WWIWIIIWHNIWHIHWWWNNIINHWHWI (.0010 mums 3 1293 00686 4684 , L This is to certify that the thesis entitled Influence of Adding Ammonia With or Without Molasses to Corn Stalklage on Performance of Dairy Cows and on Stalklage Fermentation presented by Antonio Hargreaves has been accepted towards fulfillment of the requirements for M.S. degcein Animal Science Major professor AMMM/ fl December 2, 1982 Date 0-7639 MSUi: MI ‘mmnfi'm ‘ ' "' ' "r, ’ , Institution mmfii’f igam Mate mm M Universm MSU RETURNING MATERIALS: Place in book drop to remove this checkout from R l lfifllggfigiih ,your record. FINES will be charged if book is returned after the date stamped below. a". 3i r INFLUENCE OF ADDING AMMONIA WITH OR WITHOUT MOLASSES TO CORN STALKLAGE ON PERFORMANCE OF DAIRY COWS AND ON STALKLAGE FERMENTATION BY Antonio Hargreaves A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1982 Guam}; ABSTRACT INFLUENCE OF ADDING AMMONIA WITH OR WITHOUT MOLASSES TO CORN STALKLAGE ON PERFORMANCE OF DAIRY COWS AND ON STALKLAGE FERMENTATION BY Antonio Hargreaves Upgrading of corn stalklage with ammonia mixtures was investigated and compared to the following treatments: 1) corn silage (control); 2) untreated stalklage; 3) stalklage treated with 2% ammonia, and 4) same as 3 plus 2% molasses. Stalklage was fed as 25% of the complete ration to 6 cows per group in mid— to late—lactation. Cows produced an average of 19.29 kg FCM/day. Treatments 1 and 2 (20.77 and 17.16 kg FCM/day) were significantly higher in milk yield than 3 and 4 (14.71 and 15.44 kg FCM/day) (P<.05). In additional experiment, stalklage (in treatment 4) was compared with corn silage at 10 and 20% of the ration dry matter and no depression in milk yield was observed. Fermentation patterns were characterized by ensiling stalklages in experimental bag silos and sampling at 0, 3, 7, l9 and 50 days. Dry matter did not differ signifi- cantly. Lactic acid increased with ammonia-molasses and time; pH for all treatments decreased with time and then stabilized. After ammoniation all fiber components decreased, but IVDMD increased (P<.Ol). DEDICATION To My Mother, Mrs. Graciela B. de Hargreaves ii ACKNOWLEDGEMENTS The author extends his deep appreciation to Dr. John T. Huber for his valued guidance and counsel during his graduate program. The author is also indebted to Dr.le3.Bergen, Dr. J.C. Waller, and Dr. D. Hillman, as members of his graduate committee, for their advice and assistance throughout the program. The author also wishes to thank Dr. Ronald Nelson for making the facilities of Michigan State University and the Dairy Teaching and Research Center available for this research, as well as for the financial assistance. My appreciation is extended to the Dairy Nutrition Secretary, Mrs. Elaine Kibbey, for her excellent job in typing this manuscript. Also to my professors and friends. The author is indebted to his employers, The Agricultural Research Institute of Chile, for the financial support during the course of his graduate program. TABLE OF CONTENTS INTRODUCTION ................................................ LITERATURE REVIEW ............................................ 2.1 Chemical Composition of Corn Stalklage ................ 2.2 Influence of Ammonia on the Chemical Composition and Dry Matter Digestibility ............................. a. Moisture ......................................... b. Plant species ..................................... c. Treatment time .................................... d. Level of ammonia ................................. e. Temperature and pressure ........................... 2.3 Influence of Ammonia on the Preservation, Digestibility of Dry Matter Components and Intake of Roughages ...... 2.4 Molasses Addition to Ammonia—Treated Roughages ....... 2.5 Ammonia-Treated Roughages and Animal Production ....... a. Beef cattle and sheep ............................ b. Ammoniated forages for dairy cows ................. MATERIALS AND METHODS ........................................ 3.1 Stalklage Management .................................. 3.2 Feeding Trial 1 ...................................... 3.3 Feeding Trial 2 ...................................... 3.4 Corn Stalklage Fermentation Study .................... 3.5 Statistical Analysis ......... . ........................ iv Page 10 13 13 14 15 15 16 18 21 21 23 25 25 26 29 3O 31 Page RESULTS AND DISCUSSION ....................................... 34 4.1 Feeding Trial 1 .............. . ........ . ................ 34 4.1.1 Chemical analysis of corn stalklages ............ 34 4.1.2 Animal performance .............................. 36 4.2 Feeding Trial 2 ........................................ 40 4.2.1 Animal performance .............................. 40 4.3 Corn Stalklage Fermentation Study ...................... 42 4.3.1 Dry Matter ...................................... 42 4.3.2 pH .............................................. 43 4.3.3 Lactic acid ..................................... 44 4.3.4 Nitrogen fraction ............................... 46 4.3.5 Fiber fractionation ............................. 47 4.3.6 In vitro organic matter digestibility ........... 49 SUMMARY AND CONCLUSIONS ...................................... 54 LITERATURE CITED ............................................. 56 Table 10 ll 12 l3 14 15 LIST OF TABLES Chemical composition of corn stalks .................. Effect of corn stalk harvest date on calf performance. Effect of corn stover level in corn silage based rations on intakes by sheep .......... . ............... Effects of anhydrous ammonia treatment of corn stalks upon apparent digestibility of fiber components ...... Chemical composition of molasses ..................... Cow weight chance and condition score change with four corn stover treatments .......................... Ammonia and ammonia—mineral—molasses corn stalklage for lactating dairy cows .... ......................... Pre—experimental period ration for lactating dairy cows ................................................. Experimental rations for feeding trial 1 ............. Composition of the supplement ........................ Experimental rations for feeding trial 2 ............. Effect of ammoniation and ammonia-molasses treatment on the composition of corn stalklage composites ...... Composition of ration composites for cows. Feeding trial 1 .. ........ . ..... . ........ . .................... Treatment and time effect on the dry matter intake (kg DM/cow/day). Feeding trial 1 .... ................ Milk production, milk composition, feed efficiency and changes in body weight. Feeding trial 1 ............. vi Page 18 19 22 24 26 27 28 29 35 38 39 Table 16 17 18 19 2O 21 22 23 24 25 26 27 Composition of ration composites for cows fed varying amounts of stalklage treated with ammonia and molasses (AMS) ........ . ....... . .............. Effects of treatment and time on the dry matter intakes of cows fed varying amounts of stalklage treated with ammonia and molasses (AMS). Feeding trial 2 ...... . .................................... Milk production, milk composition, feed efficiency and changes in body weight of cows fed varying amounts of stalklage treated with ammonia and molasses (AMS). Feeding trial 2 ................. . Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on dry matter content(%) in corn stalklage ........ . ................................ Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on pH in corn stalklage .......... Effect of ammonia, ammonia-molasses, inoculum and time of ensiling on lactic acid in corn stalklage Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on total nitrogen in corn stalk— lage ................ . ............................. Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on insoluble nitrogen in corn stalklage ......................................... Effect of ammonia, ammonia— molasses, inoculum and time of ensiling on soluble nitrogen ......... .... Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on neutral detergent fiber in corn stalklage . ..... . ....................... . ..... Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on acid detergent fiber in corn stalklage ..... ........ .... ............. . ........ . Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on hemicellulose in corn stalk- lage ....... . ................................ . ..... Page 40 41 42 43 44 45 47 48 48 50 50 51 Table 28 29 3O 31 Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on cellulose in corn stalklage .... Effect of ammonia, ammonia-molasses, inoculum and time of ensiling on acid detergent fiber ....... .... Total ash of corn stalklage ........................ Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on in vitro organic matter diges— tibility in corn stalklage .. ....................... viii Page 51 52 52 53 1. INTRODUCTION During the last few years the amount of crop residues such as cereal straws, corn stalks, corn cobs and husks fed to ruminant animals has increased to reduce feeding costs and because of somewhat inadequate supplies of conventional roughages. Current reports estimate that around 80% of the corn crop is harvested for grain which means that more than 150 million metric tons of corn residue are produced in the United States each year (Klopfenstein, 1978). Approximately 50% of the corn plant dry matter remains in the field after grain harvest (Berger et a1., 1979). This residue consists of 54% stalks, 12% leaves, 21% cobs and 13% husks (Klopfenstein and Owen, 1981). Based on these estimates, nearly 81 million metric tons correspond to corn stalks and leaves which offer good potential sources of energy for ruminants. Although this material could be grazed directly, one may reduce field losses and obtain higher palatability through ensiling. Corn stalks are characterized by low crude protein, high lignin and neutral detergent fiber, and a dry matter digestibility of no more than 55% (Klopfenstein, 1981). When harvested as mature corn, degree of lignification of the plant is at its maximum. Ruminants are able to digest polysaccharides that are contained in both the hemicellulose and cellulose, but these fractions are poorly utilized because of high lignin and silica which impede microbial digestion. Animal performance is limited by the low digestibility and poor intake. Researchers have been attempting to upgrade these low—quality roughages by physical or chemical treatments, or both. This research is justified not only because of low digestibility, but also because of environmental pollution by burning residues. Nitrogen and phosphorus are limiting nutrients in corn stalks. Considerable improvements on corn stalk nutritive value have been obtained by chemical treatment with ammonia (Oji et al., 1977; Saenger et al., 1982). Ammonia increases energy availability and nitrogen content of corn stalks, thereby improving the efficiency of utilization. However, supplementation with sugar might be advantageous for an improved silage fermentation. Corn stalk consists of the stem or main axis of the plant and its leaves. Corn stalks stored as silage is called corn stalklage. Most of the studies on ammonia—treated or untreated corn stalklage have been with beef cattle and sheep with promising results. Studies with lactating dairy cows are limited. The lactating dairy cow is an efficient food producer. Ammonia addition to corn stalklage may enhance nutritive value so that it may substitute for a portion of the forage in dairy cow rations. The objectives of this study were as follows: 1. To evaluate corn stalklages treated with ammonia or ammonia plus molasses as forage sources for lactating cows. 2. To characterize fermentation patterns of treated and untreated corn stalklage. 2. LITERATURE REVIEW Crop residues have received considerable attention during the last few years. They are considered low quality forages inefficiently utilized by ruminants because of their high content of poorly digestible fiber. This poor digestibility is due to the high lignin and cell wall contents of these forages (Klopfenstein, 1973). Van Soest and Wine (1967) divided forages by extraction with a neutral detergent solution into two groups. These are: l) soluble cell contents comprised of sugars, organic acids, starch, lipids, protein, nucleic acids and most of the inorganic constituents; and 2) the insol- uble residue or cell walls containing hemicellulose, cellulose, cutin and lignin. The proportion of cell walls in forages and the proportion of lignin in the cell walls increases with: 1) advancing maturity; 2) with the elevation of evapotranspiration rates and ambient temperatures, and 3) with declining day length under which the plants are grown (Osbourn, 1978; Deinum et al., 1968). Digestion of the cell wall fraction is entirely dependent upon the enzymes secreted by the micro— organisms in the rumen and/or large intestine, and is reduced as the forage plant matures and lignification increases (Anderson, 1978; Osbourn, 1978; Van Soest and Marcus, 1964). Several crop residues have been studied with the goal of increasing their availability for livestock. Corn residue, for example, has been widely tested as a ration component for cattle and sheep. 2.1 Chemical Composition of Corn Stalklage The quality of corn residues varies with location, water avail- ability and maturity at harvest (Klopfenstein, 1981). Corn stalk decreases in digestibility and content of cell solubles with time after physiological maturity of the corn plant (Van Soest and Marcus, 1964; Ward, 1978; Berger et al., 1979; Klopfenstein and Owen, 1981). Both in vitro dry matter digestibility and carbohydrate content of the stalks decrease with maturity throughout the harvest season and are related to rainfall (Cummins, 1970; McDonnell and Klopfenstein, 1980). After corn grain harvest, the remaining stover is usually high in dry matter (Holloway et al., 1977) and the crude protein is low (Klopfenstein and Owen, 1981). Chemical composition of corn stalks and stalklage is summarized from several authors (Table 1). TABLE 1. Chemical composition of corn stalks, % of dry matter Item Crude Nuetral Acid Acid Source protein detergent detergent detergent fiber fiber lignin % 95 % % Corn stalks 5.06 85.70 53.30 9.1 Saenger et a1. 1982 Corn stalks 4.00 75.00 — 7.0 Klopfenstein and Owen,1981 Corn stalklage 5.16 91.65 71.28 10.05 Holloway et a1. 1977 Corn stalklage 8.20 64.90 42.50 7.90 Colenbrander et al.,l971 Average 5.61 79.31 55.69 8.51 Protein content of corn stalks is quite low (Boehlje and Vetter, 1979). Diets containing mostly stalks might be deficient in protein for the gestating cow, but in both protein and energy for the lactating cow (Ward, 1978). Calf diets containing corn stalks required supplemental protein and energy (Orcasberro et al., 1978). Furthermore, corn residues are deficient in most minerals, particularly phosphorus; and very low in vitamin A (Kendall, 1977; Ward, 1978). It is therefore necessary to supplement these nutrients to prevent deficiencies. Also, supplemental magnesium was critical for beef cows fed corn stover (Vona et al., 1980). Corn stalks vary in digestibility depending upon moisture, variety, temperature and maturity (McDonnell and Klopfenstein, 1980; Klopfenstein and Owen, 1981). Dry matter digestibility values of 38% to 65% have been reported with an average of 43% (Klopfenstein and Owen, 1981). In vitro dry matter disappearance of the stalks decreased linearly with time (Kamstra et al., 1958; Cummins, 1970; Berger et al., 1979; Paterson et al., 1981; Klopfenstein, 1981). Time between grain harvest and corn stalk harvest markedly affects dry matter digestibility, soluble carbohydrate content and cellulose digestibility (Johnson and McClure, 1968; Cummins, 1970; Ward, 1978), as well as animal gains (Paterson et al., 1981). Berger et a1. (1979) reported that steers fed corn stalklage harvested within 72 hours of grain harvest gained significantly more than steers fed stalklage har— vested one month later, when moisture content dropped to 30% (Table 2). Hence, manipulation of harvest time offers the opportunity to obtain higher quality forage at no extra expense (Klopfenstein and Owen, 1981). TABLE 2. Effect of corn stalk harvest date on calf performancea Item Corn Early harvest Late harvest silage corn stalklage corn stalklagec Number of steers 79 43 43 Initial weight, kg 252.3 251.8 251.4 Daily gain, kg 0.95 0.65 0.48 Daily feed intake, kg D.M. Corn silage 7.04 -——- ——-— Corn stalks -——— 5.4 4.8 Supplement 0.78 1.07 0.95 Total 7.82 6.47 5.75 Feed efficiency (kg/kg) Corn silage 3.38 ———— ———— Corn stalks ——-- 3.78 4.55 Supplement 0.38 0.75 0.90 Total 3.76 4.53 5.45 aAverage of 2 years. bStalklage harvested same day as 25% moisture corn grain. CStalklage harvested 3 to 4 weeks after 25% moisture corn grain. From Klopfenstein, T. 1981. In Upgrading Residues and Byproducts for Animals. John T. Huber (Ed.), p. 43. The decrease in in vitro dry matter digestibility as the plant matured was attributed to decreased cell solubles, increased structural carbohy— drates, and greater lignification (Berger et al., 1979). Lamm and Ward (1977) have found that corn stalks which remained in the field during winter decreased in crude protein from 6.6% (as per— centage of organic matter) to 5.8%, and that in vitro organic matter digestibility decreased from 62.1% to 48.8%, compared to fall—harvested corn stalks. A negative correlation was shown between in vitro dry matter digestibility and neutral detergent fiber, and a positive rela— tionship between loss of solubles with time and IVDMD (Berger et al., 1979). Van Soest (1968) pointed out that chemical composition of forages (and feeds in general) determines the availability of nutrients and true digestibility. Additional factors which contribute to apparent digesti- bility, metabolizable energy and net energy of feeds arise mostly from the digestive physiology of the animal. Cell solubles can be separated from cell wall constituents by a neutral detergent solution (Deinum, 1973; Theander and Aman, 1980) which provides a clear differentiation in availability of forage dry matter (Van Soest, 1968) and is highly related to animal intake. Cell contents of a forage are almost completely digested, and is independent of the lignin, hemicellulose and cellulose content. Lignin is primarily responsible for the incomplete digestibility of cellulose and hemicellulose. Such action occurs through its direct linkage to the structural carbohydrates (Van Soest, 1964; Van Soest, 1968; Falen et al., 1968; Deinum, 1973; Johnson and Pezo, 1975; Fahey et al., 1979; Fahey et al., 1980). Plant cell wall content is the most important factor that affects organic matter digestibility of roughages for ruminants. It represents the portion of the plant over which lignin exerts its influence and determines the space—occupying capacity of a feed. Hence, it is not surprising that high cell wall content is associated with low intakes (Van Soest, 1968; Deinum, 1973; Thomson and Beever, 1973). Furthermore, characteristics of forages which determine digestibility also affect intake and explain the close relationship between digestibility and intake (Blaxter et al., 1961; Demarquilly, 1965; Demarquilly and Jarrige, 1971). Growing lambs fed different levels of corn stover with corn silage (Keys, 1980) efficiently utilized corn stover at about 25% of the dry matter, but gains and efficiency decreased when stover was increased to 50%. Similar trends have been reported by others (Oh et al., 1971; Meno et al., 1972; Colenbrander et al., 1973; McDonnell et al., 1979). TABLE 3. Effect of corn stover level in corn silage based rations on intakes by sheep Level in the diet Dry Matter Cell wall Intake Average Corn Corn Soybean digestibility content daily silage stover meal gain ------- % of DM-———————- % % g DM/kg 9 BW-75 O 90.4 9.6 68.6 47.0 58.1 150 25 64.6 10.4 68.9 64.1 58.7 138 50 38.7 11.3 59.1 63.9 46.2 74 75 12.8 12.2 57.7 75.1 45.2 62 Source: Keys, J.E. 1980. 75th Ann. Meet. ADSA, Virginia, 153. Through plant breeding, lignin content has been decreased in experimental corn; therefore, better utilization of corn stover might be obtained. Colenbrander et a1. (1973) fed Holstein heifers two corn stalklages, one from the brown midrib 3 mutant gene for low lignin and the other from a commercial hybrid (normal). Silage from the mutant stover was significantly lower in lignin, cell wall content, acid detergent fiber and higher in crude protein. Average daily gain, efficiency of gains, intake and digestibilities of dry matter, cell walls, acid detergent fiber, hemicellulose and cellulose were higher for heifers fed the mutant stover (Colenbrander et al., 1973). The importance of both intake and digestibility in utilization of poor quality roughages has been reported by many investigators (Campling et al., 1962; Oh et al., 1971; Ellis, 1978; Lippke, 1980). There is a strong relationship between ration composition and food intake (Conrad et al., 1964; Bines, 1976). For diets of low digestibility, intake is regulated by rumen capacity, rate of passage and dry matter digestibility; whereas, metabolic size, production and digestibility become the con— trolling factors on intake of rations higher in digestibility (Conrad et al., 1964; Oh et al., 1969; Jones, 1973; Bines, 1976; Journet and Remond, 1976; Ellis, 1978). There is also a relationship between intake by sheep of low-protein roughage and their nitrogen status (Egan 1965a; Oh et al., 1969). When nitrogen status of sheep was gradually improved by infusing nitrogen supplements (casein or urea) into the duodenum, daily digestible energy intake increased. When urea was added to the ration, voluntary feed consumption, microbial activity, dry matter digestibility, concentration of rumen volatile fatty acids and microbial protein, and blood urea were increased (Egan, 1965b; Oh et al., 1969) when steers were fed oaten chaff ad libitum. Intake was 23% higher with N supplementation (urea or casein) than without (Redman et al., 1980). Huber and Thomas (1971) reported that intakes of corn silage by lactating cows and total dry matter were directly related to the protein content of the ration between 8.5 and 12.5% crude protein equivalent. 10 2.2 Influence of Ammonia on the Chemical Composition and Dry Matter Digestibility of Roughages Ammonia is a ubiquitous constitutent of the soil, the atmosphere and the waters of the earth. It is a colorless gas under standard condi- tions, whose pungent odor is easily discernible at concentrations above 50 ppm. It represents the 3 valence state of nitrogen, and can exist in a number of additional valence states (N.R.C., 1979). Ammonia is a polar substance and exhibits strong hydrogen-bonding characteristics. A promising method to treat low—quality roughages is with ammonia. Anhydrous ammonia (NH gaseous) and solutions of ammonia in water 3! (NH OH, aqueous) have both shown positive effects in improving the nutri— 4 tive value of low—quality roughage (Sundstol et al., 1978). Lignin is bound tightly to the plant polysaccharides at several sites to give a strong structure which, as was discussed before, decreases digestibility of the plant fiber by rumen or caecum microorganisms. The attachment to lignin prevents swelling of the plant fiber in an aqueous media to a condition suitable for penetration by microbial polysacchari— dases (Harkin, 1973). Also, non—glycosidic crosslinks inhibit digestion of cellulose derivatives because cellulases normally cannot approach closer than 2-3 sugars away from the crosslink (Harkin, 1973). Ammonia treatment does not affect the particle size of the material but it does increase swelling of cell walls. During swelling there is a marked increase in the water—holding capacity (fiber saturation point) of the cell walls (Tarkow and Feist, 1969; Chandra and Jackson, 1971; Harkin, 1973; Oji, 1979) due to the cleavage of uronic ester crosslinks. 11 Also, ensiling anaerobically produces organic acids (lactic and acetic) that lower the pH and cleave acid—labile lignin—carbohydrate bonds (Harkin, 1973). Oji (1979) reported that alkali reduces the strength of intermolecular hydrogen bonds which bind cellulose, thus causing swelling. Alkali causes intracrystalline, as opposed to intercrystalline swelling of cellulose which enhances rumen microbial accessibility and greater digestibility of roughages (Oji, 1979). Addition of 3 g NH3 per 100 g tall fescue hay resulted in ammonolysis of ester bonds and acetyl groups attached to xylan chains present in lignocellulosic materials (Buettner, 1978; Oji, 1979; Kuhl, 1982). The acetyl groups increase with the maturity of the grass, and accompany a fall in the digestibility, particularly of the pentosan fraction. They may be as high as 2.7% of the dry matter (Bacon et al., 1975). Morris and Bacon (1977) found that the removal of acetyl groups, using sodium ethoxide, leaving sugars and lignin unchanged, increased the digestibility of cell walls. Ammonolysis readily changes acetyl groups to acetic acid (Oji et al., 1977). Ammonia also increases solubilization of hemicellulose (Klopfenstein, 1978; Holloway et al., 1977; Itoh et al., 1975; Solaiman et al., 1978; Kuhl, 1982) of different roughages. Ammonia often does not reduce the lignin content of treated roughages (Garret et al., 1974; Waller, 1976; Klopfenstein, 1978; Garret et al., 1979; Streeter and Horn, 1981). However, lignin in cereal straw was reduced after treatment with ammonia (Horton, 1981; Harbers et al., 1982) and in corn stover after treatment with ammonium polyphosphate (Colenbrander 12 et al., 1971). Furthermore, alkali treatment of barley straw decreased the content of cis-p—coumaric, cis-ferulic and diferulic acids, which are phenolic components of plant cell walls, causing cell wall digestibility to increase (Hartley and Jones, 1978). In general, crop residues do not contain more than 5% crude protein (dry matter basis); and, as it was discussed earlier, corn stalklage was about 5.6%. One of the most important advantages of using ammonia is the value of the residual nitrogen as nonprotein nitrogen (Klopfenstein and Owen, 1981). Ammonia treatment increases crude protein content of low— quality roughages (Martynov, 1972; Lawlor and O'Shea, 1979; Breaux et al., 1981; Harpster et al., 1981; Johanning et al., 1981; Males, 1981; Oji and Mowat, 1981; Saenger et al., 1981). Ammonia—treated corn silages increased in water insoluble nitrogen (Henderson and Bergen, 1972; Huber et al., 1973; Huber et al., 1979a; Huber et al., 1979b; Foldager and Huber, 1979), due to a combination of decreased breakdown of original plant protein; increased synthesis of silage microbial protein; and binding of ammonia by silage particles (Huber et al., 1979b; Bergen et al., 1974). With 15N labelled ammonia, Huber et al. (1979b) found that 41% of the increase in water insoluble nitrogen was due to indirect action of ammonia on plant material. They (Huber et al., 1979b) proposed a reduction in proteolysis by plant enzymes during Unsearly stages of fermentation as suggested by Bergen et a1. (1974), and subsequently established by Johnson et a1. (1982). Ammonia addition to corn silage also delays decreases in pH and increases lactic acid (Britt and Huber, 1975; Huber et al., 1973; 13 Huber et al., 1979b). This greater buffering capacity caused a higher lactate content (Huber et al., 1973). Factors which affect response to ammoniation are moisture, plant species, treatment time, level of ammonia, temperature and pressure (Oji, 1979). a) Moisture In an experiment by Solaiman et a1. (1978), chopped wheat straws of five dry matter levels (10,20,30,40 and 50%) were sprayed with NH4OH at 3.3% of DM and then sealed. Water content had significant linear effects on in vitro dry matter digestibility and total nitrogen content. High moisture material resulted in distribution problems when anhydrous ammonia was injected; so if straw is dried, this should be done before rather than after ammonia treatment (Sundstol et al., 1978). Higher digestibility with higher moisture was demonstrated by Borhami and Sundstol (1982). b) Plant Species Response of low quality roughages to alkali treatment is fairly specific. Variation between and within species influences the effec— tiveness of NH3 treatment. Kiangi et a1. (1981) compared different sources of ammonia for treatment of corn stover, rice and wheat straws at rates of 0, 25 and 50 g NH3/kg DM. They found that anhydrous ammonia was the most effective reagent for improving IVDMD and in vitro organic matter digestibility (IVOMD) of corn stover; that aqueous ammonia was the most active in increasing IVDMD and IVOMD of wheat straw; and the two forms of ammonia were similarly effective on rice straw. Kernan et a1. (1981) found different responses within the same l4 crop and among cultivars and locations. They concluded that crop residues of annual grass families responded more to ammonia treatment than did those of annual legume families. Legumes generally have a higher lignin content than grasses and the chemical bonds between lignin and polysaccharides differ. In grasses, ester links occur between lignin acid and a xylan chain (hemicellulose), whereas in legumes they are mainly glycosidic bonds (Harkin, 1973; Oji, 1979). Response to alkali treatment is smaller in legume straws due to the resistance of glyco— sidic bonds to alkaline hydrolytic cleavage (Kernan et al., 1981). Initial quality of roughage has been suggested as influencing response to ammoniation. The improvement obtained with ammonia is more pronounced for roughages with low initial digestibility (Waiss et al., 1972; Oji, 1979; Sundstol et al., 1978). c) Treatment Time Time of treatment is an important factor influencing the effect of ammoniation. Chandra and Jackson (1971) concluded that there is a slight decrease with time in the amount of residual alkali present in sprayed straw which indicates that alkali continues to react with straw at a slow rate (Jackson, 1977). There is an interaction between time and temperature during ammoniation of low—quality forages. For low— quality roughages treated with 3 to 4% ammonia at different ambient temperatures, the following treatment times were recommended (Sundstol et al., 1978). 15 Temperature Length of Treatment 0 weeks <5 >8 5—15 4—8 15—30 1—4 >30 <1 Time of treatment is more critical at low than at high temperatures. Ammonia reaction time may be up to 20 days longer than sodium hydroxide (Klopfenstein and Owen, 1981). d) Level of Ammonia Sundstol et a1. (1978) proposed that the optimum level of ammonia is 3—4%, since no further improvement in digestibility is obtained with higher concentrations. Similar observations were reported by Felix and Diarra (1982) and Felix et a1. (1982). However, Sundstol et a1. (1979) found that the nitrogen content and enzyme solubility increased with increasing NH level up to 7.0%. On the other hand, 3 maximum digestibility was at 2.6% NH3, 620C and 4 days incubation, or 5.9% NH3, 300C and 3—7 days incubation (Waagepetersen and Thomsen, 1977). These data suggest that besides the time-temperature interaction, there is a dosage—time—temperature interaction influencing the effect of ammoniation. e) Temperature and Pressure At moderate temperatures, ammonia reacts slowly with coarse roughage (Oji, 1979), but the reaction is extremely slow below 50C (Sundstol et al., 1978). Ammoniation at elevated temperatures and/or l6 pressure will bring the greatest improvements in digestibility (Oji and Mowat, 1979; Oji, 1979; Oji et al., 1979). Oji (1979) concluded that smaller concentrations of ammonia are required when treating at high than at low temperatures. High temperatures and pressureshavebeen reported to yield products low in digestibility and nitrogen utilization (Oji, 1979). Nonetheless, greatest increases in nutritive value are achieved when the roughages are pressure-cooked with alkali solutions (Ololade et al., 1970). 2.3 Influence of Ammonia on the Preservation, Digestibility of Dry Matter Components and Intake of Roughages Antifungal effects constitute another advantage of ammonia. Spoil— age and decomposition from microbial action is inhibited after treating forages with ammonia, especially those of high moisture content (Huber, 1981; Kuhl, 1982). These effects were observed in rice straw (Waiss et al., 1972); in corn silage (Huber et al., 1979a); in whole high moisture shelled corn (Mowat et al., 1981); and in alfalfa hay (Knapp et al., 1974; Knapp et al., 1975). Several researchers have demonstrated that both in vitro and in vivo digestibilities of dry matter are increased after treatment with ammonia (Anderson and Ralston, 1973; Horton, 1978b; Bales et al., 1979; Kernan et al., 1979; Buettner et al., 1982; Paterson et al., 1982; Owen et al., 1982). Mechanisms for improvement of digestibility have been discussed earlier. 17 Nevertheless, the effect of ammoniation on nitrogen utilization is somewhat controversial. Some reports have shown that the apparent nitrogen digestibility decreases after treatment with ammonia (Garret et al., 1974; Garret et al., 1979; Oji et al., 1977; Oji and Mowat, 1979), whereas others have shown an increase (Laguta, 1962; Dokukin and Tracenko, 1967; Sundstol et al., 1978; Horton and Steacy, 1979). Waiss et a1. (1972) mentioned that the production of toxic 4-methy1— imidazole has been observed in the ammoniation of molasses, but they were unable to demonstrate its presence in ammonia-treated rice straw. The cause of the reduced nitrogen digestibility has not been clarified. Oji (1979) reported a color change in ammoniated materials due to oxidation of phenols. Presumably, some nitrogen bound to pheno— lic compounds could partially account for the decreased nitrogen digesti- bility. However, the addition of supplements to ammoniated straws improved the utilization of the nitrogen bound through ammoniation (Horton and Holmes, 1976; Horton, 1978a). The digestibility of other organic components generally increases after ammoniation. The effect of anhydrous ammonia treatment of corn stalks upon apparent digestibility of fiber components is presented in Table 4. After treating with ammonia, apparent digestibilities of NDF, ADF, hemicellulose and cellulose increased 19.6, 13.7, 34.4 and 16.2%, respectively (P<.05). These results suggest that a major effect of ammonia on corn stalks is increasing digestibility of the hemicellulose fraction of fiber (Saenger et al., 1982). 18 TABLE 4. Effects of anhydrous ammonia treatment of corn stalks upon apparent digestibility of fiber components Treatment Apparent Digestibility of NDF ADF Hemicellulose Cellulose Lignin O ———————————————————— 6-—————---———————————-—— 1) NH —stover + corn supplement 66.0 58.4 78.7 72.3 22.8 2) Stover + soybean meal 56.3 52.4 59.1 62.8 19.4 3) Stover + urea 56.0 52.3 57.4 62.5 24.5 4) Stover + corn supple— ment 53.3 49.4 59.1 61.3 24.7 % change (1 vs. 4) 19.6 13.7 34.4 16.2 0 Source: Saenger et al., 1982. J. Animal Sci. 54(2):419-425. Some authors have shown decreased consumption of ammonia-treated roughages (Rounds et al., 1976; Davis, 1981; Males and Falen, 1982). In contrast, several groups demonstrated that treatment with ammonia improved voluntary intake of roughages (Horton and Steacy, 1979; Saenger et al., 1980; Nelson and Klopfenstein, 1980; Herrera-Saldana et al., 1982; Nelson et al., 1982a; Pace et al., 1982). Feeding the ammoniated material with fermented feed (50:50), cottonseed meal, or other supplement also improved intakes and rates of gain. 2.4 Molasses Addition to Ammonia—Treated Roughages Molasses is an inexpensive source of carbohydrate for ruminants and has been used in ruminant rations for many years to increase pala— tability, improve pelleting characteristics, avoid dustiness in conven— tional dry—mixed rations (Karalazos and Swan, 1976), and increase rumen l9 microbial activity (Bently et al., 1954). a) Chemical Composition and Nutritive Value of Molasses Table 5 shows the chemical composition of molasses from three different sources. The level of potassium is rather high. However, the ruminant is able to rapidly excrete an excess of dietary potassium. TABLE 5. Chemical composition of molasses Dry Matter Sugar Crude Protein Ash Potassium Source % % NX 6.25, % % a. 78.0 50.0 1.3 11.0 4 Karalazos and Swan, 1976 77.6 55.5 1.9 10.5 — Lofgreen and Otagaki,1960 44.0 86.2 3.0 10.8 - Sharma et al., 1972 66.5 63.9 2.1 10.8 4 Average Net energy content of molasses for dairy cows is about 75% that of corn grain (Karalazos and Swan, 1976). When Lofgreen and Otagaki (1960) fed molasses at a level of 10% and 30% of the total ration for lactating dairy cows, the net energy of molasses for lactation was 68.1 and 23.1 Mcal per 100 lb, respectively, compared to 54.2 Mcal for the basal ration. They concluded from this very large energy loss, together with a significant decrease in percentage of butterfat and solids-not—fat in the milk that 30% molasses in the entire ration is too high for lactating dairy cows because it changes volatile fatty acids in the rumen. As the level of molasses in the diet increases, 20 microorganisms of high butyrate and low propionate fermentation patterns increase (Sutton, 1979). A depression in the digestibility of crude fiber was reported when molasses was added to the diet, which was greater than that caused by equivalent carbohydrate as starch (Elias and Preston, 1969). The decrease in dry matter digestibility was not statistically significant when 10 or 20% of the barley dry matter was replaced by molasses or condensed molasses solubles (CMS) (Karalazos and Swan, 1977). Potter et a1. (1971) reported improved dietary nitrogen utilization by steers fed 2.5% molasses (dry matter basis) in a diet containing either urea or soybean meal. No further improvements were found when molasses was increased to 10%. The authors suggested that the addition of molasses to the urea ration provided an essential factor for maximum growth and synthesis of protein from nonprotein nitrogen of microorgan— isms. Large decreases in dry matter intake observed with animals fed high levels of molasses (Komkris et al., 1965; Sharma et al., 1972) were improved by the addition of forage (Karalazos and Swan, 1976). At low forage, digestibility was not limiting capacity but food intake fell rapidly. This may be explained by a physiological change due to the physical characteristics of molasses (Karalazos and Swan, 1976). They also proposed the low rate of rumen turnover on molasses—based diets a main reason for poor voluntary intakes. The effect of adding molasses and ammonia to silage has been studied by several authors. Soper and Owen (1977) treated chopped whole corn 21 plant (32% dry matter) with an ammonia—molasses—mineral solution and found a lower loss of dry matter and increased preservation of crude protein. Based on quality measurements such as lactic acid content, titratable acidity, freedom from observable mold, dry matter preservation, and temperature stability, the treated silage was higher in quality than untreated, after both had been exposed to air for 48 hours. In another experiment, Heidker et a1. (1982) treated high moisture sorghum grain with inoculum (.l% Silabac), molasses (1.0%), and a com— bination of the two. The grain treated with molasses had higher final lactic acid and the fastest decline in water soluble carbohydrates. The slowest decline was noted for grain treated only with the inoculum. ProSil (liquid suspension of anhydrous ammonia, minerals and molasses) treatment of corn silage has been studied in a series of trials by Henderson et a1. (1971) and Beattie et a1. (1971) at Michigan State University. Crude protein content of 35% dry matter silage was increased 90% by the addition of 27 kg of ProSil per ton. Forty percent of the increase was organic protein, 55% ammonia salts and 5% unidentified nitrogen (Henderson et al., 1971). ProSil significantly increased lactic acid, but changes in acetic acid and butyric acid were small and nonsignificant. 2.5 Ammonia—Treated Roughages and Animal Production a) Beef Cattle and Sheep When ammoniated feeds are used in production experiments, rate of gain and feed efficiency increase for sheep (Rounds et al., 1976; 22 Al—Rabbat and Heaney, 1978; Klopfenstein and Owen, 1981) and for beef cattle (Horton et al., 1979; Paterson et al., 1981; Nelson et al., 1982b; Saenger et al., 1982). Klopfenstein and Owen (1981) reported that at ad libitum intake, dry matter digestibility of corn stalks was increased from 36.8% for sheep fed untreated stalks to 47.0% for sheep fed stalks treated with 2 g ammonia/100 g dry matter. Saenger et a1. (1982) fed four forms of corn stover to mature, pregnant beef cows during 70 days. Results showed a marked decrease in body weight loss of cows on ammonia-treated stover compared to control (Table 6). Holloway et a1. (1977) fed mature, dry, pregnant beef cows with: dry corn stover, ammonia—treated stover, corn stalklage, and corn silage. Cows fed corn silage lost considerably less weight and less condition than those fed the other forages. Cows fed dry corn stover, ammonia—treated stover, and corn stalklage lost similar amounts of weight and condition, although ammonia—treated stover tended to maintain better condition. TABLE 6. Cow weight change and condition score change with four corn stover treatments Treatment Cow Weight Change Condition Scorea % NH3 stover + corn supplement 7.6 .00 Stover + soybean meal —20.7 -.14 Stover + urea —27.l —.57 Stover + corn supplement —44.0 —.64 aVisual condition score: 1=very thin; 3=average flesh; 5=very fat. Source: Saenger et al., 1982. J. Animal Sci. 54(2):419—425. 23 b) Ammoniated Forages for Dairy Cows Ammoniated feeds have been used in dairy rations although not extensively. Sundstol et a1. (1978) summarized experiments on dairy cows. Animals receiving ammoniated straw (only a small part of the ration) produced an average 19.7 kg FCM, whereas the controls produced 19.9 kg. They concluded that dairy cows showed poor response when ammoniated feeds formed a small amount of the ration. Similar results were obtained by Heinrichs and Conrad (1982) and Greenhalgh et a1. (1976). Huber (1981) reported milk yields of cows fed different ammonium salts were similar to controls fed soybean meal. However, fat—corrected—milk was higher when either urea or ammonium salts were added to the diet, suggesting that milk fat production was increased with NPN. Huber et a1. (1980) fed lactating dairy cows corn silage, corn stalklage, ammonia—treated corn stalklage, ammonia—mineral—molasses treated corn stalklage (14% CP), and ammonia—mineral-molasses treated corn stalklage (16.8% CP). At 25% of the ration dry matter results presented in Table 7 show that dry matter intake was significantly higher for corn silage than other treatments and milk yields were higher for corn silage and AMS—stalklage (16.8% CP). The better response of cows on ANS—stalklage (16.8% CP) than other stalklages may be due to increased digestibility. They concluded that treatment of corn stalklage with an ammonia—mineral- molasses suspension improves its usefulness for lactating dairy cows. Based on available evidence, one can reasonably conclude that ammoniation increases the energy availability of low quality roughages equal to that of medium—quality hay primarily through improved fiber digestion. 24 TABLE 7. Ammonia and ammonia—mineral molasses corn stalklage for lactating dairy cows Item Dry Matter Intake Milk Yield kg/day kg/day Corn silage 20.0 26.6 Corn stalklage 16.9 24.2 Ammonia stalklage 16.7 23.7 AMS—stalklage (14% CP) 16.5 23.9 AMS—stalklage (16.8% CP) 16.3 25.7 Source: Huber et al., 1980. 75th Ann. Meet. ADSA, p. 152. Ammoniation of corn stalklage improves dry matter digestibility and increases nitrogen content. However, the effect of ammoniation on nitrogen utilization of low quality forages is controversial. Effect of ammoniation on intake of forages has been controversial, but one can conclude that 25% or more of the ration dry matter as corn stalklage fed to dairy cows, depresses intake regardless of type of ammonia treatment. The variation in results suggest that more research is needed to improve the effectiveness of ammoniation on corn stalklage. Changes on the nitrogen fractions and energy fractions and digestibility after treatment with ammonia need to be elucidated,as well as fermentation patterns after ensiling. It is also desirable to determine the useful— ness of varying levels of ammoniated corn stalklage in diets for dairy cows in terms of effects on milk yield and efficiency of feed utilization. 3. MATERIALS AND METHODS 3.1 Stalklage Management Stalks were harvested 2 weeks after high moisture corn harvest from an area of 7.1 hectares at Michigan State University Experimental Station located South of campus from October 15 to 30, 1981. The average yield of corn stalklage was 8.5 tons per hectare with a 42% dry matter content. Therefore, around 3.4 tons of dry matter were produced per hectare. The total 60.2 tons of stalklage was finely chopped and placed in three concrete upright silos (3X12 m). Each silo contained around 20 tons of material. Silo 1 contained untreated corn stalklage and therefore constituted a control treatment. Stalklage in Silo 2 was treated with 2.1% (of DM) aqueous ammonia (28% NH3) as it passed through the blower to be elevated into the silo. The correct amount of ammonia to add to each was determined and was delivered with a pump calibrated according to unload- ing rates of wagons. In Silo 3, a 50:50 mixture of aqueous ammonia and molasses (mixed before ensiling) was added. The amount of ammonia was similar to that added to Silo 2, and as indicated, molasses level was also 2.1% (of DM). At ensiling samples of treated and untreated material were taken for subsequent analysis of dry matter, of nitrogen and fiber fractions and of in vitro dry matter digestibility. Stalklages remained in the 26 silos at least 60 days before they were opened for use in feeding trials. 3.2 Feeding Trial 1 Twenty four Holstein lactating dairy cows in mid— to late—lactation were fed a standard ration (Table 8) for 14 days before the experiment began. During this period, milk yields were recorded and used as the primary factor for assigning to treatment. Cows were allotted in a randomized block design to four treatments balanced for age, stage of lactation and breeding group. Treatments were as follows: A. Corn silage (Control (CS). B. Untreated corn stalklage (US). C. Corn stalklage treated with 2% aqueous ammonia (AS). D. Corn stalklage treated with 2% ammonia plus 2% molasses (AMS). The experimental period was 60 days. TABLE 8. Pre—experimental period ration for lactating dairy cows Dry Amount in Amount in Crude matter diet diet protein % % of DM as is % Alfalfa haylage 44 17.00 38.64 2.42 Alfalfa hay 88 11.00 12.50 1.71 Corn silage 28 28.00 102.71 2.44 High moisture corn 68 25.08 36.88 2.26 Supplement 90 11.92 13.24 5.24 Minerals and salt 100 6.24 0.24 ———— Total 100.00 204.21 14.07 27 Corn stalklage was added to experimental rations to supply 25% of the total ration dry matter (Table 9). Supplement contained 44% crude protein and varied in amount so as to obtain approximately 14% CP in each ration. Average body weight of the cows was 613 kg and the initial milk yield was 19.30 kg/day. Cows were weighed for two consecutive days at the beginning and at the end of the experiment. Milk yields and weighbacks for the complete ration were recorded daily. A composite sample from two consecutive milkings was taken each week and analyzed for total solids, butterfat, and protein content at the DHIA Laboratory. TABLE 9. Experimental rations for feeding trial 1 (% of dry matter) A B C D (CS) (US) (AS) (AMS) Ground hay ' 10 10 10 10 Corn silage 50 25 25 25 Corn stalklage 0 25 25 25 High moisture corn 3O 27 33 33 Supplement* 10 13 6 6 *The composition of the supplement is in Table 10. Feed was sampled twice weekly. Dry matter (DM), crude protein (CP), and in vitro dry matter digestibility (IVDMD) were determined on the complete ration mixtures, and on components of the ration. Additionally, stalklages were analyzed for insoluble (IN) and soluble nitrogen (SN), neutral detergent fiber (NDF), acid detergent fiber (ADF), hemicellulose 28 (HC), cellulose (C), acid detergent lignin (ADL), ash (A) and lactic acid (LA). TABLE 10. Composition of the supplement Item Percentage of the DM Crude protein 44.0* Crude fat 1.6 Crude fiber 8.0 Calcium 3.0 Phosphorus 1.6 Salt (NaCl) 2.0 Vitamin A (USP units/1b) 15,000.0 Vitamin D3 (USP units/lb) 3,000.0 Zinc (ppm) 250.0 Copper (ppm) 50.0 Manganese (ppm) 100.0 Magnesium (%) 0.5 *This includes not more than 4.4% equivalent crude protein from non- protein nitrogen. Determination of DM was by drying a weighed sample at 600C for 48 hr. Crude protein (Nx6.25) was by the Kjeldahl procedure (AOAC,1965) IN and SN by Bergen et a1. (1974) and IVDMD by the modified Terry—Tilly procedure (Goering and Van Soest, 1970). Fiber fractionation (NDF, ADF, HC, C, and ADL) was according to Goering and Van Soest (1970) and LA by the procedure of Barker and Summerson (1941). Ash was determined by 29 burning off all organic material in a muffle furnace at 6000C for 5 hr. Ash remains in the crucible. 3.3 Feeding Trial 2 Fifteen lactating Holsteins in early lactation were fed a standard ration as in Trial 1 (Table 8) for 10 days and allotted to three treat— ments (S cows per treatment) in a randomized block design. Treatments were again balanced for age, stage of lactation, and breeding group, and the trial was for 30 days. Rations (listed in Table 11) were as follows: A. Corn silage — Control (C82). B. 10% of the ration dry matter as AMS (10 S). C. 20% of the ration dry matter as AMS (20 S). Average body weight of cows was 554 kg and initial milk yield was 26.7 kg/day. Milk weights and composition and feed samples were managed as in Trial 1. TABLE 11. Experimental rations for feeding trial 2 (% of dry matter) A B C (c52) (10 S) (10 S) Ground hay 10 10 10 Corn silage 50 4O 3O Corn stalklage 0 10 20 High moisture corn 30 31 33 Supplement* 10 8 6 *See Table 10. 30 3.4 Corn Stalklage Fermentation Study Double plastic bags of 30 kg capacity were used as experimental silos for stalklage harvested 19 days after high moisture corn harvest. Corn plant material was finely chopped with a silage harvestor, weighed into 17—20 kg lots, and placed in bags. Bags were evacuated with a heavy duty vacuum, sealed, and stored at room temperature until opened for processing. The following treatments were compared: 1. Untreated stalklage (US) 2. Corn stalklage treated with 2% (of dry matter) aqueous ammonia (AS) 3. Corn stalklage treated with 2% ammonia and 2% molasses (of dry matter) (AMS) 4. Corn stalklage treated with bacterial inoculum (Lactobacillus brevis, l:_p1antarum and Pedoecocos acidilactici applied at .5 g/kg (IS) Duplicate bags for each treatment were opened and samples processed on days 0, 3, 7, l9 and 50 of fermentation. Samples were analyzed for the same components as in Trial 1, using the following scheme. Duplicate sample (40 g) was cut with a laboratory chopper and used for DM determination (600C oven for 48 hr). Part of this sample was used for total nitrogen (TN) determinations. Another (15 g) was mixed with 100 ml distilled water on which pH was immediately measured using a lab pH meter (Digital Ionolyzer Model 801A). The mixture was then homogenized with a lab Omni Mixer in an ice bath for 2 min, filtered through a double layer of cheesecloth and centrifuged at 15,000 rpm 31 for 15 min. The residue was discarded and the supernatant retained for determining soluble nitrogen and lactic acid. Insoluble nitrogen was calculated by difference between TN and SN. A third sample was air dried and ground through the Wiley Mill to a 1 mm particle size for determination of IVDMD (Goering and Van Soest, 1970) and fiber analysis (NDF, ADF, HC, C and ADL) (Goering and Van Soest, 1970) and ash. Results were corrected for dry matter content. 3.5 Statistical Analysis 3.5.1 For Feeding Trials 1 and 2 a) The effect of treatments on milk yield was analyzed by analysis of variance using initial milk yield (4% fat corrected) and initial body weight as covariates, according to the following model: Y = u + COV + COV + T + error 1 2 where: Y = milk yield u ~ = overall mean COVl = initial milk yield COV2 = initial body weight T = effect of treatment The analysis and the program are presented in Appendix Table l. b) The effect of treatment and time on dry matter intake was analyzed using a split—plot, repeated measurement design. The following statistical model was used: Y1 = u + T + A/T + t + T-t + Z where: Y1 A/T 32 dry matter intake overall mean effect of treatment effect of animal within treatment or error a, used to test treatment effects effect of time effect of the interaction treatment x time residual error (includes the animal within treatment over time), or error b, used to test the time effect and treatment x time The analysis and program are presented in Appendix Table 2. c) Scheffe's test as described by Gill (1978) was used to compare the means between treatments for the Feeding Trial 1. replicates, Fermentation Study This experiment was analyzed as a factorial 4 x 5 with two four treatments and five times. The statistical model was: Y = u + T + t + T-t + error where: Y Tot Error = dry matter, pH, total nitrogen, soluble nitrogen, insoluble nitrogen, neutral detergent fiber, acid detergent fiber, hemicellulose, cellulose, acid detergent lignin, lactic acid, in vitro dry matter digestibility overall mean effect of treatment effect of time effect of interaction between treatment and time II = residual error 33 In those cases where the interaction treatment x time was signifi- cant, Dunnett‘s test as described by Gill (1978), was used to compare the means against the control (Appendix Table 3). 4. RESULTS AND DISCUSSION 4.1 Feeding Tria1_1 4.1.1 Chemical Analysis of Corn Stalklages Compared to the untreated stalklage (US), dry matter was slightly higher when ammonia (AS) and ammonia plus molasses (AMS) were added (2.41% and 5.28%, respectively), but differences were not significant (Table 12). Ammonia and ammonia—molasses treatments significantly increased total (P<.01), insoluble (P<.Ol) and soluble (P<.05) nitrogen. The higher insoluble nitrogen obtained after treat— ment with ammonia was partially due to a reduction in proteolysis by plant enzymes during the early stages of fermentation (Bergen et al., 1974; Huber et al., 1979; Johnson et al., 1982) and to linkage of ammonia with insoluble components of plant tissue (Huber et al., 1979). Neutral detergent fiber, acid detergent fiber, hemicellulose and cellulose did not differ between US, AS and AMS, even though they showed a slight decrease with ammonia treatments. Lignin was signifi— cantly decreased (P<.05) in AMS. Horton (1981) also found a consistent trend of lower lignin and hemicellulose in ammonia-treated cereal straws, but the differences were not significant. Harbers et a1. (1982) reported decreased lignin in wheat stems following treatment with 3.2% NH OH, as suggested by less dense staining under light microscopy. 4 Furthermore, they reported less dense, but more diffuse, staining 34 TABLE 12. Effect of ammoniation and ammonia—molasses treatment on the composition of corn stalklage compositesa USb ASb Change AMSb Change Effect % % Dry matter (%) 35.20 36.05 + 2.41 37.06 + 5.28 N.S. Total nitrogen (%) 0.957 2.022 +111.29 1.827 + 90.91 P<.01 Insoluble N (%) 0.664 1.506 +126.8l 1.389 +109.19 P<.Ol Soluble N (%) 0.293 0.516 + 76.11 0.438 + 49.49 P<.05 Fiber components (%) NDF 75.40 73.30 — 2.79 72.60 — 3.71 N.S ADF 41.68 40.79 — 2.14 41.03 - 1.56 N.S Hemicellulose 33.72 32.51 — 3.59 31.57 — 6.38 N.S Cellulose 31.03 29.89 — 3.67 30.15 -— 2.84 N.S. Lignin 9.46 8.28 - 12.47 7.96 — 15.86 P< 05 Ash 17.81 17.96 + 0.84 17.31 — 2.81 N.S Lactic acid(% DM) 3.10 3.03 — 2.56 3.17 + 2.26 N.S pH 4.51 4.63 + 2.66 4.46 - 1.11 N.S. IVOMD (%)c 46.60 54.12 + 13.90 54.78 + 14.93 P<.Ol aSamples were taken twice a week from upright silos. bStalklage designations were: US, untreated; AS, ammonia added (2.1% of DM); AMS, ammonia and molasses added (2.1% of DM) for each. CDMD was corrected by OMD by subtracting ash content from 100. throughout cell walls indicating loosening of the lignin—polysaccharide binding. We conclude,therefore, that ammonia solubilized part of the hemicellulose and lignin in our corn stalklages, but cellulose was not affected. In contrast, Colenbrander et in cell wall components after treatment ammonium polyphosphate. Lactic acid was lowered in AS, but to more soluble carbohydrates available However, differences were not significant. a1. (1971) found no differences of corn stover silages with increased in AMS, probably due for growth of microorganisms. Addition of ammonium 36 polyphosphate did not change lactic acid production, but when urea was added, a linear increase in lactic acid was obtained (Colenbrander et al., 1971). Huber et al. (1979b) reported decreased lactic acid when 1.92% anhydrous ammonia was added to corn silage. The pH did not differ among corn stalklages, but AS was slightly higher and AMS slightly lower than US. This may be associated with higher lactic acid in AMS. Both AS and AMS were higher in in vitro dry matter digestibility (IVDMD) (P<.Ol). These findings agree with several other authors (Oji and Mowat, 1979; Oji et al., 1979; Saenger et al., 1982). Total ash was not different between silos but values are somewhat high, even though within the normal range for corn stalklage. 4.1.2 Animal Performance Analysis of composites of samples taken twice weekly for dry matter, crude protein and IVDMD of the experimental rations are in Table 13. As expected, dry matter and crude protein of rations were not different, but IVDMD of stalklages treated with ammonia was signi— ficantly lower (P<.Ol) than the control corn silage. Dry matter intakes of the complete ration (Table 14) are weekly averages of daily observations statistically analyzed as repeated measurements in a split—plot design (Gill, 1978). There were no significant differences between treatments, even though intakes tended to be lower for cows fed treated stalklages (AS and AMS) than controls (UC and US), compared to corn silage controls. Huber et a1. (1980) 37 TABLE 13. Composition of ration composites for cows. Feeding trial la cs US AS AMS Effect Dry matter (%) i 40.4 41.0 40.3 41.2 N.S. Crude protein (%) 13.82 13.89 13.86 13.93 N.S. IVDMD (%)b 61.69C 56.14d 55.31d 52.32e p<.01 aSee Table 9. In vitro dry matter digestibility. CdeMeans within a row without a common superscript are different (P<.Ol). Dunnett's test (Gill, 1978). reported a depression in intakes when lactating cows were fed untreated, ammonia—treated and ammonia—mineral—molasses—treated stalklages at 25% of the ration dry matter. The lower digestibilities (P<.05) of AS and AMS rations than CS and US may in part explain the lower intakes. Also, a negative effect of ammonia on palatability may have affected the intake, similar to the observations of Rounds et al. (1976), Davis (1981) and Males and Falen (1982). Huber et al. (1979a) also reported depressed intakes by dairy cattle fed corn silage treated with .375% gaseous ammonia (82% N) but the effect was not consistent. Furthermore, the estimated net energy for lactation (Table 14) was lower for AS and AMS. In our study, there was a high variation in DM intakes within groups as denoted by the large coefficient of variation (CV = 28%). Even though the trends we noted were similar to those reported by others, treatments did not differ significantly. All cows averaged about 19.3 kg fat—corrected milk per day (Table 15) with those fed AS and AMS significantly lower than those fed CS or US 38 TABLE 14. Treatment and time effect on the dry matter intake (kg DM/ cow/day). Feeding trial la Trt. % NElC Trt. 1 2 3 4 5 6 7 8 Meansb B.W. (Mcal) CS 18.36 16.70 17.16 17.79 17.40 17.75 17.49 17.86 17.56i2.48 2.86 30.12 US 19.37 17.05 17.64 18.38 18.46 18.16 17.06 17.27 17.92i2.52 3.02 29.34 AS 15.51 15.11 15.54 15.98 16.33 15.61 15.29 15.20 15.57i2.20 2.50 25.36 AMS 15.36 14.56 15.39 15.52 15.47 15.33 14.90 15.49 15.25i2.20 2.64 24.85 Time a 17.22 15.89 16.47 16.96 16.94 16.76 16.22 16.51 Means aSix cows per treatment for 60 days. bTreatment effect on DM intake was not significant (P<.05). gEstimated net energy lactation (NEl). Significant time effect (P<.05). (P<.Ol). In a previous experiment (Huber et al., 1980), cows fed rations with 25% of the dry matter as ammonia or ammonia—mineral—molasses treated stalklage (AMMS, 14% CP) produced significantly less milk than those fed a control (corn silage) ration or one containing stalklage treated at a high level of AMMS (16.8% CP). Cows fed untreated stalklage were intermediate between two groups. The higher milk production obtained with untreated stalklages than those treated with ammonia may have been due to the higher digesti— bility of ration dry matter, probably because of the addition of more nat— ural protein supplement and, also, the higher values of NEl for those rations. This may also explain the higher, though not significantly, dry matter intakes. Milk components were all within normal ranges, and did not differ significantly between groups (Table 15). However, milk protein tended 39 TABLE 15. Milk production, milk composition, feed efficiency and changes Feeding trial 1. in body weight. Treatments cs 08 AS AMS Initial milk yield 19.46 19.71 18.85 19.12 (kg FCM/cow/day)c Milk yield (kg FCM/ 20.77a 17.16ab 14.71b 15.44b cow/day)e predicted milk yieldf 24.00 22.80 17.57 16.82 (kg/day) Milk composition protein (%) 3.64:_.10 3.56:_.13 3.37:_.11 3.54: .10 Fat (%) 3.86:_.09 3.80:_.1O 3.99:_.07 4.10:_.09 Feed efficiencyd 1.18 0.96 1.06 0.99 Changes in body weight (kg) l3.86:5.55 3.73:5.55 9.05:6.09 5.55:5.55 abMeans within a row with an uncommon superscript are Interval (Gill, 1978) (P<.05). CInitial fat—corrected-milk used as covariate. dCalculated as total milk yield/total dry matter intake. eCovariate adjusted production. fCalculated by estimated net energy lactation. to be lower and milk fat higher when stalklage was fed. different. Scheffe's This effect might be explained by the higher fiber and/or lower milk production. Efficiency of conversion of feed to milk was not significantly different between treatments; however, cows fed stalklage had slightly lower efficiency. All groups gained weight which might be expected for cows in mid— to late—lactation, and treatments did not affect weight changes. 40 4.2 Feeding Trial 2 4.2.1 Animal Performance Corn stalklage used for this experiment was the same as the AMS in treatment in feeding trial 1 (Table 12). Dry matter content, crude protein and in vitro dry matter digestibility of rations are in Table 16. As in feeding trial 1, analyses were from composites taken twice weekly. Dry matter, crude protein and IVDMD did not differ between treatments, even though the ration with 20% AMS (20 AMS) was slightly higher than others in IVDMD. TABLE 16. Composition of ration composites for cows fed varying amounts of stalklage treated with ammonia and molasses (AMS). Feeding trial 2a Rationb Corn lO AMS 20 AMS Effect silage Dry matter (%) 39.2 41.2 40.9 N.S. Crude protein (%) 13.92 14.01 13.99 N.S. IVDMD (%)b 62.26 62.00 64.49 N.S. aSee Table 11. In vitro dry matter digestibility No significant treatment effects were found for dry matter intakes (Table 17), although a slight depression was observed on 20 AMS, probably because of the lower levels of NEl in this ration. However, when dry matter consumption was expressed as percent body weight, values were similar to experiment 1 with stalklage rations lower than the corn silage. 41 TABLE 17. Effect of treatment and time on the dry matter intakes of cows fed varying amounts of stalklage treated with ammonia and molasses (AMS) (kg DM/cow/day). Feeding trial 2a Ration Week Treatment % NElC 1 2 3 4 meansb B.W. (Mcal) Corn silage 15.62 15.61 15.52 16.25 15.75:,17 2.95 27.01 10 AMS 20 AMS Time means 13.84 15.39 15.45 16.14 15.21::49 2.69 24.79 13.62 14.56 14.95 15.08 14’55i:33 2.63 23.91 14.36 15.18 15.30 15.83 aFive cows per treatment for 30 days. Differences were not significant (P<.10). CEstimated net energy lactation. TABLE 18. Milk production, milk composition, feed efficiency, and changes in body weight of cows fed varying amounts of stalklage treated with ammonia and molasses (AMS). Feeding trial 2. Treatmentsb Corn silage lO AMS 20 AMS Initial milk yield 22.85 22.39 23.24 (kg FCM/cow/day)a Milk yield (kg FCM/ 25.50 25.30 28.10 cow/day) Predicted milk (kg/day)d 20.98 17.97 16.79 Milk composition Protein (%) 2.75:.25 2.651325 2.64:.25 Fat (%) 3.18:412 3.37:.12 3.32:,12 Feed efficiencyC 1.62 1.66 1.93 Changes in body weight 13.45:10.1 —7.05:10.1 16.05:10.1 (kg) aInitial fat corrected milk used as covariate. None of the differences between treatments were significant (P<.10). cCalculated as total milk yield/total dry matter intake. Calculated by estimated net energy lactation. 42 Initial FCM yields of cows averaged about 22.83 kg/day (Table 18). The main objective of this experiment was to determine whether treated stalklage (AMS) at lower levels than experiment 1 (20% or 10% of dry matter) would maintain as high milk yields as the corn silage control. The data show that AMS could be added at up to 20% of the ration dry matter without a decrease in milk production. Predicted milk yield is lower than the observed productions. This situation leads to the conclusion that corn stalklage can be fed during short periods of time with no detrimental effects. The slightly higher milk yields for treatment 20 AMS may be related to the higher, although not significant, IVDMD of the complete ration. Milk fat was within the normal range, but milk protein was lower than standard for all treatments. A possible explanation might be a consistent error in protein determination. As in experiment 1, stalklage treatments were slightly higher than the corn silage control in percent milk fat, lower in percent milk protein, but similar in feed efficiency and body weight changes. 4.3 Corn Stalklage Fermentation Study 4.3.1 Dry Matter Initial dry matter of stalklages was not significantly altered by treatments or time of ensiling (Table 19). This result is consistent with other studies(Huber et al., 1979b; Johnson, 1981). However, dry matter of treated stalklages decreased slightly as fermentation progressed, possibly due to a loss of volatile organic 43 acids during oven drying, coupled with the evolution of CO2 and the production of water during fermentation (Johnson, 1981). TABLE 19. Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on dry matter content (%) in corn stalklagea Treatment Days of Ensiling Treatment 0 3 7 19 50 mean ————————— % of dry matter————-———— usb 38.81 42.67 41.70 40.15 40.08 40.68 Asb 42.88 42.41 40.23 40.70 39.89 41.22 AMSb 41.42 37.54 42.55 39.06 39.39 39.99 ISb 42.08 42.68 37.55 41.41 40.80 40.90 Time mean 41.30 41.33 40.51 40.33 40.04 aValues are averages from two experimental silos with two determinations per silo. Stalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. Treatment x time interaction is significant (P<.05). 4.3.2 pH Values for pH are presented in Table 20. Initially (days 0 and 3) treatments with ammonia resulted in a much higher pH than the control and inoculum—treated stalklages (P<.05). All stalklages decreased with time, but on days 19 and 50 AS and AMS were still somewhat higher than US and IS. Similar results have been obtained in other studies (Britt and Huber, 1975; Johnson, 1981; Boman, 1980) with corn silage treated with ammonia. 44 TABLE 20. Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on pH in corn stalklagea Treatment Days of Ensiling Treatment 0 3 7 19 50 meanb ush 7.13 4.45 4.38 4.38 4.49 4.969 Ash 8.87d 6.20d 5.44d 4.85 4.87 6.04C AMSh 8.76d 6.13d 4.93 4.82 4.75 5.88c ISh 7.13 4.49 4.51 4.45 4.42 4.99 Time meanf 7.97 5.31e 4.81e 4.62e 4.63e aValues are averages from two experimental silos with two determinations per silo. bSignificant treatment effect (P<.Ol). CSignificantly higher than the control (P<.05). dSignificantly higher than the control (P<.05). eSignificantly lower than day 0 (P<.Ol). fStandard error of any time mean is i30401. gStandard error of any treatment combination mean is i10359. Treatment x time interaction is significant (P<.Ol). hStalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. 4.3.3 Lactic Acid Lactic acid values in corn stalklage are shown in Table 21. There were significant treatment and time of ensiling effects (P<.Ol). As can be observed, AS and IS were initially lower in lactic acid than US and AMS. Because of the extra sugar provided for microbial growth, lactate in AMS was highest at the end of fermentation. Ammon— iation delayed lactic acid production, but values were similar to the control by 50 days of fermentation. Oji et a1. (1977) reported negligible levels of lactic acid in ammonia—treated stover suggesting 45 TABLE 21. Effect of ammonia, ammonia—mola—ses, inoculum, and time of ensiling on lactic acid in corn stalklagea Treatment Days of ensiling Treatment 0 3 7 19 50 meanb —————————— % of dry matter——--——-—- usi 1.22 3.43 3.89 4.59 4.00 3.429 i e e AS 1.49 1.53 2.32 3.77 3.83 2.59e AMsi 2.17d 1.99e 4.68d 4.74 5.85d 3.88 Isi 2.52d 2.28 3.89 3.82 3.34 3.17 Time meanC 1.85h 2.31 3.69f 4.23f 4.25f aValues are averages from two experimental silos with two determinations per silo. bSignificant treatment effect (P<.Ol). CSignificant time effect (P<.Ol). Significantly higher than control (P<.05). eSignificantly lower than control (P<.05). fSignificantly higher than day 0 (p<.01). 9Standard error of any treatment combination mean is :.0875. Standard error of any time mean is :30979. ‘Treatment X time interaction is significant (P<.Ol). lStalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. an absence of fermentation during storage. Boman (1980) and Beattie (1970) reported the highest values of lactic acid in corn silage treated with an ammonia—mineral—molasses suspension. Furthermore, there was a trend for increased lactic acid with time of ensiling (Boman, 1980) which is consistent with results of this experiment. The delayed increase in lactic acid on ammonia treatments is attributable to an increased buffering, thus requiring more lactic acid to reduce pH to 4.5 or below (Johnson, 1981). Stalklage treated with IS was slightly 46 lower in lactic acid than the control, but differences were not signi— ficant. The values of LA by day 0 of ensiling were rather high maybe because stalklage laid in the field for at least 5 hr after being chopped. These are some of the first studies where fermentation of corn stalklage has been characterized at different times after ensiling. The decrease in pH to about 4.5 and increase in lactic acid to 3 to 5% (of DM) suggests that a substantial lactic acid type of fermentation occurs in stalklage, which is enhanced with addition of ammonia and molasses. 4.3.4 Nitrogen Fraction The effect of treatment and time of ensiling on total, insoluble and soluble nitrogen are in Tables 22, 23 and 24. Ammonia, and ammonia plus molasses significantly increased total, insoluble and soluble N (P<.Ol). Time of ensiling did not affect any nitrogen fraction of stalklages. Similar observations have been observed with corn silages (Huber et al., 1979a,b; Johnson, 1981) and corn stalklages (Saenger et al., 1982; Oji, 1979) treated with ammonia. Huber et al. (1973) observed a sparing effect of the original plant protein when ammonia was added to corn silage resulting in higher insoluble N. Bergen et a1. (1974) and McDonald (1979) reported that much of the corn plant protein breakdown during ensiling is due to plant enzymes and that proteolysis is greatly diminished in ammonia—treated corn silages. Also, the higher insoluble N in ammonia—treated corn materials is partly due to a binding of ammonia by insoluble compounds in plant tissues (Huber et al., 1980). 47 TABLE 22. Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on total nitrogen in corn stalklagea Treatment Days of Ensiling Treatment 0 3 7 19 50 meanb ————————— % of dry matter—————-———— h Cf US 0.879 0.888 1.006 0.819 0.941 0.906 Ash 1.575e 441 .732e 1.895e 1.895e 1.708d h e e e e d AMS 1.908 944 .980 1.771 1.705 1.862 ISh 1.149 834 .069 0.912 0.986 0.990c Time meang 1.378 277 .447 1.349 1.383 aValues are averages from two experimental silos with two determinations per silo. Significant treatment effect (P<.Ol). cheans within a column without a common superscript are different (P<.Ol). eSignificantly higher than control (P<.Ol). fStandard error of any treatment combination mean is :3054. 9Standard error of any time mean is +.O604. Treatment x time interaction is significant (P<.05). hStalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. The higher values of water soluble nitrogen (WSN) in stalklage after ammonia treatment was due to addition of nitrogen as soluble ammonia. Similar results were observed by Johnson (1981). 4.3.5 Fiber Fractionation Effects of treatments and time of ensiling on neutral deter— gent fiber (NDF), acid detergent fiber (ADF), hemicellulose (HC), (ADL) are in Tables 25, 26, 27, cellulose (CL) and acid detergent lignin 28 and 29. Fractions of NDF, ADF, and ADL were decreased after ammonia 48 TABLE 23. Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on insoluble nitrogen in corn stalklagea Treatment Days of ensiling Treatment 0 3 7 19 50 meanb —————————— % of dry matter———————-— usi 0.620 0.641 0.727 0.548 0.692 0.646cg Asi 0.937 0.649 0.996e 1.201e 1.292e 1.015d AMSi 0.938e 1.236e 1.279e 1.131e 1.191e 1.155d 151 0.849 0.475f 0.796 0.634 0.758 0.702c Time meanh 0.836 0.750 0.950 0.879 0.983 aValues are averages from two experimental silos with two determinations per silo. ‘ Significant treatment effect (P<.Ol). CdMeans within a column without a common superscript are different(P<.05). eSignificantly higher than control (P<.Ol). fSignificantly lower than control (P<.05). gStandard error for any treatment combination mean is :30601. hStandard error for any time mean is +.O672. ‘Treatment x time interaction is significant (P<.05). lStalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. TABLE 24. Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on soluble nitrogen in corn stalklagea Treatment Days of ensiling Treatment 0 3 7 19 50 meanb —————————— % of dry matter——-———-—— 08h 0.260 0.247 0.279 0.271 0.249 0.261Cf Ash 0.638e 0.792e 0.736e 0.694e 0.603e 0.693d AMSh 0.970e 0.708e 0.701e 0.640e 0.514e 0.707d lsh 0.300 0.359 0.273 0.278 0.228 0.288c Time meahg 0.542 0.526 0.497 0.471 0.400 aValues are averages from two experimental silos with two determinations per silo. bSignificant treatment effect (P<.Ol). CdMeans within a column without a common superscript are different(P<.Ol). eSignificantly higher than control (P<.Ol). fStandard error for any treatment combination mean is :30361. gStandard error for any time mean is :10403. Treatment x time interaction is not significant. hStalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. 49 treatment (P<.Ol). Hemicellulose was also decreased (P<.05) (Table 26), but CL was least affected. The AMS treatment decreased CL (P<.05) (P<.05) but AS and IS did not. Similar results have been reported for ammoniated corn stover by Kiangi et a1. (1981) and Saenger et a1. (1982) and for stalklage treated with ammonia polyphosphate (Colenbrander et al., 1971). In tall fescue hay (Buettner et al., 1982), NDF, HC and lignin were decreased by ammoniation, but the ADF and CL were not. Lignin was also decreased by ammoniating wheat straw (Streeter and Horn, 1981). One can reasonably conclude from the present study that ammoniation increased solubilization of HC, ADL and silica although the values for lignin are higher than reported in the litera— ture. Cellulose solubility was not greatly altered. Similar conclu— sions were reached by Jackson (1977) and a review article. Time of ensiling affected NDF and ADF (P<.Ol) (Table 24, 25) but not HC, CL and ADL. Ash values are presented in Table 30. Ash content was used to correct by organic matter. 4.3.6 In Vitro Organic Matter Digestibility Values of IVDMD were corrected by OM because of the high values of ash. Effects of treatment and time of ensiling on in vivo organic matter digestibility (IVOMD) are presented in Table 31. Ammonia— tion and time of ensiling increased IVOMD (P<.Ol). Several studies have also demonstrated increased IVOMD in corn stalklage treated with ammonia (Oji and Mowat, 1979; Paterson et al., 1981; Saenger et al., 1982) and have associated this effect with a reduction of the fiber components after ammoniation. The significant effect of time of ensiling on IVOMD 50 TABLE 25. Effect of ammonia, ammonia-molasses, inoculum and time of ensiling on neutral detergent fiber in corn stalklage Treatment Days of ensiling Treatment 0 3 7 19 50 meanb —————————— % of dry matter————————— ush 73.72 73.20 71.20 70.85 70.52 71.90f Ash 70.84 68.36d 67.98d 67.28d 67.10d 68.31c AMSh 71.47 67.71d 68.27d 66.08d 66.37d 67.98c 1sh 70.50 70.51 69.63 68.86 69.40 69.78 Time meang 71.63 69.94 69.27 68.27e 68.35e aValues are averages from two experimental silos with two determinations per silo. bSignificant treatment effect (P<.Ol). CSignificantly lower than control (P<.05). dSignificantly lower than control (P<.05). eSigniflcantly lower than time 0 (P<.05). fStandard error for any treatment combination mean is :3597. gStandard error for any time mean is :3667. Treatment x time interaction is not significant. hStalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. TABLE 26. Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on acid detergent fiber in corn stalklagea Treatment Days of Ensiling Treatment 0 3 7 19 50 meahb —————————— % of dry matter--~-—-——- 059 44.27 44.63 43.49 43.06 43.25 43.74e ASg 43.98 42.38C 40.76C 40.45C 40.08c 41.53C AM59 42.59c 41.94C 41.72c 40.77c 40.64c 41.53C ISg 40.87c 41.99C 41.91c 41.60c 41.97C 41.67C Time meanf 42.93 42.73 41.97 41.47d 41.48d aValues are averages from two experimental silos with two determinations per silo. bSignificant treatment effect (P<.Ol). cSignificantly lower than the control (P<.05). Significantly lower than day 0 (P<.05). eStandard error for any treatment combination is i32676. Standard error for any time mean is :12992. Treatment x time interaction is significant (P<.05). gStalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. 51 TABLE 27. Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on hemicellulose in corn stalklagea Treatment Days of Ensiling Treatment 0 3 7 19 50 mean —————————— % of dry matter—————-——~- usf 29.45 28.57 27.72 27.79 27.26 28.16d ASf 26.86C 25.98c 27.22 26.84 27.03 26.78c AMSf 28.87 25.77c 26.55 25.31c 25.74C 26.45C ISf 30.18 28.52 27.73 27.26 27.43 28.23 Time meahe 28.84 27.21 27.31 26.80 26.86 aValues are averages from two experimental silos with two determinations per silo. Significant treatment effect (P<.05). cSignificantly lower than control (P<.05). dStandard error for any treatment combination mean is :3680. Standard error for any time mean is :.761 Treatment x time interaction is not significant. fStalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. TABLE 28. Effect of ammonia, ammonia-molasses, inoculum and time of ensiling on cellulose in corn stalklagea Treatment Days of Ensiling Treatment 0 3 7 19 50 meanb ————————— % of dry matter——--———-— usf 30.97 30.61 30.92 30.93 31.11 30.91d Asf 30.46 30.09 30.20 29.29 29.39C 29.89 AMSf 31.04 29.26 28.96c 28.91C 28.87C 29.41C Isf 29.91 30.03 29.81 29.89 29.62 29.85 Time meane 30.60 30.00 29.98 29.76 29 75 aValues are averages from two experimental silos with two determinations per silo. bSignificant effect of treatment (P<.05). CSignificantly lower than control (P<.05). dStandard error for any treatment combination mean is i3464. eStandard error for any time mean is :3518. Treatment x time interaction is not significant. fStalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. 52 TABLE 29. Effect of ammonia, ammonia—molasses, inoculum and time of ensiling on acid detergent lignin in corn stalklagea Treatment Days of Ensiling Treatment 0 3 7 19 50 meanb —————————— % of dry matter--————-— f d us 12.83 13.30 12.73 12.58 12.60 12.81 f AS 11.40 10.59C 10.26C 10.26C 9.01C 10.31C f AMS 10.62c 10.49c 10.87c 9.99C 9.56c 10.30c f IS 11.18 11.09C 11.13 11.22 11.22 11.17 Time meane 11.51 11.37 11.24 11.01 10.60 aValues are averages from two experimental silos with two determinations per silo. Significant treatment effect (P<.Ol). cSignificantly lower than control (P<.05). dStandard error for any treatment combination mean is :3749. eStandard error for any time mean is :3837. Treatment x time interaction is not significant. fStalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. TABLE 30. Total ash of corn stalklage Treatmenta Days of Ensiling Treatment 0 3 7 19 50 mean USa 13.32 11.88 10.90 16.72 13.54 13.27 Asa 14.83 12.76 13.70 13.20 13.85 13.67 AMSa 13.36 13.33 13.17 13.15 14.76 13.55 ISa 15.66 13.05 14.52 14.49 15.89 14.72 Time mean 14.29 12.75 13.07 14.39 14.51 aStalklages designated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated with bacterial inoculum. 53 might be explained by the possible cleavage of acid—labile lignin- carbohydrate bonds (both benzyl ethers and esters) by the lower pH caused by the production of organic acids after anaerobic fermentation in the silo (Harkin, 1973). TABLE 31. Effect of ammonia, ammonia—molasses, inoculum, and time of ensiling on in vitro organic matter digestibility] in corn stalklagea Treatment Days of Ensiling Treatment 0 3 7 19 50 meanb ——————— % of organic matter——-—--— 081 43.15 42.16 41.35 44.61 45.05 43.459 Asi 44.73 45.39 46.81d 47.12d 47.82d 46.38d AMsi 40.69 42.63 45.38d 45.94 46.40 44.20 Isi 39.90e 39.28e 40.54 40.52e 44.76 40.98 . C h f f Time mean 42.12 42.36 43.52 44.56 46.02 aValues are averages from two experimental silos with two determinations per silo. bSignificant treatment effect (P<.Ol). CSignificant time effect (P<.Ol). eSignificantly higher than control (P<.05). Significantly lower than control (P<.05). Significantly higher than day 0 (P<.05). gStandard error of any treatment combination mean is :,539. hStandard error of any time mean is :.603. 'Treatment x time interaction is not significant. lStalklagesdesignated as follows: US, untreated; AS, treated with 2% ammonia; AMS, treated with 2% ammonia and 2% molasses; IS, treated 'with bacterial inoculum. 3Dry matter digestibility was corrected by organic matter as follows: 1) 100 — ash = OM 2) (DMD/OM) x 100 = OMD (%) 5. SUMMARY AND CONCLUSIONS The value of anhydrous ammonia and molasses, as an additive to corn stalklage at ensiling was studied in two feeding trials with lactating dairy cows and in a fermentation study with experimental bag silos. when ammonia treated and ammonia—molasses treated corn stalklage were fed at 25% of the ration dry matter to lactating dairy cows, milk production was significantly (P<.05) depressed and tended to decline the dry matter intake. This is consistent with a previous experiment (Huber et al., 1980). Cows fed stalklage showed a slight increase in milk fat and a decrease in milk protein probably as a result of the increase of dietary fiber. At lower stalklage intake levels (10% or 20% of DM) cows fed stalklage treated with ammonia—molasses maintained milk yields with no depression in dry matter intake. The present study is one of the first to characterize fermentation of corn stalklage at different times after ensiling. We conclude that corn stalklage underwent a normal fermentation pattern with lactic acid lower and pH higher than corn silage of similar dry matter. A decrease in pH to about 4.5 and increase in lactic acid to 3 to 5% of dry matter suggest that a substantial lactic acid—type of fermentation occurs in corn stalklage, which is enhanced with addition of ammonia and molasses. Microbial inoculum and ammonia alone had little effect on corn stalklage fermentation. 54 55 Total and soluble nitrogen were increased by ammoniation. This increase was proportional to the ammonia additions. Insoluble nitrogen was also increased by ammoniation as shown by others (Huber et al., 1979a,b) for corn silage, and by Saenger et al. (1982) and Oji (1979) for corn stalklage. Ammoniation decreased neutral detergent fiber, acid detergent fiber and lignin (P<.Ol). Hemicellulose and cellulose were also reduced (P<.05) but cellulose was least affected. Similar conclusions were reached by Jackson (1977) in a review article. Ammoniation and a longer time of ensiling had a beneficial effect on in vitro dry matter digestibility. In summary, the beneficial effect of ammoniation upon nutritive value of low quality forages if fairly clear. Energy derived from the digestion of structural carbohydrates makes an important contribution to the energy supply of the ruminant. Increases in the content of structural carbohydrates in forage are associated with declining digestibility and reduced intake. Lignin and hemicellulose restrict the digestion of cellulose; therefore, removing lignin or lignin linkages with polysacchardies by ammoniation increased the amount of plant cell—wall polysaccharides digested by bacterial cellulases. This finding has special relevance when low—quality roughages are fed in productive rations for ruminants. LI TERATU PE C I TED LITERATURE CITED Al-Rabbat, M.F. and D.P. Heaney. 1978. The effects of anhydrous ammonia treatment of wheat straw and steam cooking of asoen wood on their feeding value and on ruminal microbial activity. I. Feeding value assessments using sheep. Can. J. Anim. Sci. 58(3):443. Anderson, D.C. 1978. Use of cereal residues in beef cattle production systems. J. Anim. Sci. 46(3):849. Anderson, D.C. and A.T. Ralston. 1973. Chemical treatment of ryegrass straw: In vitro dry matter digestibility and compositional changes. J. Anim. Sci. 37(1):l48—152. A.0.A.C. 1965. Official Methods of Analysis (10th edition). 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