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(fiesta Date LIBRARY Michigan Stat: University This is to certify that the thesis entitled RUMEN LIQUOR AS A PROTEIN SOLVENT presented by Ricardo A. Celma Alvarez has been accepted towards fulfillment of the requirements for M.S. Dairy Science degree in y 'Major professor - Dr Roy S. Emer 3 October 19, 1979 0-7 639 2 lie.“ OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: A .ueéa<=;~: Place in book return to remoVe ¢*’.~a‘+‘5.-. mud“ charge from circulation records ;adutiann1vmwmnn " if ‘2, __' - .> m «cyan» ”flit” RUMEN LIQUOR AS A PROTEIN SOLVENT By Ricardo A. Celma Alvarez A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy Science 1979 ABSTRACT RUMEN LIQUOR AS A PROTEIN SOLVENT By Ricardo A. Celma Alvarez Rumen fluid was collected from rumen-fistulated Holstein cows fed either corn silage or alfalfa hay. The rumen fluid was either autoclaved or filtered and used as solvent on protein solubility tests for corn meal, alfalfa hay and casein. Protein solubility was not different (p:>»0.05) due to either autoclaved or filtered rumen fluid. Protein solubility of corn, alfalfa and casein were 52.17 : 11.46% 43.48 1 11.46% and 79.02 i 11.46%, respectively. The difference between corn and casein was significant (p<fi.0.05). Protein solubility of all the protein sources at pH 6.0, 6.5, and 7.0 was 54.14 I 1.95%, 55.05 I 1.95%, and 47.49 i 1.93%, respectively. The difference between pH means was significant (p41 0.05). Linear effect of pH was significant (pa: 0.05), but the quadratic effect was not (p:> 0.05). Exposure to the solvent for 60 minutes or 120 minutes gave different protein solubility (pl: 0.01). I would like to dedicate this work to my wife, Cristina Pohlenz de Celma, whose love and support gave me the strength to fulfill this goal and to my mother, Mrs. Estela Alvarez de Celma, for her love and understanding. I would also like to express my sin- cere thanks to Ing. Wolfgang Pohlenz and Mrs. Gertraud Ernst for their support and confidence in me. ACKNOWLEDGEMENTS I would like to express my sincere gratitude and appreciation to Dr. Roy S. Emery, for his advice, and to Drs. J. w. Thomas and M. T. Yokoyama, who served on my committee. I would also like to thank Drs. John Gill and Clay Anderson for their advice on statistical analysis and my fellow graduate students who have helped in various ways. ii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . vii INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . 4 Protein Solubility . . . . . . . . . . . . . . 4 Factors That Affect the Degree of Protein Solubility . . . . . . . . . . . . . . . . . . 4 a. Chemical composition . . . . . . . . . . 4 b. Sample size . . . . . . . . . . . . . . 5 c. Solvents . . . . . . . . . . 7 d. Ionic strength . . . . . . . . . . . . . 12 e. Temperature . . . . . . . . . . . . . . 12 f. pH . . . . . . . . . . . . . . . . . . . 14 g. Time of extraction . . . . . . . . . . . 15 h. Stirring . . . . . . . . . . . . . . . . 16 Protein Solubility and Ammonia Concentration in The Rumen . . . . . . . . . . . . . . . . . . . . 18 Bypass Protein . . . . . . . . . . . . . . . . 23 a. OeSOphageal groove closure reflex . . . 27 b. Heat treatment of protein sources . . . 27 0. Chemical treatment . . . . . . . . . . . 28 d. Antibiotics . . . . . . . . . . . . . . 29 iii Page MATERIALS AND METHODS . . . . . . . . . . . . . . . 51 Animals and Management . . . . . . . . . . . . . 51 Rumen Fluid Processes . . . . . . . . . . . . . . 51 Solubility Tests . . . . . . . . . . . . . . . . 52 Statistics . . . . . . . . . . . . . . . . . . . 55 RESULTS . . . . . . . . . . . . . . . . . . . . . . 56 Main Effect of Cow, Forage and Period . . . . . . 56 Main Effect of Method . . . . . . . . . . . . . . 57 Main Effect of Feedstuff . . . . . . . . . . . . 57 Main Effect of pH . . . . . . . . . . . . . . . . 58 Main Effect of Time . . . . . . . . . . . . . . . 40 Method-Feedstuff Interaction . . . . . . . . . . 41 Feedstuff—pH Interaction . . . . . . . . . . . . 41 Feedstuff-Time . . . . . . . . . . . . . . . . . 42 DISCUSSION . . . . . . . . . . . . . . . . . . . . . 46 Cow's Effect . . . . . . . . . . . . . . . . . . 46 Forage Effect . . . . . . . . . . . . . . . . . . 47 Periods . . . . . . . . . . . . . . . . . . . . . 47 Methods . . . . . . . . . . . . . . . . . . . . . 48 Feedstuffs . . . . . . . . . . . . . . . . . . . 48 pH . . . . . . . . . . . . . . . . . . . . . . . 49 Time . . . . . . . . . . . . . . . . . . . . . . 50 Method-Feedstuff . . . . . . . . . . . . . . . . 5O Feedstuff-pH . . . . . . . . . . . . . . . . . . 50 Feedstuff-Time . . . . . . . . . . . . . . . . . 51 iv RECOMMENDATION . . . . . . . . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . APPENDIX A . . . . . . . . . . . . . . . . . . . . Macro-Kjedahl Procedure N- Determination . . . APPENDIX B . . . . . . . . . . . . . . . . . . . . Total Nitrogen Contained in Control (Blank) Solutions . . . . . . . . . . . . . . . . . . . APPEIqDIX C O O O O O O O O O O O O O O O O O O 0 Example of Calculations to Obtain the Amount of Soluble NitrOgen . . . . . . . . . . . . . . . APPEl~lDIXD.................... Curriculum Vitae . . . . . . . . . . . . . . . Page 52 $1383 66 67 67 68 68 Table 10 ll 12 15 LIST OF TABLES Solvent Composition . . . . . . . Effect of Solvent on Nitrogen Solubility . Effect of Ionic Strength of Mineral Sol- vents on Nitrogen Solubility . . . Sources of Variation and Degrees of Freedom. Statistical Tests Used on the Different Traits . Main Effect of Cows, Forages and Periods on Percent Protein Solubility. . . . . Effect of Either Autoclaved or Filtered Rumen Fluid as Solvent on Percent Pro- tein Solubility . . . . . . . . . Percent Protein Solubility of Corn, Alfalfa Hay and Casein . . . . . . . . . . pH Effect on Percent Soluble Protein Protein Solubility When Protein Sources were Incubated for Two Intervals . . . . Protein Solubility for 5 Protein Sources Different Rumen Fluid Prepara— Using Two tions . . PS for Corn, 0 O O O O O O O O O O O Alfalfa, and Casein at Three Different pH Levels . . . . . . . . PS for Corn, Intervals Alfalfa, and Casein at Two 0 0 O 0 O O O O 0 vi 0 O Page 10 13 54 55 56 57 58 38 4O 41 42 LIST OF FIGURES Figure Page 1 Effect of nitrogen concentration per 100 ml of solvent on protein solubility . . . . 6 2 Effect of physiological state on potential retention of nitrogen in relation to di- gestible organic matter intake . . . . . 24 5 Protein solubility of three different pH values . . . . . . . . . . . . . . . . . 59 4 Protein solubility for three different pro- tein sources at three different pH‘s . . 45 5 Protein solubility for three protein sources at two intervals . . . . . . . . . . . . 45 vii INTRODUCTION The ever growing human p0pu1ation demands the pro- duction of huge amounts of food from different sources. Milk is one of the best food sources, but its supply is becoming shorter than its demand for human consumption. While the number of dairy cows is diminishing, the produc- tion per cow has been increasing due to improved genetic potential, better management techniques and a higher rate of feeding. Protein nutrition is critical for the dairy cow. For many years, it was believed that the supply of amino- acids (A-A) from ruminal microorganisms, which are capable of synthesizing high quality microbial protein from non- protein-nitrogen (NPN) sources such as: urea, biuret, ammonia, etc. was sufficient for fulfilling the A-A re- quirement of the dairy cow. Actually the demand on the high producing dairy cow to produce at the t0p of her gene- tic potential makes it necessary to supply an extra source of dietary A-A's to satisfy her requirements for milk pro- duction, growth, and foetal development. Because milk production is the purpose of dairy cattle, the supply of nutrients to the mammary gland is Vital. It follows that the quality of the protein--that is, l the A—A supply reaching the lower gut and becoming avail- able--is important. The main sources of A-A's to the lower gut are: 1) the rumen microbial population which contributes A-A's from its own protein structure, and 2) bypass protein, i.e. that dietary true protein that will pass through the rumen without being degraded. This bypass protein will supply A-A's in addition to that provided by the microbial population. Thus, there are two main sources of A-A's for satisfying the A-A's requirements of the high producing dairy cow. One of the main factors that influences the amount of bypass protein is the physical property of the dietary protein. This factor is protein solubility (PS) which is characteristic of each protein source that gives a measure of the disappearance of nitrogen (N) from the solid phase of a feed when incubated in an inanimate aqueous solution (Bull, et al., 1977). The amount of dietary protein degraded in the rumen has been correlated with the solubility of the protein in the rumen fluid (Crawford et al., 1978; Bull et al., 1977; Craig et al., 1978; Henderickx et al., 1965). Different ig zitgg methods have been used for evaluating the amount of soluble nitrogen (SN) in different feedstuffs (Crooker et al., 1975; Lyman et al., 1955; Nohlt et al., 1975; Burroughs et al., 1950a) using a wide variety of aqueous media, such as autoclaved rumen fluid (ARF), Burroughs mineral mixture (BMM) solution (10%), modified Burroughs mineral mixture (MBMM), distilled water, NaCl solutions, etc. The objective of this work was to attempt to find a more accurate method, i.e. a method that gives a closer estimate of PS before any protein degradation occurs in the rumen. LITERATURE REVIEH Protein Solubility Factors That Affect the Degree of Protein Solubility a. Chemical composition Since the investigation done by T. B. Osborne (1924) in England with plant proteins it has been known that plant proteins are compounded principally of four main groups characterized by their solubility pr0perties. Those proteins that solubilize in water are gigg- mins. Globulins will dissolve in saline solutions but are insoluble in water. The plant protein fraction that is neither soluble in water, saline solutions, or alcohol but is soluble in very dilute acids or alkalies is the glutelin fraction. Prolamines are usually soluble in relatively concentrated (70%) alcohol (Clark, 1975; Crooker et al., 1975; Nohlt et al., 1975). These plant protein fractions form most of the protein structure of the plants; there are other fractions such as albuminoids, histones, and protamines but they are rather a small portion. Wohlt et al. (1975) demonstrated that feeds whose major protein fractions were composed of albumins and 4 globulins had a higher solubility (52% SN in unprocessed and 42% SN in processed protein sources) than those com- posed primarily of prolamins and glutelins (25% SN in un- processed and 18% SN in processed protein sources). b. Sample size Some of the earliest i3 ziggg studies of PS were done without considering total nitrogen concentration in the solvent (Lyman et al., 1955; Smith et al., 1959). Saturation of a solution has been shown to be an important factor for measuring PS as demonstrated by Nohlt et a1. (1975), who in order to determine the effect of protein concentration on solubility placed 25, 50, 100, 250 and 500 mg. of N of either casein or soy protein per 100 ml. of Burroughs mineral mixture (BMM) at pH 6.5 at 40° C for 60 minutes. The solutions were agitated by magnetic stir- rers in a dry bacteriological incubator at minimum rate to insure the movement of a stirring bar. Large amounts of nitrogen reduced the solubility of a given protein when the saturation point was approached (see Fig. 1). In the case of casein, which is 96% soluble, a decrease of solubility was observed with concentrations above 250 mg/100 ml. When soybean meal was added at different con- centrations, SN increased linearly with the amount added and the solubility remained constant. Johlt concluded Figure 1. mg N soluble/100 m1 solvent 400 F’ 500 '- casein 200 +- -f-¥-w‘ soy lOO -' l l I 100 200 500 400 500 m1 mg N added/100 ml solvent Effect of nitrogen concentration per 100 ml of solvent on protein solubility (Nohlt et al., 1975)- 7 that since most of the protein sources are less soluble than casein, solubilities can be accurately measured at concentrations of 25 mg/100 ml of solvent. Another important factor may be sample particle size as pointed out by Lyman et al., 1955; Smith et al., 1959; Wohlt et al., 1975; and Crooker et al., 1978, be- cause of the area exposed to the action of the solvent. 0. Solvents Differences in chemical and physical properties of different solvents used in KEEEQ PS tests have resulted in distinct solubility values for a given protein source (Burroughs et al., 1950a; Little et al., 1965; dohlt et al., 1975; Crooker et al., 1975; Crawford et al., 1978; and McDougal, 1949). If we are going to simulate the PS in the rumen, we should recognize the specific physical and chemical properties of the rumen fluid, as suggested by Jancarik et a1. (1970). Different solvents have been used such as: auto- claved rumen fluid (ARF), cold or hot distilled water (dH20), 0.01 N or 0.02 N NaCl, 0.01M NaOH, 0.05M NaPOB, 0.05M NaCl, 70% ethyl alcohol, 0.8M trichloroacetic acid (TCA), 0.15M NaCl, BMM, modified Burroughs mineral mix- ture (MBMM), McDougal's artificial saliva (MCD). See Table 1. Table 1. Solvent Composition‘ Solvent Salt BMMa MCDEP . (gr per 10 1 of distilled water) Mg012°6H20 .... 1.5 MgSO4-7H20 1.15 .... Na2HPOZ+ 10.41 56.80 CaC12°2H20C 0.25 0.55 KCl 5.75 5.70 NaCl 5.75 4.70 NaHCO5 26.25 98.00 (NH4)2804 18.75 .... c d Na2SO4 FeSO,+ 0.04 .... CoC1Ao6H20 0.01 C ZnSO4°7H20 0 04 . tn304~320 0.05 Cu504'5H20 0.02 . ‘Crocker et al., 1978. aBurrough Mineral Mixture diluted to 10% with distilled water. McDougal's artificial saliva. CBubble C02 through solution after addition of CaClg°2H20 until solution clears. dReplaces (NH )SO4 on an equimolar basis for NagSOn (20.155 g%) for modified Burrough's mineral mixture (MBMM). Little et al., 1965; Crooker et al., 1978, and Henderickx and Martin, 1965, concluded that protein sources differ in soluble nitrogen; however, solubility in any one solvent was not necessarily related to the solubility in other solvents (see Table 2). Solubility of nitrogen in dH20 and ARF was lower than in 0.02N NaOH. Heat treatment of soybean oil meal reduced NS in all the solvents. Those protein sources that were readily con- verted to ammonia ig zgtgg at about the same rates were soybean oil meal, soy protein, linseed oil meal and casein. In contrast, heated soybean oil meal, corn gluten meal or zein were slowly converted to ammonia. Little et a1. (1965) found no consistent relationship between NS and level of ammonia in incubation flasks. However, nitrogen soluble in ARF and level of free ammonia at 2 hours had the highest correlation (r = 0.95). The correlation for dH20 and NaOH were r = 0.58 and r = 0.52, respectively. Crooker et a1. (1978) demonstrated that the differ- ence among the average PS of various feedstuffs with MBEM, MCD and ARF was significant (p A£.Ol)(Table 2). However, a significant difference among PS values obtained with BMM and ARF “was not observed. The difference may be due to the differences in solvent composition (see Table 1). ARF was most closely simulated by BNM in extracting N from hominy, wheat and citrus pulp. ARF and BMM extracted about equal amounts of nitrogen from buckwheat. Distiller's lO Table 2. Effect of Solvent on Nitrogen Solubility Solvent ARFa BMM5_ MBMMc MCDd 0.02N dH20e Reference Feedstuff NaOH No. -------- % soluble nitrogen ------- Soybean oil meal 19 ---- ---- ---- 81 16 62 H. soybean oil mealf lO ---- -—-- ---- 5O 11 62 Linseed oil meal 45 ---- ---— --—- 68 59 62 Corn gluten meal 15 ---- ---- ---- 52 ll 62 Purified soy protein 7 --—- ---- ---- 99 2 62 Purified casein 81 ---- ---~ ---- 98 2 62 Purified zein 5 —--- ---- ---- 99 0 62 Distillers dried grains with solubles 22.6 20.4 22.8 22.7 -- -- 54 Wheat 20.8 21.7 26.4 29.2 -- -- 54 Citrus pulp 24.2 25.0 56.9 57.6 -- -— 54 Sunflower meal 24.0 54.1 58.9 59.5 —- -- 54 Buckwheat 50.5 54.1 57.1 59.8 -- -- 54 Oats 18.5 56.8 45.8 48.9 -- -— 54 Purified casein 78.1 79.8 ---- ---- -- -- 106 Isolated soy protein 15.9 14.6 ---- ---- -- -- 106 aARF - Autoclaved rumen fluid. b CMBMM - modified BMM. d edH20 - distilled water. H. soy protein oil meal — heated on forced air oven at 110° C for 24 hours. BMM - Burrough's mineral mixture. MCD - McDougal's artificial saliva. ll dried grains with solubles had similar NS measures in all solvents. The variations among solvents in NS may be due to the various inter and intra molecular forces acting be- tween the proteins and the various ionic species contained in each solvent (Cohn, 1945). This can be observed in the values obtained between BMM and MBMM as solvents since they only differ in two ionic species NHL,+ and Na+. Among correlations between percent SN in each min- eral solvent for each feed and percent SN from ARF; solu- ble nitrogen from NaCl were correlated (r = 0.8) while BMM, MBMM, and MCD have much less correlation with ARF (r = 0.21, 0.12 and 0.06) this higher correlation between NaCl and ARF was due to the results obtained with oats. If oats were omitted from correlations then Bkh exhibited the highest correlation (r = 0.74) with NaCl, MCD and MBNM having slightly lower correlations (r = 0.71, 0.68, and 0.65)(Crooker et al., 1978). Nohlt et a1. (1975), with ig 13339 studies, showed that casein Was more soluble (p 41.01) than soy protein (79.0 vs 14.5%). They also obtained a difference (p A..05) in the percent of NS of the same nitrogen source when either ARF or BMM was used as solvent. Protein solubility in ARF was less than in mineral mixture (46.0 vs 47.2%). Other factors that have been found to produce dif- ferences on NS are: ionic strength, temperature, pH, 12 length of extraction, motion of stirring (Crooker et al., 1975; Crooker et al., 1978; Peter et al., 1971; Wohlt et al., 1975, and Burroughs et al., 1950a). d. Ionic strength Ionic strength may be defined as: dj = fiZciZE; where dj = ionic strength; 0 = Molar concentration; Z = Valance and i = ionic Species. Ionic strength is varied by appropriately changing the amount of distilled water added. Rumen fluid has an average ionic strength of 0.15 as calculated by Salobir et a1. (1970). In their work they had a range of ionic strength from 0.10 to 0.22 and identified that protein solubilities of soybean, peanut, and sunflower meal in sodium chloride solutions had similar values to those obtained with ARF at ionic strength of 0.15. However, using other solvents (BMM, 0.15M NaCl and MBMM) ionic strength within the range of 0.11 to 0.19 had no significant effect on the amount of nitrogen extracted from various protein sources as shown by Crooker et a1. (1978) (see Table 5). e. Temperature This factor should resemble that value usually ob- served in the rumen, i.e. 58° - 42° C (Burroughs et al., l3 s m s .eommmz seas mammn Hmaosflzvm so emomagmm on A mzv I magpxfla amnmmfis mnmsoaasm cmwwflvoao .quHpsHom ocfisoago ssflwom 6 .Hopm; ccaaapmfld spa; SCH on umpsaflc manpxfis Hmmmqfla mnmsommsmo .mQOflpomapxm ozp mo amps esp ma msam> momma .mofloomm oedoa n H can 00833» n N .qoapwnpqoomoo 9832 u 0 0905.1. .mHNfloNse u npwsmmpm oesow u .30 66368 Hops; doaaflpmflc mo pmsoam on» mmflmqmno haopmanmommmm an Umflsm> npmmompm oflQOHm .mnma ..Hm pm pmxooao. ¢.me 0.5m o.wm 6.5m n.5m m.mm m.mm ma.o e.me m.mm m.mm 5.5m e.mm H.6m 0.4m ma.o o.m¢ m.nm ©.mm m.mm ©.mm 6.9m m.mm HH.O 025mg m.om m.mm m.mm 0.0m o.mm 0.6m ¢.mm mH.o ¢.©H m.mm H.Hm m.mm o.wm o.mm m.am ma.o m.ma H.3m ¢.mm m.dm o.wm w.mm m.mm Ha.o eaomz 0.4m m.em o.mm m.mm s.em 6.0m o.mm ma.o m.mm m.mm H.em m.mm m.mm m.am m.wa mH.o ®.mm H.¢m m.mm m.©m m.mm m.mm m.om Ha.o 022m llllllllllllllllllll nmowompfln mandaom & unnulialsupniuuuliunull I: .mnev mpmo among Heme mass hmflsom among mmaczaom an» soupm pso>dom ixodm Hosoaqum mahpflo spas mQHmuw oHQOH cease mumaaflpmflm mumpmcmom .sefiaaesaom semeseaz so muso>aom Hmnosfla Mo mnpmsospm oHQOH Mo pomwmm .m magma 14 1950a; Nohlt et al., 1975; Lehninger, 1970; and Marvin, 1977)- f. pH pH of the rumen is usually within the range of 5.8 to 7.0 (Andrew, 1977). The pH level is influenced by the diet consumed. A decreased pH after feeding has been ob— served which is specially associated with the ingestion of appreciable amounts of rapidly fermentable sugars. A negative correlation exists between the concentration of volatile fatty acids (VFA) and lactic acid, on the one hand, and the pH of the contents on the other (as cited in Andrew, 1977). Fasting usually decreases the concen— tration of VFA and the ruminal pH increases above pH 7 to a value close to that of blood (Phillipson, 1942). Lactic acid fermentation is associated with pH values of 5.5 or less when sheep or cattle are consuming diets high in starch or sugar and low in fiber, producing acidosis that might cause death. Urea introduced in ex- cessive quantities into the rumen can produce alkalinity due to an excessive formation of ammonia, which may also be fatal (Andrew, 1977). The buffering capacity of the rumen does not depend entirely on the saliva secreted, as exchange across the rumen wall of unionized acid with bicarbonate accounts for 15 about one half the acid absorbed (Ash and Dobson, 1965). Turner and Hodgetts (1955) explained the role of salivary bicarbonate and phosphate in buffering the rumen contents. Buffering capacity of rumen contents is due principally to its bicarbonate content. Wohlt et al., 1975, demonstrated 33 33359 that with an increase of pH from 5.5 to 7 there was an increase (p .01) in average solubility (26.7% to 57.4%). The solubility of two protein sources (purified casein and isolated soy protein) was less at pH 5.5 (p AL.01) than at either pH 6.5 or pH 7.5. There were no significant dif- ferences in solubility between pH 6.5 and 7.5 regardless of solvent (ARF vs BMM) or protein source. g. Time of extraction The time that a protein is exposed to the activity of the rumen—reticulum contents depends upon the level of feed intake, physical features of the particle, and asso- ciative effects of other ration ingredients (Satter et al., 1977). The longer the residence in the reticulo-rumen the higher the amount of nitrogen solubilized and the higher the degree of degradation of dietary protein. Johlt et al., 1975, used two solvents (ARF and BMM) exposing different protein sources to their action. They measured the amount of NS at 50, 60, 90, 120 minutes. 16 Solubility at 50 minutes was less (p44-.01) than at 60 minutes in both solvents. At 60 minutes there was no dif- ference between solvents. In ARF, solubility decreased as time passed and slowly increased in BMM. h. Stirring Using different varieties of beans, Smith et a1. (1952), ground them with a hammer-mill (1 mm screen) and diluted the meal with water in a relation of 40:1 (waterzmeal) with a pH of 6.5, different methods were used for stirring the water: meal slurry as follows: 1. Mechanical shaker (Precision Scientific Company) with a reciprocating motion at room temperature (25° C). 2. Mechanical stirrer in a beaker at room tempera- ture (25° C). This system gives less agitation than method 1. 5. Same method as in No. 2, but at 50° C. 4. Very vigorous agitation with a stirrer which had pr0peller blades that were nearly equal to the diameter of the flask, and rapid rotation of the blades gave very effective shearing action, the temperature used for this method was 25° C. The amount of nitrogen extracted from method 5 and ‘t‘was almost the same, with method 5 being slightly greater. 1'7 Method 2 was always lower than methods 5 and 4 em- phasizing the importance of controlling the shearing action. Increasing the temperature from 25° C to 50° C was approximately equal to vigorous stirring, thus showing that the speed of stirring is an important factor for lg vitro PS studies. Possible explanations for this are: l. The cell structure of the beans may not have been well enough destroyed by the hammer-mill for easy liberation of the protein. A part of the protein may be attached to insolu- ble carbohydrate particles, and vigorous stirring or beating of the solvent is required to bring the protein into dispersion. The heating (of the solvent) or stirring may be supplying the energy necessary for dispersing coarse protein particles or complexes. The action of the hammer—mill may weld some other— wise soluble protein into insoluble meal parti— cles. Another factor retarding protein dispersion and common to the first three suggestions may be the formation of a hydrated shell around each parti- cle of protein which gives a case hardening ef- fect that retards penetration of water and rate 18 of dispersion. This phenomena is observed in reverse in the drying of protein. Other workers (Crooker et al., 1978; Wohlt et al., 1975; Lyman et al., 1955) have used different systems for stirring the solution to be utilized on PS studies such as: a) water bath in a shaker, b) magnetic stirrers, c) mechanical stirring, thus influencing on the difference given by the different authors for a given protein source. Protein Solubility and Ammonia Concentration in the Rumen How much dietary nitrogen is required to obtain the Optimal benefit out of the rumen microbial population and their growth? This has been a question that several work- ers have tried to answer. For instance, Satter et a1. (1975) With ig Kiggg studies showed that microbial pro— tein synthesis is highly dependent upon the amount of energy available and demonstrated that the maximum micro- bial protein production was obtained at a concentration of 5 to 5 mg NHS-N/100 ml of rumen fluid, which is approxi- mately equal to a dietary crude procein content of 12.5 to 15%. Henderson et al., 1969; Allison, 1970; Bryant et al., 1961, with ip lipgg studies found 5.0 to 6.0 m8 N 3—N/100 ml the maximum utilizable ammonia concentration in the rumen for microbial protein synthesis. Bull et a1. 19 (1975) reported a value of approximately 20 mg NHB-N/100 ml with 3g 31339 studies. Hume et a1. (1970), using the tungstic acid pre- cipitable nitrogen technique in sheep, obtained maximum nicrobial protein synthesis in the rumen with an ammonia nitrogen concentration of 15.5 mg/100 ml. No further benefit on dry matter disappearance and/or organic matter digestibility was obtained by increasing ammonia concen- tration in the rumen. Miller (1975), with ig 1139 studies showed that the maximum microbial protein synthesis per unit of substrate fermented was obtained with concentra- tion of approximately 15.0 mg NHB-N/lOO m1 of rumen fluid. Other workers (Mehrez et al., 1977), with mature rumen fistulated sheep using the polyester bag technique, obtained a value of 25.5 mg/100 ml of rumen fluid as the minimum NHB-N concentration for maximum rate of fermenta- tion. IQ zizg studies with soybean meal vs starea (Edwards et al., 1979) demonstrated that the maximum microbial pro- tein was PrOduced when ammonia concentration in the rumen was 76 mg/100 ml of rumen fluid, this value was obtained with starea as the dietary nitrogen supplement. Optimal ammonia concentration in the rumen may be defined as that which results either in the maximum pro- duction of microbial protein or in the maximum rate of fer- mentation. Orskov et a1. (1974) showed with barley fed mature wethers that the microbial protein produced per 20 unit of substrate fermented was not altered as a result of urea supplementation while the extent of rumen fermenta- tion and digestibility was increased. Production of NH —N depends on the degree of proteol- 5 ysis in the rumen, which is influenced by proteolytic rate, rumen turnover, and PS (Isaacs et al., 1972). High corre- lations were noted between PS and ammonia concentration in the rumen, i.e. as protein solubility increases the amount of free NHB-N increases (Lewis, 1957; Nohlt et al., 1976; Sniffen, 1974; Annison et al., 1954; Henderickx et al., 1965; Hudson et al., 1969; Little et al., 1965; Peter et al., 1971; Sherrod et al., 1962; Chalmers et al., 1954; El-Shasley, l952a,b and Mangan, 1972). Hume (1970b) demonstrated that readily degraded protein is superior to non-proteic nitrogen (NPN) in supporting formation of microbial protein suggesting that ruminal microorganisms also require a supply of dietary polypeptides and/or A-A's for their growth. Little et al., 1965; Belasco, 1955; Burroughs et al., 1950b, showed that some readily available nitrogen is beneficial to rumen function while small amounts of solubilized feed protein may leave the rumen without being degraded, most of the soluble protein will be broken down. The net result of dietary protein degradation is the effect of: a) Initial period, where the highly SP is degraded and b) Slower breakdown of the less SP which is extended beyond the initial period 21 (Crawford et al., 1978). This was confirmed by Bull et al., 1977. They pointed out that ruminal degradation of a protein is necessarily dependent on the ability of the protein to "solubilize" in the rumen medium, solubility per se does not insure degradation and the rate of the two processes are not necessarily equal. The NEE-N that is not incorporated into microbial protein will be absorbed through the rumen wall and either carried out by the venous blood and excreted as urinary ni- trogen (Lewis et al., 1957; McDonald, 1948; McDonald, 1952) or recycled as urea via the saliva (Marvin, 1977) or pass to the lower gut where it could be utilized by the intes- tine microflora (Bergen, 1978). Fate of dietary nitrogen has been studied in vivg using nitrogen isotOpes in mature sheep (Mathison et al., 1971; Nolan, 1975; and Pilgram et al., 1970) and in mature bovines (Al-Rabbat et al., 1971a, 1971b) showing that NH -N is indeed the central intermediate in the degrada- 5 tion and assimilation of nitrogen in the rumen and that NHa-N is in the preferred or required nitrogenous nutrient of many species of rumen bacteria (Bryant, 1970). Some rumen microorganisms use peptides and some A-A's directly. Other products of the fermentation of dietary protein are VFA's (El-Shasley, 1952a, 1952b; Sherrod et al., 1964). In order to maintain Optimal conditions of the ru- minal population for digestibility of dry matter (3M), 22 organic matter (0M), crude protein (CP), nitrogen free extract (NFE) and total digestible nutrients (TEN) a mini- mum ammonia concentration of 5 mg/100 ml is required as shown by Wohlt et al., 1978. Satter et a1. (1975) also recommend 12.5% CP in the diet for maintaining the maximum growth potential of the rumen microbiota. If dietary 0P does not satisfy the minimum requirements for the rumen microflora then fermentation will be limiting and the rate of passage of feed to the lower gut will decrease (Campling et al., 1962). Degradation of protein in the rumen increases with PS and this results in higher losses of feed nitrogen as NHB-N. The effect of this is possible decrease of feed protein reaching the lower gut (Nohlt et al., 1976; Isaacs et al., 1972). Studies of the effects of PS on nitrogen metabolism in ruminants have shown that as PS increases the level of plasma urea nitrogen increases and the excretion via urinary nitrogen increases (Nohlt et al., 1976). Plasma urea nitrogen concentration and the amount of urinary nitrOgen had a linear correlation (r = 0.97) (Thornton et al., 1972). Increasing PS also increased the water intake (may be due to the higher concentration of urinary nitrogen), feed intake, nitrogen intake, nitrogen absorption through the wall of the rumen and gastro- intestinal tract, fecal nitrOgen excreta, and decreased gross energy digestibility of the feeds (flohlt et al., 1976; and Blaxter et al., 1962). 25 Bypass Protein Orskov (1970) demonstrated that microbial protein synthesis was able to support maintenance, slow growth and early pregnancy, but not fast growth, late pregnancy or early lactation (see Fig. 2). In order to satisfy the protein requirements of animals whose production level can- not be sustained by the out-put of the rumen microbiota, an extra source of dietary protein that will pass through the reticulo-rumen without being degraded but will be ab- sorbed as A-A's for their absorption in the lower gut is required. This extra dietary protein source has been termed "bypass protein." It has been calculated that the normal range of bypass protein is between 20 to 60% (aver- age 40%) of the dietary protein (Chalupa, 1975; Hogan, 1975). Rumen microbial protein tends to remain rather con- stant in patterns of essential amino-acids regardless of dietary source of nitrogen (true protein or NPN) (Hatfield, 1977; Purser and Buechler, 1966) i.e., changes on micro- bial protein are quantitative but not qualitative. So, if any change in protein quality that reaches the lower gut is to be attained, it should be through the bypass protein. Bypass protein is influenced by solubility and degradation characteristics of dietary protein (Amos et al., 1971; Little et al., 1967; McGregor et al., 1978). 24 g N retained/100 g DOM 0.0 ; [BarlylLate[Maintenance[EarlylLatelEarly Peak Late I Growth I [Pregnancy I Lactation Figure 2. Effect of physiological state on potential re- tention of nitrogen in relation to digestible organic matter intake (Orskov, 1970). 25 Other factors that influence the amount of bypass protein besides resistance to ruminal degradation are the rate at which the rumen contents degrade protein and the flow rates of liquid and solid phases through the rumen because degradable fractions disappear from the rumen by degradation or passage whereas undegradable fractions dis- appear only by passage (Crawford et al., 1978; flaldo et al., 1972). Thus the amount of dietary protein bypassing the rumen can be depicted by the ratio Kr/(Kr+Kp) where Kr and Kp are rate constants for turnover of ruminal con- tents and ruminal proteolysis, respectively (Broderick, 1978). Using ig zitgg methods, McGregor et a1. (1978) showed that the A-A profile of the undegraded protein which by- passes the rumen may be different from the A-A profile of the dietary protein as originally ingested. In most of the feedstuffs studied there were marked differences be- tween the A-A profile of the total protein and the A-A profile of the insoluble protein fraction. Some A-A's such as valine, leucine and iso—leucine are rather located in the soluble fraction of the feed protein. Perhaps those feedstuffs with more soluble protein may be able to support a higher rate of cellulose digestion than feedstuffs with less of these A-A's in the soluble protein fraction (McGregor et al., 1978). Other A-A's that have been identified as required for certain rumen microorganisms 26 are methionine and cystein (Allison, 1970; Bryant, 1970; Buttery, 1976). Thus, A-A requirements for ruminants is equal to the A-A requirements at tissue level plus that of the microbial population in the rumen (McGregor et al., 1978). The metabolizable protein (MP) concept was developai to recognize different degradability of protein sources as well as synthesis of microbial protein in rumen fermenta- tion. These factors were used to predict the amount of amino-acids which can be absorbed postruminally and used to meet the protein requirements of the individual (Bur- roughs et al., 1975). It has been shown through numerous studies that changes in MP are achieved by postruminal in- fusion of protein or A-A's that have increased animal per- formance, e.g.: Clark (1975) supplied additional high quality pro- tein postruminally increasing both milk production by 1 to 4 kg/day/cow and milk protein by 10 to l %. Chalupa (1975) showed that nitrogen retention usual- ly was increased when a mixture of methionine, lysine and threonine was supplied postruminally to growing cattle, this was duplicated by the same worker in 1976. Different systems have been proven to increase the amount of available A-A's, such as: 27 a. OeSOphageal groove closure reflex (Orskov et al., 1969a; Orskov et al., 1969b; Orskov, 1972; Standaert, 1979). OeSOphageal groove closure is a normal function in young ruminants, passing suckled liquid from the esophagus through the reticular groove and omasal canal into the abomasum. Factors that are believed to influence this re- flex are age, posture of the animal, temperature of the liquid, chemical composition of the suckled liquid and salts contained in the solution (sodium salts; c0pper salts; silver and zinc salts). In mature ruminants the oesophageal groove is not closed very easily. According to Jester (1926) the oesophageal groove mechanism regresses with age due to a failure of the groove to develop prcportionately with the rumen and reticulum. Also, its vagal innervation regressed with age. 0es0phagea1 groove closure requires more investigation at this moment. b. Heat treatment of protein sources A reduction of protein degradation has been observed when heat has been applied to the feed before consumption by ruminants. This reduction is thought to be due to a decrease in solubility of the protein (Danke et al., 1966; Gree et al., 1954; Hudson et al., 1969; Little et al., 1963). Heating could be disadvantageous. Overheating decreases the availability of A-A's in the lower gastro-intestinal 28 tract (Goering et al., 1972; Goering and Waldo, 1974; Goering et al., 1975; Hill and Noller, 1965). The maillard reaction between free amino groups of protein and sugar aldehyde groups is responsible for the decrease in digesti- bility (Goering and Waldo, 1974; waldo et al., 1975a and 1975b). Heat damage can occur without oxidizing fat or sugar (Bjarnason and Carpenter, 1969, and 1970). Nitrogen retention and animal performance have been increased when the protein source has been exposed to heat and pressure in such a way that protein is not degraded in reticulo-rumen but the A—A's of that protein remain avail- able for post-ruminal digestion (Goering et al., 1974; Hudson et al., 1969; Sherrod and Tillman, 1964). c. Chemical treatment Chemicals which form reversible cross-linkages be— tween the amino and the amide groups make the A—A less degradable in the reticulo-rumen. Later on in the abomasum with the HCl produced in this organ, these linkages are broken down and the A-A's are available for proteolysis and intestinal absorption. Two main chemical compounds have been tested, formaldehyde and tannins. Formaldehyde has been used extensively in practical feeding (Walker, 1974; Fraenkel-Conrat et al., 1946, 1948). Formaldehyde treat- ment of plant protein has resulted in better feed efficiency (Chalupa, 1975). Nitrogen retention has increased as a 29 result of formaldehyde treatment of casein. flool produc- tion and muscle growth have also been increased (Barry, 1972, 1975; Faichney, 1971; Hemsley et al., 1975; Reis et al., 1969; and Wright, 1971). Animal performance was also enhanced when they received forages that were treated with formaldehyde at ensiling time (Waldo et al., 1975a, 1975b; Brown and Valentine, 1972). Tannins are found in forages and seeds, they may be responsible for some of the natural protection observed in these proteins (McLeod, 1974). Chemically, tannins have been classified as either hydrolysable or condensed. Stu- dies done by Zelter et a1. (1970) indicated that complexes formed by condensed tannins may not be hydrolysed to re- lease amino acids in the abomasum. Hydrolysable tannins have the prOpriety to form cross-linkages with proteins through hydrogen bonding (Ferguson, 1975). Saba, Hale and Theurer (1972) showed that sorghum varieties with high tannin content are less degradable in the rumen than sor- ghum varieties with low tannin content. However, other investigators (Manson et al., 1975) demonstrated that high tannin sorghum has a lower net energy and apparent protein digestibility than normal sorghum. d. Antibiotics Antibiotics have been studied as means for protein protection against rumen microbial degradation, the results 30 have not been encouraging (Hogan and Weston, 1969; Schelling et al., 1972). MATERIALS AND METHODS Animals and Management Two mature Holstein cows fitted with rumen fistula were kept in an individual stall and fed at 8 a.m. every day either alfalfa hay or corn silage gd libitum. A l5-day period was allowed for adaptation to the diets. After the adaptation period, rumen fluid was collected and utilized as explained below. When collection of rumen fluid of the first period was over, the cows' diets were reversed and again a l5-day period for adaptation was permitted before starting the second collection period of rumen fluid. Rumen Fluid Processes Two l.of rumen fluid from each cow were collected 2 hours after feeding, strained through 4 layers of cheese cloth and poured into one 1. flasks. The samples were transported to the laboratory where they were centrifuged: first at 1,500 A g for 10 minutes followed by a second centrifugation at 15,000 X g for 20 minutes. After the centrifugation process was done, the rumen fluid supernatants were either autoclaved at 121° C, with 31 32 15 lbs/in2 pressure for 45 minutes or filtered using an all glass millipore filter apparatus #4 (47mm); first with a pore size of 0.8 m and a second filtration through a filter with pore size of 0.45 m. Rumen liquor of both treatments (autoclaved or filtered) was stored overnight in a cooler room at 4° C. Solubility Tests The percent soluble nitrogen of three different pro- tein sources (alfalfa hay, cornmeal and casein) was deter- mined in autoclaved rumen fluid (ARF) and/or filtered rumen fluid (FRF). Rumen fluid was processed as described above. The nitrogen sources were ground with a Wiley mill to pass through a 1 mm mesh screen, allowed to air equili- brate overnight, and analyzed for total nitrogen, using the Macro-Kjedahl method (Appendix A), and expressed on a dry matter basis. The solvents were allowed to warm up until they reached room temperature (25° C) and then preheated in a water bath set at 40° C. Solvents and nitrogen sources were mixed at a con- centration of 20 mg N/80 m1 of rumen fluid in 250 ml flasks. The pH was adjusted with ortho (85%) phosphoric acid or 2N sodium hydroxide to three different pH's: 6.0, 6.5 and 7.0. 53 The mixtures were placed in a Dubnoff shaking water bath at 40° C at a shaking rate of 50 strokes per minute. Fifteen ml samples were withdrawn after 60 minutes and 120 minutes and then centrifuged at 1500 X g for 10 minutes. After centrifugation, a 4 m1 aliquots of the supernatant was used for soluble nitrogen determination using the macro-Kjeldahl method (Appendix A). The final value of SN was the average of two determinations, after subtracting blanks (Appendix B). An example of the calcu- lations performed is given in Appendix C. Statistics The experimental design was a quadruple split-plot with repeated measurement with three factors in space and one factor on time with 2 x 2 change over in whole plots. Main effects and their interactions were tested by analysis of variance (ANOVA) (Gill, 1979) and differences between means with more than one degree of freedom by the Fisher's variance ratio or F-distribution (see Table 4). The dif- ference between two means was tested using various appro- priate statistical tests (see Table 5). 54 Table 4. Sources of Variation and Degrees of Freedom Sources of Variation Q: H 9) Cow (0) Period (P) Forage (Fo) Error A Method (M) Fo x M CM + PM = Error B Feeds (F) Fo x F M x F CF + PF + CMF + PMF + FoMF pH (H) Fo x H M x H F x H CH + PH + FoMH + CMH + PMH = Error D 58 Time (T) Fo x T M x T F x T H x T CT + PT + FoMT + GMT + PMT + FoFT + CFT + PFT + MFT + FoMFT + CMFT + PMFT + FoHT + CHT + PHT + MHT + FOMHT + CMHT + PMHT + FHT + FOFHT + CFHT + PFHT + MFHT + FOMFHT + CMFHT + PMFHT = Error NNNNHHOHi—‘H H 0 Error 0 NR) #m ru n)+e FJre 65 ts ad.f. = degrees of freedom. 55 Table 5. Statistical Tests Used on the Different Traits Trait Test Used Method (M) Dunnet's Feedstuffs (F) Bonferroni's pH's polynomial orthogonals Time (T) Dunnet's M x F Bonferroni's F x pH Tukey's F x T Tukey's RESULTS Rumen fluid from cow A seemed to yield more soluble protein (SP) on the mean of the three protein sources ex- amined than that from cow B (55.9% vs 47.21% soluble pro- tein). There was also a higher protein solubility with rumen fluid from cows fed alfalfa hay (58.76% SP) compared to corn silage (44.56% SP). The mean values for soluble protein in period I was 55.1% and for period II it was 50.01% (see Table 6). These period differences could not be evaluated statistically. Table 6. Main Effect of Cows, Forages and Periods on Per- cent Protein Solubility Cow A B Compo site Forage/period I II I II Mean ............... % psa -_---------_--___---_--- Alfalfa hay 64.65b ---------- 52.86 58.76 Corn silage ----- 47.15 41.56 ----- 44.56 Mean, cow 55.9 47.21 Mean, period I 55.1 Mean, period II 50.01 a% PS: Percent protein solubility. bMean. 56 57 Autoclaved rumen fluid (ARF) was similar to fil- tered rumen fluid (FRF) in percent protein solubility when used as a solvent (p.> .05), see Table 7. Table 7. Effect of Either Autoclaved or Filtered Rumen Fluid As Solvent 0n Percent Protein Solubility Method Autoclaved Filtered -------------- % Pea --------—---—- g 51.89 I 9.06bI 51.22 i 9.06bI ‘ a% PS: percent protein solu- u bility. No statistical difference (p :>.05). I + g Mean - DE. Protein solubility of corn, alfalfa hay and casein were 52.17 i 11.46, 45.48 I 11.46 and 79.02 t 11.46, re- spectively. The difference between corn and casein was significant (1);: .05), see Table 8. The large size of the SE's of the main effect on feedstuff sources (Table 8) may be due to the degreesof freedom used to divide the sum of squares (SS) of the main effect of feedstuffs plus the SS of the interactions considered within this block. 58 Table 8. Percent Protein Solubility of Corn, Alfalfa Hay And Casein _, Feedstuff Corn Alfalfa Hay Casein .................. % psa --------___-----_- 52.17 i 11.46 bl 45.48 I 11.46b’c 79.02 i 11.46° a% PS: percent protein solubility. b’CDifferent subscript shows significant difference (p 41.05). IMean : SE. pH level affected protein solubility, pH at 6.0, 6.5 and 7.0 yielded 54.14 I 1.95%, 55.05 i 1.93%, and 47.49 i 1.95%, respectively. There was a significant dif- ference (p»zfi.05) between the mean values. Increasing the pH decreases (p.4..05) the percent of soluble protein (see Table 9 and Figure 5). Table 9. pH Effect on Percent Soluble Protein ApH 6.0 6.5 7.0 .................. % p33 ---_-----___-_---- 54.14 t 1.93bI 55.05 i 1.95° 47.49 I 1.95d ___ a% PS: percent protein solubility. b’C’dDifferent subscripts p‘4-.05; Linear response p 41.05; Quadratic response p.>..05. IMean i SE. Percent protein solubility Figure 5. 70 60 50 40 30 20 10 39 l l I 6.0 6.5 7.0 pH level Protein solubility of three different pH values. 40 Time of exposure to the action of the solvent gave 50.19 t 0.7% and 52.92 i 0.7% protein solubility at 60 minutes and 120 minutes, respectively, which was signifi- cantly different (p 41.01). See Table 10. Table 10. Protein Solubility When Protein Sources were Incubated for Two Intervals Time 60 minutes 120 minutes ------ % PSa ------ 50.19 i 0.7b 52.92 i 0.7C a% PS: percent protein solu- bility. b,c Means with different sub— script are different (p 41.01). PS of corn, alfalfa hay and casein dissolved in either autoclaved or filtered rumen fluid was not differ- ent to those results obtained in feedstuff alone, i.e., corn was different to casein (p.4..05) but corn was simi- lar to alfalfa (pf) .05) and alfalfa was similar to casein (p.>>.05), see Table 11. 41 Table 11. Protein Solubility for 5 Protein Sources Using Two Different Rumen Fluid Preparations Method Feedstuff Autoclaved Filtered Mean ------- % PSfi—-----—- Corn 55.54 i 16.21bI 50.8 i 16.21b 52.17 i 11.46b Alfalfa Hay 40.10 i 16.21b’C 46.85 i 16.2lb’c 45.48 i 11.46”:C I+ 16.21C 76.01 i 16.21c 79.02 i 11.46C 9.06d 51.22 d Casein 82.05 l+ l+ Mean 51.89 9.06 a% PS: percent protein solubility. b’C’dDifferent subscript show significant difference (p.4..05). I, + mg lean - 0.1). The interaction between feedstuff and pH gave the following results with respect to PS (see Table 12): a. The PS for corn meal, alfalfa hay or casein were not significantly different (p :>.05) when each one was at either pH 6.0, 6.5, or 7.0. b. There was no difference (p 2>.05) between corn and alfalfa hay when compared at pH 6.0, 6.5, or 7.0. c. Alfalfa hay and casein were different (p.4l.05) at each pH level (pH 6.0, 6.5, or 7.0). d. Difference between corn and casein was signifi— cant (p‘éL.05) when compared at the same pH (6.0, 6.5, or 7.0) (see Figure 4). 42 Table 12. PS for Corn, Alfalfa, and Casein at Three Dif- ferent pH Levels 2- _ I :- _: I j: M ,pH Feedstuff 6.0 6.5 7.0 ....... % psa -----_- Corn 57.75 i 5.54b 50.9 i 5.54b 27.89 i 5.54b Alfalfa 50.86 i 5.54b 42.11 i 5.54b 57.46 i 5.54b Casein 75.85 i 5.54° 86.15 i 5.54C 77.11 i 5.54C 3% PS: percent protein solubility. b’CDifferent subscripts show p.4L.05. Feedstuff-time interaction showed that the percent protein solubility of corn and alfalfa hay at either 60 minutes or 120 minutes of exposure to the action of the solvent was not different (p 2>a05) for each feedstuff. However, protein solubility of casein at 60 minutes and 120 minutes was different (p44;.005), see Table 15 and Figure 5. 43 lOOI- 9O _ 80 _. 70 #- Casein 60 —. SO ,a—1I- Alfalfa O—H Corn 40 u. Percent Soluble Protein 20 _. lO — I I I 6.0 6.5 7.0 pH Figure 4. Protein solubility for three different protein sources at three different pH's. Casein: y = 1755.01 + 557.08x — 42.6x2 Corn: y = 418.51 - 109.59x + 7.66x2 Alfalfa: y s 475.86 - 120.08x + 8.203x2 x = pH 44 Table 15. PS for Corn, Alfalfa, and Casein at Two Intervals Time Feedstuff 60 Minutes 120 Minutes ------ % PSa -----— Corn 51.55 i 1.22b 52.99 i 1.22b Alfalfa 45.20 i 1.22C 45.75 t 1.22C Casein 76.02 t 1.22d 82.05 i 1.22e ag/ m PS: percent protein solubility. b’CMean with different subscripts are different p (p 4.05). II d’eMean with different subscripts are different (p L .005). 45 lOOI— \o C) I 80:- Casein 60 _. +H Alfalfa 50 _. -H—. Corn 4O _. Percent Protein Solubility 2O - lO ';/’ I I I I 60 120 minutes Time .Figure 5. Protein solubility for three protein sources at two intervals. DISCUSSION Cow's Effect Protein solubility of corn, alfalfa hay and casein was greater with rumen fluid from cow A than that from cow B (see Table 6). This was consistent throughout the entire experiment, i.e., when cow A was fed either alfalfa , hay or corn silage as forage, its rumen fluid yielded a higher protein solubility values. Since rumen fluid sam— ples from both cows were treated equally, i.e., they had the same standardized pH, temperature, degree of agitation, length of extraction time, sample and particle size of the protein material tested; none of these factors could have been responsible for the difference in protein solubility between rumen fluid from different cows. another possible factor is ionic strength but, according to Crooker et al., 1975; Crooker et al., 1978; and Salobir et al., 1970, pro— tein solubility was not affected by an ionic strength from .10 to .22. They also calculated that rumen fluid has an average ionic strength of .15, therefore, it is possible to assume that ionic strength is not the factor responsible. Thus, the difference in protein solubility may be the re— sult of other factors that are unknown now. 46 47 Forage Effect Protein solubility of the various protein sources was greater when they were incubated in rumen fluid from cows fed alfalfa hay than that from cows fed corn silage (see Table 6). Rumen fluid from cows fed corn silage had a higher density due to high soluble starch content which form gels that do not filter easily. Rumen fluid from cows fed alfalfa hay was easier to filter than that from cows fed corn silage. Thus the difference in protein solu- bility may be due to differences in solvent composition, as it is with the values obtained between Burrough's mineral mixture (10% solution) and modified Burrough‘s mineral mixture (10% solution) as solvents since they only differ in two ionic species NH: and Na+. Periods Protein solubility mean during period I was 55.1% and that in period II was 50.01% (see Table 6). These mean values are close enough to assume no difference. If one considers this a difference then it may be due to changes in the chemical composition of both forages through time. This change in chemical composition may similarly change rumen fluid composition. 48 Methods Protein solubility was similar in both solvents, autoclaved rumen fluid (ARF) vs filtered rumen fluid (FRF). This should be interpreted to mean that physical and chemi- cal characteristics of both solvents were similar. Thus, ARF and FRF may be used as solvents for protein solubility tests although it is suggested by the author of this work to keep using ARF because it is easier to prepare. Bur- rough's mineral mixture is another alternative as solvent. This mineral mixture behaves similarly to ARF as solvent when pH is 6.5 and incubated for 60 minutes at 40° C (Nohlt et al., 1975; and Crooker et al., 1978) but, Bur- rough's mineral mixture has no obnoxious odor as does ARF and it is more available in most laboratories and loca- tions since no ruminants are required to obtain it. Feedstuffs Amount of soluble protein of corn, alfalfa hay and casein was 52.17%, 45.48%, and 79.02%, respectively. There was a significant difference between corn and casein (see Table 8). Casein solubility was similar to that obtained in ARF by Nohlt et al., 1975. However, corn and alfalfa hay had 15.5% soluble protein (SP) and 24.55% SP when incu- bated in Burrough's mineral mixture (10% solution) (fiohlt et al., 1975; Crooker et al., 1978, and Crawford et al., 49 1978). The difference between the results obtained in this work and those of other investigators for corn and alfalfa may be due to type of protein of various batches of the same feedstuff and/or to the effect of different solvent. Nohlt et al., 1975, and Crooker et al., 1978, compared Burrough's mineral mixture (10% solution) vs ARF (obtained from cows fed timothy hay) and found that percent soluble protein yield was about the same in both solvents at a pH of 6.5 incubated for 60 minutes at 40° C. If pH, temperature or time were different, ARF and Bur- rough's mineral mixture would yield different protein solu- bility (p 21.05). Since rumen fluid from cows fed either alfalfa hay or corn silage was used in this work and, corn and alfalfa hay were exposed to pH 6.0, 6.5 and 7.0, and, 60 minutes and 120 minutes of exposure to the solvent action, the difference between the quoted works and this one might be explained. LH The effect of pH in protein solubility as noted in Table 9 may be due to the type of protein that is being tested, i.e., its amino-acid (AA) composition. Since pH influences the acid—ionization or dissociation constant (Ka) of each AA, any pH change in the solvent will affect the titration curve proper of each AA. Thus, the nitrogen 50 (protein) sources would have different solubilities at different pH's (Lehninger, 1970). Time Time of exposure of the various protein sources in the solvent at 60 minutes yielded 50.19% and at 120 minutes released 52.92% SP. The difference due to time was signifi- cant (see Table 10). This may be due to the solvent had more Opportunity to dissolve that protein fraction that is attached to other structures of the feedstuff such as carbohydrates, lipids, etc. Method-Feedstuff Protein solubility in the method-feedstuff inter- action was as expected (see Table ll), i.e., protein solubility was similar to that obtained in the main effect of feedstuff alone. Hence, ARF and FRF were similar as solvent. Feedstuff-pH Corn and alfalfa hay released more protein at pH 6.0 and less protein at pH 7.0. Casein yielded more pro- tein at pH 6.5 and less protein at pH 6.0 (see Table 12). This may be due to the same reasons explained before on the main effects of pH. 51 Feedstuff-time Corn and alfalfa hay yielded about the same amount of soluble protein when each one was exposed for either 60 minutes or 120 minutes to the solvent. Casein yielded more protein at 120 minutes than at 60 minutes (p £L.OOS) of exposure to the solvent (see Table 15). This may be due to: a) Most of the soluble protein from corn or that from alfalfa hay was readily soluble, and the other pro- tein was either attached to other structures, such as: carbohydrates, lipids, etc. or, it required more time to solubilize. b) Most of the casein was readily soluble in the media during the first 60 minutes, but it required more time to solubilize completely. RECOMMENDATION It is suggested to continue using autoclaved rumen fluid or Burrough's mineral mixture (10% solution) as solvents for quantifying protein solubility. Latter re- search may find better laboratory systems to quantitate protein solubility. \J] m BIBLIOGRAPHY Allison, M. J. (1970). "In physiology of digestion and metabolism in the ruminant," p. 456 (A.T. Phillipson, editor) Newcastle upon Tyne: Oriel press. Andrew, T. P. (1977). Ruminant digestion in Swanson, Duke's physiology of domestic animals. Comstock Publishing Associates, a division of Cornell Uni- versity Press, Ithaca and London, pp. 250-286. Al-Rabbat, M. F., Baldwin, R. L., and Weir, N. C. (l97l,a). "In vitro 15 Nitrogen.~-Tracer techni- que for some kinetic measures of ruminal ammonia." J. Dairy Sci. 54:1150-1161. Al-Rabbat, M. F., Baldwin, R. L., and Weir, N. C. 1971,b). "Microbial growth dependence on ammonia nitrogen in the bovine rumen." J. Dairy Sci. 54: 1162-1172. Amos, H. E., Little, C. 0., Ely, D. G., and Mitchell, G. E., Jr. (1971). "Abomasal protein and amino- acids in steer fed different protein supplements." Can. J. Anim. Sci. 51:51. Annison, E. F., Chalmers, M. I., Marshall, S. B. M., and Synge, R. L. M. (1954). "Ruminal ammonia for- mation in relation to the protein requirement of sheep. III. Ruminal ammonia formation with various diets." J. Agri. Sci. 44:270. Ash, R. N., and Dobson, A. (1965). "The effect of ab- sorption on the acidity of rumen contents." J. Barry, T. N. (1972). "The effect of feeding formalde- hyde treated casein to sheep on nitrogen retention and wool growth." N. Z. J. Agric. Res. 15:107. Barry, T. N. (1973). "Effect of treatment with formal- dehyde and intraperitoneal supplementation with D - L methionine on the digestibility, and voluntary in- take of silage of sheep." Proc. N. Z. Soc. Anim. Prod. 52:48. 53 10. 11. 12. 13 14. 15. 17. 18. 19. 20. 54 Belasco, I. J. (1955). "The comparison of urea and protein meals as nitrogen sources for rumen micro- organisms." J. Anim. Sci. 12:907. Bergen, W. G. (1978). "Postruminal digestion and ab- sorption of nitrogen compounds." Federation Pro- ceedings vol. 57, No. 5, pp. 1225-1227. Bjarnanson, J. and Carpenter, K. J. (1969). ”Mechan— isms of heat damage in proteins. 1. Models with acylated lysine units." British J. Nutrition 25:859. Bjarnanson, J. and Carpenter, K. J. (1970). "Mechan- isms of heat damage in proteins. 2. Chemical changes in pure proteins." British J. Nutrition 24:515. Blaxter, K. L. and, Martin, A. K. (1962). "The utili— zation of protein as a source of energy in fattening sheep." British J. Nutrition 16:597. Broderick, G. A. (1978). "In vitro procedures for estimating rates of ruminal protein degradation and proportions of dietary protein escaping the rumen endegraded." J. Nutrition 108:181—190. Brown, D. C. and Valentine, S. C. (1972). "Formalde— hyde as a silage additive. I. The chemical compo- sition and nutritive value of frozen lucerne, lucerne silage and formaldehyde treated Lucerne silage." Austr. J. Agric. Res., 25:109. Bryant, M. P. and Robinson, I. M. (1961). "Studies on the nitrogen requirements of some ruminal cellu~ lolytic bacteria." Applied microbiology 9:96. Bryant, M. P. (1970). "Microbiology of the rumen," pp. 484-515. In Duke's Physiology of domestic ani- mals. 8th ed. B. J. Swenson (Ed , Cornell Univ. Press, Ithaca. Bull, L. S., Helferich, N. G. Hilenshade, T. S. and, Sweeny, T. F. (1975). ”Protein solubility, source and ruminant protein synthesis." Proceedings XIII Conference on rumen function, Chicago, Ill. Bull, L. 8., P003, M. I. and Bull, R. C. (1977). "Protein solubility and NPN for dairy cows. --A prob- lem of energy metabolism." Distillers feed research council proceedings 52:25. 21 22. 25 24 25. 26. 27 28. 29. 50. 55 Burroughs, W., Norma A Frank, Gerlaugh P. and Bethke, R. i. (l950,a). "Preliminary observations upon factors influencing cellulose digestion by rumen microorganisms." J. Nutrition 40:9. Burroughs, w., Long, J., Gerlaugh, P., and Bethke, R. M. (l950,b). "Cellulose digestion by rumen microorganisms as influenced by cereal grains and protein rich feeds commonly fed to cattle using an artificial rumen." J. Anim. Sci., 9:525. Burroughs, W., Nelson, D. K. and Mertens, D. R. (1975). "Protein physiology and its application in the lactating cow: The metabolizable protein feeding standard." J. Anim. Sci., 41:955. Buttery, P. J. (1976‘. "Protein synthesis in the ru- men: Its application in the feeding of nonprotein nitrogen to ruminants." pp. 145-168. In, Princi- , ples of cattle production, H. Swan and w. H. 1 Broster (Ed.), Butterflorths, Boston. Campling, R. C., Freer, M. and Balch, C. C. (1962). "Factors affecting voluntary intake of food by cows. 5. The effect of urea on voluntary intake of oat straw.” Bri. J. Nutrition 16:115. Chalmers, M. I., Cuthbertson, D. P., and Synge, R. L. M. (l954,a). "Ruminal ammonia formation in rela— tion to the protein requirement of sheep. I. Duo- denal administration and heat processing as factors influencing fate of casein supplements." J. Agric. Sci., 44:254. Chalupa, w. (1975). ”Amino Acid nutrition of growing cattle." pp. 175-194. In, Tracer studies on non- protein nitrogen for ruminants. II. Int. Atomic Energy Agency, Vienna. Chalupa, N. (1976). ”Approaches to determining amino— acids requirements in producing ruminants." pp. 99-109. In, Reviews in rural Science II, T. M. Sutherland, J. R. Ncwilliam and R. A. Ling id.) Univ. of New England Publ. Unit, Armidale, NS", Australia. Clark, J. H. (1975). "Lactational responses to post- ruminal administration of proteins and amino-aCids." J. Dairy Sci., 58:1178. Cohn, E. J. (1945). "The solubility of proteins. In proteins, amino-acids and peptides as ions and 56 dipolar ions." E. J. Cohn and J. T. EdSall, ed. Reinhold publishing corporation, New York. Craig, w. M. and Broderick, G. A. (1978). "Rela- tionship of N-solubility to ruminal degradation of cottonseed meal protein." Abstracts 70th An- nual Meeting. July 9-15, 1978, MSU, E. Lansing. Pub. by American Society of Animal Science, Ab— stract No. 495. Crawford, R. J., Jr., Hoover fl. H., Sniffen, C. J. and Crooker, B. A. (1978). "Degradation of feed- stuff nitrogen in the rumen vs nitrogen solubility in three solvents." J. Anim. Sci., Vol. 46, No. 6, p. 1768. Crooker, B. A., Sniffen, C. J. and Hoover, H. N. (1975). "Factors affecting protein solubility mea— . surements in feedstuffs." J. Dairy Sci., 58:1240. e Crooker, B. A., Sniffen, C. J., Hoover, N. H. and Johnson, L. L. (1978). "Solvents for soluble ni- trogen measurements in feedstuffs.” J. Dairy Sci., 61:457—447. Danke, R. J., Sherrod, L. B., Nelson, E. C. and Till- man, A. D. (1966). Effects of autoclaving and steaming of cottonseed meal for different lengths of time on nitrogen solubility and retention in sheep." J. Anim. Sci., 25:181. Edwards, J. S. and Bartley, E. E. (1979). ”Soybean meal or Stares for microbial protein synthesis or milk production with rations above thirteen percent natural protein." J. Dairy Sci. 62:752. El-Shasley, K. (1952a). "Degradation of protein in the rumen of the sheep. 1. Some V.F.A.'s including branched—chain isomers found in vivo." Biochem. J. 51:640. El-Shasley, K. (1952b). ”Degradation of protein in the rumen of sheep. 2. The cation of rumen micro- organisms on amino-acids." Biochemistry J. 51:647. Ferguson, K. A. (1975). "The protection of dietary proteins and amino-acids against microbial fermen- tation in the rumen." In digestion and metabolism in the ruminant. I. W. McDonald and A. C. I. warner, ed., Univ. of New England. 40. 41 45. 44. 45. 46. 47. 48. 49. 50. 51. 57 Fraenker- Conrat, M. and Olcott, H. S. (1946). "Re- action of formaldehyde with proteins. II. Partici- pation of the guanidyl groups and evidence of cross- linking." J. Amer. Chem. Soc. 68: 54. Fraenkel-Conrat, h. and Olcott, H. S. (1978). "The reaction of formaldehyde with proteins. V. Cross- linking between amino and primary amide or guanidyl groups." J. Am. Chem. Soc. 70:2675. Faichney, G. J. (1971). "The effect of formaldehyde- treated casein on the growth of ruminant lambs." Aust. J. AgriC. Res. 22:461. Gill, J. L., (1978). Design and analysis of experi— ments in the animal and medical sciences. The Iowa State University Press, Iowa. Goering, H. K., Gordon, C. H., Hemken, R. w., waldo, D. R. VanSoest, P. J., and Smith, L. N. (1972). "Analytical estimates of nitrogen digestibility in heat damaged forages. J. Dairy Sci. 55: 1275. Goering, H. K., VanSoest, P. J. and Hemken, R. w. (1975). "Relative susceptibility of forages to heat damage as affected by moisture, temperature and pH.” J. Dairy Sci. 56:157. Goering, H. K. and vValdo, D. R. (1974). "Processing effects on protein utilization by ruminants." Proc. Cornell Nutr. Conf., p. 25. Gree, N. M. and, Neurath, H. (1954). "The prOteins." Vol. II, part 3., Academic press Inc., New York, p. 1057. Hatfield, E. E. (1977). "Responses of ruminants to dietary amino-acids." Distillers feed research council. Proc. vol. 52, Cincinnati, Ohio. Hemsley, J. A., Reis, P. J. and Downes, A. M. (1975). "Influence of various formaldehyde treatments on the nutritional Value of casein for wool growth." Aust. J. Biol. Sci. 20:961. Henderickx, H. and Martin, J. (1965). "In vitro stu- dies of the nitrogen metabolism in the rumen.’ Comt. Rend. Researches Sci. Ind. Agr. Bruxelles Henderson, C., Hobson, P. N. and Summers, R. (1969). In Proceedings of the Fourth International SympOSium 58 on the continuous Cultivation of Micro-organisms, pp. 189-204. (I. Malik, ed.) Prague:Czechoslo- vakia. Hill, D. L. and Noller, C. H. (1965). "Ehe apparent digestibility of protein in low moisture silages." J. Anim. Sci. 22:850. Hogan, J. P. and Weston, R. H. (1969). "The effects of antibiotics on ammonia accumulation and protein digestion in the rumen." Aust. J. Agric. Res. 20:559. Hogan, J. P. (1975). "Quantitative aspects of nitro- gen utilization in ruminants." J. Dairy Sci. 58:1164. Hudson, L. W., Glimp, H. A., Little, C. 0. and Wool- folk, P. G. (1969). "Effect of level and solubil- ity of soybean protein on its utilization by young lambs." J. Anim. Sci. 28:279. Hume, I. D., Moir, R. J. and Somers, M. (1970). "Syn- thesis of microbial protein in the rumen. I. Influ- ence of the level of nitrogen intake." Aust. J. Agric. Res. 21:285. Hume, I. D. (1970b). ”Synthesis of microbial protein in the rumen. III. The effect of dietary protein." Aust. J. Agric. Res. 21:505. Isaacs, J. and Owens, F. N. (1972). ”Procein soluble in rumen fluid." J. A. Sci. 55:267 Jancarik, A. and Proksova, M. "The Breakdown of pro- tein in the rumen in relation to the physical and chemical characters of the rumen juice." Nut. Proc. 8th. Intern. Congr., Prague. Excerpta medica, Amsterdam, pp. 504-506. Lehninger, A. L. (1970). "Biochemistry." Worth Publishers, New York. Lewis, D. (1957). "Blood urea concentrations in re- lation to protein utilization in the ruminant." J. Agric. Sci. 48:458. Little, C. 0., Burroughs, N. and Woods, w. (1965). "Nutritional significance of soluble nitrogen in dietary proteins for ruminants." J. Anim. Sci. 22:558. 63. 65 66 67 68. 70 71 72 75 74 59 Little, C. 0, and Mitchell, G. B., Jr. (1967). "Abomasal vs oral administration of protein to wethers." J. Anim. Sci. 40:411. Lyman, C. M., Chang, W. Y. and Couch, J. R. (1955). "Evaluation of protein quality in cotton seed meals by chick growth and by a chemical index method." J. Nutr. 49:679. Mangan, J. L. (1972). "Quantitative studies on ni- trogen metabolism in the bovine rumen. The rate of proteolysis of casein and ovalbumin and the re- lease and the metabolism of free amino-acids." Brit. J. Nutr. 27:261. Manson, W. E., Shirley, R. L., Bertrand, J. E. and Palmer, A. Z. (1975). "Energy values of corn, bird resistant and non-bird resistant sorghum grain in rations fed to steers." J. Anim. Sci. 57:1451. Marvin, P. B. (1977). Microbiology of the rumen in l Swenson, Duke's physiology 9f domestic ggimals. Comstock publishing associates, a division of Cornell University Press, Ithaca and London, pp. 287-504. Mathison, G. W. and Milligan, L. P. (1971). "Nitrogen metabolism in sheep." Brit. J. Nutr. 25:551-566. . McDonald, I. N. (1948). "The absorption of ammonia from the rumen of the sheep." Biochem. J. 42:584. McDonald, I. N. (1952). "The role of ammonia in ruminal digestion of protein." Biochem. J. 51:86. McDougal, E. I. (1949). "Studies on ruminant saliva. l. The composition and output of sheep's saliva." Biochem. J. 45:99. McGregor G. A., Sniffen, C. J. and Hoover, W. H. (1978). ”Amino-acids profiles of total and soluble protein in feedstuffs commonly fed to ru- minants." J. Dairy Sci. 61:566-575. McLeod, M. N. (1974). "Plant tannins--Their role in forage quality." Nutr. Abstracts Rev. 44:805. Mehrez, A. Z., Orskov, E. R. and, McDonald, I. (1977). "Rates of rumen fermentation in relation to ammonia concentration." Brit. J. Nutr. 58:457. 75. 76. 77- 78. 79. 80. 81. 82. 85. 85. 60 Miller, E. L. (1975). "Symposium on nitrogen utili- zation by the ruminant. Evaluation of food as sources of nitrogen and amino-acids." Proc. Nutr. Soc. 52:79. Nolan, J. V. (1975). "Quantitative models of nitrogen metabolism in sheep." pp. 416-451, In Digestion and metabolism in the ruminant. I. W. McDonald and A. C. I. Warner (Ed.), Univ. New England. Publ. Unit., Armidale, NSW, Australia. Orskov, E. R. and Benzie, D. (1969). "Using the oeso- phageal groove reflex in ruminants as a means of bypassing rumen fermentation with high-quality pro- tein and other nutrients." Proc. Nutr. Soc. 28:5OA. Orskov, E. R. and Fraser, C. (1969b). "The effect on nitrogen retention in lambs of feeding protein sup- plements direct to the abomasum, Comparison of liquid and dry feeding and of various sources of proteins." J. Agric. Sci., Camb. 75:469. Orskov, E. R. (1970). "Proc. of the 4th nutrition Conference for feed manufacturers. Univ. Nottingham, Churchill, London." Orskov, E. R. (1972). "Reflex closure of oes0phagea1 groove and its application in ruminant nutrition." S. Afr. J. Anim. Sci. 2:169. Orskov, E. R., Fraser, C., McDonald, I. and Smart, R. I. (1974). "Digestion of concentrates in sheep. 5. The effect of adding fish meal and urea together to cereal diets on protein digestion and utilization by young sheep." Brit. J. Nutr. 51:89. Osborne, T. B. (1924). "The vegetable prOteins." Longmans, Green and Co., London. Peter, A. P., Hatefield, E. E., Owens, F. N. and Garrigus, V. S. (1971). "Effects of aldehyde treat- ment of soybean meal on in vitro ammonia release, solubility and lamb performance." J. Nutr. 101:605. Phillipson, A. T. (1942). "The fluctuations of pH and organic acids in the rumen of the sheep." Jo Expo B101. 193186-198. Pilgram, A. F., Gray, F. V., Weller, R. A. and Belling, C. B. (1970). "Synthesis of microbial protein in the sheep's rumen and the proportion of dietary 86. 87. 88. 89. 90. 91. 92. 95 94 61 nitrogen converted into microbial nitrogen." Brit. Purser, D. B. and Buechler, S. M. (1966). "Amino- Acid composition of rumen microorganisms." J. Dairy Sci. 49:81. Reis, P. J. and Tunks, D. A. (1969). "Evaluation of formaldehyde-treated casein for wool growth and nitrogen retention." Austr. J. Agric. Res. 20:775. Saba, N. J., Hale, N. H. and Theurer, B. (1972). "In vitro fermentation studies with a bird resistant sorghum grain." J. Anim. Sci. 55:1076. Salobir, K. A. and Muck, O. (1970). "fhe importance of solubility of proteins in feed for the evalua— tion of their propriety for ruminants.” I. Proc. lst. Yugoslav Intern. Conf. Anim. Production. p. 529. Satter, L. D. and Roeffler, R. E. (1975). "Nitrogen requirements and utilization in dairy cattle." J. Dairy Sci. 58:1219-1257. Batter, L. D. Whitlow, L. N., and Beardsley, G. L. (1977). "Resistance of protein to rumen degradation and its significance to the dairy cow." Distillers feed research council proceedings. Vol. 52:65. Schellin , G. I., Mitchell, G. E. and Tucker, R. E. (1972 . ”Prevention of free amino—acid degrada- tion in the rumen." Fed. proc. 51:681. Sherrod, L. B. and Tillman, A. D. (1962). "Effects of varying the processing temperatures upon the nu- trition values for sneep of solvent-extracted soy- bean and cottonseed meals." J. Anim. Sci. 21:901. Sherrod, L. B. and Tillman, A. D. (1964). ”Further studies on the effects of different processing temperatures on the utilization of solvent extracted cotton-seed meal protein by sheep." J. Anim. Sci. 25:510. Smith, A. K., Belter, P. A. and Johnson, V. L. (1952). "Peptization of soybean meal protein; effect of method of dispersion and age of beans.” J. Am. Oil Chemists Soc. 29:509. 62 Smith, C. R., Jr., Earle, F. R. and Wolff, I. A. (1959). ”Comparison of solubility characteristics of selected seed proteins." J. Agric. Food Chemist. 96 97. Sniffen, C. J. (1974). ”Nitrogen utilization as re- lated to solubility of NPN and protein in feeds.” Proc. Cornell Nutr. Conf., p. 12. 98. Standaert, F. E. (1979). "Rumen bypass of protein through esophageal groove closure in lactating cows." Thesis for M. Sci., Department of dairy sci., MSU, E. Lansing, Mich., 48825. 99. Thornton, R. F. and Wilson, B. J. (1972). "Factors affecting the urinary excretion of urea nitrogen in cattle. High plasma urea concentrations.” Austr. J. Agric. Res. 25:727. 100. Turner, A. w. and Hodgetts, V. E. (1955). ”Buffer systems in the rumen of the sheep. . pH and bicar- bonate concentration in relationship Ito p002." Austr. J. Agric. Res. 6:115-124. 101. waldo, D. R., Smith, L. N., and Cox, E. L. (1972). ”Model of cellulose disappearance from the rumen.” J. Dairy Sci. 55:125-129 102. waldo, D. R., Keys, J. E., Jr. and Gordon, C. H. (1975a). "Preservation efficiency and dairy heifer response from unwilted formic and wilted untreated silages." J. Dairy Sci. 56:129. 105. Maldo, D. R., Keys, J. E., Jr. and Gordon, C. H. (1975b). ”Formaldehyde and formic acid as a silage additive." J. Dairy Sci. 56: 229. 104. Walker, J. F. (1974). "Formaldehyde." 5rd ed., Reinhold, New York. 105. Hester, J. (1926). ”Die physiologie und pathologie der Vormagen beim Rinde.” R. Schoetz, Berlin. 106. Nohlt, J. E., Sniffen, C. J. and Hoover, V. H. (1975). "Measurements of protein solubility in common feed- stuffs." J. Dairy Sci. 56:1052. 107. Wohlt, J. E., Sniffen, C. J., Hoover, J. H., Johnson, L. L. and walker, C. K. (1976). ”Nitrogen metabo- lism in wethers as affected by protein solubility and amino acid profile." J. Anim. Sci. 4221280. 65 108. Johlt, J. E., Clark, J. H. and Blaisdell, F. S. (1978). "Nutritional value of urea vs preformed protein for ruminants. II. Nitrogen utilization by dairy cows fed corn based diets containing sup- plemental nitrogen from urea and/or soybean meal." J. Dairy Sci. 61:916. 109. wright, P. L. (1971). "Body weight gain and wool growth responses to formaldehyde treated casein and sulfur amino acids." J. Anim. Sci. 55:157. 110. Zelter, S. Z., Leroy, F. and Tissier, J. P. (1970). Annales de Biologie Animale. Biochimie, Biophysi- que 10:111. APPENDIX A Macro-Kjedahl Procedure N— Determination >4 Weighing 2S 4 4 Jet Feces 5 g 5 g 25 ml Dry Feces 1-2 g 5 g 25 m1 Urine 5 cc 4.5 g 20 m1 wet silage 4—5 g 25 ml Blank (put also in the blank 15 ml a piece of filter paper) Pro-Sil 0.5 g 5 g 18 ml Molasses l g 4 g 18 ml DIGESTION 1. Onto dhatman filter paper weigh proper amount of sample. Fold filter paper; put paper and contents into Kjeldahl Flask. To each Flask add: a) Proper amounts of K2804 - 5 g. 6-6.5 g of mixture b) Slightly less than 1 g of CuSOA. in each flask c) Proper amounts of H2804 - 25 ml d) 5 boiling beads to all flasks Digest on #2 position on burners: Digest until turns a blueish—green color; boil until solu— tion only fills inside ring of the burner; then cool: Add 250 cc of water to each flask: (Keep flask pointed towards burner when pouring water, protects self from vapors; also water may boil in acid- salt dilution reaction) 6. Let sit until cool. Distillation: 1. Put 25 cc 4% Boric Acid in bottom of each beaker. 2. Place each flask under condenser outlet. 5. To each Kjeldahl flask add 60 cc of 50% NaOH solution. (Pour on a slant, so it settles on bottom) 4. Add pinch of zinc. . , 5. Attach flask on condensor: Turn burner to #5 until b011- ing then #2 and finally to #1 position. STAND BEHIND DOOR AND WAIT FOR REACTION. 64 65 6. Distill 200 cc of solution (Remove distilled flasks and substitute flasks with 200 cc water; set Kjeldhals off burner, then turn off. TITRATE with .10 N HCl. CALCULATE (Using .1 N HCl) 1. Solids (silage, feces) - - 0 ’ l , sample weight x DM decimal ' g N b dry hatter R) Neighed liquids (Pro-Sil, molasses) __ ml x414 _ a sample weight ‘ 8 N A wet 5. Liquids by volume (urine) m1 x .14 sample volume in ml = g N/lOC m1 4. The factor .14 is N(.l) x .014 x 100). If normality is other than .1 the factor must be recalculated. APPENDIX B Total Nitrogen Contained in Control Blank) Solutions Method Rumen Fluid From Autoclaved Filtered --—— g. N/lOO m1 -—-- Alfalfa Hay 0.055 0.0472 Corn Silage 0.585 0.0567 66 APPENDIX C Example of Calculations to Obtain the Amount of Soluble Nitrogen I. Obtain 20 mg of nitrogen from casein. DATA Feedstuff: casein Percent dry matter (DM): 90.70% Total nitrogen (% DM): 15.07% ): Total nitrogen (% as fed 15.668% Solution —49§—— 100 o 146 f - -‘1 15.668 x = - 5 g 0 Casein wi 1 pro- vide 20 mg of N II. Obtain amount of SN in rumen fluid. DATA Total N: 20 mg of N from casein Total solvent: 80 ml Temperature: 40° C Time of incubation: 60 minutes Amount of supernatant after last centrifugation: 4 m1 Method used for determination of total SN: macro-Kjeldahl Titration with .lN HCl for the casein solution: 1.5 m1 Titration with .1N HCl for the blank solution: 1.0 ml Equation for total N determination: (m1 of .1N HCl for problem - ml of .1N HCl for blank) x .14 sample volume in ml = g N/lOO m1 Solution: (1.5 - 1.02.14 = 0.0175 8 N/lOO m1 4 ml Calculate percent SN: 94%%22 x 100 = 87.5% SN 67 APPENDIX D Curriculum Vitae Name: Ricardo Antonio Celma Alvarez Nationality: Mexican Languages: Spanish and English B.S.; D.V.M3 at the National University of Mexico (March, 1975 M.S.: Michigan State University Works Place Activity Year ’ Research National Insti- Physiopathology 1969-1971 Assistant tute of Animal Research Private vet. Private Small Species 1975—1979 Clinic Professor National Uni- Teaching-- 1974-1977 versity of Animal Mexico Nutrition Professor National School Teaching-- 1975-1976 ENEP for Pro- Animal fessional Edu- Nutrition cation 68 “QTIT‘IIGHWIWIWWilliam[1111111115s 31293 03082 0728