FERMENTATWN PROXPUCTS AND MGESTIBILITY OF ALFAKFA, CER?’ER~CUT CORN SUDAN AND SU-DAX SRAGES TREATED WITH THREE DIFFKRENT ADDITWES Thesis for the Degree of M. S. MlCHIGAN SKATE UNWERSH’Y ioseph A. Atekwana 196-5 Fermentation Products and Digestibility of Alfalfa, Center-cut Corn, Sudan and Sudax Silages Treated with Three Different Additives Fermentation Products and Digestibility of Alfalfa, Center-cut Corn, Sudan and Sudax Silages Treated with Three Different Additives by Joseph A. Atekwana AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1965 ABSTRACT Additive effect on the fermentation products and dry matter digestibility of silages was studied. Forages ensiled in miniature metal silos under 10 lb. per square inch pressure were alfalfa, center-cut corn, sudan grass and sudax. Urea (42%N) and two commercial silage additives, were used. The experiment was a factorial design with four treatments on four forages and one replicate. The forages from pre- and post-fermentation were analyzed for dry matter, pH, crude protein, volatile fatty and lactic acids. Digestion trials, using the modified nylon bag technique, were carried out by incubating the silages in the rumen of a fistulated Holstein steer. Silages treated with urea resultedbin significantly (Pz:.OS) higher protein, pH, acetic and lactic acid con- tents. The effects of the other additives were erratic. Unfermented forages had significantly (P4 .05) higher percent apparent dry matter digestion than the fermented ones. There was an increase in dry matter digestion with increased time in the rumen. The highest dry matter digestion occurred between the 24 and 36 hour periods. J. A, Atekwana Correlation coefficients of 0.91 and 0.84 were observed between pre-and post-fermentation dry matter con— tent and between crude protein and 24 hour period digestion reapectively. There was a negative correlation (coeff. -0.58) between the 12 hour post fermentation digestion period and lactic acid content. Fermentation Products and Digestibility of Alfalfa, Center-cut Corn, Sudan and Sudax Silages Treated with Three Different Additives by Joseph A. Atekwana A THESIS Submitted to Michigan State University in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1965 ACKNOWLEDGEMEQTS The author wishes to.express his unqualified appre- ciation to Dr. H.W; Newland, Associate Professor, Department of Animal Husbandry, for his.help.in planning the study, and his invaluable time spent correcting the manuscript. The author also expresses deep appreciation to Mr. B.E. Brent, Chemist, Animal Husbandry Nutrition Laboratory, without whose help chemical analyses and calculation of the results would have been difficult. Thanks is also due the Staff of the Dairy Science Department Laboratory.for allowing the author the use of their facilities including the gas Chromatograph in the volatile fatty acid determination. The same thanks are also extended to Dr. S.T. Dexter, Professor Crop Science Depart- ment for.permitting the use of his_miniature metal silos for the silage fermentation. Thelauthor_is most grateful to Dr. D.E. Ullrey, Associate Professor. Animal Husbandry Department and Dr. R.W; Luedke, Professor, Biochemistry Department: members of his academic committee..for proof- reading and suggesting corrections in the manuscript. Special thanks are here extended to Mr. Arikpo E. Ettsh, whose companionship created~a continuous congenial atmosphere in which to work. It would be impossible to name all other friends who lent the author a helping hand during the study. To these sincere friends the author is deeply grateful. 11 TABLE OFfiEONTENTS Page INTRODUCTION ......COOOOOOOOOCOO000............O....... 1 REVIEW’OF LITERATURE 0.000....0............OOOOOOOCCOOC 4 l. Silage Fermentation ........................... 4 I AC1d PrOdUCtiOn eeeeeeeeeeeeeeeeeeeeeeeeeeee 5 II Heat PIOdUCtion .eeeeeeeeeeeeeeeeeeeeeeeeeoe 9 III ‘Additives eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoee 11 (a) Urea ......OCCOOOOCOOO00......COCCOOCOC. 11 (b) Mineral acids .......................... 15 (c) Calcium Phosphate and Calcium Carbonat. ......C....................... 17 (d) Molasses and Miscellaneous Additives ... 21 (e) Conclusions ............................ 24 2. Digestibility Trials .......................... 25 EXPERMTE mm ......O'OOOOOOO......OOOOOOOOOOO 37 (EXPERIMENT 1, TRIAL 1) A.a Silage-Fermentation .......................... 37 B. Chemical Analyses ......OOOCOOOOOOOO0.0.0.0... 4]; (a) Dry Matter, pH, and Crude Protein ........ 41 (b) Valatil. fatty aC1d8 . C. O. . ‘0. .. ... ... C .0 O 42 (c) Laetic aCid ......O.........OCCCC......... 45 EXPERIMWT 1' TRI“ II ...0.00.0.0........OOOOOOOOOOOOO 45 mERIMENT II’ DIGESTIBEITY 00............OOOOOOOOOOOO 45 PROCEDURES 0............OOOOOOOOOCOOOO0.0.0....00000000 49 RESULTS ......OOOOOOOOOC.......OOOOOOOOOOOOOOOOOO...... 57 Overall effect of fermentation .................... 57 Mditiv. effCCt on dry matter eeeeeeeeeeeeeeeeeeeee 59. Additive effect on pH ............................. 61 Additive effect on crude protein .................. 61 Additive effect on volatile fatty acids ........... 64 (a) Acetic ......O..............‘O...’......... 64 (b) Propionic acid ............................ 67 iii TABLE OF CONTENTS (Continued) ...... Page (C) Laetic aCid .000............OOOOOOOOOOOOOOI 69 DigeStibility trial 00............OOOOQOOQOOOOCOCOO 70 DISCUSSION 0.0.0..........COOOOOOOOCOOOOOOO...0.000.... 85 SWARY ......OOOOOOCOOCOQOOO0.0.........OOOOOOOOOOOCOC 91 BIBLIOGRAPHY eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeOOOOOO 94 iv II. III, IV, VI, VII. VIII. IX XI, XII. XIII. XIV, LIST 0;: TABLES EXperimental design ........................... (a) Experimental forages and additives ........ (b( Additive application scheme ............... Experimental design - digestibility trial ..... Overall effect of fermentation on nutrient content, pH and digestibility of all Silages eOeeeeeeoeeoeeoeeeeeeeeeeeeeeeeeeeeeeee Additive effect on dry matter during fermentation ......0.00.00.00.00.0.00.00.00.00. The pH depressive effect of additives as measured after fermentation ................... Additive effect on crude protein after fermentation O.......000............OOOOOOIOOOO Additive effect on acetic acid production during fermentation in micromoles per gram .... Additive effect on propionic acid production during fermentation in micromoles per gram .... Additive effect on lactic acid production during fermentation in micromoles per gram .... Dry matter digestibility as affected by‘ time intervals and additive treatment ......... (a) Trial I: Before fermentation ............. (b) Trial I: After fermentation .............. Dry matter digestibility as affected by time intervals and additive treatment ......... (a) Trial II: Before fermentation ............. (b) Trial II: After fermentation Percent of dry matter digestibility as affected by fermentation and additives °€""" Overall silo pressures .....,............a..... Dry matter recovery in silage run-off Trial II. Page 38 38 39 48 58 60 62 66 68 71 73 73 74 76 76 77 79 82 83 LIST OF FIGURES FIGURE Page 1. Cross-section of a filled miniature 811° ....LOOOOOOOCOOOOOOOOOCO......OOCOO,..O... 43 11. Progression in‘preparing the nylon bag ........ 51 III. Time effect on apparent dry matter digestion (Pooled averages of all four Bilag“) ...........................C...‘...... 63 IV. Percent dry matter digestibility of all four silages as affected by fermentation and addit1VOB ...eeoo0.00.00.00.00...0.00.00.00.00. 84 vi LIST OF_PLAT ES Page A close-up View of pressure guage in Operation showing 10 lb. per square inch ................... 44 Complete set up for a typical six-day run ........ 46 Injection of 3 millimoles of sample into gas chromatograph for volatile fatty acid determination 00......0......00.0.0000300000000000 47 Nylon ba s containing a set of 18 forage samples ?16 eXperimental and 2 controls) P arranged on a metal ring previous to placement into the rumen 0‘0.............OOOOOOOOOOOOO0...... 53 Flushing one ring ofi~bags with tap water“ juSt after {mova1_fr9m:tb¢ rm¢n QLQOQCQQoeeeeeee 55 A group of bags on one ring left to drain after flushing ......OOOOOOOOOOOOOOOQOOOOOOOOOOOOO 56 vii INTRODUCTION The problem of equitable distribution of animal feed through the various seasons of the year is one with which livestock farmers are familiar. Sometimes, farmers in the temperate regions of the world have more forage than they can profitably handle in early summer and in winter: their fields are completely covered with snow. The same is true of farmers in the tropics where there are two distinct seasons - the dry and wet seasons. During the wetyseason, there is an abundant supply of forage and in the dry season, the fields are virtually scorched by the sun. Silage making has become one of the best weapons of the farmers against these seasonal irregularities in live- stock feed supply. The farmer has also realized that in cutting, hauling, ensiling and storing forage, he loses some of the vital nutrients. Consequently, he has tried many ways to salvage this nutrient loss during silage making. One of the well known ways he has done this is to treat the silage with various additives. Some of the commonly used additives have been mineral acids like sulfuric,_hydrochloric and phosphoric. Others like urea, calcium carbonate and yeasts have also been used with varying success. Some farmers have also used microorganisms for good silage production. 2 In using these additives, the silage makers have often asked the question of how much and what kinds of nutrients are lost during the ensiling process. They also want to know how much of these nutrients the additives can save the most. Various researchers have addressed themselves to some of these pertinent questions and have come up with answers which are by no means conclusive. It was, therefore, in the search of further answers to these questions that this research has been conceived. The data which are to be presented in this manuscript deal with the effects of some of these additives on the chemical processes and transforma- tions occurring in the ensiled products. Two rather recent commercial additives — Bio-zyme referred to as "P'and Silo Guard referred to as 'Q’- have been used in addition to urea. The ensiled materials treated with these additives were alfalfa, center-cut corn, sudan grass and sudax - A cross between sorghum and sudan grass, It would have been necessary to carry out some feed- ing trials with livestock to find out the effect of additive treatment on the metabolism of these silages. But this was not possible because the quantities of the silages made in miniature silos were inadequate. However, some digestibility - dry matter disappearance - trials were.carried out using the modified nylon bag technique in the rumen of a fistulated steer. These data too, will be presented. The researcher also believes that grass silage mak— ing will become an important operation in livestock production, 3 eSpecially among the developing nations of the world where grasses abound, concentrates are in short supply and the com- petition is acute between man and beast for food. REVIEW’OF LITERATURE Roughage occupies a unique place in the nutrition of ruminants. Among the various forms of roughage that comprise livestock rations, silage is of prime importance. It is then no wonder that the production of silage has received considerable attention since man began the domestication of animals. 1. Silage Fermentatigg Some of the earliest work done on silage is recorded in Food & Life, the Year Book of Agriculture, 1939. It is stated in this publication that silage production as we know it today was started by a Frenchman named Goffart in 1875. Woll, the author of the article stated that the idea of modern silage was brought to the United States by Morris in Maryland in 1876. Watson et.al. (1937) and W011 (1939) both maintained that livestock farmers should prefer silage over hay because in harvesting and storing forages or hay they lose about 20 percent of the nutrients (including total loss by fire) as compared to almost negligible losses as silage. Allen 25 21. (1937) observed that the rapid changes taking place in the ensiled mass were influenced by (a) external temperatures, (b) moisture content, (c) botanical Species, (d) age or 4 5 stage of maturity of the silage material and also (e) by the season during which the silage crOp was harvested. They also said that the compaction of silage during filling of the silo to exclude as much air as possible enhanced silage quality. Allen gt al. also observed that the main chemical changes in the silage took place within the first ten days. This point is supported by numerous other investigators (Russell, 1908: Watson, 1937: Virtanen, 1938 and Barnett, 1954). Writing about the sequence of changes taking place in the ensiled material, Russell (1908) reported that the general chemical changes known to occur in silage were the conversion of sugars to carbon dioxide and water, the pro- duction of acetic, butyric and lactic acids and the produc- tion of non-protein material from proteins by the process of deamination. I, Acid Production Most of the investigations into the changes that silage material undergoes have centered around the chemistry associated with production of volatile and non-volatile fatty acids and the period of most rapid fermentation. Esten and Mason (1912) concluded that the most important period of corn silage fermentation began shortly after the material was ensiled and was usually completed within a few days. Allen, gt a1. (1937) studied the chemical and bacteriological changes occurring in grass silage, and 6 reported that most changes were initiated by the microflora that was usually carried on the fresh crop. He observed that the coliform bacteria and some rods, mostly lacto- bacilli, appeared to be prevalent in the fresh material and hence in the silage. He went on to report that the.coliforms were of a type that would not thrive above 30°C. In this case, the coliforms were the dominant bacteria in the early days of silage fermentation before the temperature rose above 30°C and from then on, lactobacilli which could thrive at 37°C and above became prevalent and multiplied rapidly just after the decline of the coliforms. The progress of fermentation is associated with higher temperatures and lower pH. Since a rapid increase in both temperature (to a certain point) and pH will inhibit the growth of the microorganisms that will produce un- desirable acids like butyric, the earlier these conditions are achieved, the better the silage quality. It.is, there- .fore, important that the acidity be rapidly increased. "The pH ofthe.silage is one of the.best indices of its valuJ“ (Watson, 1937). Low pH can be achieved by directly adding acids to the silage or adding fermentable carbohydrates which lactobacilli will use in.lactic acid production. Thus, acid and molasses addition at the time of ensiling will produce an excellent product if care is taken to achieve great compaction. Ferguson & Watson (1937) observed that when the pH did not exceed 4.5, the quantity of butyric acid, an indication of putrefaction, was negligible. They 7 also reported that acetic acid was the main constituent of the volatile fatty acids while lactic comprised most of the non-volatile fatty acids. However, they noted that in cases where butyric acid exceeded 0.75 totl percent by weight of the fresh silage, care had to be taken to feed the silage outdoors or in the open air. Thus, according to Ferguson & watson, the aim for good silage was to produce a minimum of butyric acid. This observation was shared by many other investigators including Hayden g; 31. (1945), Herman g; 31. (1941) and Virtanen (1933) as quoted by Watson (1939). Virtanen (1939) reported that Edin and Sandberg (1922) were the first to determine the pH in silages. Virtanen (1938) was stimulated by Edin & Sandberg's work to start a systematic investigation of the processes taking place in silage and their effects on silage quality. Among the_detri- mental processes, he cited protein breakdown and the ferment— ation caused by the coliform and the butyric acid bacilli. Irwin (1956) working with orchardgrass silage, Langston (1958) and Kanpton and San Clemente (1959) working with thirteen different grass silages reported on their findings which were in agreement with those of Virtanen (1938). Barnett (1954) reported that the process of ensiling meadow and corn crops was characterized by the fermentation of carbonhydrates which resulted primarily in lactic and acetic acids. In their studies of the use of urea to increase the crude protein content of corn silage for fattening steer, Bentley gt al. (1955) reported that organic acids, especially acetic and lactic, sad a high feed replacement value and that production of these acids should be encouraged whenever a silage fermentation scheme was planned. This view was also expressed by Dobrogosz and Stone (1957) when they used metabisulfite to increase acid production in alfalfa silage. In this same study 0, 8 and 12 pounds of metabisulfite were used per ton of silage,and the authors concluded that the utilization of sugars and the production of acids in the silages was inversely correlated with the amount of meta- bisulfite added to the silage. Many other workers have continued to investigate acid production in silages. Klosterman £5 31. (1960) analyzed the organic acids produced in whole corn plants ensiled in large glass jars and treated with various neutralizing agents. These neutralizers consisted of (1) 0.5 percent low magnesium limestone plus 0.5 percent urea, (2) 0.5 percent urea, and (3) 1.0 percent urea. The acidity of these silages as determined by the pH was 4.30, 4.10, and 4.40, respectively. The acetic acid content of the three silages was 2.13, 1.93, and 1.71 percent on dry matter basis, reSpectively. The lactic acid content was 12.05, 8.71 and 12.00 percent, reSpectively. Klosterman gt 2;. (1962) treated ear corn silage with different additives and increased the moisture content by adding water. They reported that the difference in acidity between the treatments, as measured by the pH, was 9 not statistically significant but that there was an increase in lactic and acetic acid production. A report by Hall 2; 3;. (l954),in which sweet potato vine silage was treated with additives showed that the silage had an "acid type'. fermentation with good odor, color and texture. Lactic acid formation increased four fold in the first four days due to the increase of lactobacilli Plantarium. The chemical changes were evidenced by a rapid drop in the pH and a rise in lactic acid production. Acidity was further increased by the addition of tubers and molasses to the vines. One of the most detailed studies on acid production in silage was done by Irwin g; 11,. (1956). They determined formic, acetic, proprionic, butyric, succinic and lactic acids during the 40th to 60th days of silage fermentation. The eXperiment was designed in such a way that poor to good quality silages were produced. Prior to ensilation, acid content on a dry matter basis was less than 1 percent of the fresh grasses. silages classed as "mediocre' had butyric acid after five to eight days. Lactic acid increased up to 10 percent on a dry matter basis within the first five days then stabilized. PrOprionic and succinic acids were negligible. II, Heat Production Most of the chemical reactions taking place within the ensiled mass is associated with heat production. This is governed by the general environment and such internal lO factors as degree of aeration and wetness of the ensiled mass. Temperatures in excess of 100°F were associated with scorched silage with excessive dry matter loss, and tempera- tures below 75°F were sometimes associated with poor quality silage, characterized by butyric acid production according to Briggs £5 31. (1959). A report by Benne and Wacasey (1960) closely agreed with Briggs findings. They showed that temperatures between 80°F to 100°F produced cold and those from lOOOF to 120°F produced warm fermentation. This would seem to conflict with Benne and Wacasey (1960) who considered eo°—1ooO F favorable to the production of good silage. These findings have stressed the effects of both ex- cessive and subOptimal temperatures on silages. At the Imperial Chemical Institute Experiment Station at Lealott's Hill, England, Watson gt al. studied |'losses of dry matter and digestible nutrients in low temperature silage with and without added molasses or mineral acids.u They found that under the low temperature conditions dry matter loss in five silages ranged from 13.7 to 31.1 percent. seepage was also blamed for most of the dry matter loss. Watson et 2;. (1937) observed that about 38.7 percent of the bases and 85 percent of volatile fatty acids were lost during drying. One of the nutrients affected most by temperatures is digestible crude protein. Bratzler e; 2;. 'l (1956) remarked that under high temperature conditions, a silage may lose as much as 30 percent of its original crude 11 k.’ r“. ‘J 1 ( ‘j protein and stil. higher percentage of this nutrient at the and or the storage period due to large carbo- . " - ‘ ,N vr‘! .__~ 1- _ r- e hjdratc loriog. :atiou 35 ii. (1333) also found that higher hanpera ures in the silage rsndered proteins indigestible. Others “40 reported studies or silage temperatures are: V 4‘ A 'fi .. .1 ." , a, O p O o mentor (1917) who acacureu a tenterature of 30 ~e0 C in the I: 5' ‘ . ;' 1 P a. ‘ -1 O.- ‘ ‘1 0'“. center or the 81107 Allen (19;?) who measured 90 g and 102 z for the tOp and bottom of the silo, respectively. Sherman and Bechdel (1918) found that corn stover F O 0 ' O Silage containing excess water had a temperature of 57.7 ‘ On the other hand Kenpton and San Clemente (1959) reported 0‘1 0 " ‘0‘} temperatures of 111 z and 131 1 in one case and lie u and A’N‘O'r O 0 e 0 lbs 2 in another in Silages with excess water. These tempera- ture readings were taken froa the too and bottom of the silos and the higwr: ones were considered letrimental to good sil- age material. III. Additives It was noted in the introduction that one of the ,0 best ways iarmers have achieved desirable silage quality has been through the use of appropriate additives. Urea, calcium carbonate, calcium phoSphate, t”new, molasses and mineral acids have been SOme of the additives used. (a) Ureg: The use of urea as a protein substitute in the ration of ruminants has assumed greater importance with the increased use of concentrates. One way of getting urea into animal feed has been through its addition to silages at the time of enSilation. 12 Wbodward and Shepherd (1944) ensiled corn silage using.lO pounds of urea per ton. They later fed some of the treated silage to milk cows with a low protein concentrate ration. Another group was fed urea to boost the protein intake, but this time the urea,was added directly to the concentrate. No significant differences were noted between treatments, and milk production remained on a high plane. This showed that whether urea was incorporated into the silago or fad along with concentrates, the not effect remained the same. It was further learned that additional increases in urea had deleterious effect on the palatability of either the silage or the concentrate. In 1944, Davis §§,31. used a uroa.solution in quantities of O, 10, 30, and 50 pounds of urea per ton of sorghum silage. Crude protein determinations prior to and after fermentation showed that there was a slight migration in nitrogen and that free ammonia was noticed from tho sil- ages with highor concentrations of urea. There was a great variation in pH. The control silage had pH 3.5 while the silage with 50 pounds of urea per ton had a pH of 7.6. The cattle that were fed these silages showed no discern- ible difference between the O and 30 pound urea silages but completely refused to eat the 50 pounds per ton urea silages. However, when free ammonia had been released they ate this silage. Cullison (1944) quoted Harris and Mitchell_(194l) as having improved the digestibility of a low protein ration for sheep by supplementing with urea. The improvement 13 was eXplained as due to the stimulation of microflora in the rumen which enhanced carbohydrate fermentation. With the addition of urea to the silage, prolonged fermentation stopped and the resulting silage was superior to that which was ordinarily made in carotene content, palatability and general feeding value for lambs and beef cattle. The use of urea as an additive to silage continued to be investigated by other workers. Means (1945) compared urea treated and untreated sorghum silages. He used silage to which 10 pounds per ton urea was added. He carried out feeding trials for a 77 day period. Three lots of beef cows and three lots of yearling heifers were used. The rations were: (1) A standard ration of 30 pounds of untreated sorghum silage, 1 pound of cottonseed meal and 5 pounds of Johnson grass hay: (2) 35 pounds of the same.untreatod sil- ages, 5 pounds of Johnson grass hay: (3).35 pounds of urea treated silages, and 5 pounds of the same hay. The heifers were fed 5 pounds less than the cows in a combination of these ingredients. The cows in Lots 1, 2, and 3.gained 9 pounds, lost 99 pounds and gained 13 pounds.respectively. Heifer calves, differed although the standard ration.produced the best gain, still the treated ration remainedsuperiorin an overall consideration. In studying the effect of urea— treated and untreated corn silage in the performance of lactating dairy cattle, Wise g; al. (1944) noticed that the treated corn silage had carmalized odor and brownish color. in terms of dry matter, it took 16.9 pounds and 15.5 pounds 14 per animal daily of untreated and treated silages, reSpect- ively. Although the results of Wise at al. seem to differ from those of Means (1944) the work of Cullison (1944) agreed with Means. Feeding results obtained by Wise agreed with those of WOOdward and Shepherd (1944). One important point about Wise's work is that the urea-treated silage had a crude protein content of 10.79% as compared to 7.48% for the untreated silages. Similar results were reported by Bentley 3; a1. (1955). In the same work, Bentley reported that urea-treated corn silage was not inferior in feeding value to corn and soybean oil meals. Contrary to this finding, Archibald and Parsons (1945) found urea-treated silage quite unsatisfactory because of the conversion of the urea to ammonia with objectionable odor. Hall gt :1. (1954).fonnd the color, odor and texture of sweet potato vine silage treated with urea to be good. One of the.most recent' studies, in the investigation of the.addition of urea to silage, was done by Klosterman,1§,al. (1960) and (1961). In one of these studies, they found that 0.5 percent urea treated silage increased the lactic acid production 75 per- cent over that which was not treated: on a dry matter basis. However, they also noticed a slight loss in dry matter in the treated silage. This brief review of the value of add? ing urea to silages and to livestock feed has shown a few outstanding things. These are: that urea has a " protein sparing'function. It arrests delayed fermentation and accentuates the production of lactic acid. Animals fed urea 15 treated “silages have generally shown consistent gains and thrift in feed conversion. (b) fling;al_§giggfi The principal inorganic acids used as silage additives and preservatives have been hydrochloric, sulfuric and phosphoric. Virtanen (1933) as quoted by Watson (1939) was one of the strong proponents for silage treatment with mineral acids. He carried out intensive eXperiments in which he used hydrochloric, phosphoric and lactic acids to depress the pH of silages to.3.6. In.this work hydrochloric acid was superior to the other two in depressing pH to the desirable range. Other workers, Stone st a1. (1943) observed that phosphoric eacidtapplied .at 16 pounds per ton of silage depressed the pH.and resulted in good silage product if care were taken to include ferment- able sugars with the ensilage. .A.classic example of the use of phOSphoric acid in silage work was reperted by Ingham gt a1. (1949). In five years.of intensive research in silage fermentation, with.mo1asses.and phosphoric acid, they arrived at some interesting.conclusions. The silages produced in each of five experiments were fed to dairy cattle and records of milk production,_physiological.effects on the.animals and palatability were kept. JThe.deeign of experiments was basically the same. In the first trial, one lot of three Holsteins and two Guernseys were fed corn silage, mixed hay and a grain mixture. A second,loteof the same number and types of dairy cattle was fed molasses - alfalfa silage, mixed hay and a grain mixture. The third 16 group of the same number of animals was fed phosphoric acid - alfalfa silage, mixed hay and the same grain mixture. The fourth group had only molasses - alfalfa silage alone and the last group was fed phoSphoric acid - alfalfa silage alone. In general the results showed that cows on alfalfa silage had a positive nitrogen balance. ,Exceptiens were observed in a 28 day trial in which limestone was added to the phosphoric acid treated silage of lot five. In this period, cows in lot five showed a negative.nitrogen balance of 13 gm. daily. However, for every pound of digestible nutrient consumed, lot five which was fed the phosphoric acid silage produced 1.63 pounds of milk as compared to 1.42 pounds.for lot four fed the molasses silage. The researdhera explained this by saying that the cows in let five seemed to have been inherent good producers. In one of the experiments in which oats substituted for alfalfa, the phosphoric acid - oat silage promoted greater protein digestibility. In conclusion,.the.authors noted that the decisions as to which additive to useiin a silage.should.be made with the local conditions.in mind. However they maintained that "silage preserved with-phosphoric acid definitely hinders,.strange as it may.seem. the reten- tion of.phoSphorusr Allen 33,31. (1937) observed that addition of mineral acids reduced protein breakdown. Similar observa- tions were made by Hermangt al. (1941). They -dug out two trench silos with capacities to hold 45.50 tons..~ These silos 17 were 8 feet wide at the bottom and 12 feet at the top. In one of the trench silos, barley silage was ensiled with 60 pounds of molasses per ton. The second had eight pounds of 75 percent phosphoric acid per ton. Except for_peripheral Spoilage, both silages did not have any discernible differences either in color, odor, or texture. Work reported by Colovas gLIQL. (1958) showed that calcium phosphate depressed the digestibility of the silages due to the release of phosphorus during decomposition. (c)_C P t d Calc Car na 3 The addition of calcium phosphate and limestone to silages is a relatively recent concept. One of its.eat1yiinvestigators is Qelovas st 31. (1958) who used twelve dairybeifers to determine the effect of pulverized limestone and.calcium phosphate on the nutritive value of dairy feeds. They fed ladino clover, bromegrass, timothy and grass-legume silages wuth_limestone and calcium phosphate at the rates of 50 and 100 grams per head daily in the silages. During the second year of the same experiment, the ration varied slightly. ,They,included 16 pounds of crude protein.concentrate mixture. The.fieeding of 100 grams of pulverized limestone daily depressed the crude protein digestibility and.energy of the silages. On the other hand,.50 grams did.not depress the above nutrient digestibility to any appreciable extent. They noted that two percent of.dica1cium phosphate did not depress the.same nutrients while-the.addition of one percent of limestone depressed the digestibility of both the protein and energy. 18 The authors then fed 2 percent calcium phosphate and 2 per— cent limestone and found that the depressive effect of limestone on crude protein was minimized. They concluded that limestone depressed while phOSphorous enhanced the digestibilities of protein and energy of silages. Klosterman gt _1. (1961) at the Ohio Agriculture EXperiment Station reported the use of limestone in the treatment of high moisture corn silages. They used 0.5 per- cent high calcium limestone and 0.5 percent urea. In an- other instance, they varied the procedure, l.0 percent high- calcium limestone was used with 60 percent water. In both trials cattle gained faster and more efficiently with the treated as opposed to the untreated silages. The production of lactic acid was significantly greater in the treated sil- ages as compared with the untreated one or the control. There was an increase of 78 percent lactic acid in the treated over untreated corn silage and as much as 125 per- cent in the high-moisture calcium-treated silages. Pre- viously Klosterman 3; a1. (1960) reported a 100 percent increase in lactic acid in silages treated with 1 percent calcium carbonate and another increase in lactic acid of 40 percent in silages treated with dolomitic limestone over that produced by the control. watson (1939) also cited Sani (1912) as having treated fodder with monocalciumphOSphate at the.rate of 6.75 pounds per ton. The color of the treated silage remained fairly green and although odor esters were emitted l9 from the silage it still retained a high protein digest- ibility. One of the latest investigations on the effect of calcium on silage quality was done by Nicholson gt a1. (.1964). Nicholson noted that Byer at 31.. (1963) and Klosterman et a1. (1962) found that the feeding value of the calcium-treated silages was higher than untreated sil- ages, but Nicholsons group could not confirm these observa- tions. Thomas at al. (1951) also found that treatment with limestone did not improve the feeding value of silages appreciably. The experiment reported by Nicholson (1964) consisted of three trials. In the first trial, he ensiled the grasses in 16 oz. glass jars equipped with pressure relief valves. The grass was chOpped into 1/2 to 1/4 inch lengths with a hand scissors. The material was left to ferment for 7 weeks at controlled temperature of 70°F. The silage juice was ex- pressed and the organic acids were determined according to methods of Wiseman and Irwin (1957). The second trial consisted of four silages fed to growdng heifers in a 76 day feeding trial. Slightly wilted grass-legume hay and corn harvested.at the milk.stage were used. In two similar tower silos, a plastic sheet was used to divide the silos into two vertical sections. Alternate .loads of 1.0 percent limestone treated.and untreated materials were filled into the silos. They used.12 Holsteins,-8 Shorthorns and 4 Jerseys as test animals. Animals were 2O assigned to each of the four silage groups and from each class and age groups at random. Each animal received 2 lb. supplement ration in addition to water, bone meal and salt which were fed ad libitum. The third eXperiment compared the feeding values of six silages. Limestone at 1.0 percent and 2 percent was used in the silage each of which was made in the plastic divided silos. Two other silages of timothy in the bloom stage were made in temporary silos and these had shredded newSpapers added at the rate of 5 percent to in— crease dry matter content. These two silages were made in silos of plastic sheets encased in snow fences. Thirty-six steers of average 655 lbs. were assigned at random to the The digestibility trials were conducted by feeding the silages to wether lambs as the only roughage. These lambs were confined to metabolism cages. Feces and urine were collected for analysis. At the end of these experiments, the authors con- cluded that addition of limestone at the time of ensiling grass or immature corn silage resulted in an increase of organic acid production during fermentation, but reduced the feeding value of the silage. They also reported that the addition of limestone to silage usually resulted in lower lactic but appreciably higher prOportions of acetic and butyric acids. Feeding the limestone treated silages in- variably resulted in lower feed intakes and reduced gains. 'The limestone did not seem to have any effect on the organic 21 matter digestibility of grasses but tended to reduce the digestibility of nitrogen and ash. Finally, steers fed paper—supplemented silage consumed less feed than those fed a control silage and lost weight when this was given as the only feed. (d) Mglgsses and.Mjsgellaneous Additives: As reviewed in the section of acid production in silage fermentation it seems clear that lactic acid is highly desirable. This is because of its high nutritive value. Many researchers have shown that some form of fermentable carbohydrate is desir- .able for lactic acid production. In most cases in which farmers have desired to increase the lactic acid content of their silages molasses has been their first choice. watson (1937) reported that the addition of about 0.75 to 1.0 percent of molasses in a ton of silage was sufficient to achieve the desired level of organic acid pro- duction. Hayden 3;,QL. (1945), found that molasses depressed the pH of the silages just as much as phOSphoric acid. Perhaps one of the most intensive silage studies involving molasses as an additive was carried out through a five year experiment by Ingham gt 31. (1949). Several silages were studied and all were compared with plain corn as the control. These silages were (1) molassesealfalfa and phOSphoric acid - alfalfa (1940-41): (2) Molasses-oat and thSphoric acid-oat 1941-42: (3) Molasses-soybean and cornmeal - soybean 1942-43: (4) Molasses - grass and ground barley grass 1943—44; and 22 (5) Molasses - timothy and ground barley - timothy 1944-45. In evaluating the silages the following factors were con— sidered: l. Feeding value - maintenance or lactation 2. Physiological effects 3. Apparent digestibility 4. Protein, calcium and phosphorus metabolism In discussing the results, the authors observed that the feeding trials did not show any statistically significant difference among the quantities.of the silage in.the three balanced rations. ,However, the molasses-alfalfa silage was more palatable and more economical (in terms of total digestible nutrients required per pound of milk produced) .than the phosphoric acid - alfalfa silage: ibut both were more economical than.the control.corn silage. There were no physiological abnormalities in the blood and urine of the cattle fed molasses—alfalfa or corn silages while in one of the lots, (phOSphoric acidealfalfa).cows had.marked increases in.urinary ammonia and acidity. Whereas.phosphoric.acid- alfalfa silages held a slight edge in crude protein digest- ibility over.the molasses silage, the latter was superior -with respect to fiber, fats.and.nitrogen.free extracts. They found that.phosphorus consumption by cows on phosphonio» acidealfalfa silage was matched by a high calcium intake. Yet, for both.ca1cium and phOSphorus, the cows had negative balances.in.three out.of four trials whereas cows an molasses—alfalfa silage retained both calcium and.phosphorus 23 very well. When palatability was conSidered in the third trial, in which molasses-soybean and cornmeal-soybean silages were used, it was found that cornmeal-soybean silage was well preserved and palatable. Plain corn silage was rated second and molasses silage was rated lowest. In the fourth trial, the feeding value of the barley- ;rnss silage was significantly greater than that of the molasses grass silage. Both silages were considered palat- able but txe animals relished the molasses-grass silage more. In the fifth and final experiment, the authors noticed that timothy silages were less palatable than the standard corn silage but the molasses-timothy silage was just as economical as corn in maintaining weight and lactation. In conclusion Ingham at al. stated that, in general, molasses-preserved grass silages had very good palatability. Also cattle fed grass silage preserved with molasses or ground grain had excellent retention of both calcium and phOSphorus. On the other hand, silages preserved with phosphoric acid definitely hindered, strange as it might seem, the retention of phosphorus. A few non-acidic additives apart from urea, calcium carbonate and molasses have also been used in silage preser- vation. Barnett g; al. (1954) reported the use of dried and wet whey as a cheap source of carbohydrate to accentuate lactic and acetic acid production. They had previously (1951) used sulfur dioxide in promoting organic acid produc- tion. Bolstedt 35 al. (1941) was quoted by Barnett (1954) 24 as having used corn meal to increase useful fermentation in grass silages. Working with high protein legume silages Krauss (1941) used hay to increase the carbohydrate and then promoted rapid organic acid production. Some workers have advocated the use of enzymes, yeasts and bacteria as additives to speed silage fermentation. Kronlik gt_a1. (1955) reported that the microbial population in green plants were absolutely necessary for good silages. Hunter (1917) observed that the pepulation of microorganisms in grass silage increased.rapidly within,thelfirst two weeks and preposed that in cases where.the silage_material had been frozen or where workers had suspected a deficiency in microbial content of silage material, deliberate effort should be made to increase them. (e) Songlgsigns From the foregoing partial inventory of related studies, it can be seen that silage fermentation is a well investigated field. It is highly desirable to have a high organic acid production. This phenomenon is a well defined process and the types as well as the levels of the acids produced.have much to do with the quality of the silage product. Medium to high.quality silages-have high lactic and acetic acid levels. Excessive production.of butyric acid at the expense of lactic and acetic is-a sign of poor quality silage. It has been reviewed how mineral.and in- organic acids, fermentable carbohydrates mainly molasses, whey and certain starchy meals have been used to rapidly achieve the desirable fermentation. Some researchers have 25 used bacterial cultures and others have used yeasts and enzymes to achieve similar purposes in silage making. In the case of bacteria the consensus seems to be that certain rods and cocci are the most prevalent microorganisms in silages. The.cocci.are believed to disappear as the silage process progresses whereas the population of rods increases. The rods include.lactobacilli which are conducive to good fermentation and high lactic acid levels. .Many of these researchers have estimated or seem to agree that optimum silage temperature should preferably lie within the 80°F to 100°? range. But this temperature range is subject to vari- ation brought by experimental conditions and the depth and size of the silos from which such temperatures are recorded. 2. Digestihility Trials When silagehhas been fermented in the 31103, and the material is fed to livestock, the next logical question to ask is how much has.fermentation done to hinder or en- hance digestibility of the forages? This problem has.long been rsoognized.and most silage investigationsrhave included some form of either.conventional digestion trial with live animals or used some artificial technigue to obtain a criterion on which to assess the digestibility of the parti- cular silage in question.. Because most conventional diges- tion trials.with live animals.have been expensive, tedious and time consuming, many recent workers, whenever possible, have used artificial techniques to achieve or to attempt to 26 reach the same goal. Crop scientists, nutritionists and feed manufacturers have used some quick means to have some data or information on the digestibility of their products. In doing so they have tried to approximate natural conditions as much as possible. To achieve this, some have used rumen fluid as inoculum to stimulate microbial growth. Others have used controlled temperatures and some have gone even nearer to nature by using fistulated animals. The preponents of these artificial techniques_in.forage and feed investi- gations have.advanced reasons why they think these methods are good. They maintain that: (l) the artificial techniques, especially using the nylon or dacron bags,.are accurate, rapid and.simple for measuring forage qualities. (2) They cut down costs and excessive tine consumption common to con- ventional trials with live animals. (3) These tedhniques are suitable means of evaluating forages by small plant breeders whose plots are too small to supply all the forage needed for metabolism trials with large animals like cattle. And (4) that there is a high correlation between conventional and nylon bag technique trials, Van Keuren‘gg‘al. (1962). Much literature is accumulating on the subject of in‘gigg and.in,y;§;g digestion of animal feeds. Fina gt‘gl. (1958) at Kansas Agriculture Experiment Station, Manhattan, carried out artificial techniques in studying rumen diges- tion ig4gigg. While admitting that it was still difficult to correlate results obtained in vitro with those obtained 27 from actual animals, he maintained that the artificial techniques were necessary and would continue to be. In their experiment, cellulose decomposition was tested by placing tubes containing 500 milligrams of cellulose with and without a rumen inoculum for ten days in the rumen of a fistulated animal. A control tube with distilled water was kept in the rumen through the same period. The procelain tubes used could allow acetic, valeric, propionic, butyric acids, urea and glucose to go through its porous walls. Microbes did not penetrate the tubes and hence the un- inoculated tubes remained uncontaminated for twenty-one days, There was no cellulose decomposition in the tube containing cellulose and distilled water after ten days. Thus, under controlled conditions Fina and his group studied cellulose digestion with the help of microorganisms through rumen inoculum. Hughtamen e; 2;. (1952) studied artificial cellu- lose digestions and the results essentially agreed with Finds. Salsbury et al. (1956) studied the rates of cellulose digestion of some plant fractions by rumen microorganisms in zitgg. The study showed that the liquification of roughages did place a limitation on the digestion of its cellulose. An eXperiment was carried out in the East African Veterinary Research Center. In this study, Todd ,§§.§1. (1956) investigated the digestibility, chemical comp- osition and nutritive value of certain forage plants at mid- altitude in the tropics. Digestibility varied with the 28 maturity stage of the grass in question and the season at which the forage was harvested. They also observed that Kikuyu Grass, (Pgnnisetgm Clandestinum) and Rhode grass (legris gayana) had high and moderate digestible crude pro- teins, reSpectively. Erwin g; a1. (1959) studied rapid methods of determining digestibility of concentrates and roughages in cattle. They used nylon bags of 4'x 8u tied up to 36 on one chain and inserted in the rumen of a fistulated steer. Four fistulated steers were used for this trial. One of the steers was on a milo, the second on an alfalfa, the third on barley and a fourth on.bar1ey straw rations. They observed that the position of the bag in the rumen did not make any difference in the.amount of substrate digested. There was a linear decrease in digestibility as weight of material increased in the bags from 10 grams through 24 grams. The greatest decrease of 15 percent in this reSpect was observed in the milo bags. The variability in digestion within the first nine hours were alfalfa 26-34 percent,.straw 16—19 percent, barley 37-54 percent and milo 27-36 percent. In conclusion, the authors.mentioned that the technique then used in Arizonian experiment stations was 20 grams in the rumen for nine hours. In the study of “the dacron bag technique in the evaluation of forages'Hopson gt 3;. (1961) put representative samples of the feed fed to lambs in dacron bags. The grasses used were bromegrass, timothy and the legume alfalfa. The time intervals of the bags in the.rumen were 6, 12, 18, 24, 29 30, 36, and 42 hours. Results showed that only the 36 and 42 hours digestion periods had a high and significant corre- lation with conventional trials using four years old wethers. Significant digestion rates were found between the three grasses. Alfalfa digestion was detectable between 6 and 12 hours. This was sooner than the rest of the forages. Different digestion rates were found between alfalfa and the other forages when alfalfa was used as the experimental animal feed. On the contrary, there was no significant change in the normal rates of digestion of the four forages when forages other than alfalfa were used in experimental animal ration. Similar results were found by Walker gt 31. (1956) when they studied the nutritive value of forages for sheep. Stewart gt __J.. (1961) estimated in m volatile fatty acid production from various feeds by bovine rumen microorganisms. They used twelve large mouthed jars and in- cubated with inoculum at 39-39.5°c in a thermostically con- trolled water bath. As quoted by Steward, Nasserman at al. (1952) carried out a similar experiment and their findings agreed closely with Stewarts. Schultz e3 3;. (1949) found similar results and in addition reported that propionic acid was glucogenic. Another study conducted in the area of forage digest- ibility was at the washington Agriculture Experiment Station. In this study, Van Keuren gt _1. (1962) investi- gated the value of the nylon bag technique for determining ID in yiyg forage digestibility. The samples of forages used were dried completely at 65°C and were then ground through a 20, 40 and 60 mesh per square inch screens. They used ten grams of feed per bag of Z'x 45“. The nylon string used in fastening the bags to a 3/4"x 8“ “lucite' or “Plexiglass“ rods had a tensile strength of 75 pounds. These samples were put in pre-weighed nylon bags and dried at 65°C for 24 hours. The bags were labelled with plastic tags. After the bags had been in the rumen of fistulated steers for pre- determined time periods they were removed, dried at 65°F for 48 hours in a forced draft oven, removed, crushed and re- dried for 24 hours. The difference between the dry weight. that went into the rumen and that which finally came out of the oven was taken as dry matter digestibility. In some of the trials, twelve bags containing five grams each were tied to one stick. Alfalfa was the substrate and the diges- tion period was 24 hours. The twelve bags were randomly assigned to positions on the stick. Justifying that it was not necessary to randomize the bags on the sticks, Van.Keuren gt 3;. quoted Erwin and Elliston (1959) as having found that the position of the bag on the chain did not matter in terms of the amount of substrate digested. However, Niles of the University of Wisconsin at Madison, in a Ph.D. thesis, did find some variations in digestibility according to the levels placed in the rumen. 31 In discussing their results, Van Keuren 33,31. stated that there were significant increases in digestibility of orchard grass after 24 hours, alfalfa up to 72 hours and a mixture of both forages after 48 hours. No interaction was found between length of time in the.rumen and fineness of _grind of the forages in influencing digestibility of the forages. Although alfalfa still showed slight digestibility at 72 hours, digestion started off.earliest, rose until the first 24 hours and then declined. Just as Hopson gt‘gl. (1951) -.found, _ the .diet . of the. .fistulatedwanimeal influenced the digestibility of the forages. There was a slight in- crease in the digestibility of the.substrate in bags which were presoaked before immersion into the rumen. The authors . explained that some material could.have moved in and out of the bag. However they asserted-that if this.movement were true, then it. .did .so. at a uniform ”ratein -all the bags .and did not affect.accuracy of the results. They concluded that goodirepeatability,was found in the degree.and patterns . of.digestion-throughout most of the trials. There were ,.significantiincreases.inndigestion of.sudan grass and orchard .-grass after the first 24 hours while.a1falfa and ladino 5 .-clover.showediquick-initialwdigestibilityibefore,24nhours .and.then declined gradually. .Fineness of grind did not “.significantly affect digestibility.. Sample sizes.affected digestion for comparable periods of_time in the rumen.. And .finally, dietary regime of.the animalwinfluenced digestibility. 32 On the correlation between artificial and conventional digestion trials, Baumgardt et a1. (1962) evaluated forages in the laboratory. They reported that in comparing con— ventional and artificial rumen digestion trial methods, the fermentation of carbohydrates by the latter method yielded data which correlated significantly (P_ (a) TRIAL I ,_ 30 1' 25 ‘P. S 20 u] ‘3 0 I5 5 '10 o ... 6 < z >_ A — AFTER FERMENTATION . a: _ B — BEFORE FERMENTATION Q 35 ,_ 30 E (b) TRIAL 1:: a: 25 E a Si 20 °\. I5 I A I0 I 1 O-———-+ O .—————"r’—0J"""”T 5 ’°,'/’ /v0 0 . I 510:5 20.25 30 35 40 TIME INTERVALS m HOURS Eig.TlI: The effect of tine on apparent dry matter digestion. .64 inherent differences in protein.content of the forages. Thus, no attempt was made to subject forage effect on pro- tein retention through fermentation to statistical analysis. (However, analysis of variance show'ed the differences in additive effect on this nutrient to be significant. ~In both trials, protein values from additive (R) were significantly higher (P<:.05) than the control and other additives, but these were-expected because the additive R was urea (42%N) applied at the rate of .5%. ,On the other.hand, adding Bio- zyme (P) resulted in proteinvalues almost-equal to the con- trol (5) while Silo Guard (0) had no consistent effect: lowering protein values in Trial I and slightly increasing it in Trial II. Additive effect on volatile fatty acids (a) Aggtig. The results showing the production of.acetic acid are presented in Table VII. Analysis of variance showed some significant additive effect on this acid. Trial I showed that urea (R) significantly (P<‘-05) increased acetic acid production over the control includingother additives. eDifferences in acetic acid production due to forages were statistically significant (p1:.05). When con- fident interval limits using thaschaffa' (1959) method were carried out between the mean acetic acids.due to each forages, it was shown.that the meanlalfalfa was significantly (Pe use censusesm men s in «K as no.“ pad on; 6.} Ac... 6%... .63 man 65.2 no.2 $5 6a.: .84 o.o~ n.» 9.5 9.5 ¢.¢ w.HH ~.c~ ¢.a 5.5 o.n m.¢ «.0 N.a s m.N~ «.mN H.0H on m.m o.¢ o.n o.m m.m w.¢ ¢.¢H 5.5 n.5 N.n n.m w.m H.m H.nH 5.0H m.nH 5.0a «N .4 o.n 5.N 5.m m.~ ¢.m N.d ¢.n ~.o o.¢ o.n m.~ o.~ m.N m.NH H.NH 5.NH m.w NH 7 2388 m a o m m a a a .6. a a m m e a .— 05a 2.. . 22 ~38 538 .28 «and 5d.so«ueusesueu Hound. “H Aaquvs was nauseous“ uses an veuoewme as auwawpuueewav wouuel_5ua A N manna 75 of 9.3 vs. 5.7 for the 12 hour period, 14.5 vs. 8.3 for the 24 hour, and 31 vs. 21.6 for the 36 hour periods for the unfermented silages in Trial I and II, reapectively. The same trend, although not to the same magnitude, showed up in the fermented silages. There was no consistent additive effect on dry matter digestibility. The data in Table XII and again as presented in Figure IV show that urea (R) and Silo Guard (Q) significantly (P<'.05) increased percent dry matter digestion of alfalfa and corn over the control (3), on unfermented forages. Both additives also showed increases in dry matter digestion on unfermented sudan and sudax but these were not significant. Except in sudax where Bio-zyme (P) increased forage digestibility over the control (5), it consistently lowered digestibility of the other forages below the control. A point of crucial consideration was the effect of the additives on the fermented silages. Silo Guard (0) and urea (R) significantly (Par.05) increased dry matter digestion of alfalfa. Bio-zyme (P) slightly depressed alfalfa dry matter digestion below the control (8), There was virtually no additive effect on the digestion of corn silage. In fact, all the additives lowered corn silage digestion by 1% below the control. Only urea (R) slightly increased per- cent dry matter digestibility of sudan and sudax over the control. Bio-zyme (P) and Silo Guard (Q) actually decreased dry matter digestibility of both silages below tn) control. Considering additive effect»on percentfdry'matter digestion of both fermented and unfermented silages, the L D. .5395 323393»: Momma—u use 09 eueuuea unaueeueese same 93.309 9.33 09939 cede nevus nose: 9 .u .9 .555 one can.“ use egg case we nausea be H33 90 emeuoeoueo e as censuses \m. «ox an; and .16 is a...» .1.» Am; uni a.m.: no.2 8%.: sea ...}: Boa 66th .22 mm 9.: 9.5 n.m 9.m 9.9 q: «.3 9.: .7: «.3 9.: n.9~ 9.: 9.: a.o~ a.ma 9: 9n m.9 «.9 in m.n «.3 «.5 5.9 9.9 «.2 5.: 0.2 9.3 9..» 9.9 m.9 9.9 «N 5.9 9.». n.5 0.9 9.~ «.9 9.9 9.5 5.m ~.a n6 n.5 94 9.9 ~.n ~.m NH .388 a e a a s a a .6. s a m m a a a «any .ww“ gm an 380 43a .el seaweeds—anew “...—omen "HH 3‘ eel-uses“. 05:23 use ...-buoys.“ end» he veuoemue ee 53393-093 nausea E «.ng 7 7 .5395 53.33353 .5993 see 09 unauoeuoesa ease 95.509 9:300 oweuom :03 needs «one: 9 .38?“ ecu 35 use madame nose «0 Houses .39 Hence we oweuseuuea e as penance: \W .032, :5 asses meninges 39,326 was mongoose men a. sm.m sm.s aw.n 65.6 .m.~ sm.o se.n no.6 am.“ pm.n sm.s no.n n~.- .0n.oa nn.- s~.~a .osa 6.6a o.n o.e « .5.» m.o 6.» m.m n.~H ~.~H m.~a a.» « a.ma n.m~ o.- a.ma on “.0 e.m ~.¢ m.s ~.n a.» ~.s o.n ~.¢ o.“ o.~ e.e H.m H.H~ a.ma ~.HH o.~a «N «.6 n.~ ~.m a.~ s.~ n.n H.e o.n n.m n.m n.~ m.H o.s o.“ s.“ a.“ 0.5 «a souuuou m a a e m a a a m a a m m s a a case .wwu xgasm agape zuoo 4sq.o m.h m.m o.oa H.m o.m m.m .H ensue m m o M4 a s dllnmr m a ha. a m m a.llnMIIJmofifluee .o>< amuse g ammo gill! .uomsuom HM [4| 9339.3 0.36 Hasuotao .HHHX 0.309 °/. APPARENT DIGESTIBILITY ON 0 O I N p e U (I! U! 0! OT) 9. -.. e...u.000...,. .... 4 ......... coo. v s o. eoooeooce... e o...¢o...... .......-.... t. O ..... .oo .0. . .. .o‘oon¢OQD~Io poo:.‘.. ~9...... Vd'Wd'lV 'IC‘III‘I ...-....- ID‘IQOQOQ- . oo.o-,oo... ~ . -..eoono¢0a.. 54090‘4- . e ............ SBMLIOOV ~9'1I911 o-Iotoooeooo 0.009.... :- ........... o . .0 u. o. .0 .- . o ..eoe-otvoa quItIOIQI'I .... :~.v000.00u¢ ...ooo-o....~ echoes-cotte. -. o....o~oo¢o.. Auqoauu-una. rig. IV.: Pooled additive effect on dry matter digest- ibility of each silage: fermented and un- fi-.. -_ 85 DISCUSSION The primary purpose of this study was to investi- gate the effects of various additives on fermentation of silages made of alfalfa, corn, sudan grass and sudax. For these findings to be useful for statistical inferences, it was necessary to work with as many silages as possible. This objective was met, in part, by the fact that the results presented above represented averages of duplicate samples from four forages each treated with three additives and one control: and the eXperiment replicated once. There was some discrepancy in dry matter content of the silages in the two trials. Since there was no apparent reason for this, from the standpoint of procedure, the author felt that the difference might have been due to a higher lactic acid content in Trial II which was lost in the drying process. This could possibly lead to lower dry matter con- tent. Adding urea to the various silages resulted in higher pH than the other additives and the control. These results were in agreement with work reported by Schmutz (1962) and Klosterman gt 31. (1961). Considering the effect of forages on pH, the values for alfalfa were the highest. Sudan grass had the lowest pH. Surprisingly, this forage had the highest moisture content (about 79%). It could be SBMLIOOV F1 "I. APPAR ENT DIGESTIBILITY a: 9- - a: 3:: -. 01) U! 9. o-o ...vn -,c-eeeeeeo . .ee.....o... -~oevv- . . , Ioeovot-ey-Cv. eo-.'.eoeceac. . . ......... .nuooo-u UGO-Ivy oeevovoovov- bet... u... eoeo‘e.e-O-. o 0 ' 00.9.0090... .. were... > -e ‘ e ‘ o . e , ' e ‘ o ' . ' e. on..ncoc. e aloe-.4 . Ae~Ov-n.o- . . .. ..a....... ...ey4-.-. ..‘.'-....o ...-...... ..cyo-.... u U"'."OOQ I‘Q‘DeOOOvl .....9....-. . IIOOC< o.n n.“ n.o n.o ~.m «.m H.m N.m n.n~ n.nn n.n~ N.«u o.n «.On 6.0 «.5 HH neauh a.« n.“ n.o H.n n.nH «.5H o.o~ ~.«H H.«N o.- o.«~ «.mn a.mu o.e~ o.~n n.su H nouns sown-usesuou seamen n a a a ... e a m r m e a m n e a m 33:34 38 an H :80 «an: eobuuuvoe one sequencesueu he oouoouue es huuuuoaueouwo house-.59: .mmm_wuomu 80 It was planned to collect run-off from the silos and determine how much dry matter was lost through this route. Due to an accident in the first trial, most of the run-off was lost. Hence, no figures were determined for this trial. However, the results of dry matter recovery in run— off for Trial II are presented in Table XIV. The results showed that the loss of dry matter through run-off ranged from .06 gm to .24 gm per c.c. Expressed as total percentage dry matter 1055 during fermentation, alfalfa was the highest with 1.67%, followed by sudan with .78% then corn and sudax with .56 and .50%, reSpectively. The high loss by alfalfa could possibly have been due to its high leafiness and succulence. Sudan coming next could have been because it was equally leafy and succulent. Consequently, the more matured and tougher sudax had the least loss through run-off. There was some relationship between dry matter loss through run- off and the moisture content of each forage. Among other things, the wetter the silage the more dry matter loss. Sudax with the least moisture (65.3%) lost the least dry matter through run-off whereas alfalfa (72.1%) lost more dry matter through run-off. Simple correlation analyses were run between the variables. There were very few high correlations. Some were considered to be of some value. There was a high correlation between dry matter content determined prior and after fermentation (coefficient of 0.91). The production of acetic acid was negatively correlated {—0.38) to that of 81 butyric acid. This showed that acetic acid production was inversely related to the production of butyric acid. An- other striking correlation was that between the results of the 12 hour post fermentation digestion and lactic acid production. The coefficient here was —0.58. As was mentioned while trying to rationalize the disparity between digestibility of fermented and unfermented silages, it shows here that the high lactic acid might have been lost during drying of the samples and, hence, the materials left were less digestible. Another high correlation of 0.86 was observed between the 24 hour post fermentation digestion and crude protein content. This seemed to be understandable since, as it was shown in Table VI, alfalfa with the highest protein content produced the least lactic acid which in turn was negatively correlated to digestibility. 82 m.m 0.0H coca mom m.m m.m 0.0 0.0 m.m h.m h.m boo h.m How v.m m.m mom .HH Hmaua mob oom m.m m.m m.m m.m H.h H.h ¢.v m.o 5.0 m.b mom 0.0H Hom m.m w.m .H HMMNB m m o M1 m a d! {a m m Iaf m m m a.lIIIIIIm 32.383 .e>< Koo—.6 g flmdw I37 . some.» om ll." Ill ensue-5m ode Admuoeeo .HHHK 0.309 8 % APPARENT DIGESTIBILITY >1 - - - - N U (n 0" a. 973 0 .. .....'.... IIOIOODO-ol III‘I‘I.'.U. ~v<00000. .. VJ'IVd'IV v -Ov004‘- -~p'.oc. on..¢.. . .... ’o.onv¢..n. ...... naeobnoooooc .t-‘voq.q-. Io¢b~v~quot v o . . ..aaooo...ou . ......-9.-.. 0.0-ovooooa. o o SBAILIOOV - u ....‘o.- 0 v o 0000'. - ono~o¢ . .q...tu.-a -V-O¢Ol.- , - - -obOvOoOon .- o u. o o o . -4-atool'.' - .......-..., ~u-o-q.o- o ...-cueuotequ uvvaovooa-o v c-ocooo¢~.. .. . on. .... . IV.: Pooled additive effect on dry matter digest- ibility of each silage: fermented and un- A: 1:--- A- 85 DISCUSSION The primary purpose of this study was to investi- gate the effects of various additives on fermentation of silages made of alfalfa, corn, sudan grass and sudax. For these findings to be useful for statistical inferences, it was necessary to work with as many silages as possible. This objective was met, in part, by the fact that the results presented above represented averages of duplicate samples from four forages each treated with three additives and one control: and the experiment replicated once. There was some discrepancy in dry matter content of the silages in the two trials. Since there was no apparent reason for this, from the standpoint of procedure, the author felt that the difference might have been due to a higher lactic acid content in Trial II which was lost in the drying process. This could possibly lead to lower dry matter con- tent. Adding urea to the various silages resulted in higher pH than the other additives and the control. These results were in agreement with work reported by Schmutz (1962) and Klosterman g1; 31. (1961). Considering the effect of forages on pH, the values for alfalfa were the highest. Sudan grass had the lowest pH. Surprisingly, this forage had the highest moisture content (about 79%). It could be 86 inferred that higher moisture content would promote acid production: hence, lower the pH. Klosterman 35 31. (1961) found increased acid (acetic and lactic) production correspond- ing to increased moisture content of high moisture ear corn. The results on crude protein were also somewhat in agreement with the findings of Sohmutz (1962) who found that high moisture corn silage treated with urea at 20 lb. a ton resulted in crude protein values of 13.6%. This was higher than the average of 10.8% protein in the urea treated corn silage found in the eXperiment reported here, where urea was added at the rate of 10 lb. per ton. Wise, 3; 31. (1944) also found that urea treated corn silage had a crude protein content of 10.79%, whereas untreated silage had 7.48%. Bentley :3; al- (1955), and Labedinsky and Corb (1960) reported increases in crude protein with urea treated silages. Apart from the unusually high acetic acid production of about 138.5 micromoles per gram of silage in alfalfa in Trial I (Table VII), the general production was well below 85 micromoles per gran. Taking acetic acid as a percent of total acids produced, this was found to be about 40%t :Lactic acid was about 55% and propionic and butyric made up 5%. Sohmutz (1962) observed that acetic could range from zero to 30% total acidity while Nicholson and Cunningham (1964) reported acetic acid to be 20.4%. lactic 79.6% and butyric 0% in untreated silages and 1% lime treated bromegrass 87 silage showed 26.1% acetic, 55.3% lactic and 18.6%'butyric. In this last reference, butyric acid content seems too high but this could have been due to the conditions of aeration of the silages. There was a difference in the amounts of acetic acid produced in the two trials. No_apparent explana- tion could be given either for the high values for alfalfa in Trial I or the overall difference between the two trials. More research may be recommended in this area to find out whether these discrepancies were due to factors like differ- ences in moisture content, or some other unidentified variables. Very few workers have reported on the production of propionic acid in silages. In this study, there was no con- sistent pattern for this acid. It was seen that some forages that produced most prepionic in Trial I produced practically none in Trial II. However, sudax was consistently low in both. It could also be said here that as researchers con- tinue to seek more precision in silage fermentation product investigation methods, more attention may be directed towards this minor acid rather than towards the oftbeaten tract of the major fatty acids, or lactic and acetic acids. The overall average lactic acid production was.about 200 micromoles per gram of silage material on a dry basis. This compares favorably with 198 micromoles found by Schmutz (1962) and results found by Klosterman g; 31,. (1961), and also by Nicholson and Cunningham (1964). Klosterman showed that there was a corresponding increase in both lactic and 88 acetic to increase in moisture. If further research should confirm these findings then there could be a hopeful future for high moisture corn silage and haylages in ruminant nutrition. The second experiment was to obtain some digestibility data on dry matter as measured by its disappearance in the rumen of a fistulated steer. The results showed that un- fennented silages were more digestible than the fermented ones. This finding seemed to go contrary to common sense. Since digestion is essentially a process of fermentation, one would expect a partially fermented silage to be on its way to.easy digestion and hence would show more dry matter disappearance. As already mentioned under the presentation of the results, this low percentage digestion could have been due to drying of the samples whereby the already broken down constituents, like the fatty acids, were driven off by heat and the material left was therefore less digestible. More work may be recommended in this area. In the event that such a work is to be done, it may be desirable to carry it out in two phases. More could be done in the same way as was carried out in this study to see if these obser- vations would be confirmed. Some means should be found whereby differences in weights as a result of digestion could be detected without subjecting the silages to drying at all, or not to dry them to the same extent as was done in this study. 89 On the basis of the results from the present study, it would be futile to draw any conclusions as to which additive was the best. Since the Bio-zyme ('P”) and the Silo Guard ('0') are rather recent commercial additives, it would seem necessary to investigate their silage improving potentials by carrying out more research. Nevertheless, some additive differences showed up. Figure IV shows the pooled effect of the additives on all forages in the two trials. In essence, this figure shows that during fermenta- tion of alfalfa, Silo Guard and urea increased digestion of dry matter over the control, while Bio-zyme actually decreased dry matter digestibility. Differences between additives for the other forages were small. Losses in dry matter as determined from silage run- off are presented in Table XIV. These losses ranged from .44% to 1.98% with the greatest loss occurring in alfalfa, followed by sudan and then corn and sudax. The high rate of dry matter losses in both alfalfa and sudan could be related to the high leafiness and per- haps high moisture content of these forages. Corn and sudax, though grainy, had high cellulose content due to their characteristic stems and advanced stage of maturity. Except in sudan where the additive R (urea) had up to 1.07% dry matter loss, additive effect on each individual forage was well within the same range or they did not differ very much. The results from this study have fallen below 90 those obtained by Schmutz (1962). Schmutz reported dry matter loss in high moisture ear corn silage to be about 5%. However his silage was fermented for periods ranging up to 12 months. 91 SUMMARY During the winter and the early part of Spring 1965, alfalfa, center-cut corn, sudan grass and sudax were treated with feed grade urea (42%N) and two commercial silage additives and ensiled in miniature galvanized metal silos for six days periods. Air tight conditions and_an average pres- sure 8.7 1b. per square inch were maintained, thus_similating, as far as possible, conditions in conventional silos. The experiment consisted of one run and one replicate. The en- siled forages were sampled before and again after fermentation and the samples were used for chemical as well as dry matter determinations. Digestion trials were also carried out on these samples in the rumen of a fistulated Holstein steer. Chemical analyses were done on dry matter, pH, crude protein, and volatile fatty acids. The digestion trials were carried out on the dry matter using a modified form of the nylon bag technique in the rumen of a fistulated Holstein steer. Of all the factors studied, the pH both before and after fermentation had the smallest standard deviation. Every other factor varied greatly. The additives used had no significant affect on dry matter in the various silages, but adding urea resulted in slightly higher dry matter than the control and other additives. There was more dry matter (ave. 35.49%) conserved in Trial I than in II (26.94%). 92 The average pH before fermentation was 5.4 and after fermentation, this was depressed to 3.7. silages treated with urea were slightly higher in pH (4.0) and this additive was more consistent in its effect on pH than the rest. The effect of additive on crude protein was signifi- cant (P<:.05). Urea treated silages consistently resulted in significantly higher crude protein content while the other additives apparently had no influence on crude protein during fermentation. As regards organic acid production in the entire fermentation experiment, lactic was produced in the greatest amount followed by acetic, propionic and butyric. There was more acetic acid produced in Trial I than in II and adding urea resulted in significantly (P<'.05) higher acetic acid production in both trials. The other additives did not sig- nificantly affect acetic acid production. I Prepionic acid production was both small and erratic Whereas that of butyric acid was only negligible. Addition of urea resulted in significantly (Pk:.05) higher lactic acid production in both trials: the.levels being 195.6 and 220 micromoles per gram of dry forage in Trials I and II, respectively. The other additives did not significantly affect lactic acid production during fermenta- tion. Average lactic acid production was higher in Trial II than I. The overall forage effect on acid production was significant (P<:.05) in both trials. In Trial I, acetic acid 93 production by alfalfa was highly significant (P<=.001). This forage showed the least lactic acid production and the overall average of lactic acid for sudan grass was theihigh- est. In the digestibility trial, additive and forage effect and the interaction between both were significant (P<.01) for the first 12 hours and (P<.1) for the 24 hour period. The effect of time interval was outstanding. There was a steady progression in digestion through time in that the digested amounts approximately doubled for every addition- al 12 hour period. This direct proportion was true.especia11y for the unfermented materials. It was observed that the unfermented material showed more dry matter digestion than the fermented ones. There was more dry matter disappearance in Trial I than in 11. Because of maturity and species differences in.the forages no attempt was made to compare the forage effect on dry matter digestion. An average pressure of 7.9 and 9.5 pounds per square inch were maintained in Trials I and Ilirespect- ively, during fermentation. Run-off was collected from Trial II and dry matter loss was determined. The average less.range from .6 to 2.4 grams per 100 ml run off. Simple.cerre1ations analysis showed high correlations between dry matter deter- minations prior to and after fermentation (9.91): 24 hour post fermentation and crude protein (0.84). There were.negative correlations (coeff. -O.38) observed between butyric and acetic acids, and (coeff. -0.58) between the 12 hour post fermentation period and lactic acid. 94 BIBLIOGRAPHY A.O.A.C. 1961. Official methods of analysis of the Association of Official Agricultural Chemists. 9th edition, Association of Official Agricultural Chemists, Publishers, Washington 4, D.C. 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