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' 11,1 1.4.1111, 1.1. 1, """'1"'11"1'111111:111'1 111111111111111.. .111111111111111-1111111‘11111 h1 u 111111111111111'11'11'1111111111111111 1 ['11'1'1'1'1' . 3.1 .. . 11".“ 1.“ 1 11 ""1 11"..‘1'1111 .111 111.111 1" 111W1'1'1111'11111'1'11171"11 11-111'1J1M 1' 1 1 1 1 1 111' "' ' ' ' '1'I"1'1' . ' ' 1'1"" " "I"1”II "'."111"'.'1"'.11'111"""1 1"" ' 1".11'1' " ' "" Wk 1'1" 11" '11"1'1~ " (11111; . '1 1... 117127111... ~."?"'1'I.1I2i.11""1‘ 11.: .I II I I1.» 1111111111111'11'11‘1'111 I I '1 1 1 I1 . l.\\\\\\\\\\\\l , “an" i m IWHW“ This is to certify that the thesis entitled METABOLIC EFFECTS OF MALIC ACID IN RUMINANTS presented by Jerry Doyle Krummrey has been accepted towards fulfillment of the requirements for Masters degree in Dairy Science mm. M Major professor ll ‘ ‘3 Date Apr-it 11, 1979 0'7 639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. METABOLIC EFFECTS OF MALIC ACID IN RUMINANTS BY JERRY DOYLE KRUMMREY A THESIS Submitted to Michigan State University in partial fUlfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy 1979 ABSTRACT METABOLIC EFFECTS OF MALIC ACID IN RUMINANTS By Jerry Doyle Krummrey In the lfl.Xl££2 study gas production, volatile fatty acid pro- duction and ph changes were used to estimate the effect of malic acid on the rumen fermentation rate. Malic acid increased gas production and volatile fatty acid production. In the milk production trial 32 lactating Holstein cows were randomly allotted to 4 treatment levels of malic acid (0, 70, 105, and 140 grams/day) fed during a 100 day treatment period. The group receiving highest malate had significantly higher milk persistency than controls (95 vs. 88%).. Early lactation cows receiving malic acid were significantly higher in total rumen volatile fatty acids. In the nitrogen balance study 6 steers (420 kilograms) were ran- domly assigned to a 3 x 3 Latin Square receiving 0, 100, or 200 milligrams/kilogram body weight of malic acid per day. Rumen pro- pionate was significantly higher in animals receiving malic acid than in controls. ACKNOWLEDGEMENTS The author expresses appreciation to his major professor, Dr. Robert M. Cook, for continued encouragement, assistance and guidance during graduate study. Gratitude is extended to the Department of Dairy Science for financial support in the form of a Graduate Research Assistantship. Thanks is rendered to Dow Chemical Company for financial backing enabling this research to be conducted. The author is grateful to Dr. Melvin T. Yokoyama, Dr. C. A. Reddy and Dr; John T. Huber for aid and advice while serving on the guidance committee. Appreciation is conveyed to Laurie Allison, Sheri Schlotz, Denise Schlotz, Karen Wernette and Kathy Wood for analysis of samples and compilation of data. Last and foremost, recognition is given to my wife, Elizabeth Jean, for her patience and understanding throughout this graduate school program. ii TABLE OF CONTENTS INTRODUCTION no...ooooouoooouooooooooooooooooooooooooooo.cooon.o... LITERATURE REVIEW o0.0.000IDIOIOIOICIOOOOOOOIOOIOO0.00000...00000.0 MATERIALS AND METHODS nococoons-oo-ooncoon-000.000.000.000...onpoo. In Vitro Experiments 000I000000000000000.0000000000000000 Milk PrOdUCtiOn Trial onono0.00.0.0...OOOOIOIOOIOOOOO0000 Nitrogen Balance StUdy to00O0.0.0.0....000.000.000.000... RESULTS I.OI0.0.00.0..000000.00IOIOOOOIOOOIOIOO00.000.00.00...CO... In Vitro Experiments 00OI.0.0.0.0..IIIOOIOOIOIOOOIOO0.000 Milk Production Trial ono...no.cooIOOOOIOOOOOOOOIIOOIOOOQ Nitrogen Balance StUdy 00000000000000.0000...Inoooooocooo DISCUSSION o0000.00.00.00.Cocoa-cococoonono0.0000000000000000000000 SUMI‘qARY AND CONCLUSIONS 00on0.0IOIIOIOOIOOIIOIIOOOOOOIIOICOCCOIOOIO LITERATURE CITED ooOOIOOIOOOOOOOIOOOOI0.00000000000000.0000.00.0000 APPENDIX coo-0.000000000000000..-o00000000000000...0000000000000... iii Page 1 3 9 9 11 15 18 18 22 43 66 68 69 11. 12. 13. 1h. 15. 16. 17. 18. 19. 20. LIST OF TABLES Physical properties Of malic aCid 00000000000000.0000.00.00. Composition Of the £2 vitro media ooooooooooooooococoon-coo- Composition of concentrates used in the production trial ... Gas PrOdUCtion in the in vitro experiments noon-00000000000. The effects of malic acid on energy utilization, early and mid lactation combined noOI.OOOOOOOOOOOOOOOOOIOOOIOI0000 The effects of malic acid on energy utilization in mid laCtatiOn COWS so.00000000000000.0000ooooooooooo-oooooooooo. The effects of malic acid on energy utilization in early lactation cows The effects of malic (weekly differences) The The The The The The The The The The The The effects effects effects effects effects effects effects effects effects effects effects effects of of of of of of of of of of of of malic malic malic malic malic malic malic malic malic malic malic malic acid on persistency of lactation acid acid acid acid acid acid acid acid acid acid acid acid on on on on on on on on on on on on iv dry matter intake ............. roughage dry matter intake .... average body weights .......... total dry matter intake . . . . o . . roughage dry matter intake .... % fat in milk ................. % protein in milk ............. % total solids in milk ........ plasma glucose ................ plasma urea ................... nmaiwmmua..u.u.u.u.u. men Ph 00000000000000.0000... Page 8 10 13 19 23 24 25 26 #0 Table 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33- 34. 35- 36. 37. 39. 40. The effects of malic acid on total volatile fatty acid concentration in men fluid for all COWS 00.000000000000000 The effects of malic acid on total acetate in rumen flUid for all COWS 00000.00OI.00000000000000.0000...00000000 The effects of malic acid on total volatile fatty acid concentration in rumen fluid for early lactation cows ...... The effects of malic acid on acetate in early lactation COWS on...non-o.o0000000000000000000000000.oooooocooaooooooo The effects of malic acid on propionate for early lactation COWS n.coo-cocoo-I...cocoon-cocooooooooooocoo-coco The effects of malic acid on isobutyrate for early lactation COWS I0.00000000IOIIOOIOIOO'IOOII000.00.00.00...00 The effects of malic acid on butyrate for early lactation COWS onOIOOOOIOIOOOIIOOOOOOOO000.00.000.00...00000 The effects of malic acid on 2-methyl butyrate for early lactation COWS coco-IIOOOOOOIIIOOO00.000.000.00...0000000000 The effects of malic acid on isovalerate for early lactation COWS no.cocoooooooooooooooooooocoo-coco...cocooooo The effects of malic acid on valerate for early lactation COWS no.00000000....onoooono00000000000000000.000.0000..000. The effects of malic acid on feed intake, feces and urine excretion o00.000.on.I0000000000000000000.coo-00.000.000.00. The effects of malic acid on daily dry matter intake ....... The effects of malic acid on dry matter digestibility ...... The effects of malic acid on acid detergent fiber .......... The effects of malic acid on total nitrogen retained ....... The effects of malic acid on digestible nitrogen retained .. The effects of malic acid on protein digestibility ......... The effects of malic acid on rumen ammonia (nitrogen balance StUdy) cocoon...a...coo.coon-00000000000000.0000noo. The effects of malic acid on plasma urea (nitrogen balance Stde) o0000.00.00.00.-00.000000000l0000000000000000 The effects of malic acid on plasma ammonia (nitrogen balance study) 00.000.ooOIIOIOIOOOIOOOIO0.000000000000000... V Page Ad 42 45 46 47 49 50 51 52 54 55 56 57 58 59 6O 61 62 Table 41. 42. 43. 45. 46. 47. 48. 49. 5o. 51. 52. 53- 54. 55- 56. 57. 58. 59. 60. 61. 62. 63. Page The effects of malic acid on volatile fatty acid concentration (nitrogen balance StUdy) oooooooo-oooooooooooo 63 The effects of malic acid on volatile fatty acid concentration (nitrogen balance study) Low level malic aCid fed ooooooooo-oo000000.000000OIIOOOQQQQQQQQQQOoooooooon 64 The effects of malic acid on volatile fatty acid concentration (nitrogen balance study) High level malic aCid fed .........................o..................o...... 65 The analysis of variance for persistency ................... 72 Sum of squares for persistency ............................. 73 Test of significance of persistency ........................ 74 The orthogonal test of persistency ......................... 76 Analysis of variance for total dry matter intake ......o.... 78 Analysis of variance for roughage dry matter intake ........ 79 Analysis of variance for total dry matter intake/100 kg bOdy weight 000000000000000000000000.IOU-Ioooooocooocoogoooo 80 The orthogonal test of significance of total dry matter intake/100 kg bOdy Weight 0000000000000.000000000000000...o. 81 Analysis of variance for roughage dry matter intake/100 kg .mdy weightC..........°.................................... 83 Analysis of variance for percent fat in milk ............... 85 Analysis of.variance for percent protein in milk ........... 87 Analysis of variance for percent total solids in milk ...... 89 Analysis of variance for plasma glucose .................... 91 Analysis of variance for plasma urea ....................... 93 Analysis of variance for rumen ammonia ..................... 95 Analysis of variance for rumen ph .......................... 97 Analysis of variance for total VFA for all cows ............ 99 Analysis of variance for total acetate for all cows ........ 102 Analysis of variance for total VFA in early lactation cows . 105 Analysis of variance for acetate for early lactation cows .. 107 vi Table 64. 65. 66. 67. 69. 70. Page Analysis of variance for propionate for early lactation COWS none.ococo-ooooooloiololuonloo-o00.000000000000000...on 109 Analysis of variance for isobutyrate for early lactation COWS oooooooooooooon.on0000.000000.IOIoOOIOOOOOOIIOOIOOO0000 110 Analysis of variance for butyrate for early lactation cows . 112 Analysis of variance for 2-methy1 butyrate for early laCtatiOn COWS coco-00000.0...ouooooooluoooogooooooo.coco... 114 Analysis of variance for isovalerate for early lactation COWS 000.000.lo00.000000000000000...0000.00.00.0000030can... 116 Analysis of variance for valerate for early lactation cows . 118 Analysis of variance for propionate in the nitrogen balance StUdy .ooooon.coco-ocooococo-coco.0.000000000000000. 119 INTRODUCTION Research in animal agriculture consists of conducting experi- ments to determine the origin of various biological and physical processes so that the system may be more fully understood. The goal is to use this new knowledge to increase the productivity and efficiency of animal agriculture. The ruminant, by virtue of the microbial population inhabiting its rumen, is unique in its ability to digest feeds that are meta- bolically less available to other animals. The bovine is able to produce milk and meat which are two high quality foods. Thus, the bovine has the capacity to convert feeds of low nutritional value to high quality food for humans. Digestion of feeds in the rumen is an important process in ruminant nutrition. If microbial fermentation is at the optimum level, maximum intake and utilization of feeds can be achieved. Certain microbial growth factors have been identified which have been shown to be able to increase feed efficiency and nitrogen retention in ruminants. This is accomplished by increasing the growth rate of rumen bacteria enabling more complete digestion of feeds to occur in the forestomach of the ruminant. The end result is a more efficient system whereby the animal receives more available nutrients than was possible with a less desirable rumen fermentation. In animal agriculture today the cost benefit ratio of microbial growth factors must be considered. Industry must know if the 1 2 increase in feed efficiency provided by supplying microbial growth factors is substantial enough to justify research, manufacturing, and marketing costs of the microbial growth factors. There has been considerable effort put forth recently in study- ing feed additives for beef and dairy cattle. malic acid has been shown to increase feed efficiency and nitrogen retention in beef and dairy cattle presumably by increasing the rumen fermentation efficiency. However, little research has been conducted with dairy cattle concern- ing the effect malic acid might have on milk production, milk composition, feed intake, body weight changes, and feed efficiency. The objective of this thesis was to develop an ip.zitrg technique to assess the effects of microbial growth factors such as malic acid on the rumen fermentation rate and to determine if supplemental malic acid increases the utilization of nutrients by ruminants and enhances milk yields in lactating dairy cows. LITERATURE REVIEW In_Vitro Technique for Studying the Human Fermentation Rate Most of the methods used for measuring in yitrg rumen microbial activity have been a measure of fiber disappearance during a specified time interval. The measurement of microbial activity by fiber dis- appearance in forty eight hours may not show differences between treatments even though there were differences at some time prior to forty eight hours. Thus, a system was needed that could quickly test the effects of chemicals on the rumen fermentation rate. Production of gas as measured manometrically has been used by Hungate (16), McBee (25), Perez (27), Quin (29), and Reid (30). Like volatile fatty acid production, gas production data needs to be considered cautiously because of its lack of specificity. Gas can be produced from a variety of substrates by a mixed culture of rumen micro- organisms. Furthermore, C02 can be released from carbonate buffered medium by the acid produced and care must be taken to account for it. Nevertheless, this parameter has been used successfully and will be important in future studies of rumen fermentation. The ig_zitrg method of using gas production rates to measure microbial net growth was developed by ElShazly and Hungate in 1965 (11). They found that if substrate was in excess and the optimum dilution was used, fermentation progressed at the maximal rate and was proportional to total microbial cells. This technique was used with some modifications for rapidly determining the effects of different 3 4 microbial growth factors on the rumen fermentation rate. 14.122.949.12 Malic acid is an important natural organic acid. It is widely dispersed among the vegetables of the world and is the most abundant of the acids found in fruit. For example, the fruit of lychee has malic acid present in it totaling eighty percent of the nonvolatile acids (6). Malic acid is found in strawberries (28), grapes (17), peaches (20), and peas (36). In the wine industry malic acid concen- trations are monitored to give an idea of the stability of the wine in question (32). Since wine is a product of certain yeasts transforming sugars to alcohol, the breakdown of malic acid by lactic bacteria reduces acidity producing a more stable wine. Malic acid is the pre- dominant organic acid in many plants (12). It is in grasses (4, 8), silages (31). and legume forages (31) in varying amounts. In the Animal Kingdom malic acid plays a key role in carbohydrate metabolism. It serves as the precursor of pyruvate and oxalacetate (19). Malic acid is used in the food industry as an anti-spattering agent for margarine, as a metal chelating agent to inactivate heavy metals, and in the extraction of pectin from fruit waste (23). Malic acid has applications in the pharmaceutical industry as a component of compounds used to treat hepatic disfunction (23). Examples of chemical uses of malic acid are as an essential ingredient of insect repellents, hydrogen peroxide stabilizers, and as an algicide. In cosmetics malic acid is used in teeth cleaning tablets, toothpastes and mouthwashes (23). Malic acid contains an asymmetric carbon. Thus, it exists in 5 both dextrorotary and levorotatory forms. The form used in this research was obtained from Dow Chemical Company and is a racemic mixture of D and L isomers. The acid found in nature is the levorotatory configuration (35). Malic acid is a key intermediate in the metabolism of bacteria (2, 10, 14, 15, 22, 24, 33). This is important to ruminants because of the symbiotic relationship between the ruminant and its microbial population. Since malic acid is a key intermediate in microbial metabolism, the concept arises as to whether it is a limiting growth factor for these microbes. Malic acid has been shown to stimulate the growth of rumen bacteria on lactate media (21). This occurs because malic acid is a source of oxalacetate which is limiting. The oxalacetate deficiency arises because of the need for glucose synthesis. A limitation of this important metabolic intermediate could limit microbial growth. Thus, malic acid increases oxalacetate which is used in propionate and glucose formation and other biosynthetic reactions to increase the rumen microbial fermentation efficiency. Studies on certain species of yeasts indicate that the L form of malic acid is utilized to a greater extent than the DL mixture (5). However, research conducted with rats show both the L and DL forms are metabolized equally with no apparent difference in utilization of either form (9). In ruminants malic acid increases propionate production when added to the rumen (34). This is associated with more efficient utilization of energy sources from the rumen. This is very important to ruminants especially if the ratio of forage to concentrate in the ration is high. The reasoning being that propionate is converted to 6 glucose in the liver in ruminants, increased propionate production will increase glucose production in the ruminant. This makes more energy available for body processes including milk production. Malic acid functions in the tricarboxylic acid cycle to supply a source of oxalacetate (19). Oxalacetate is necessary for the production of carbohydrate from all precursors except glycerol and L—glycerophosphate (19). The precursors are propionate and lactate produced from fermentation of forages and concentrates (34). Feedinngrials Experiments have shown that the L isomer of malic acid is twice as effective as the DL mixture in improving nitrogen retention in sheep (34). This is expected since it is the L form that occurs in nature. The supplementation of malic acid to steers on high forage rations containing urea increased protein digestibility, and nitrogen retention (34). The addition of malic acid to sheep on high forage rations increases nitrogen retention (34). However, adding malic acid to steers on a high concentrate diet was not successful in increasing digesti- bility of nitrogen or dry matter or retention of nitrogen (34). A study was recently completed at Utah State University (1). It was found that feeding 107 grams malic acid per head daily gave increases in milk production. Two other studies have fed malic acid to lactating dairy cattle (34). In the first study cows receiving 70 grams malic acid per head per day produced 4.5 pounds more milk per day than controls. The solids corrected milk was also significantly increased and the 7 treatment group gained more weight. Feeding 28 grams malic acid per head daily gave no significant increase in milk production or weight gain. In the second study cows receiving 70 grams were more efficient in converting energy to weight gain and milk production. This treat- ment group gained more weight and produced more milk than controls or cows receiving 35 grams malic acid daily. In conclusion previous studies of the effects of feeding malic acid to lactating dairy cattle have been with cows in average production and only a limited number of animals were used. Also, previous studies have not considered metabolic indices which may indicate the mechanism of action of the compound. This study was undertaken to develop and utilize an in_3it£g technique for measuring the effects of malic acid on the rumen fermentation rate and then use applied research to determine the effects of malic acid on lactating dairy cattle. A nitrogen balance experiment was also performed to help elucidate the mechanism of action through which malic acid increases productivity in ruminants. Table 1. Physical prOperties of malic acid. Formula Molecular weight Melting point Physical shape Forms Solubility Ethanol Ether Chloroform Heat of combustion Heat of solution Viscosity, 50% aqueous solution Odor Specific gravity, Dio HOCHCOOH CHZCOOH 134.07 Racemic DL-form, 131-13200 White crystals Natural L-levorotatory Synthetic DL-racemic mixture 39.16 grams/100 ml 1.41 grams/100 ml 0.04 grams/100 ml -320. 1 kcal/mole -4.0 kcal/mole 6.5 Odorless 1.601 MATERIALS AND METHODS I. In Vitro Experiments Variations occurred in the amount and kind of substrates utilized and in the composition of the culture media (Table 2). The source of the rumen fluid was a mature, nonlactating Holstein cow equipped with a permanent rumen fistula. The cow was on an all corn silage ration with access to a trace mineralized block throughout the experimental sampling period. Rumen fluid samples were collected by removing whole rumen contents via the fistula and squeezing it through cheesecloth into a dewar. The dewar had been warmed with hot water to prevent changes in rumen fluid temperature. The lid was placed on the flask and rumen fluid was immediately transferred to the laboratory. In the laboratory one hundred milliliter aliquots of rumen fluid were transferred to one-pint flasks containing substrates. The rumen fluid was stirred gently to insure uniform aliquots. The flasks were preincubated at 39°C. The flasks were gased with co2 to exclude oxygen and then closed with a rubber stopper provided with a tube having a three-way stOpcock. The stopcock connected the flask to a ten milliliter glass syringe. The stopcock allowed the syringe to be emptied without exposing the incubation to the atmosphere. Gas measurements started after a thirty minute equilibration period. The time interval between rumen sampling and incubation was approximately fifteen minutes. 10 Table 2. Composition of the in vitro media. Experiment Component I II III IV Buffer, ml* 200 200 200 200 Rumen fluid, ml 100 100 100 100 Sodium bicarbonate, mg -- 3000 1000 500 Cellulose, g -- 5 5 -- Amylose, g -- 5 -- -- Concentrates, g -- -- 5 5 Experiment Component V VI VII Buffer, ml* 200 200 200 Rumen fluid, ml 100 100 100 Sodium bicarbonate, mg 1000 3000 3000 Cellulose, g -- -- 5 Amylose, g -- 7-5 2-5 Concentrates, g 5 -- -- * Hungate buffer: 1 part A & 1 part B & 4 parts double distilled water. A = 0.3% KHZPO 0.6% NaCl, 0.3% (NH 1+9 0.06% MgSOu, 0.06% CaCl “>230“, 2 B = 0.3% KZHPO4 11 Gas production was measured using a ten milliliter water- lubricated glass syringe. The volume of gas forced into the syringe was read every five minutes. The incubation time was two hours (not including the thirty minutes of preincubation). The ph of each incuba- tion mixture was measured before and after each incubation period (initial ph was 7.0 for each flask). One sample was taken prior to incubation and treated as the others to serve as the zero time control. When the incubation was complete, samples from each flask were collected, placed in ice, and later centrifuged at 10,000 x gravity for fifteen minutes. The supernatant was recovered for volatile fatty acid determination. II. Effects 9: Malic Acid 9n Milk Production Thirty-two lactating Holstein cows (16 in mid and 16 in early lactation) were randomly allotted to four treatment groups of eight cows each. The statistical design was a randomized complete block design. Treatment groups were balanced for milk production, age, days after calving and breeding groups. Treatments consisted of four levels of malic acid (0, 70, 105, and 140 grams/day) fed during a one hundred day treatment period. Feeding Regime: The cows were fed once daily in a stanchion-type barn. Water was free choice. Corn silage and alfalfa hay were mixed together in relative proportions of sixty kilograms corn silage to five kilograms alfalfa hay. This mixture was identified as mix-three and was fed to the cows ad libitum. Concentrate was fed at the rate of one kilogram concentrate for each two and one half kilograms milk. The concentrate 12 was placed into the feed bunk on top of mix-three so that the concen- trate was totally consumed. Weighbacks of feed occurred each morning at 6 am. Cows were fed between 8 am to 11 am each morning. Cows were fed quantities of mix-three to enable a ten percent weighback to occur each day. Feed records were checked every two- three days to insure that a ten percent weighback was occurring. The amount of concentrate fed was adjusted every three days based on changes in milk production. Three different concentrates were fed: D200, D208 and D209. The composition of each concentrate is shown in Table 3. The early lactation cows were adapted to nonprotein-nitrogen during the first four weeks after they calved. D208 concentrate was used for this and as the four weeks elapsed, D208 (contains NEN) replaced D200 as the concentrate source. The malic acid was in D209 and was fed as follows: 0, 4, 6, or 8 pounds D209 was fed so that each cow received 0, 70, 105, or 1&0 grams malic acid per day. Thus, the amount of D209 a cow received during the one hundred day treatment period stayed the same and it was D208 that fluctuated depending on the animal's previous three days milk production. D208 and D209 were of similar composition except for the malic acid content of D209. The late lactation cows had a three week adaptation period to NPN starting approximately one hundred fifty five days into their lacta- tion. All groups were fed the same basal ration of mix-three and concentrate during treatment. Milk Data: Individual milk weights were recorded twice daily. Milk com- position (percent fat, protein and total solids) was determined weekly 13 Table 3. Composition of concentrates used in the production trial (kilograms). D200 Ground Shelled Corn 392.4 Ground Oats 192.8 Soybean Meal 247.2 Deflourinated Phosphate 15.9 Trace Mineralized Salt 9.1 Vitamin A & D* 4.5 Sugar Cane Molasses 45.4 D208 Ground Shelled Corn 469.9 Ground Oats 235.9 Soybean Meal 104.3 Trace Mineralized Salt 9.1 Deflourinated Phosphate 13.6 Limestone 4.5 Urea 20.0 Vitamin A & D* 4.5 Sugar Cane Molasses 45.4 D202 Ground Shelled Corn 446.8 Ground Oats 224.1 Soybean Meal 104.3 Trace Mineralized Salt 9.1 Deflourinated Phosphate 13.6 Limestone 4.5 Malic Acid 34.9 Urea 20 0 Vitamin A & D* 4.5 Sugar Cane Molasses 45.4 * Vitamin A contained 4409 international units per kilogram. Vitamin D contained 441 international units per kilogram. 14 for each cow. A composite milk sample was.taken on Tuesday afternoons and Wednesday mornings. Milk was analyzed for fat by the Milkoscan 300 machine, protein by the Orange G Dye Binding method and for total solids by drying 25 grams in a forced air oven for 3 hours. Herd Management: All cows were weighed seven days after the beginning of treatment and biweekly thereafter. The cows were housed in a stanchion-type barn which was completely enclosed. The milking parlor was approximately twenty meters from the cows and was attached to the barn where the cows were housed. The cows were milked in a double-eight herringbone milking parlor twice daily at 4:00 am and 3:00 pm. After being milked, the cows were allowed to exercise in an outside lot for one hour before returning to their stanchions. Herd health and husbandry programs were conducted by the Michigan State University Dairy Research Barn management personnel. Rumen Fluid and Blood Collection and Analysis: Rumen fluid and coccygeal tail vein blood samples were collected biweekly for each animal during the treatment period. The animals were sampled two hours after feeding. Rumen fluid samples were taken with a stomach tube, speculum and suction pump. Rumen fluid was analyzed for volatile fatty acids and ammonia. Ph was determined. Volatile fatty acids (VFA) were determined with a Hewlett-Packard gas chromatograph, model 5730A, equipped with a model 7671A automatic sampler and an Integrator-Recorder, model 3880A. The column was packed with graphited carbon, Carbowax B. Nitrogen was the carrier gas. The acid standards contained 0.1N each of acetic, propionic, 15 isobutyric, butyric, 2-methylbutyric, isovaleric and valeric acids in double distilled water. The carrier gas flow rate was forty milliliters per minute. The temperature program was initiated at 155°C for four minutes and prOgressed to 190°C at the rate of 40 per minute. Rumen fluid samples were first centrifuged at 10,000 x gravity for fifteen minutes and then the supernatant stored at -u°c until analyzed. Rumen ph was determined using a standard Beckman ph meter. Rumen ammonia concentrations were determined by using the phenol- hypochlorite colorimetric procedure (26), plasma urea was determined by using the phenol-hypochlorite colorimetric procedure (18), and plasma glucose by the glucose oxidase and peroxidase method (37). Feed Sampling and Analysis: The silage was sampled three times per week. A composite sample was made every two weeks and analyzed for dry matter and crude protein (3). Concentrates were also analyzed for dry matter and crude protein (3). The dry matter of silages and concentrates was determined by placing duplicate representative samples in an oven set at 90°C for twenty four hours (3). Samples were alloWed to cool in a dessicator and then weighed. Crude protein content of silages and concentrates was determined by the Macro-Kjedahl procedure (3). III. Nitrogen Balance Trial Six Holstein steers (420 kilograms) fitted with rumen cannulae were used in an experiment designed to test the effects of feeding malic acid on ration digestibility and nitrogen utilization as well as 16 rumen ammonia, plasma urea, plasma ammonia and volatile fatty acid concentrations. The experimental design was a 3 x 3 Latin Square. There were two animals per treatment. Malic acid was fed at the level of 0, 100 or 200 milligrams per kilogram body weight per day. Feeding Procedure: Steers were fed once daily at 8:00 am. The diets were composed of shelled corn and corn silage (ad libitum) on a 1:1 dry matter basis. Urea was supplemented to increase total dietary protein to 12% on a dry matter basis. A mineral supplement was also fed. Each morning the steers were fed individually according to how much feed had been consumed the previous day. The malic acid was carefully mixed with the diet of each individual steer. Management: After a five-week adaptation to the corn-urea diet, malic acid was fed for three periods of twelve days each. Feed intakes were recorded throughout each twelve day period. Urine and feces were collected for the last seven days of each twelve day period. When the experiment was completed, each steer had received all malic acid treatments. The steers were housed in metabolism stalls. Feces were collected each morning and afternoon. Blood and rumen fluid samples were taken before feeding, and then every two hours for twelve hours on the last day of each treatment period. Rumen Fluid and Blood Collection and Analysis: Rumen fluid samples were taken with an aspirator and plastic tube 17 through the rumen fistula. Rumen fluid was analyzed for ammonia and volatile fatty acids as previously described. The coccygeal tail vein blood samples were analyzed for plasma urea and plasma ammonia (18, 26). Feed Sampling and Analysis: Daily feed samples were taken and stored at -4OC. Feeds were composited for each period and analyzed for dry matter, nitrogen and acid detergent fiber (3). Feces and Urine Sampling and Analysis: Daily fecal samples were stored at -4°C. Composite samples were made for each seven day period for each animal and then analyzed for dry matter, nitrogen, and acid detergent fiber ( 3). Daily urine samples were stored frozen at -4OC. A composite sample was made for each seven day period for each animal. Urine samples were analyzed for nitrogen (3). RESULTS I. In Vitro Experiments Experiment I was a time study to determine the point after feeding when the rumen fermentation rate was maximal. Whole rumen contents were incubated for two hours. The highest rate of gas production occurred from samples obtained two hours after feeding. The first three hours after feeding samplings gave the highest gas production. By five hours after feeding, the gas production had diminished (Table 4). Thus the rumen microbial population was multiplying at its maximum capacity at two hours after feeding. This shOUId be when microbial growth factors such as malic acid would be in short supply. It is important to the rumen microbial population to have all essential nutrients present at optimum levels in order to achieve maximum growth rates. This enables more complete digestion of feeds to occur in the rumen because the number of bacteria is increasing at the maximal rate and more feed is broken down and absorbed. This creates a more effi- cient rumen fermentation. Two hours after feeding was the period selected for sampling of rumen fluid for demonstrating the effects of malic acid on the rumen fermentation. Experiment II compared the gas production rates when amylose or cellulose were substrates. As expected the amylose fermentation was much faster than the cellulose (Table 4). When solka floc was used as the only substrate source, the fermentation rate was too slow to detect differences between treatments. In the rumen the starch 18 19 Table 4. Gas production in the in vitro experiments. Experiment Treatment Total gas produced Time after feeding (ml) ‘(hourS)’ I --- 3.0 0 --- 4.0 0 -—- 27.8 1 --- 27.6 1 --- 31.2 2 --- 31.3 2 --- 23.8 3 --- 17.0 3 -—- 11.2 4 --- 13.7 4 --- 22.0 5 --- 20.6 5 --- 21.2 6 --- 21.6 6 --- 22.0 7 ——- 21.5 7 --- 20.1 8 --- 21.2 8 --- 18.1 9 --- 18.8 9 --— 20.5 10 --- 20.1 10 II Control 0 0 Control 2.4 0 5 grams starch 159.0 0 5 grams starch 151.3 0 5 grams cellulose 2.8 0 5 grams cellulose 4.0 0 Control 52.7 2.5 Control 65.4 2.5 5 grams starch 205.5 2.5 5 grams starch 192.1 2.5 5 grams cellulose 74.6 2.5 5 grams cellulose 62.7 2.5 Control 32-7 5 Control 39.7 5 5 grams starch 172.3 5 5 grams starch 167.7 5 5 grams cellulose 35.0 5 5 grams cellulose 31.5 5 Control 25.2 7.5 ContrOl 24.7 7.5 5 grams starch 154.0 7.5 5 grams starch 154.0 7.5 5 grams cellulose 26.9 7.5 5 grams cellulose 25.4 7.5 Table 4. Experi- ment III IV VII Continued Treatment Control Control 5 grams cellulose 5 grams cellulose 5 grams concentrate 5 grams concentrate Control Control 100mg malic acid 100mg malic acid 300mg malic acid 300mg malic acid 500mg malic acid 500mg malic acid Control Control 100mg malic acid 100mg malic acid 300mg malic acid 300mg malic acid 600mg malic acid 600mg malic acid Control Control 600mg malic acid 600mg malic acid 900mg malic acid 900mg malic acid 1200mg malic acid 1200mg malic acid Control Control 600mg malic acid 600mg malic acid 900mg malic acid 900mg malic acid 1200mg malic acid 1200mg malic acid Total gag produced Length E ( WK») um ) .8 .4 .3 .7 .3 .5 aarrc>co uocotcfié wN\OO\\»OOH \O\O\O\O\O\O\O(D \O\ka) VVF? g: Incubation (hours) NNNNNNNN HHHHHHHH HHHHHHHH NNNNNN NNNNNNNN 21 fermenting bacteria multiply much faster than the cellulose fermentors. Fermentors of readily available carbohydrate rapidly digest soluble sugars and starches whereas the cellulose fermentors must first attach to the fibrous substrate. Also, the cellulolytic population does not fluctuate nearly as much as the amylolytic. This enables members of the latter to increase more rapidly as substrates become available for digestion. Experiment III was a time study to determine at what time after feeding the carbohydrate fermentors were at their peak in terms of fermentation rate. Two and one half hours after feeding was when the rumen fluid sampled gave the maximum fermentation of amylose (Table 4). It became clear that fluctuation in the activity of rumen fluid samples from day to day made it difficult to identify amounts of carbo- hydrate or cellulose desired for optimum fermentation in the in_xitrg experiments. Total microbial cell counts or some other method is needed to determine the population density of the rumen fluid before levels of substrate required per flask can be defined. After the in 21229 technique had been refined, the effects of malic acid on the rumen fermentation rate was tested. Experiments IV, V, VI, and VII determined the effects of malic acid on the rumen fermentation rate. The levels used ranged from one hundred milligrams to twelve hundred milligrams malic acid added per bottle of incubation media. These amounts of malic acid were arrived at by considering levels fed during the production trial. Malic acid was fed at 0-140 grams per head per day. Thus, 70 grams malic acid in a 70 kilogram rumen was 0.001 grams malic acid per milliliter of fluid. The total volume of the in_xi§rg incubation media was 300 milliliters so 70 grams fed translated into 0.3 grams malic acid per flask. Malic acid increased 22 the gas production and volatile fatty acid production over controls. In all in vitro experiments volatile fatty acid production was directly correlated to gas production. II. Milk Production Trial The effect of malic acid on energy utilization for both early and mid lactation cows is shown in Table 5. Treatments A, B, C, and D correspond to 0, 70, 105, and 140 grams malic acid fed per head per day respectively. Cows fed the high level malic acid were most efficient in terms of milk produced per megacalorie of feed ingested. When malic acid was fed the efficiency was greater for the mid lactation cows than for the early lactation cows (Tables 6 & 7). However, differences between treatments were not significant. The high level of malic acid significantly increased the persis- tency of lactation over controls (Table 8). Since malic acid enhances milk production the cost of manufacturing and marketing malic acid as a feed additive should be examined to determine if it is profitable to use malic acid as a commercial feed additive. There were no significant differences between treatments for total dry matter intake (Table 9). There also were no significant differences between treatments for roughage dry matter intakes although stage of lactation tended to influence roughage dry matter intake (Table 10). This was because the early lactation cows were producing more milk so were receiving more concentrate in the ration than mid lactation cows. Consequently, the early lactation cows consumed less roughage. The control group of cows weighed the lightest (Table 11). After'balancing the animals for milk production, breeding groups, age Table 5. The effects of malic acid on energy utilization, early and mid lactation combined. FEED CONSUMED SILAGE (kg) CONCENTRATE (kg) TOTAL (kg) EIERGY CONSUMED SILAGE (0.51 Meal/kg) CONCENTRATE (1.76 Meal/kg) TOTAL (Mcal) ENERGY REQUIREMENTS BODY WEIGHT Tkg7 NE FOR MAINT. (Meal) ENERGY AVAILABLE FOR MILK (ENERGY INTAKE - MAINT.) (Meal) EFFICIENCY MILK PRODUCED (kg) kg MILK/Meal TREATMENTS C) N N K» but—3 wo N bur-5H O H \nNka) 620.4 10.5 Table 6. lactation cows. 24 The effects of malic acid on energy utilization in mid TREATMENTS A B C D FEED CONSUMED SILAGE (kg) 5.9 25.3 23.5 4.4 CONCENTRATE (kg) 9.4 9.2 10.3 9.7 TOTAL (kg) 35.3 34.5 33-8 34-1 ENERGY CONSUMED SILAGE (0151 Mcal/kg) 3.1 12.8 11.9 2.4 CONCENTRATE (1.76 Meal/kg) 6.6 16.2 18.2 12.0 TOTAL (Mcal) 9.7 29.0 30 1 9.4 ENERGY REQUIREMENTS BODY WEIGHT (kg) 581.6 650.8 674.8 640.8 NE FOR MAINT. (Mcal) 10.0 10.9 11.2 10.8 ENERGY AVAILABLE FOR MILK (ENERGY INTAKE - MAINT.) (Meal) 19.7 18.1 18.9 18.6 EFFICIENCY MILK PRODUCED (kg) 21.6 20.4 22.8 22.2 kg MILK/Meal 1.10 1.13 1.21 1.19 25 Table 7. Effects of malic acid on energy utilization in early lacta- tion cows. TREATMENTS A B C D FEED CONSUMED SILAGE (kg) 20.2 22.4 23 8 20.2 CONCENTRATE (kg) 12.6 11.6 12.0 12.1 TOTAL (kg) 32.8 34 0 35.8 32.3 ENERGY CONSUMED SILAGE (0.51 Meal/kg) 10.2 11.4 12.1 10.2 CONCENTRATE (1.76 Meal/kg) 22.2 20.5 21.1 21.4 TOTAL (Meal) 32.4 31.9 33.2 31.6 ENERGY REopIREMENTS BODY WEIGHT (kg) 559.3 567.7 600.8 600.0 NE FOR MAINT. (Meal) 9.6 9.7 10.3 10.3 ENERGY AVAILABLE FOR MILK (ENERGY INTAKE - MAINT.) (Meal) 22.8 22.2 22.9 21.3 EFFICIENCY MILK PRODUCED (kg) 31.1 28.7 29.3 29.9 kg MILK/Meal 1.36 1.29 1.28 1.40 26 .mo. a e6 ecceacasmam * mtm n H088 chUQSm *o.ma r.ar a.em c.mw a.oa c.0m o.ma m.ra N.aa r.ma n.aa m.m0a n.06a m.m0a m.aoa a m.am m.ma a.aa m.ma o.nm c.6r o.rr a.mr H.ma o.ca a.ca o.aa m.ma m.na N.N6a o c.aw N.aa m.ma a.mm H.ar m.rr o.rw H.0m a.rw m.ma o.mm m.cm a.am c.mm m.ooa m N.@@ N.ra e.or s.ar c.sm c.aa N.mr n.am a.ma N.Nm a.ea o.ma H.Na m.ea o.ra s .63. i: 9 Na 2 3 m1 m a b {w a D N a arrears are: .coapmwomfi mo hocopmHMHom so wfiom oaams mo mpoommc one .m manna Table 9. 27 The effects of malic acid on dry matter intake (kg) for all cows (cow # on left side of column). 1430 1359 1397 1407 1415 1458 1387 1385 AVG. 18.4 18.5 18.9 19.2 17.4 18.8 17.3 21.7 18.8 1345 1435 1456 1376 1282 1321 1417 1377 19.1 21.2 16.3 17.9 17.3 17.4 21.6 16.6 18.5 1350 1448 1352 1449 1400 1263 1418 1419 20.3 20.0 17.1 20.5 17.8 20.1 19.2 17.2 19.0 1442 1328 1364 1410 1269 1369 1302 1390 21.1 17.7 16.1 18.2 18.2 16.8 18.7 17.9 18.1 Standard error = 0.5 Table 10. 28 for all cows (cow # is on the left side of column). The effects of malic acid on roughage dry matter intake (kg) 1430 1359 1397 1407 1415 1458 1387 1385 AVG. 7.5 7.6 6.3 9.5 9.7 11.2 9.3 11.3 9.1 1345 1435 1456 1376 1282 1321 1417 1377 7.6 9.6 8.0 8.5 9.8 10.1 12.8 7-9 9-3 1400 1263 1418 1419 9.9 8.6 6.4 10.9 9-3 10.9 10.0 7-7 9.2 1442 1328 1364 1410 1269 1369 1302 1390 9.1 6.7 7.2 8.2 9.8 9.8 9.5 8.4 8.6 Standard error = 1.4 Table 11. 29 The effects of malic acid on average body weights (kg) for treatment period for all cows (cow # is on the left side of the column). 1430 1359 1397 1407 1415 1458 1387 1385 AVG. 488.7 631.9 539.7 577-0 589.8 592-4 571.5 572-9 570-5 1345 1435 1456 1376 1282 1321 1417 1377 576.4 606.1 546.9 541.1 635.6 707-7 672.8 586.9 609.2 1350 1448 1352 1449 1400 1263 1418 1419 671.6 566.6 597.8 567.4 585-? 772.1 698.7 642.6 637.8 1328 1364 1410 1269 1369 1302 1390 558-9 584.2 679.8 577.1 636.1 583-3 724.? 619.2 620.4 30 and days after calving, it was impossible to also evenly distribute the cows among the treatment groups according to weight. Since weight was a less important factor than those mentioned, it was given less attention. After treatments started early lactation cows lost and then regained weight. Late lactation cows were constantly gaining weight during treatment. Cows generally loose weight during the first third of the lactation due to the drain of energy from body tissue reserves caused by high milk production. Cows gain this weight back during the last two-thirds of the lactation. All groups receiving malic acid had significantly lower total dry matter intake as a percent of body weight than the controls (Table 12). No significant linear dose response was observed although the trend existed. Since the heavier cows received malic acid and produced more milk but consumed less feed as a percent of body weight, malic acid increased the feed efficiency of the cows. Roughage dry matter intake (percent of body weight) was not significantly different between treatments (Table 13). The percent fat in milk was not significantly different between treatments (Table 14). Since percent fat in the milk is a major factor in determining the price the farmer receives for milk, it is important to know malic acid does not decrease the fat test. There were no significant differences between treatments for percent protein or total solids in milk (Table 15 & 16). Plasma glucose concentrations were not different between treat- ments (Table 17). Malic acid could have been converted to propionate in the rumen, the propionate converted to glucose by the rumen microbes, and the glucose used up by the rumen microbes in various biosynthetic Table 12. 31 body weight (cow # is on the left side of column). The effect of malic acid on total dry matter intake (kg/100kg) 1430 1359 1397 1407 1415 1458 1387 1385 AVG O 3.76 2-93 3-49 3-33 2.94 3.17 3.02 3-78 3-30 1345 1435 1456 1376 1282 1321 1417 1377 3-31 3.50 2.98 3.30 2.71 2.45 3.20 2.88 3.04* 1350 1448 1352 1449 1400 1263 1418 1419 3.02 3-53 2.85 3.61 3.04 2.60 2.74 2.67 3.01* 1269 1369 1302 1390 3.77 3.02 2-37 3.14 2.85 2.87 2.58 2.89 2.94* Standard error’z 0.129 * Significant at p .05. 32 Table 13. The effects of malic acid on roughage dry matter intake (kg/100kg) body weight for treatment period for all cows (cow # is on the left side of column). A B C D 1430 1.527 1345 1.312 1350 1.473 1442 1.625 1359 1.197 1435 1.588 1448 1.525 1328 1.150 1397 1.165 1456 1.468 1352 1.066 1364 1.057 1407 1.651 1376 1.575 1449 1.917 1410 1.418 1415 1.642 1282 1.548 1400 1.590 1269 1.537 1458 1.930 1321 1.430 1263 1.409 1369 1.685 1387 1.623 1417 1.900 1418 1.425 1302 1.313 1385 1.977 1377 1.353 1419 1.202 1390 1.363 AVG. 1.589 1.522 1.451 1.394 Standard error = 0.072 Table 14. 33 The effects of malic acid on % fat in milk (cow # is on the left side of column). 1430 1359 1397 1407 1415 1458 1387 1385 AVG. 3.08 3.07 2.65 3-38 3.39 4.10 3.89 3-38 3-37 1345 1435 1456 1376 1282 1321 1417 1377 3.34 3.31 3.66 3.61 3.12 3.82 3.81 3-45 3.52 1350 1448 1352 1449 1400 1263 1418 1419 3.58 3.26 2.77 3.88 3.58 3.82 3-55 3.48 3-49 1328 1364 1410 1269 1369 1302 1390 3-37 2.81 3.66 3.62 3.61 3.10 3.69 3.76 3-45 Standard error'= 0.10 Table 15. The effects of malic acid on % protein in milk (cow # is on the left side of column). 1430 1359 1397 1407 1415 1458 1387 1385 AVG. 3-03 3.06 3-03 3.10 3.41 4.12 3-71 3.69 3-39 1345 1435 1456 1376 1282 1321 1417 1377 2.86 3.22 3°35 3-33 3.26 3-93 3.68 3.38 3-36 1350 1448 1352 1400 1263 1418 1419 3.14 3.44 2.98 3-25 3.66 3.94 3-39 3-71 3.44 1328 1364 1410 1269 1369 1302 1390 3.17 3-13 3.19 3-07 3.32 3-79 3.79 3-79 3.41 Standard error = 0.07 Table 16. 35 is on the left side of column). The effects of malic acid on % total solids in milk (cow # 1430 1359 1397 1407 1415 1458 1387 1385 AVG. 11.61 11.77 10.71 12.17 12.15 13.43 12.96 12.57 12.17 1345 1435 1456 1376 1282 1321 1417 1377 11 12. 12. 12 -93 08 73 .56 .08 .51 .25 .34 1350 1448 1352 1449 1400 1263 1418 1419 12.65 12.09 11.12 12-93 12.60 13.01 11.68 12.65 12.34 1328 1364 1410 1269 1369 1302 1390 12.02 12.02 12.66 12.31 12.46 12.67 13.23 12.43 Standard error = 0.18 36 - Table 17. The effects of malic acid on plasma glucose concentration in milligrams/100 ml of plasma (cow # is on the left side of column). 1430 41.8 1345 48.1 1350 45.7 1442 43.6 1359 50.7 1435 45.5 1448 46.1 1328 44.4 1397 45.9 1456 43.0 1352 43.1 1364 49.8 1407 48.2 1376 44.8 1449 4442 1410 47.2 1415 44.3 1282 53.1 1400 45.6 1269 52.1 1458 52.3 1321 49.9 1263 50.3 1369 46.8 1387 48.6 1417 51.6 1418 55.0 1302 48.1 1385 51.7 1337 49.6 1419 51.0 1390 43.1 AVG. 47.9 48.2 47.6 46.9 Standard error = 1.12 37 reactions. Plasma urea and rumen ammonia levels were not different between treatments (Tables 18 & 19). In the rumen malic acid is a potential source of oxalacetate and alphaketoglutarate needed for trapping free ammonia (6). This nitrogen is then incorporated into rumen microbial protein. When low quality protein is fed to dairy cattle, isobutyrate and other precursors directly involved in the synthesis of essential amino acids are limiting microbial growth in the rumen (13). It has been demonstrated that certain branched chain fatty acids stimulate production in ruminants and malic acid may function at the trans- amination reaction step to help trap free ammonia and direct it into microbial protein synthesis (6). However, if malic acid had increased the utilization of free rumen ammonia, plasma urea and rumen ammonia levels should have been lowered by the malic acid. Less ammonia would have been converted to urea by the liver and plasma urea concentrations would have been lowered. If malic acid enhanced ammonia uptake by the rumen microbes for microbial protein synthesis, rumen ammonia levels should have been lowered by malic acid. Further investigation will be conducted in the form of a nitrogen balance trial to determine the role of malic acid in the rumen. Rumen ph was not different between treatments (Table 20). Total volatile fatty acid concentrations in the rumen were not significantly different between treatments (Table 21). Total rumen acetate levels were not significantly different (Table 22). It was found that volatile fatty acid concentration was increased significantly in the early lactation cows fed malic acid but not in the mid lactation cows. Thus, all groups receiving malic Table 18. The effects of malic acid on plasma urea concentrations in milligrams/100 ml of plasma (cow # is on left side of column). 1430 1359 1397 1407 1415 1458 1387 1385 AVG. 14.66 13.15 13-57 13.52 16.64 17.49 15.28 15-35 14.96 1345 1435 1456 1376 1282 1321 1417 1377 13.31 14.80 13.77 14.34 15.92 12.98 12.39 15.88 14.17 1350 1448 1352 1449 1400 1263 1418 1419 11.83 16.48 13.76 14.04 12.76 14.83 17.03 17.23 14.74 1442 1328 1364 1410 1269 1369 1302 1390 16.45 13.22 11.59 13.15 12.49 13.84 16.18 15-95 14.11 Standard error = 0.58 39 Table 19. The effects of malic acid on rumen ammonia concentration in milligrams/100 ml of rumen fluid (cow # is on the left side of column). A B C D 1430 10.50 1345 15.27 1350 13.53 1442 23.78 1359 13.97 1435 17.46 1448 16.55 1328 13.37 1397 19.88 1456 22.59 1352 17.33 1364 14.90 1407 13.91 1376 19.06 1449 21.90 1410 20.97 1415 22.62 1282 18.22 1400 13.96 1269 15.68 1458 23.16 1321 14.13 1263 18.56 1369 23.55 1387 16.62 1417 19.54 1418 15.14 1302 16.74 1385 25.28 1377 25.41 1419 23.97 1390 13.09 AVG. 18.24 18.96 17.62 17.76 Standard error = 1.50 40 Table 20. The effects of malic acid on rumen ph (cow # is on the left side of column). A B C D 1430 7.1 1345 7.0 1350 7.1 1442 7.1 1359 7.3 1435 6.9 1448 7.0 1328 6.4 1397 7.0 1456 7.1 1352 6.6 1364 7.1 1407 7.3 1376 6.8 1449 7.1 1410 7.1 1415 6.9 1282 7.0 1400 6.9 1269 7.2 1458 7.0 1321 7.0 1263 7.1 1369 6.4 1387 7.2 1417 7.1 1418 7.3 1302 7.0 1385 6.9 1377 6.8 1419 6.8 1390 7.2 AVG. 7.1 7.0 7.0 6.9 Standard error’z 0.07 41 Table 21. The average concentration of total VFA content in mmoles/100 ml rumen fluid (cow # is on the left side of column). 1430 7.848 1345 9.056 1350 8.743 1442 8.318 1359 6.612 1435 10.556 1448 9.461 1328 10.556 1397 8.504 1456 8.699 1352 11.233 1364 7.604 1407 6.796 1376 9.639 1449 7.622 1410 9.127 1415 9.638 1282 9.723 1400 9.857 1269 7-533 1458 9.552 1321 8.458 1263 9.111 1369 12.069 1387 8.674 1417 8.996 1418 7.079 1302 9.907 1385 10.520 1377 11.095 1419 11.153 1390 8.355 AVG. 8.518 9.528 9.282 9.184 Standard error = 0.41 Table 22. 42 The effects of malic acid on the average concentration of total acetate content in mmoles/100 ml rumen fluid (cow # is on left side of column). 1430 1359 1397 1407 1415 1458 1387 1385 AVG. 4.663 4.282 4.819 4.125 6.114 6.205 5.884 6.831 5-365 1345 1435 1456 1376 1282 1321 1417 1377 5-545 6.014 5.519 5.666 6.261 5.571 5-927 6.828 5.916 1350 1448 1352 1449 1400 1263 1418 1419 5.409 6.216 6.263 4.912 5.908 6.049 4-571 6.895 5-778 1442 1328 1364 1410 1269 1369 1302 1390 5-405 5-605 4.619 5-765 4.628 7.410 6.342 5.198 5.622 43 acid in early lactation had significantly increased production of volatile fatty acids (Table 23). This increase in total volatile fatty acids was due to significant increases in acetate, isobutyrate, butyrate, 2-methyl butyrate and isovalerate Tables 24, 26, 27, 28, and 29). Propionate and valerate were also higher but treatment effects were not significant (Tables 25 & 30). The mode of action of malic acid is not clear. It increases volatile acid production in early lactation cows. Malic acid could trap free ruminal ammonia and increase microbial protein synthesis or it may supply a source of oxalacetate for gluconeogenesis by the rumen microbes. The nitrogen balance trial was next undertaken to determine more definitely the effect of malic acid on the rumen fermentation. III. Nitrogen Balance Trial Malic acid has been shown to increase nitrogen retention in grow- ing steers and heifers and enhance milk yields in lactating cows. It has been proposed that malic acid promotes more efficient utilization of ammonia by rumen microbes. However, a more efficient uptake of ammonia by the rumen microbes was not reflected in the data taken from the lactating cows. Thus, the nitrogen balance trial was designed to examine the effects of malic acid on the plasma urea, plasma ammonia, rumen ammonia and volatile fatty acid production as well as nitrogen metabolism and digestibility. The fistulated steers (420 kilograms) consumed approximately eighteen kilograms feed per head per day (Table 31). Their urinary excretion rate was about eight kilograms per head per day (Table 31). Their fecal excretion rate was approximately ten kilograms per head per day (Table 31). 44 Table 23. The effects of malic acid on the average concentration of total VFA content in mmoles/100 ml of rumen fluid for early lactation (cow # is on left side of column). A B C D 1430 7.848 1345 9.056 1350 8.743 1442 8.318 1359 6.612 1435 10.556 1448 9.461 1328 10.556 1397 8.504 1456 8.699 1352 11.233 1364 7.604 1407 6.796 1376 9.639 1449 7.622 1410 9.127 AVG. 7.440 9.488* 9.265* 8.901** Standard.error'= 0.48 * Significant at p .05 **Significant at p .10 45 Table 24. The effects of malic acid on the average concentration of acetate in mmoles/100 ml rumen fluid for early lactation (cow # is on left side of column). A B C D 1430 4.663 1345 5.545 1350 5.409 1442 5.405 1359 4.282 1435 6.014 1448 6.216 1328 5.605 1397 4.819 1456 5.519 1352 6.263 1364 4.619 1407 4.125 1376 5.666 1449 4.912 1410 5.765 AVG. 4.472 5.686* 5.700* 5.348* Standard error = 0.18 * P -05 Table 25. 46 The effects of malic acid on the average concentration of propionate in mmoles/100 ml rumen fluid for early lactation (cow # is on left side of column). 1430 1359 1397 1407 AVG. 1-979 1.306 2.400 1.486 1.793 1345 1435 1456 1376 1.963 3.052 1.709 2~37? 2.275 1350 1448 1352 1449 1.840 1-759 3-115 1.438 2.038 1442 1328 1364 1410 1-597 3.092 1.653 1.752 2.024 Standard error'= 0.28 Table 26. 47 The effects of malic acid on the average concentration of isobutyrate in mmoles/100 ml rumen fluid for early lactation (cow # is on the left side of column). 1430 1359 1397 1407 AVG. .057 -055 -055 .062 ~05? 1345 1435 1456 1376 .077 .066 .074* 1350 1448 1352 1449 .082 .083 .071 .071 -077* 1442 1328 1364 1410 .072 .067 .072 .082 .073* Standard error = 0.004 * P .05 Table 27. The effects of malic acid on the average concentration of butyrate in mmoles/100 ml rumen fluid for early lactation (cow # is on the left side of column). 1430 1359 1397 1407 AVG. .975 .811 .981 .903 .918 1345 1435 1456 1376 1.184 1.160 1.142 1.211 1.174* 1350 1448 1352 1.152 1.156 1.409 .979 1.174* 1442 1328 1364 1410 1.005 1.282 .985 1.309 1.145* Standard error'= 0.053 * P .05 49 Table 28. The effects of malic acid on the average concentration of 2-methyl butyrate in mmoles/1OO ml rumen fluid for early lactation (cow # is on the left side of COIUmn). A B C D 1430 .059 1345 .118 1350 .083 1442 .087 1359 .051 1435 .082 1448 .084 1328 .077 1397 .065 1456 .084 1352 .111 1364 .109 1407 .083 1376 .079 1449 . 079 1410 .069 AVG. .065 .091* .088* .086** Standard error’= 0.008 *P -05 **p .10 50 Table 29. The effects of malic acid on the average concentration of isovalerate in mmoles/1OO ml rumen fluid for early lacta- tion (cow # is on the left side of column). 1430 .042 1345 .069 1350 .064 1442 .054 1359 .045 1435 .044 1448 .060 1328 .049 1397 .037 1456 .060 1352 .058 1364 .062 1407 .042 1376 .056 1449 .054 1410 .062 AVG. .042 .057* .059* .057* Standard error = 0.004 *P .05 Table 30. 51 The effects of malic acid on the average concentration of valerate in mmoles/100 ml rumen fluid for early lactation (cow # is on the left side of column). 1430 1359 1397 1407 AVG O .072 .071 ~139 .096 -O95 1345 1435 1456 1376 .102 .139 .105 .183 .132 1350 1448 1352 1449 .112 .104 .207 .094 o 129 1442 1328 1364 1410 .099 .185 .105 .107 0124 Standard error = 0.018 52 Table 31. The effect of malic acid on feed intake, feces and urine excretion. TREATMENT L H H L PERIOD II FEED (kg) 17.0 18.6 14.8 6 17.6 FECES (kg) 9.6 7.3 7.1 .8 10.6 URINE (kg) 5.4 8.5 6.4 2 7.1 TREATMENT C L L C PERIOD III FEED (kg) 14.2 21.0 18.7 23.0 18.6 FECES (kg) 8.2 9.8 8.9 13.6 11.2 URINE (kg) 6.4 12.2 6.3 6 1 6.9 TREATMENT H C C H PERIOD IV FEED (kg) 13.6 20.1 12.1 2.4 18.0 18.6 FECES (kg) 6.1 10.0 4.7 5 8 10.2 8.7 URINE (kg) 6.9 16.2 7.1 8 6 6.7 5.3 53 Percent dry matter digestibility of the ration increased with the level of malic acid fed but the treatment differences were not signi- ficant (Table 33). The digestibility of acid detergent fiber in the ration increased as the level of malic acid fed increased but the treatment differences were not significant (Table 34). Animals receiving malic acid showed trends of increased nitrogen retention as a percent of total nitrogen (fed and absorbed) but treat- ment differences were not significant (Tables 35 and 36). Percent protein digestibility of the ration was not different between treatments (Table 37). Rumen ammonia, plasma urea and plasma ammonia concentrations were not significantly different between treatments (Tables 38, 39 and 40). Rumen prOpionate was significantly higher in animals receiving malic acid than controls (Tables 41, 42 and 43). These results demonstrate that malic acid is affecting the rumen fermentation not by aiding in the incorporation of free ammonia but by stimulating the production of the volatile fatty acid propionate. This would increase gluconeogenesis in the rumen and liver and thus, increase the efficiency of the rumen fermentation. 54 Table 32. The effect of malic acid on daily dry matter intake (kg) . ANIMAL CONTROL Emit)?” HIGH 435 6.0 7.8 5.6 436 8.3 8.9 8.6 437 5.0 8.0 6.8 438 11.4 5.1 8.4 439 9.7 7.4 9.1 440 7.9 8.1 7.7 AVERAGE 8.0 7.6 7.7 Standard error = 1.6 55 Table 33. The effects of malic acid on percent dry matter digestibility. ANIMAL CONTROL TREAEEOVVM HIGH 435 71.4 74. 3 77. 2 436 75.0 78.7 83.7 437 79-4 77-3 79.4 438 74. 2 77.0 66.6 439 74.6 74.7 74.6 440 71.8 72.1 78 . 1 AVERAGE 74.4 75.7 76.6 Standard error = 1.33 56 Table 34. The effects of malic acid on percent acid detergent fiber digestibility. ANIMAL CONTROL TREAtggNT HIGH 435 56.7 60.3 62.7 436 61.8 63.6 69.8 437 74.9 67.8 63.0 438 57.4 68.0 43.8 439 52-3 58-1 55-3 440 45.7 46.6 65.1 AVERAGE 58.1 60.7 60.0 Standard error = 2.6 Table 35. The effects of malic acid on nitrogen retained as a percent of nitrogen fed (total nitrogen retained). ANIMAL CONTROL TREAtggNT HIGH 435 29.4 38.7 27.1 436 24.9 49.9 45.0 437 17.7 44.6 11.6 438 34.4 21.9 36.6 439 49.3 37.4 46.2 440 42.0 33.0 42.6 AVERAGE 33.0 37.5 34.9 Standard error*= 3.94 58 Table 36. The effect of malic acid on nitrogen retained as a percent of nitrogen absorbed (% digestible nitrogen retained). ANIMAL CONTROL TREAtgfiNT HIGH 435 42.8 57.8 37.2 436 36.1 64.7 58.8 437 24.1 59.6 16.1 438 52.6 31.1 61.9 439 67.3 51.2 65.8 “40 59-5 49-5 56-9 AVERAGE 47.1 52.3 50.7 Standard error = 5.05 59 Table 37. The effect of malic acid on percent protein digestibility. ANIMAL CONTROL TREAEEENT HIGH 435 68.6 66.9 72.9 436 69.1 77.1 69.7 437 73.6 74.8 72.2 438 65.4 70.4 59.1 439 73.2 73.0 70.2 440 70.6 68.0 74.8 AVERAGE 70.1 71.7 69.8 Standard error = 1.53 60 Table 38. The effect of malic acid on rumen ammonia (mg %). ANIMAL CONTROL TREAEWT HIGH 435 15-19 19-77 13-94 436 18.43 17.99 14.12 437 17.33 19.47 20.07 438 17.35 13.83 19.02 439 8.74 11.32 15.44 440 15.58 13.00 11.78 AVERAGE 15.44 15.90 15.73 61 Table 39. The effect of malic acid on plasma urea (me %>- ANIMAL CONTROL TREAEESNT HIGH 435 7.30 8.95 6.81 436 10.95 10.18 5.88 437 7.27 10.60 11.08 438 6.77 7.11 5.59 1+39 7-91 9-37 8.97 440 6.77 4.76 8.66 AVERAGE 7.83 8.50 7.83 62 Table 40. The effects of malic acid on plasma NH (ug/100 ml). 3 ANIMAL CONTROL IITREAESSNT HIGH 435 116.7 88.1 123.6 436 125.8 88.3 74.0 437 138.8 101.1 70.2 438 83.5 134.7 110.7 439 62.7 115.7 98.4 440 97.4 91.1 138.1 AVERAGE 104.2 103.2 102.5 63 Table 41. The effect of malic acid on rumen volatile fatty acid production (millimoles/lOO ml). ‘7‘ CONTROL 435 436 437 438 439 440 AVERAGE ACETATE 6.780 7.110 6.654 5.322 3.682 6.231 5.963 PROPIONATE 1.727 1.877 1.844 1.643 1.320 1.361 1.629 ISOBUTYRATE 0.092 0.159 0.187 0.082 0.053 0.108 0.114 BUTYRATE 1.309 0.992 1.049 1.331 0.828 0.894 1.067 2-MEI‘HYL BUTYRATE 0.090 0.229 0.209 0.095 0.068 0.136 0.138 ISOVALERATE 0.084 0.124 0.154 0.056 0.034 0.094 0.091 VALERATE 0.171 0.268 0.302 0.255 0.079 0.202 0.213 TOTAL 10.253 10.759 10.399 8.784 6.064 9.026 9.215 Table 42. The effect of malic acid on rumen volatile fatty acid production (millimoles/100 ml). 435 436 337 Low 438 __ 439 440 AVERAGE ACETATE 6.803 4.587 5.868 6.199 7.000 5.490 5.991 PROPIONATE 2 . 022 1 . 301 1 .883 1. 894 2 . 416 1 .897 1 . 902* ISOBUTYRATE 0.131 0.155 0.156 0.133 0.192 0.080 0.141 BUTYRATE 1.520 0.973 1.123 0.948 1.117 1.197 1.146 2-MEI'HYL BUTYRATE 0.149 0.141 0.131 0.139 0.183 0.172 0.152 ISOVALERATE 0.099 0.121 0.135 0.092 0.151 0.066 0.111 VALERATE 0.187 0.208 0.242 0.238 0.323 0.202 0.233 TOTAL 10.911 7.486 9.538 9.643 11.382 9.104 9.676 *P -05 65 Table 43. The effect of malic acid on rumen volatile fatty acid production (millimoles/100 ml). I'HGH 435 436 437 438 439 440 AVERAGE ACETATE 7.671 5.733 6.272 4.700 5.477 7.159 6.169 PROPIONATE 4.175 2.660 1.923 1.738 1.459 3.296 2.542* ISOBUTYRATE 0.125 0.080 0.111 0.195 0.136 0.148 0.132 BUTYRATE 1.358 1.526 1 .203 0. 659 0.857 1.278 1 .147 2-METHYL BUTYRATE 0.162 0.139 0.137 0.075 0.115 0.200 0.138 ISOVALERATE 0.132 0.072 0.139 0.087 0.087 0.144 0.110 VALERATE 0.414 0.224 0.152 0.099 0.151 0.377 0.236 TOTAL 14.037 10.434 9.937 7.553 8.282 12.602 10.474 * p -05 L DISCUSSION In the rumen alpha-ketoglutarate is considered to be the universal compound for trapping free ammonia for formation of amino groups for microbial protein synthesis. However, there has always been a question as to what is the source of alpha-ketoglutarate in the rumen. It is not from citrate since anaerobic bacteria do not oxidize compounds to carbon dioxide and water and Krebs cycle enzymes are not present in the rumen to any great extent. However, succinate is a major metabolic intermediate in the rumen and malic acid can provide a source of succinate. Allison has shown that succinate can be directly carboxylated to form alpha-ketoglutarate (2). Also, Wolin demonstrated that certain rumen bacteria, when grown on lactate media, have increased growth when supplied malic acid (22). This is due to an oxalacetate deficiency. Oxalacetate is drawn off for formation of various cellular constituents namely carbohydrate. Malic acid is thus providing a source of oxalacetate. The aspartate transamination, like the glutamate transamination, may be important but its significance is not known at this time. Glutamate and aspartate carbon are utilized for the synthesis of several essential amino acids. Thus, malic acid, for the reasons previously mentioned, is a key intermediate in rumen fermentation and deserves further study both ‘in vitro and in vivo. 66 67 REACTIONS IN THE RUMEN H20 e- GLYOXYLATE + ACETATE —-— MA —L- FUMARATE e- AV/OAA CITRATE [GLUTAMATE {a 113 SUCCINATE alpha Kg lpha KS \_/ V 00/ fASPARTATE] ATP, NH NH ,e- / 3 PROPIONATE ASPARAGINE GLUTAMATE NH . ATP LYSINE,METHIONINE 3 THREONINE GLUTAMINE ORNI INE , PROLINE ARGININE, HISTIDIN E SUMMARY AND CONCLUSIONS In 31339 rumen fermentation experiments, one milk production study, and one nitrogen balance trial were conducted to investigate the effects of malic acid on rumen fermentation and milk production. 1. The in yitgg experiments showed that malic acid increases the fermentation rate by increasing gas and volatile fatty acid production. 2. malic acid significantly increased the persistency of lacta- tion over controls (95 vs. 88%). The early lactation cows receiving malic acid had increased total volatile fatty acid production due to significant increases in acetate, isobutyrate, butyrate, 2-methyl butyrate and isovalerate. Propionate was also higher but treatment effects were not significant. 3. The nitrogen balance trial was undertaken to determine the mode of action of malic acid in ruminants. Malic acid treatments significantly increased rumen propionate over controls. In conclusion further research is needed to determine if malic acid could be incorporated into rations for growing beef cattle. This study indicates malic acid increases propionate production which has been shown to increase growth rates of growing beef cattle. Malic acid increased lactation persistency seven percent. Manufacturing and marketing costs for malic acid would have to be lower than the income from the additional milk produced to make malic acid profitable for lactating dairy cattle. 68 LITERATURE CITED 10. 11. LITERATURE CITED Alferez, J. C. A comparison of four levels of malic acid for milk production of dairy cows. Master's thesis, Utah State University Department of Animal Science, Logan, Utah, 84322. Allison, M. J. and I. M. Robinson. Biosynthesis of -ketaglutarate by the reductive carboxylation of succinate in Bacteroids ruminicola. Journal of Bacteriology, October 1970, pp. 50-56. Andrejew, Anatole, Marie-Therese, Orfanelli and Desbordes. Use of malate by various mycobacteria. C. R. Hebd Seances Acad. Sci. Ser D Sci. Association of Official Agriculture Chemists. Official methods of analysis. 1965 (10th edition), Washington, D. C. Boland, R. L. and B. Garner. Determination of organic acids in tall fescue (Festica arundinacea) by gas-liquid chromato- graphy. Journal of Agricultural and Food Chemistry (1973): 21, 661-665. Bujak,l§p. §;,, Stanislaw, and Dabkowskil. Nutritional require- ments of yeasts Schizosacchasomyces acidodevoratus decompos- ing L malic acid. Acta Microbial Polon, 11 (4), 373-381, 1962. Chan, H. T. Jr. Nonvolatile acids in lychee. Journal of Food Science (1974): 39 (A): 792-793. Cook, R. M. Personal Communication. Michigan State University Dairy Science Department. East Lansing, Michigan, 48823. Cummings, G. A. and M. R. Teel. Effect of nitrogen, potassium and plant age on certain nitrogenous constituents and malate content of orchardgrass. (Dactylis glomerata). Agronomy Journal, 1965 , 57: 127-129. Daniel, J. W. The metabolism of L and DL malic acids by rats. Food Cosmet. Toxicology, 1969, 7: 103-106. Divies, C. and M. H. Siess. Study of L malic acid catabolism by Lactobacillus casei cells immobilized in polyacrylamide gel lattice. ANN Microbiol (Paris), 127B (4), 525-539. 1976. 69 12. 18. 19. 20. 21. 22. 23. 24. 25. 26. 70 El-Shazly, K. and R. E. Hungate. Fermentation capacity as a measure of net growth of rumen microorganisms. Applied Microbiology, January 1965, Volume 13: 62-68. Fauconneau, G. Levels of organic acids in plants. Ann. Agron. Supp., 1: 1-13. Felix, A. Effect of supplementing corn silage with isoacids and urea on high producing dairy cows. Ph.D. thesis, Michigan State University Dairy Science Department, East Lansing, Michigan , 48823. Flesch, P. and D. Jerchel. PrOpagation of Bacterium gracile in natural culture mediums containing L malic acid. Mitt. Klosterneuburg , 10A: 1-1 3 , 1960 . Hare, R. The classification of the anaerobic cocci. Internati. Congr. Microbiol. Proc., Sixth, 1: 55-60, 1955. Hungate, R. E., et. al., D. W. Fletcher and R. W. Dougherty. Microbial activity in the bovine rumen; its measurement and relation to bloat. Applied Microbiology, 3: 161. Johnson, L. A. and D. E. Carroll. Organic acid and sugar content of Scuppunong grapes during ripening. Journal of Food Science (1973). 38: 21-24. Kulasek, G. Determination of urea in plasma using urease and phenol reagent. Po. Arch. Wet., 1972, 15: 801. Lehninger, A. L. 1970. Biochemistgy, Worth Publishers, Inc., New York, New York, 10011. Li, K. C. and J. C. Woodroff. Gas chromatographic resolution of nonvolatile fatty acids in peaches. Journal of Agricultural and Food Chemistry, 1968, 16: 534-535. Linehan, B., gt, al., C. C. Scheifinger, and M. J. Wolin. Nutritional requirements of Selenomonas ruminantium for growth on lactate, glycerol, or glucose. Applied and Environmental Microbiology, 1978, 35: 317-322. London, J. and E. Meyer. Malate utilization by a group D streptococcus. Journal of Bacteriology, 102 (1), 1970. Manufacturing Chemist and Aerosol News. December 1964. Matsushima, Kin'Chi. Action of poly L malic acid on various proteolytic enzymes. Ag. Biol. Chem. 34 (11), 1741-1744. McBee, R. H. Manometric method for the evaluation of microbial activity of rumen with application to utilization of cellulose and hemicellulose. Applied Microbiology, 1953, 1: 106. 27. 28. 29. 30. 31. 32. 33- 34. 36. 37. 71 Okuda, H. and S. Fuji. A direct colorimetric determination of ammonia. Tokushima J. Exp. Med., 12, 11, 1965. Perez, C. B. and C. D. Story. The effect of nitrate in nitrogen fertilized hays on fermentation in vitro. Journal of Animal Science, 19: 1311, 1960. Plocharski, W. and J. Zaleski. Effects of mineral fertilizing on the level of nonvolatile acids in strawberries. Chemi Zywnosci (1973) 23, 279-290. Inst. Sadovnictiva, Skierniewice, Poland. Quin, J. I. Studies on the alimentary tract of Merino sheep in South Africa. VII Fermentation in the forestomachs of sheep. Onderstepoort. J. Vet. Sci., 18: 91, 1943. Reid, R. L. and B. Clark. Relationship of forage digestibility and intake data to in vitro and in_vivo fermentation indices. Journal of Animal Science, 19: 1312, 1960. Rumsey, T. S., gt. al., C. H. Noller, C. L. Rhykerd, and J. C. Burns. Measurement of certain metabolic organic acids in forages, silages, and ruminal fluid by gas-liquid chromato- graphy. Journal of Dairy Science, 1967, 50: 214—219. Ryan, J. J. and J. A. Dupont. Identification and analysis of the major acids from fruit juices and wines. Journal of Agricultural and Food Chemistry (1973), 21: 45-49. Schmedt, H., 33. al., G. Huskens and D. Jerchel. 0n the degrada- tion of the CT“ malic acid by Bacterium gracile. Arch. Mikubiol., 43 (2), 162-171, 1962. Stallcup, O. T. Personal Communication. Department of Animal Sciences, University of Arkansas, Fayetteville, Arkansas. The Merck Index. Wagner, H. G. and F. Porter. The effect of maturity and variety on the content of the major organic acids of the green pea (Pisum sativum). Journal of the Science of Food and Agri- culture71973), 24: 69-75. Washko, M. E. Determination of glucose by the glucostat proce- dure. Clinical Chemistry, 7: 542 (1961). APPENDIX 72 Table 44. The effects of malic acid on the analysis of variance for persistency of lactation for all cows. AOV - Persistency Observation d.f. s.s. m.s. Trt 3 233-92 77-97 S of L 1 1530.42 1530.42 (Trt)x(S of L) 3 91.47 30.49 Block 3 653.61 217.87 Error(8-1)(4-1) .21 891.28 42.44 Total 31 Treatment C 1 2 3 1 2 2 2 2 Block 3» 2 2 2 2 4 2 2 2 2 SS 379.32 + 3252 + 397.82 + 320.42 + TL 2 , 2 2 2 4359-“ “t “00-9 '2 332'2 + 380'3L -261961.31 - 233.92 - 1530.42 88 263,817.12 - 263,725.65 = 91.47 TL 73 Table 45. The effects of malic acid on the persistency (blocked according to milk production) (cow # is on the left side of column) and sum of squares of persistency is for all cows. 1 2 3g 4 1397 85.5 1359 95.8 1430 100.0 1407 98.0 1345 87.6 1435 98.5 1456 94.6 1376 117.1 1352 89.4 1350 92.0 1448 103.5 1449 95.4 1328 88.1 1442 104.7 1410 106.3 1364 101.8 1385 85.9 1415 73.6 1387 78.1 1458 87.4 1377 69.2 1417 91.1 1282 74.3 1321 85.8 1419 81.4 1263 77.8 1418 82.0 1400 91.0 1302 82.6 1390 88.1 1269 94.4 1369 94.3 x 669.7 721.6 733.2 770.8 SSB = 669.72 + 721.62 + 733.22 + 770.82 8 SSB = 262,614.92 - 261,961.31 = 653.61 SS = 135598032 “I" 1337002 "' 2619961031 :- L 16‘ SSL = 263,491.73 - 261,961.31 = 1530.42 74 - Table 46. The effects of malic acid on test of the significance of persistency of lactation for all cows. H: t = 0 (trt) i Mst f: MS; vs f, t—1, (t-1) (r-1) f-77.7_181+ f 02513921.:102‘1'8 - 420L114”- 0 f .10, 3, 21 = 2.36 Standard error of treatment means 42. yi-t 111-EE- = ——§-u-li : 2.3 Treatment A B C D 88.2:2.3 89.6:2.3 89.3:2.3 95.0:2.3 Did blocks decrease experimental error used to test treatment effects? MS f: "MS-:- _—. 242724 = 5.13 vs. f , r-1, (t-l) (r-l) f .025, 3, 21 a 3.82 f 0005: 3: 21 = 5°73 What about treatment block interaction? It must be not significant in order to use MS to test treatments. E S _ 12 207.22 _ 8 08 SM ‘ (23392783361) ‘ '1 _ 8.98 f ‘ 891.28 - 8.98/20 VS f .25, 1, 20: 1.40 f: 8.98/44.1 a 0.20 .'. treatments and blocks don't interact 75 Test of interaction of treatments and stage of lactation .. TXL_M_ e _ f — MSE — 42.44- 0-72 VS i ~50: 3: 21 - ~815 . . no interaction exists between treatments and stage of lactation 76 Table 47. The effects of malic acid on the orthogonal test of persis- tency of lactation for all cows used to determine which treatments are different. A vs all others ‘3: 1: 19 1 (+3 (705.6) 2 (716.8 + 714.4 + 760))2 = 5535.4 Egg—'3 = 57.7 15:—21% = 1.36 B vs C & D 0, -2, 1, 1 (+2 (716.8) - (714.4.. 760))2 = 1664.6 1664.6 _ 39.7 _ "48" - 3‘“? 42.4 - 0-82 C vs D 0, 0. 1 -1 (714.4 - 760)2 = 2079.4 2 .4 0. 01 = 31%??? = 3.1 P .10 D vs A3_B, C ‘3: 1: 1: 1 (3 (760) - (21368))2 = 20,506.2 20 06.2 _ 213.6 _ __ng———._ 213.6 “2.4.. 5.0 P .05 C.V. = f 1’ 1’1-I. 32- 1, 21 (705.6 - 760)2 = 2959.4 77 2 9.4_ 18 _ ‘2IK_" 185.0 442.4- 4.36 P .05 Linear Dose Response 0, '1, O, 1 (-716.8 + 760)2 = 1866.2 f .10, 1, 21 s 2.96 1866.2 _ 116.6 _ T“ 42.4" 2'75 P '12 78 Table 48. The effects of malic acid on the analysis of variance for total dry matter intake (all cows). Observation d.f. s.s. m.s. Trt 3 13.3 4.4 S of L 1 6.2 6.2 (Trt) x (S of L) 3 10.9 3.6 Block 3 75.3 25.1 Error 2;. 260.9 12.4 Total 31 SSy = 54,094.6 - 53,728.0 = 366.6 2 2 2 SST = (331.04 +4325.6 g 335.72 + 318.8 )_= 53,728 = 13.3 ssB = 53.803-3 - 53.728 = 75.3 SS _ 662.612 + 648.612 _ 728 _ 6 2 L ‘ 16 53. - . SSsz = 53,758.4 - 53,728 - 13.3 - 6.2 = 10.9 H: ti = 0 (For DMI) f = %§% vs f , t-1, (t-1) (r-1) “'2 0.35 f1, 50, 3, 21 = 0.8 No differences Std. error for Total DMI fl = 1024 r 79 Table 49. The effects of malic acid on the analysis of variance for roughage dry matter intake (all cows). Observation d.f. s.s. m.s. Trt 3 11.7 3.9 S of L 1 108.1 108.1 (Trt) x (S of L) 3 23.5 7.8 Block 3 80.2 26.7 Error 21 1725.4 82.2 Total 31 SSy = 14,667.8 - 12,718.9 = 1948.9 SST :- 12,730.6 - 12,718.9 = 11.7 SS 12.799-1 - 12,718.9 = 80.2 B 2 2. _ Q90 + 348.04 ) _ SSL - 1677 - 12,718.9 — 108.1 SSTxS = 9111038 230445211 — 12,718.9 - 11.7 - 108.1 - 23.5 H: t = (ForR DMI) MST f: fivs f ,t-1, (t-1) (r-1) f=8%%=0.05 f 50, 3, 21:0.8 No differences H: L = 0:0 f=l§§—:%=1.3 f .25,1,21=1.40 Stage of lactation tends to influence roughage DMI Std. error for R. DMI = 3.20 8O - Table 50. The effects of malic acid on analysis of variance for total dry matter intake/100 kg body weight for all cows. Observation d.f. s.s. m.s. Trt 3 0.613 0.204 S of L 1 0.949 0.949 (Trt) x (S of L) 3 0.155 0.052 Block 3 0.037 0.012 Error 2;_ 2.795 0.133 Total 31 SSy = 307.461 — (302.912) = 4.549 _ 26.4572 + 24.3672 + 24.1062 + 93.5242 8 SST _ — 302.912 a 0.613 2 2 2 SSB = 24.6172 + 24.886 8 24.780 + 24.171 _ 302.912 = 0 037 51.9822 + 46.4722 SSL 16 - 302.912 = 0.949 53 _ 13-524§ + 13-103: + 13.035§ + 12.320 sz ' 12.933 + 11.264 + 11.071 + 11.204 1: 2 2+ - 302.912 - 0.613 - 0.949 = 0.155 H: t = 0 (For total DMI/100 kg BW) f: _ VS. f 9 13.1! (t-1) (11'-l) 1.48 2.36 0.204 m= 1-53 f -25. 3. 21 f .10, 3, 21 Std. error (for total DMI/100 kg bw) 81 Table 51. The orthogonal tests of total dry matter intake/100 kg body weight used to determine which treatments are different. A vs allgothers '39 1: 1: 1 (3 (26.457) - (24.367 + 24.106 + 23.524))2 a 54.376 :55#ZZ§-— 0.566 g=f§%-= 4.26 P .05 B vs C & D O, “2, 1, 1 (2 (24.367) - (24.106 + 23524))2 = 1.219 1.21 _ .025 _ 27378'“ 0'025 .133 ' 0'188 (24.106 — 23.524)2 = 0.159 .339 _ .021 _ 2 x 8 _ 0.021 .133 _ 0.159 C.V. = f , 1, 21 f1 .05, 1, 21 = 4.32 f1 .10, 1, 21 = 2.96 f1 .25, 1, 21 = 1.40 D vs all others (3 (23.524) - (26.457 + 24.367 + 24.106))2 lag—293: .198 %%= 1.49 P .25 18.992 24.367)2 = 4.368 349%: 0.273 942—71: 2.05 P .25 /'\ N O\ 1:- kn \) l A vs C (26.457 24.106)2 = 5.527 .27_ 0. _ 1%.. 0.345 $5.133 _ 2.59 P .25 Linear Dose Respgnse O, -1, O, 1 (24.367 - 23.524)‘2 = 0.711 0.711 _ .044= 0. 16 ‘ 0'0““ 0.133 331 f .50, 1, 21 = .47 No linear response A vs D (26457 - 23.524)2 = 8.602 8.602 _ .538 = P . O '7fi§_-_ 0.538 .133 4.04 1 B vs D (24.367 - 23.52402 :: 0.711 0.711_ .044: 16 -0.044 .133 0.33 83 Table 52. The effects of malic acid on roughage dry matter intake kg/100 kg body weight for treatment period for all cows (cow # in on the left side of column), and the analysis of variance for roughage dry matter intake/100 kg body weight. A B 1430 1.527 1345 1.312 1350 1.473 1442 1.625 1359 1.197 1435 1.588 1448 1.525 1328 1.150 1397 1.165 1456 1.468 1352 1.066 1364 1.057 1407 1.651 1376 1.575 1449 1.917 1410 1.418 1415 1.642 1282 1.548 1400 1.590 1269 1.537 1458 1.930 1321 1.430 1263 1.409 1369 1.685 1387 1.623 1417 1.900 1418 1.425 1302 1.313 1385 1.977 1377 1.353 1419 1.202 1390 1.363 AVG. 1.589 1.522 1.451 1.394 x2 20.811 18.760 17.295 15.874 Tests for roughage DMI/100 kg B.W., all cows Observation d.f. s.s. m.s. Trt 3 0.173 0.058 s of L 1 0.153 0-153 (Trt) x (S of L) 3 0.259 0.086 Block 3 0. 356 0. 119 Error 9;, 0.872 0.042 Total 31 , 2 SS = 72.740 - (27.-.2511.) = 1.813 V 32 2 2 2 SST = 12.732 + 12.174 5 11.6072 + 11.148 _ 70,927 = 0.173 2 2 2 583 = 109538 + 12.1972 8 12.971 . 12.835 _ 70 927 = 0.356 2 _ 22.714 .+ 24.9272 _ _ SSL _ 16: 70.927 _ 0.153 SS _ 5.542 + 5.9432 + 5.9812 + 5.252 + sz ‘ 7.1722 + 6.2312 + 5.6262 + 5.8982 4 _ 70.927 - 0.173 - 0.153 = 0.259 84 H: ti = 0 (for roughage DMI/100 kg BW) MST E f:°—8—3§=1.38 f .50, 3, 21:0.815 f .25, 3, 21 = 1.48 Standard error for roughage DMI/100 kg BW = 0.072 Table 53. 85 The effects of malic acid on % fat in milk (cow # is on the left side of column) and the analysis of variance for'% fat in milk for all cows. 1430 3.08 1345 3.34 1350 3.58 1442 3.37 1359 3.07 1435 3.31 1448 3.26 1328 2.81 1397 2.65 1456 3.66 1352 2.77 1364 3.66 1407 3.38 1376 3.61 1449 3.88 1410 3.62 1415 3-39 1282 3.12 1400 3.58 1269 3.61 1458 4.10 1321 3.82 1263 3.82 1369 3.10 1387 3.89 1417 3.81 1418 3.55 1302 3.69 1385 3-38 1377 3-45 1419 3-48 1390 3.76 AVG- 3-37 3-52 3-49 3:45 X2 92.217 99.285 98.293 96.149 Tests for % fat Observation d.f s.s. m.s. Trt 3 0.100 0.033 S of L 1 0.633 0.633 (trt) x (S of L) 3 0.381 0.127 Block 3 0.844 0.281 Error .21 1.725 0.082 Total 31 SSy = 3359443; (119609 : 385.944 '.' 382.261 = 3.683 26.942 + 28.122 + 21.922 + 27.62'2 SST = 8 - 382.261 a 0.100 2 2 2 883 ... 25.572 + 28.11 E 27.79 + 29.13 _ 382.261 ___ 0.844 0 + 2 SSL = 1 - 382.261 = 0.633 SS _ 12.182 + 13.922 + 13.492 + 13.462 + TxS " 14.762-+ 14.202-+ 14.435 + 14.162 4 0.381 - 382.261 - 0.1 - 0.633 a 86 H: ti = O (for'% fat) MST r: —— vs. f . t-1 (t-1)