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I ’1‘ “ .21 23‘ ‘23 3253213? 3 11 32, 3,23,22,21312} f2- ‘ . 3?, 1 3, 2 lbs, ‘1': T;__;__.:.~..» :: ;" 1' '12:" _. “My ml ,3 2.2‘1" 1'1‘1'1‘." “2,31 -;.., ... w%"'§;"r'"""~2* Iii-'1‘“ " . 3' i: [)ate This is to certify that the thesis entitled THE CHEMICAL COMPOSITION OF SEWAGE GROWN AQUATIC PLANTS AND THEIR DIGESTIBILITY BY SHEEP presented by STEPHEN ROBERT BAERTSCHE has been accepted towards fulfillment of the requirements for M.S. degvein Animal Husbandry LIB R A R Y- Michigan State University 444% ,M/a/ /4’// / THE CHEMICAL COMPOSITION OF SEWAGE GROWN AQUATIC PLANTS AND THEIR DIGESTIBILITY BY SHEEP By STEPHEN ROBERT BAERTSCHE A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Husbandry 1978 (Si/03575? f ABSTRACT THE CHEMICAL COMPOSITION OF SEWAGE GROWN AQUATIC PLANTS AND THEIR DIGESTIBILITY BY SHEEP By Stephen Robert Baertsche Aquatic plants have been used for many purposes. Different species of marine algae have been utilized in 1 fertilizers and as stabilizers in plastics, ice cream, and candy. Recently, due to their relatively high protein con- tent, animal nutritionists have begun to evaluate the possi— ble use of various aquatic plants as alternative sources of livestock feed. The quality and the quantity of available nutrients from these aquatic plants has been of primary interest in recent years. Analytical values for certain aquatic plants show high values for crude protein and min- erals. In this study two different aquatic plants, glagg- phgra algae and Elodea canagensis, were examined in regard to both their chemical composition and their digestibility by sheep. EXPERIMENT I — CHEMICAL COMPOSITION. Samples of the two aquatic plants were collected from three Michigan State University sewage treatment lakes, sun dried, ground through a 20~mesh screen with a Wiley milL.and analyzed for proximate constituents. Dry matter and crude protein values showed the least variation, with values ranging from 92.9% to 95.1% for dry matter and 17.8% to 18.1% for crude protein. Ether extract and gross energy values were lowest for the two aquatic plants when compared to dehydrated alfalfa and an alfalfa soybean mixture. Mineral analyses revealed higher concentrations of both macro- and microminerals for both of the aquatic plants on a dry matter basis. Algae contained 5.3% calcium and elodea 4.5% calcium while alfalfa contained 1.7% calcium. All samples had similar amounts of neutral detergent fiber, while the alfalfa contained a higher percentage of acid detergent fiber. Permanganate lignin values were highest for algae at 5.2% and alfalfa at 5.1% EXPERIMENT II - DIGESTION TRIAL. A 4 x A Latin square design was employed using four crossbred wether lambs. The diets consisted of 100% alfalfa meal, 95% alfalfa - 5% soybean meal, 70% alfalfa - 30% algae, and 70% alfalfa - 30% elodea on a dry basis. Digestibility coefficients were calculated for dry matter, crude protein, gross energy, and acid detergent fiber. Dry matter and crude protein digest- ibilities were significantly higher for the alfalfa-algae ration when compared to the alfalfa-elodea ration, but were not significantly different when compared with other rations. Digestible energy and acid detergent fiber coefficients were significantly higher for the alfalfa-algae ration when com- pared with the alfalfa-elodea and alfalfa—soybean meal ration. Nitrogen retention, rumen fluid pH, blood urea nitrogen, and rumen NH3 were also examined. No significant differences were found between any of these treatment means. ACKNOWLEDGEMENTS I would like to express my appreciation to the following people whose efforts, knowledge, and understanding have aided me in my graduate program and the preparation of this theses. Dr. M.T.lhkoyama for his guidance in my research work, critical reading of this manuscript, overall counseling in my academic work, and his patience while I was engaged with extension responsibilities. Dr. R.H. Nelson and the Animal Husbandry Department for the use of the facilities and animals. Drs. W.G. Bergen and H.D. Ritchie for the added super- vision of this experiment and numerous other consultations. Special thanks to E.L. Pink for her laboratory assistance. My parents, Mr. and Mrs. Wendell Baertsche, for their love, understanding, and the valuable experiences that Were given me while growing up on our family farm. Most of all, my wife, Vicky, for her support, love, and diligent work in preparing this thesis. ii TABLE OF CONTENTS LIST OF TABLES I. II. III. IV. VI. INTRODUCTION. LITERATURE REVIEW MATERIALS AND METHODS Experiment I. Harvest of plants Feed sample collection. Dry matter. Crude protein Gross energy. Ash Fiber analysis. Mineral analysis. Experiment II General design. Equipment used. Feeding program Preliminary period. Preparatory treatment Collection period Rumen fluid pH. Blood urea nitrogen Statistical analysis. RESULTS AND DISCUSSION. Experiment I. . . . Proximate components. Fiber components. Mineral content Experiment II . . Digestibility coefficients. Nitrogen retention. Rumen fluid pH. Rumen fluid NH3 Blood urea nitrogen GENERAL CONCLUSIONS BIBLIOGRAPHY. iii Page iv 15 15 15 16 16 16 17 17 18 21 23 23 23 25 25 26 26 27 27 28 29 29 29 32 34 37 38 42 44 44 44 46 48 LIST OF TABLES Table 1 2 mm: .q Experimental Design for Experiment II. . Chemical Compositions of Rations .. . . Fiber Analysis of Treatment Rations . . Mineral Contents of Alfalfa and Aquatic Plants Digestibility Coefficients for Rations . Digestibility Coefficients Determined by Difference . . . . . . . . . . . . . Nitrogen Retentions. . . . . . . . . . . Rumen pH, Rumen NH , Blood Urea Nitrogen Values. . . . . . . . . . . . iv 40 43 45 INTRODUCTION For the livestock producer, it is becoming an increas- ing problem to purchase or produce cereal grains and leguminous crops economically and not in direct competition with human needs. One solution to this problem would be the alternative utilization of aquatic plants such as algae and elodea which have recently been shown to have a possible feed potential. Research by Hintz and Heitman (1967) has found that certain species of algae contain as much as 73% crude protein. Because of this high crude protein content, research is currently being conducted to evaluate certain aquatic plants as potential protein sources. The high fiber content of many aquatic plants has caused their digestibility in monogastric species to be low (Hintz and Heitman, 1967). However, when mixed with common forages such as alfalfa and cereal grains, ruminant animals such as cattle and sheep performed as well as controls fed 100% alfalfa or 100% grain (Linn.gt gl., 1975). Another important characteristic of aquatic plants is their high mineral content. Linn.gt,gl. (1975) evaluated 21 different species of aquatic plants and found an average content of 1.62% for calcium and 0.27% for phosphorus. It has also been determined that aquatic plants are consider- ably higher than alfalfa in microminerals (Linn, gt gl., 1975). The purpose of this study was to evaluate the chemical composition and digestibility of two different species of 1 2 aquatic plants. The two aquatic plants, algae and elodea, were grown and harvested from sewage treatment lakes at Michigan State University. The plants were washed, sun- dried, and then stored for chemical analyses.. Trials incorporating these plants at 30% of the total dry matter were also carried out with lambs to investigate the digest- ibility of each of the plants. LITERATURE REVIEW Because of their possible potential as a protein supplement and as a livestock feed, several species of algae and other aquatic plants have been evaluated and studied for a number of years. These autotrophic, aquatic plants use carbon dioxide and solar energy, synthesize protein, contain variable levels of vitamin C and B complex, and some species are even able to fix gaseous nitrogen (Oswald 23 gl., 1959). Kleiber (1961) calculated that algae are 1000 times more efficient in the utilization of solar energy than cereal crops. In fact, studies by Oswald gt gl. (1959) have shown that Chlorella algae could yield more than ten times the amount of protein than soybeans on a per unit basis. Besides being more efficient in utilizing solar energy and land area, these aquatic plants, at the same time, serve the function of removing organic matter and other waste from water, which would result in environmental pollution. Algae has been used for many different purposes. Marine algae is processed to obtain iodine, and some species and genera are utilized in the production of agar (Chapman, 1962). Several alginates (polymers of manuronic acid) which are obtained from marine algae are used as thick— eners or stabilizers in various products such as ice cream, plastics, and candy (Maass, 1962). Marine algae has been 3 L; used by developing and certain.Asian countries as a fertil- izer and a source of human food. (Schmid and Hoppe, 1962; Zaneveld, 1959). In recent years, Marimura and Nobuko (1954) have suggested that due to its high protein content, unicellular algae such as Chlorella be used as a food source to alleviate protein deficiencies. Other abstract uses include suggestions by Boiko gt g1. (1962) and Lachance and Vanderveen (1963) that species of unicellular algae be stored for space travel because of its high protein content and light weight. Even though sewage-grown algae could be used as an alternative protein supplement, there have been very few studies reported in which it has been fed to the ruminant. Hintz 33 El. (1966) performed a study with three different species of algae Chlorella, Scendesmus obliques, and Scendesmus quadricauda which were grown on sewage and fed to cattle, sheep, and hogs. The mixture fed was shown to contain 51% crude protein which was 73% digestible for cattle and sheep and 54% digestible when fed to pigs. Their results showed that the algae supplied sufficient protein to supple- ment barley for growing-finishing pigs. Lambs receiving an alfalfa—algae pelleted ration also gained better than when alfalfa was fed alone (P<.01) on a dry summer range (Hintz _e_;t_ £11.. 1966). In another study, algae was shown to be an adequate protein supplement for pigs fed barley (Hintz and Heitman, 1967). In this regard, lysine is a limiting amino acid in 5 barley (Reimer gt gl., 1964). Chlorella algae was found to be rich in lysine and equal to dried skimmilk powder when added to wheat flour and fed to rats (Mitsuda gt g;., 1961). Fink and Herold (1955) found that Sendesmus obliguus was as good as milk protein for growth of rats, and Witt §t_gl. (1962) found that replacing 75% of the fish meal of a barley-fish meal ration with Sendesmus obliguus did not decrease the grthh rate of pigs. 0n the basis of feeding trials with lambs, Hintz 23 al., (1966) showed that mixing alfalfa with algae at proportions of 60% alfalfa to 40% algae in the ration produced better gains in comparison to lambs grazed on dry summer range pastures (P<.05). Hintz and Heitman (1967) found algae supplemented with certain B-vitamins and substituted for fish meal produced equal gains and feed conversion efficiency when fed to pigs. No significant differences (P<.05) were found in carcass characteristics between pigs fed on the algal diets and those fed diets containing the fish meal. Digestibility studies indicated that the algae was low in digestible energy, but that its crude protein was 70% digestible. Hintz and Heitman (1967) also demonstrated the need for B—vitamin supplementation when Chlorella algae was used in feeding trials with swine. This response to vitamin B12 was interesting because Round (1965) reported that Chlorella synthesized vitamin B12 and Fisher and Burlew (1953) reported Chlorella pyrensidosa contained 10-45 mg. of vitamin B12 per pound. It would be interesting to 6 conduct further studies to determine why swine given algae in their diet respond to vitamin B12 With better gains. Hintz and Heitman (1966) suggested several possible reasons for this reSponse, (1) algae interferes with vitamin B12 formation, or (2) low utilization because of the low algae digestibility, (3) the incidence of coprophagy may be de- creased because of the high concentration of algae in the which would not feces, and (4) true B versus pseudo B1 12 be available to the animal. 2 The adverse effect of aquatic vegetation on the environ- ment is an increasingly serious worldwide problem which is affecting normal lake and water ecosystems and their use by man. Bates and Hentges (1976) reported that in 1970, the state of Florida Spent more than one million dollars on partially effective efforts to keep its 4000 square miles of infested waters free of aquatic weeds. The development of sound control methods will require innovative thinking and creative research. The control measures to combat serious aquatic weed infestation may be placed into three broad classifications: chemical, biological, and mechanical with eventual use for livestock or human consumption. Gerloff 23 gl. (1965) and Boyd (1968, 1969) found that chemical composition of aquatic plants varied over 100% de- pending upon season, location, environment, and level of nutrification. If and when such variations would occur, animal feed formulations would have to be adjusted. Also Bates and Hentges (1976) stated that freedom from herbicide 7 and pesticide residues, naturally occurring or environment- ally induced plant toxins and pathogenic organisms is essen- tial if the material is to be safely utilized. Hentges gt gl. (1972) found that the fiydrella spp. appeared to be as well tolerated by cattle and sheep as water hyacinth, but neither was adequate as 100% of the ration. It was most effective when provided at less than 33% of the organic matter in pelleted diets. Ensilage studies have shown that the wet press residue of water hyacinth will make an excellent silage when combined with additives which provide fermentable carbohydrates and absorb moisture,1hereby preventing run-off of nutrients (Baldwin 25 al., 1974). Bates and Hentges (1976) concluded from their studies that dehydrated aquatic weed press residues have a nutritional value as a ruminant feed, but that it must represent only a small portion of the total diet and be carefully compounded with supplemental feed ingredients to balance its deficiencies. Alfalfa is recognized as the most valuable forage crop with annual yields of over two to three tons per acre (Akeson and Stahmann, 1966). In fact, it has been calculated that 300,000 square miles of alfalfa could supply the mini- mum protein requirements of the human race with a large quan- tity left over for livestock (Morrison and Pirie, 1961). In contrast, water hyacinth under intensive cultivation could easily produce three times as much protein per acre (Boyd, 1970; Steward, 1970). This represents a tremendous 8 potential for aquatic plants if harvesting costs could be kept at a minimum. Linn gt gl. (1975) conducted chemical analyses on 21 species of dried aquatic plants which were harvested from inland lakes in Minnesota and found that all contained sufficient quantities of nutrients to be considered as livestock feedstuffs. Although considerable variation existed among the 21 species, 14 species contained more than 10% protein and all species contained less than 40% crude fiber. Ca and P contents averaged 1.62% and .27% respectively. Neutral detergent and acid detergent fiber contents of the 21 species averaged 42.3% and 32.6% respec- tively. Linn.§t g1. (1975a) ensiled the mixed aquatic plant species (approximately 50% Myciophyllus, 30% Ceratophyllum, 10% Potamogeton, 5% Vallisneria, and 5% unknown) with organ- ic acids (acetic, formic, propionic), corn, or alfalfa. After 47 days of fermentation the silages had pH values be- low 4.1 and lactic acid values above .6% of the dry matter. Ensiling mixtures of aquatic plants and alfalfa resulted in silages with similar characteristics as the aquatic plant silages alone. Addition of alfalfa to sterilized aquatic plants at ensiling resulted in a silage of similar character- istics as the alfalfa silages. Linn gt gl. (1975b) in another study, completed a Digestibility trial with sheep utilizing the aquatic plants harvested from the Minnesota lakes. Their studies were 9 conducted with two Species of dried aquatic plants (Mygio- phyllum exalbescens and Potamogeton_pectinaces) and an en- siled mixture of aquatic plants (approximately 50% Myrig- phyllum, 30% Ceratgphyllum, 10% Patamogeton, 5% Vallisneria, and 5% unknown) to determine the digestibility of aquatic plants by lambs. Both the dried Myriophyllum exalbescens and Potamogeton pectinaces were found to be unpalatable (less than 6000 g. of dry matter were consumed daily). This problem of palatability could be attributed to the "bitter principle" which was reported by Marimura and Nobuko (1954) when algae and aquatic plants were fed to humans in their eXperiment. Linn.§t al. (1975) also found that mixing an equal proportion (50:50) of the two species with dehydrated alfalfa resulted in dry matter and crude protein digestibil- ities, as determined by difference, of 43.8% and 46.0% for Myriophyllum and 43.4% and 44.1% for Potamggeton. Energy digestibility was found to be higher for the Myriophyllum than Potamogeton. In the same study, lambs fed diets of ensiled aquatic plants, aquatic plants plus corn, or aquatic plants plus alfalfa silage had dry matter digestibilities for the complete diet of 41.4%, 42.0% and 38.5% respectively. Lambs fed the ensiled diets of alfalfa or alfalfa plus corn had dry matter digestibilities of 61.9% and 66.2% respective- ly. Nitrogen and energy digestibilities were lower for lambs fed the rations that contained the aquatic plants than for lambs fed alfalfa silage or alfalfa silage plus corn. Rumen fluid pH was greater and molar percentages of acetic 10 acid were lower for lambs fed rations that contained aquatic plants than for those fed alfalfa silage (P<.05). Propionic acid was greatest in rumen fluid from lambs fed the aquatic plant plus corn ration. Research by Baldwin 23 gl. (1974) found water hyacinth press residues ensiled with three concentrations of preserv— atives and evaluated the physical and chemical properties of the products along with cattle acceptability. Favorable fermentation of water hyacinths and preservatives was achieved and the silage had the desired acidity, aroma, and texture. Cattle immediately accepted the silages. Although the plants ensiled in each of five experiments were harvested at dif- ferent times of the year and at different stages of plant growth, and at different locations, the results of the preservative comparisons on chemical composition and cattle acceptability were consistent in all experiments. In further experimentation, Baldwin gt al. (1975) harvested two aquatic plants consisting of Panglograss (Digitaria decumbens) and water hyacinth (Eichornia crassipes) which were fed to sheep to compare voluntary feed intake and nutrient digestibility. They found that dry matter in- take of panglograss silage was higher (P<.05) than for water hyacinth silage. They also found that the digestibility of dry matter (P<.01) was higher for panglograss silage. Heffron et al. (1977) harvested aquatic plants from Cayuga Lake in New York which was dried and milled and in- corporated into a pelleted ration replacing 35% by weight 11 of the alfalfa meal fraction. The ration was fed to pregnant goats and sheep for 130 days and results showed no signifi- cant differences (P<.05) in feed intake, rate of weight gains or ration digestibility between the animals fed the aquatic ration and those fed the control ration. Heffron.g§ gl. (1977) found the aquatic ration to be significantly higher in ash and lower in fat, fiber, and energy than was the control ration. Ewes and nannies fed the aquatic rations had normal offspring and pathologic and histologic examina- tion of the animals' tissue revealed no apparent differences between those fed the aquatic and control rations. Salveson (1971) and Stephens (1972) also worked with pelleted aquatic plants containing 33% of total ration dry matter. Results using dried press water hyacinths met the maintenance requirements for organic matter, dry matter, digestible protein and digestible energy of yearling steers. In some experiments,however, voluntary feed intake by cattle of processed aquatic plant' products was lower than expected (Hentges, 1970; Salveson, 1971; Stephens, 1972). Economically, it is not feasible to dehydrate these aquatic plants with present known methods; therefore, the ensiling of these plants has been an alternative. Linn gt g1. (1975) found the water content as high as 90% in fresh aquatic plants and this resulted in feeding prob- lems. They used partially dried plants and then ensiled with alfalfa hay to ensure adaquate carbohydrate for fer- mentation. Hentges gt gl. (1073) in other studies attempted 12 to ensile unprocessed fresh hyacinths, chopped pressed hyacinths and chopped pressed hyacinths plus molasses. All attempts at ensiling failed because of inadequate fer- mentation and spoilage. Even though more expensive in proc- essing, dehydration followed by pelleting seems to be the most effective means in which to ensure adaquate consumption and dry matter intake for cattle and sheep. Bagnall g1 gl. (1977) evaluated harvesting methods for aquatic plants. They chopped, mechanically dewatered, dehydrated, and pelleted the plants to determine whether they could be processed effectively and efficiently in existing processing systems and components. Hydraulic pressing removed 60% to 80% of the water and 18% to 32% of the dry matter. They also found that the pressed products were difficult to dry rapidly and pellet. The resistance of complex algae cell walls to digest- ibility has been one of its major problems preventing utilization of algae as a human or livestock feed (Shefner gt gl., 1962). Their studies showed -even ruminants were not able to efficiently digest the extracellular carbohydrate, and the nonprotein, nonfat organic matter. Hintz gt gl. (1966) reported that algae is not a high energy feed, because of the low digestibility of the carbohydrate fraction and the high ash content. However, it appears that algae have considerable potential as a livestock feed, because of the high content of crude protein plus significant amounts of carotene, phosphorus, and calcium. 13 Gunnison and Alexander (1975) stated that the cell wall probably is the major determinant of the resistance or susceptibility of algae to microbial decomposition. Although considerable work has been done to determine which components of the cell walls of algae is resistant to microbial decomposition, (Ballesta and Alexander, 1971; Bloomfield and Alexander, 1967), Gunnison and Alexander (1975) have demonstrated ggqyitgg that algae were species specific to microbial destruction. They observed that Staurastrum sp., Fisherella musciola, and Pediastrum duplex were particularly resistant to attack under conditions where other algae were readily destroyed and their contents liberated. Several studies have been done to investigate environ- mental factors and their effects upon algae and aquatic plant growth. Hartel (1975) studied the environmental con- trol of algal standing crops in two nonstratified prairie lakes in South Dakota and Minnesota for 3 years. In both lakes physical factors (light, temperature, wind stress, and rainfall) were more frequently correlated with changes in algal standing cr0ps than were nitrogen and phosphorus. Both lakes showed occasional positive correlations with nitrogen. Phosphorus was positively correlated during only one season in the deeper of the two lakes and never in the shallower. This limiting factor concept has been useful in the understandingof‘lake algae dynamics because it frequently indicated causes for changes in population 14 density (Hutchinson 1944, Lund gt g;., 1963, Megard 1972). Population changes in nature can rarely be explained on the basis of only one factor (Hall, 1971). Therefore, several different environmental factors such as wind, temper- ature, and mineral content of the water play a role in algae population of the lakes. MATERIALS AND METHODS Two different experiments were performed: (1) a com- plete chemical analyses of the two aquatic plants, Cladophora algae and Elodea canandensis, which were used in the study, and (2) a digestion trial to determine ration digestibility, nitrogen retention, plus rumen fluid pH, rumen ammonia, and blood urea nitrogen values. EXPERIMENT I - Chemical Compgsition A. Harvest of Aquatic Plants and Alfalfa In the summer of 1976,454 kg of two aquatic plants, Cladophora algae and Elodea canandensis were harvested mech- anically from three of four Michigan State University sewage treatment lakes (ponds # 1,2, and 3). These samples were washed with water to remove sand, snails, and other extraneous debris that had adhered to the algae and elodea from the lakes. A wringer washing machine was used to wash the plants and remove excess water. Both species of plants were then sun—dried or air dried over screens. Some of the plant material was forced air dried, with no heat ap- plied and stored in sealed plastic containers for future use. Alfalfa meal was harvested and pelleted in June, 1976, on the Harold Lietzke farm in St. Johns, Michigan. 15 16 B. Collection of Feed Samples For Analyses All samples of the aquatic plants and alfalfa were randomly collected at several areas from each storage con- tainer and a composite was made for each plant. Samples were ground through a 20 mesh screen using a Wiley Mill1 prior to all chemical analyses with the exception of obtain- ing percent dry matter values. C. Dry Matter Percent All plant samples were analyzed for percent dry matter by recording initial wet weight and then drying the samples in an oven at 65° C for 24 hours or longer. After complete drying, weights were recorded as percent of wet sample. Dry matter values for the aquatic plants were taken on the pelleted rations and would be greater than if taken directly from the lakes. D. Crude Protein and N Levels All plant samples were analyzed for N content using a semi-micro Kjeldahl digestion method with a Sargent Specto- Electro titrator for NH3 titration. A 10% copper sulfate solution was used as a catalyst to assist in breaking down the organic matter. Potassium sulfate was added to raise the boiling point of the digestion process. The carbon and 1Thomas — Wiley Mill, Arthur Thomas 00., Philadelphia, Pa. 17 hydrogen of the organic matter were oxidized to carbon dioxide and water while the nitrogen was converted to am- monium sulfate. The procedure used was Official Methods of Analysis of the Association of Official Agricultural Chemists (1970). E. Gross Energy of Feed Samples Gross energy values for each ration were obtained by utilizing the Parr1 Adiabatic Oxygen Bomb Calorimeter. A previously weighed sample of each ration was placed into a combustion capsule. The capsule was placed in an oxygen bomb containing 25 to 30 atmOSpheres of oxygen. The oxygen bomb was covered with 2000 g of water in an adiabatic calorimeter. After the bomb and calorimeter had been ad- justed to the same temperature, the sample was ignited with a fuse wire. The temperature rise was measured under adi- abatic conditions. By multiplying the hydrothermal equiv- alent of the calorimeter times the temperature rise minus some small corrections for the fuse wire oxidation and acid production, the caloric content of the sample was calculated. F. Ash Values of Feed Samples Ash percentage was determined by igniting pre—weighed plant samples at 6000 C in a muffle furnace to burn off all of the organic material. The inorganic material which does 1Parr Instrument Co., Moline, Illinois 18 not volatize at this temperature is regarded as ash. Cal- culations were made on a dry matter basis with the weight of the residual ash expressed as a % of the original dried sample. G. Ether Extract Determination of Feed Samples Ether extract values were evaluated based on the prin- ciple that ether is continuously volatized, then condensed and allowed to reflux through the feed sample, extracting ether soluble materials. The extract was then collected in a beaker. When the process was completed, the ether was evaporated under a hood and collected in another container and the remaining ether extracted residue was dried and weighed. The final calculations were made on a dry matter basis with the weight of the ether extract expressed as a % of the dried original sample. H. Fiber Analysis Values of Feed Samples Neutral Detergent Fiber- this procedure attempts to divide the dry matter of feeds very near the point which separates the nutritively available and soluble constituents from those which are incompletely available or dependent on a microbial fermentation. The specific procedure used was described by Van Soest and Wine (1967). A previously weighed sample was placed in a Berzelius beaker for refluxing. The following reagents were added in order: neutral detergent solution, decalin, 19 and sodium sulfite. The mixture was heated to boiling for 5 to 10 minutes and then reduced and refluxed for 60 ninutes. Previously tared crucibles were placed on a filtering apparatus. Beakers were swirled and contents were poured into each crucible and a vacuum was applied. The remaining mat was washed twice with acetone, and dried at 1050 C overnight and weighed. Calculations were made on the dry matter basis with the weight of the dried NDF fraction eXpressed as a % of the original dry sample weight. Acid Detergent Fiber — this fraction suposedly repre— sents ligno—cellulose in feedstuffs. The residue also in- cluded silica, however. The difference between the cell walls and acid detergent fiber is an estimate of hemicellu- lose, although this difference does include some protein attached to cell walls. The acid-detergent fiber is used as a preparatory step for lignin determination. The procedure used was that of Van Soest (1963). A previously weighed sample was placed into a Berzelius beaker for refluxing. Reagents of acid-detergent solution and decalin were added and the mixture was heated to boiling for 5 minutes. The heat was then turned down and the material was refluxed for exactly 60 minutes. The volume was then filtered on a previously tared crucible to which a vacuum had been applied. The remaining mat was washed twice with acetone and then dried at 1050 C overnight and weighed. The calculations were made on a dry matter basis 20 with the weight of the dried ADF fraction expressed as a % of the original dried sample. Permanganate Lignin - this procedure of fiber deter- mination utilized the acid detergent fiber procedure as a preparatory step. The detergent removed the protein and other acid-soluble material which would interfere with the lignin determination. The principle of the procedure is that the acid detergent fiber residue is primarily ligno- cellulose of which the cellulose is dissolved by the per- manganate solution. The remaining residue consists of lignin and acid-insoluble ash; however, with samples contain- ing large amounts of cutin this also is measured as part of the lignin. This is an indirect method for lignin, utilizing permanganate, and allows the determination of cellulose and insoluble ash in the same sample. The insoluble ash is an estimate of silica content, which in many grasses is a factor in reducing digestibility. The crucibles from the acid detergent fiber procedure were placed in a glass tray with one end of the tray 2—3 cm higher so the acid could drain away. To each crucible, 30 to 40 ml of the permanganate solution was added. The mats of material were broken up with a stirring rod to allow better sample contact with the solution. The samples were left in contact with the solution 90 minutes. New solution was continually added at all times during the digestion process. At the end of digestion time, 21 the permanganate solution was promptly suctioned off. Ap- proximately 20 ml of demineralizing solution was then added and allowed to stand until the solution color changed. At the end of this time, this solution was filtered off and the digestion was considered complete by the completely white color indicated. The calculations were made on a dry matter basis with the weight of the dried lignin fraction expressed as a % of the dried ADF fraction. I. Mineral Analysis of Alfalfa and Aquatic Plants Determination of Ca, Mg, Mg, Fe, Cu, B, Zn! Al - these elements were evaluated using atomic absorption spectrometry. Atomic absorption is aneunlytical method based on the ab- sorption of ultraviolet or visible light by atoms in the vapor state. When a sample solution is aspirated into the flame, the solvent is evaporated or burned, and the sample compounds are thermally decomposed and converted into a gas of the individual atoms that are present. The large major- ity of these are in the ground state although a few of the atoms become excited and emit light. The neutral atoms absorb light from the hollow—cathode source that emits the characteristic wavelength of the single element to be deter- mined. The analysis was performed on an IL 252/IL 353 Atomic Absorption/Emission Spectrophotometer1 and values were re- ported on a dry matter basis. The procedure used was found 1Instrumentation Laboratory Inc., Lexington, Mass. 22 in (A.D.A.C., 1970). Determination of Phosphorus - this procedure was based on the principle that the orthophosphate ion reacts with ammoniumnnlybdate to form a phosphomolybdate compound. The phosphomolybdate compound is reduced to molybdenum blue with 1 - amino - 2 napthol - 4 - sulfonic acid. The blue color formed is in direct proportion to the orthophosphate present. Calculations for % phosphorus are shown in the equation below: mg phosphorus in aliquot x 10 mg aliquot ash solution x wt ashed sample The procedure used was by (Fiske and Subbarow, 1925). Determination of Na - the evaluation of Na was deter- mined using flame emission spectrometry. Flame emission spectrometry will produce characteristic emission spectra for the various metallic elements. Measurement of a selected spectral line by means of a spectrometer provides the basis for a very useful quantitative analytical method especially for Na. The analysis was performed on an IL 252/ IL 353 Atomic Absorption/Emission Spectrophotometer1 and values were reported on a dry matter basis. The procedures used were (A.D.A.C., 1970) and Instrumentation Laboratory Manual, 1975. 1Instrumentation Laboratory Inc., Lexington, Mass. 23 EXPERIMENT II - Digestion Trial A. Design of Study A 4 x 4 Latin square design was employed to compare the digestion coefficients for dry matter, crude protein, digestible energy, and acid detergent fiber. Other parameters measured included rumen fluid pH, blood urea nitrogen, and rumen ammonia values. The four treatment diets included 100% alfalfa, 70% alfalfa - 30% algae, 70% alfalfa -30% elodea, and 95% alfalfa - 5% soybean meal. The experimental design and rations are shown in Table 1. B. Equipment Used Metabolism Cages - sheep digestion cages were used which permitted the feeding of a known amount of feed and water and the quantitative collection of urine. Urine Containers - plastic containers were used to collect the daily urine volumes under the metabolism cages. 5 liter plastic bottles were used to store the urine during the collection period. Feces Collections - feces were collected in collection bag harnesses and emptied both morning and night and wet weights were taken. Covered plastic buckets were used to store the feces during the collection period. Scales - a portable Toledo scales was used to weigh the feed, feces, and urine. Preparation of Feed - previously weighed mixtures of the various rations were delivered to the Harold Lietzke pelleting mill at St. Johns, Michigan 24 TABLE 1 EXPERIMENTAL DESIGN FOR RATIONS AND METABOLIC TRIAL Feeding period 1 2 3 4 Lamb Number 1 A1 B c D 2 D A B C 3 C D A B 4 B C D A — 100% alfalfa 70% alfalfa - 30% algae 70% alfalfa — 30% elodea 95% alfalfa - 5 % soybean meal 1Ration Code: UOUZJCD 25 for processing. After pelleting the rations, the pellets were stored in dry plastic containers and sealed to avoid moisture and other contamination. C. Feedipg Program A daily aliquot (1.3 kg) of the pelleted rations was weighed out the afternoon before it was to be fed. This made for quicker feeding during the morning and an accurate method for measuring any uneaten feed that remained. All animals were fed at the same time each day (between 7:00 and 8:00 a.m. and 5:00 and 6:00 p.m.). Fresh water was given to the lambs both in the morning and night during the collection period. Trace mineralized salt was provided free choice to all the lambs during the entire study. Four Suffolk wethers weighing 27.2 kg were housed at the MSU Beef Cattle Research Center during the entire experiment. The metabolic study began in November and was terminated in February. Environmental conditions were uniform through- out the entire study. D. Preliminary Period The purpose of the preliminary period was to acclimate the lambs with the metabolism cages , make the necessary equipment adjustments to insure that the feces and urine were collected properly, and adjust the animal to its intake of feed in relation to the excretion of feces and urine. A preliminary period of 14 days for each lamb was used to 26 assure maximum consumption of each ration until the condi- tions of the experiment were met. E. Preparatopy Treatment All lambs were shorn, vaccinated with Type D toxoid for enterotoxemia, drenched with Loxon for external parasites, and all feet were trimmed. Rumen cannulas were inserted in each lamb 1 month in advance of the collection period. F. Collection Period The collection period for feces and urine ran for 7 consecutive days with the feed intake carefully controlled. Each afternoon before the collection was initiated, the cages and collection area was cleaned thoroughly. Each collection period began on the morning after the animal had been eating a constant amount of feed for at least 10 days. During the collection period, a random sample of the feed that was weighed out for feeding was saved for analysis. This sample was saved two days before the collection of feces and was ended two days before the collection of feces stopped. Feces and urine were removed from their containers, weighed, and stored in a freezer at 4° C. All feces defecated during the collection period were saved and stored in a freezer. Urine collection containers had 20 ml of 1 M H2804 added each day. All the urine was collected and an aliquot of the total was saved for chemical analysis. Calculations 27 used for digestion coefficients and N balance: 1.) Apparent digestion coefficients were found for the following nutrients: APPARENT DIGESTION = nutrient in feed - nutrient in feces COEFFICIENT nutrient in feed x 100 2.) N-Balance - a balance is the relation of material in the feed to the output of the same material. For most nutrition work,the feed, feces, and urine are considered. Balance = material in feed - material in feces and urine F. Rumen Fluid pH and Rumen Ammonia Rumen fluid samples were taken from each lamb at the end of each collection period and were analyzed for pH level by a Beckman Model 4500 digital pH meter. This rumen fluid was then strained through cheesecloth and random samples were analyzed for rumen ammonia values in mg %. The Orion Ammonia Ion Electrode model 95-10 was used for this analysis. G. Blood Urea Nitrogen Blood samples were collected in 10 ml heparinized vacutainers from the jugular vein of each of the lambs. The samples were then centrifuged at 3,000 rpm to separate plasma and cell contents and the plasma obtained was frozen. Urea nitrogen was determined using the Conway procedure (Conway, 1960). Conway dishes were prepared by adding 1 ml boric 28 acid solution to their inner well and 1 ml of glycerol to the depression around the outside of the plate. Exactly .5 ml of the plasma was pipetted into the one side of the outer well and then diluted with distilled water. A urease solution was added to the plate to convert the urea in the sample to NH3. After the enzyme reaction, K2003 was added to all the urease plates to release the ammonia. The plates were allowed to diffuse one hour on the rotator. They were then titrated, recorded, and calculated in mg/100ml. H. Statistical Analyses All of the data from the digestion study, N balance, and the measured rumen and blood parameters were analyzed for treatment differences by the Latin square analysis of variance method on the Hewlett Packard 9825 A. Separation of mean values was conducted using the Studentized range test found in Statistical Tables by(Rohlf and Sokal, 1974). RESULTS AND DISCUSSION EXPERIMENT I — Chemical Composition The concentrations of selected constituents in the dried aquatic plants, dehydrated alfalfa meal, and dehydrated alfalfa meal plus soybean meal are presented in Table 2. The dry matter values given for the aquatic plants were those obtained after the plants were sun sun dried. These would differ greatly from dry matter values obtained when the aquatic plants are taken directly from the water. Dry matter values of the freshly harvested plants were 10 - 15%. However, after being sun dried, the aquatic plants were both similar in dry matter content to the alfalfa and alfalfa—soybean mixture. Individual values ranged from 92.9% for the alfalfa- elodea mixture to 95.12% for the alfalfa meal. Concentrations of ash in the dry matter of aquatic plants plus alfalfa meal were considerably higher than the alfalfa or alfalfa plus soybean meal diets. Part of the reason for this large difference is explained by the fact that aquatic plants plus alfalfa meal were considerably higher than the alfalfa or alfalfa plus soybean meal diets. Part of the reason for this large difference is explained by the fact that aquatic plants included more than just plant materials. 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