusively for producing finished beef animals ready for slaughtering while 22%, especially government owned beef farms, breed stock for stall feeding and also act as site for indigenous gene conservation. Only 11% of the farms purchase poor grade small animals, and then improve them through intensive feeding before selling these animals to othe r farmers for finishing (Table 5 1 ). However, there is no reference point to determine when the animals are ready for selli ng to other farmers.
64 Breeds, Breeding M ethods and A nimal P erformance These farms have Zebu, Brahman, and crosses between Zebu and Brahman. However, 44% of the farms have Zebu and Brahman, while 33% have Zebu, Brahman, and crosses between Zebu and Brahman, 11% have crosses between Zebu and Brahman, whereas another one farm (11) keep only the Zebu (Table 5 1) In most of these farms (67%) the population of animals is below 1,000 whereas only 33% of the farms have cattle population greater than 1,000. This is attributed to the size of business capital which limits number of animals a farm can have as a breeding or starter stock. Only 44% of the farms have a breeding season and this is usually from January to March so that calving takes place somewhere around O ctober at the start of the rains to ensure good pastures for calves. The other 56% do not observe a breeding season because they mostly buy animals from other farmers and keep them for upgrading before selling. While within the 56%, others have limited kno wledge on the importance of having a strategic breeding season. In addition, natural service (bull) is the method used in all farms that p ractice cattle breeding (Table 5 2 ). This is because, natural service is considered cheap, easy to apply, more effecti ve and efficient. Other alternative methods of breeding like artificial insemination (AI) have been mentioned and described by these commercial farmers as expensive (as it needs expertise, facilities for storing semen) and therefore, not preferred. The av erage pregnancy rates on most farms (67%) ranges between 70 80% with one farm (11%) recording as high as 81 90%. It is worth noting that 11% of the visited importance of doing so Again, none of the visited farms observe and record average birth weight, these farmers also do not know the potential birth weights of the breeds of animals they are keeping
65 when kept under optimal management (Table 5 2 ) Furthermore, many farms (56%) involved in br eeding beef cattle, registered calf mortality rate of <10% for the whole of 2011, whereas the remainder reg istered no death at all Again, majority of farmers (89%) do not observe or record weaning weights of their stock and also do not know the potential weaning weight of the breeds they are keeping (Table 5 3 ) This Lack of animal performance record keeping may limit improvement in management as farmers do not realize whether applied management methods are enhancing animal performance to its potential thu s may result in low productivity and profitability of the enterprise. Finally, average mature weight of the animals ranged from 300 500 kg on 78% of farms that sell finished beef animals to abattoirs. While at one farm, mature weights were not recorded. Furthermore, it was a challenge to obtain number of animals sold per annum, because on some farms, this information is regarded as a business secret and is not shared to anyone. However, from the six farms which provided the information, the range of anima ls sold per year was between 300 and 1 000 (Table 5 3 ). Nutritional Management On majority of the farms (56%), animals graze on the rangeland and thereafter offered supplement in form of concentrates and grasses. About half of the remaining farms (22%), ani mals are fed in stalls all the time (zero grazing), whereas on the other farms (22%), animals simply graze on rangeland witho ut any supplementation (Table 5 4 ). Furthermore, out of the farms that practice zero grazing and supplementation, a majority (78%) do not grow any forage for their animals. They simply buy from the local businesses who source the grass from the rangelands at the end of each rainy season. This grass is preserved in form of hay before selling when feed is scarce more especially during t he dry period of the year. A few commercial beef producing farms
66 (22%) grow forages like Rhodes grass, Napier grass and maize, and all these farms, cultivated forages only occupy less than 5% of the total land area with Rhodes grass occupying large hectara ge because of its relative low cost of producti on and high production (Table 5 4 ). Forage production is good during the rainy season (December April) and is poor during dry season (May November). This is due to the fact that forage production is only b ased on rain as a source of water. Most of these forages are preserved in form of hay, with a few in form of silage because hay is easier and cheaper to make as it does not require special facilities (like pit, plastics for wrapping) than does silage (Tabl e 5 5) In addition, majority (78%) of the visited farms do supplementation of some form. The most common supplement is maize bran because it is purchased at a low price and in abundant supply. However, other farms provide their animals with molasses (by p roduct after making sugar from sugarcanes) as supplem ents (Table 5 5 ). Parasites and Diseases Management The most common parasite reported is the Tick on 56% of visited farms, followed by worms (33%) and tsetse fly (11%). Ticks and tsetseflies are controll ed by spraying chemicals whereas worms are controlled by oral administration of drugs (dewormers). Foot and Mouth Disease (FMD) was reported common at 34% of the visited commercial farms (especially those located in the southern part of Malawi), while 22% of the farms reported East Cost Fever (ECF) as a problem (especially in farms located in the central part of Malawi). Other diseases like Heart water was reported at 22% of the visited farms while another 22% of farms indicated pneumonia and birth complica tions as among the common health problems encountered. It is interesting to note that 22% of the visited farms did not express any problem with these diseases. Heart water, FMD, ECF, pneumonia are being controlled by a combination of drugs (vaccinations) a nd
67 husbandry practices such as isolation and hygiene i n the pens (Table 5 6 ). Farmers wait for veterinary assistance in cases when complications occur during birth. Animal H ousing and H andling F acility All the visited farms have open pens for housing their animals. At least 89% of the visited farms have cluches for handling animals. While only 11% have no animal handling facility and this limits inspection on animal health and performance (Table 5 6 ). Conclusion and Recommendation The beef industry in Malaw i is not well developed. This is evident by lack of proper strategic breeding and animal performance monitoring, inadequate nutritional management and insufficient farm mechanization. However, there is great potential for improvement due to availability of well adapted breed of cattle (Zebu and Brahman) to Malawi, abundant feed availability (green grasses in wet season and crop residues in dry season) which can improve animal performance if properly conditioned, and also availability of enthusiastic farmers who can adopt new technologies upon receiving adequate training in beef cattle management. In view of this, it is imperative to develop standards of beef cattle management through studies in breed performance under Malawi conditions. More focus should be on reproductive performance, growth performance, disease management and characterization of feed quality and quantity across the nation to enhance proper diet formulation. Thereafter, the developed standards should be effectively disseminated to commercial beef producers for implementation. This approach can uplift beef production thereby increasing its
68 T able 5 1. F arm ownership, objectives and cattle breeds presented in percentages of the nine visited beef producing far ms Item Gov a Private Beef production Breeding & gene conservation Upgrading animals Zebu only b Zebu & Brahman Zebu Brahman c All breeds d Ownership 22 78 Objective 67 22 11 Cattle breeds 11.1 44.4 11.1 33.3 a Government b Malawi zebu cattle breed c Cross between Malawi zebu and Brahman d Farms keeping Malawi zebu, Brahman and also crosses between Malawi zebu and Brahman.
69 Table 5 2. C attle population, breeding seas on, breeding methods, pregnancy rate and birth weight expressed as percentage of 9 beef producing farms Item <1000 >1000 Yes No Jan Mar a NM b Not applicable 70 80 % 81 90 % Not observed Not recorded Not known Cattle population 67 33 Have breeding season 44 56 Breeding season 100 Breeding method 89 11 Conception rate 11 67 11 11 Birth weight (BW) 100 Breed potential BW 100 a B etween months of Janu ary and March b Natural mating
70 Table 5 3. Mortality rates, weaning and mature weight and number of animals sold per annum expressed as percentage of 9 visited beef producing farms of Malawi. a ww = weaning w eight Item 0% = 10% Not applicable Not observed Not known 300 500 <500 >500 Not disclosed Mortality rate 33 56 11 Weaning weight ww a 11 89 Breed potential WW 100 Mature weight (kg) 11 11 78 Animals sold per annum 33 33 34
71 Table 5 4. Animal feeding and feed production expressed as percentage of 9 visited beef producing farms of Malawi a Grazing and Stall feeding Item Stall feeding only Grazing only G & S a None Rhodes, Napier & Corn < 5% Rhodes grass Corn/ maize Feeding practice 22 22 56 Cultivated forages 78 22 Land allocated to pastures 100 Pasture occupying largest land 100 Most productive forage 50 50
72 Table 5 5. Fo rage preservation and feed supplementation practices expressed as percentage of 9 beef producing farms of Malawi Item Yes No Hay only Silage only Hay & silage Not applicable Corn bran Cheap Forage preservation 78 22 Form of forage preservat ion 56 0 22 22 Supplementation 78 22 Common supplements 100 Why corn bran common supplement 100
73 Table 5 6. A nimal housing, disease and parasite management expressed as percentage of 9 beef pr oducing farms of Malawi Item Open pen Clutch None Ticks Worms Tsetsefly Drugs & good hygiene FMD a HW b Pneumonia & BC c ECF d Animal housing 100 A nimal handling facility 89 11 Common pests / parasites 56 33 11 Pests / parasite control 100 Common diseases 34 22 22 22 Disease control 100 a Foot and Mouth Disease b Heart Water diseases c Pneumonia and Birth Complication d East Cost F ever diseases
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79 BIOGRAPHICAL SKETCH Chunala Alexico Njombwa was born in D owa M alawi There he grew up on a small family farm, growing tobacco, peanuts a nd corn. Chunala attended primary and secondary education at Mondwe and M adisi in Dowa District respectively Thereafter, he graduated from Mzuzu University with a b achelor degree in the field of f orestry in 2007 Upon graduation, he got a teaching job at a government secondary school in Malawi After that he joined Bio Energy Resources Limited, where he worked as a Senior Planting Technician in 2008 before joining the Department of Agricultural Research S ervices in 2009. In 2010, Chunala moved to Florida US to pursue his m aster s degree in animal s ciences at University of Florida with financial support from United States Agency for International Development (USAID ) under t he supervision of Dr. Cliff Lamb His major focus has been on forage evaluation and nutrition of beef cattle.