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LSU Historical Dissertations and Theses Graduate School

1953

The Effect of Antibiotic and Vitamin B12 FeedSupplements on Lactating Dairy Cows.Muhammad Obayedul HaqLouisiana State University and Agricultural & Mechanical College

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Recommended CitationHaq, Muhammad Obayedul, "The Effect of Antibiotic and Vitamin B12 Feed Supplements on Lactating Dairy Cows." (1953). LSUHistorical Dissertations and Theses. 8045.https://digitalcommons.lsu.edu/gradschool_disstheses/8045

THE EFFECT OF ANTIBIOTIC AND VITAMIN B12 FEED SUPPLEMENTSON LACTATING DAIRY COWS

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial Fulfillment of the requirements for the degree of Doctor of Philosophyin

The Department of Dairying

Muhammad Obayedui Haq B# Se*i Dacca University* (Pakistan)i 1942 B. Ag*, Dacca University, (Pakistan), 1944 M.S#, Louisiana State University, (USA), 1950*August, 1952

UMI Number: DP69423

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MANUSCRIPT THESES

Unpublished theses submitted for the masterTs and doctorfs degrees and deposited in the Louisiana State University Library are available for inspection. Use of any thesis is limited by the rights of the author. Bibliographical references may be noted, but passages may not be copied unless the author has given permission. Credit must be given in subsequent written or published work.

A library which borrows this thesis for use by its clientele is expected to make sure that the borrower is aware of the above restrictions,

LOUISIANA STATE UNIVERSITY LIBRARY

kQKmwimmmTThe author wishes to express hie sincere appreciation

to his major professor. Dr# L. L* Rusoff, and to the mem**

bars of his advisory committee,* Dr* J. B* Frye, Jr*, Head of the Dairy Department, and Dr# J* G* Lee, Jr., Depart­ment of Biochemistry, Louisiana State University for their counsel and guidance during the progress of this research, and the editing of this manuscript* Acknowledgment is also extended toz Prof* A* J* Gelpi, Jr., and Dr* Cecil Branton of the Dairy Department, and Dr*, C* ¥• Upp of the Poultry Department for their constructive criticism of the manu­script, and to Mr».B* J* Burch, Jr., Mr. G. D* Miller, and Mr* Paul Henderson of the Dairy Department for their co­operation and help which they' rendered.

The author expresses his gratitude to the Government of East-Bengal, Pakistan, and to its Department of Agri­culture for awarding him a scholarship and granting him study leave to continue his studies.

TABLE OF CONTENTS

TITLE PAGE ACKNOWLEDGMENT TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABSTRACT

I. INTRODUCTION II. REVIEW OF LITERATURE

A. Antibiotics1.

2.

3<4<5<

Origin and historya). Aureomyeinb). TyrothricinChemistrya). Aureomyeinb). Tyrothricin Mechanism of action Mastitis and antibiotics Animal nutritiona). Dairy cattleb). Swine e). Poultryd). Other animals

B. Vitamin1. History and isolation2. Chemical and physical properties 3# Mode of action4* Assays of vitamina). Microbiological assay of vitamin B125. Nutritive values in:Dairy cattle Chickens Rats Hogs Human6. Occurrence of vitamin Bioa). Milkb). Bloodc). Feed stuffs

*5:c d e

iii

PAGE1iiill

ixx1333334 4 445 7 7 911121313

1717191920 212425 27 273132

LUF£i3438383d3839394041414141424343I I MT '¥

44444647484850505153545455575858596064667182

d). TissuesEXPERIMENTAL METHODSA. Experiment I

1. Statement of the problem 2* Experimental design3. Experimental animals4. Eat >ions fed5* Collection of samples 6* Analyses of milk samplesB* Experiment II

!• Statement of the problem Z• Experimental design 3« Experimental animals 4* Collection of samples 5# Analyses of samples

RESULTS AND DISCUSSIONA« Experiment I

1* Milk production 2* Fat production 3# Titratable acidity 4* The pH 5* Total Solids 60 Solids-not-fat 7* Protein a. Casein S» Ash 9* Calcium 10* Phosphorus11 • Bacterial count12 i Vitamin B12

B« Experiment IIli Milk production 2# Fat production 3i Vitamin &xz content

GENERAL DISCUSSION SUMMARY AND CONCLUSIONS REFERENCESAPPENDIXAUTOBIOGRAPHY

iv

TABUS1

23

4

5

6

7

6

9101112

13

141516 17

LIST OF TABUS

Vitamin B^2 content of milk of different species*Vitamin B^2 content of goat's milk*Vitamin B*i2 content of milk from the Holstein# Guernsey and Jersey breeds of cattle*Vitamin Bxst la colostrum and normal milk from Holstein and Jersey cows*Vitamin content of whole blood from various species of animals*Vitamin Bio content of the blood of dairy calves before and following Ingestion of colostrum*Food and feed stuffs containing vitamin activity for normal mammals*Vitamin Bi2 content of the tissues of chicks fed various levels of vitamin B^2*Experimental design*Composition of the ration*Level of vitamin Bjjj supplement in the rations.Average daily pounds of k% fat-corrected milk, (F*C*M*)•Butterfat produced by the animals before the experimental feeding and during the experi­mental period*Tltratable acidity of the milk*Total solids present In the milk*Sollds-not-fat in the milk*Percentage of protein and casein in the milk from the different groups of animals*

v

PAGE

2930

31

32

33

34

35

36 3S4042

45

4743 4951

52

TABLE PAGE16 Percentage of ash in the milk* 5319 Calcium values of the milk* 5420 Phosphorus values of the milk* 5521 Number of bacteria per c*c* of milk* 5622 Vitamin content of the milk* 5723 Average amount of k$ F*C*M* of the various groups of cows* 5924 Percentage of butterfat in the milk* 6025 Amount of vitamin in the milk* 63

vl

APPENDIX TABLESTABLEla The date of birth, age* number of lactation, and the stage of lactation of each cow on experiment I*lb The date of birth, age, number of lactation, and the atage of lactation of each cow on experiment XI*2a The amount of k$> F*C.M* for each animalbefore experimental feeding and during the experimental period of 60 days*3a Average daily k% F«C*M* for each animal prior to and at the completion of the experimental period of 60 days*4a The average daily 4# F*C*M* of each animal on experiment II*5a The number of c*c* of 0*1 N NaOH solution required to titrate the acidity produced in each tube*5b The number of c*e* of 0.1 N NaOH solution required to titrate the acidity produced in each tube of the milk samples*6a The number of e*c* of 0*1 N NaOH solution required to titrate the acidity*6b Number of c*c* of 0*1 N NaOH solutionrequired to titrate the acidity produced in each tube of the milk samples*7a Number ofc*c* of 0.1 N NaOH solutionrequired to titrate the acidity*7b Ntmber of c*c* of 0.1 N NaOH solutionrequired to titrate the acidity produced in each tube containing milk (experiment II).Ba The number of c*c* of 0*1 N NaOH solution required to titrate the acidity.

vil

PAGE

63

64

65

67

66

94

96

97

99

100

102

103

TABLE PAGESb The number of c.c. of 0,1 N NaOH solution required to titrate the acidity produced in each tube containing milk (experiment II)* 1059a Analyses of variancef milk production (experiment I)* 1069b Analyses of variance# fat production (experiment I)* 10610 a Analyses of variancet milk production (experiment II)* 10?10 b Analyses of variance, fat production (experiment II* 107

viil

1

2

X.XST OF FIG^reS

lelchmenali 4’leichsiannii 4

• le-icbmawall ) *.«• laiahinannli

IX

page

9596

101

104

ABSTRACT

A* Twenty lactating Holstein cows were divided into four comparable groups on the basis of lactation and gestation* Bach group consisted of 5 animals* They re-* ceived a 15*5# digestible protein basal ration* Group I served as the control* group II received a supplement containing 130 mg* of tyrothricin* group UI received a supplement containing 130 mg* of aureomyein* and group IV received a supplement containing 0*33 mg* of vitaminBi2 oow dally for * pariod of 60 m of th®animals had access to pasture during the experimental period of 60 days* Daily milk production was recorded for each animal* Milk samples were collected from each animal prior to the experimental feeding and at intervals of 15 days* These samples were analysed for butterfat* total solids* solids-not-fat* protein* casein* titratable acidity* pH* ash* calcium* phosphorus* bacterial count and vitamin content*

No adverse effects were observed during the feeding of antibiotics* The animals were in good appetite and showed no evidence of diarrhea*

The milk records showed no difference in production whether or not the animals received antibiotics or vita- min Bj^* The amount of milk produced daily over that of the initial period were 41*#5* 40*02* 39*72* and 40*04 pounds fat-corrected milk for the control, tyrothricin*

x

aureomyein, and vitamin 8 ^ groups, respectively*The percentage of butterfat in the milk samples of

the different groups was very similar* The control group averaged 3*36#, the tyrothricin group averaged 3*54#, the aureomyein group averaged 3 *28# and the vitamin 3 ^ group averaged 3*24#* These differences were not signi­ficant*

The total solids content of the samples of milk of the different groups varied from 11*46 to 11*66#, and the solids-not-fat from 3*16 to 9*25#* The tyrothricin group had the highest value*

The protein content of the milk for the different groups varied from 2*69 to 3*26#, and the casein content from 1.72 to 2*43#* The tyrothricin group averaged the highest in protein and in casein contents*

The titratable acidity of the milk of the differ** ent groups varied from 0*130 to 0*175#, the averages being 0*156, 0*156, 0.152, and 0*155# for the control, tyrothricin, aureomyein, and vitamin B^2 groups, respec­tively* The pH of the milk samples was similar* The values were 6*61, 6*62, 6.60, and 6*60 for groups I, II, III, and IV, respectively*

The ash content of the milk for the control group averaged 0*6913#, the tyrothricin group, 0*7023#, the aureomyein group, 0*6857#, and the vitamin B^2 group, 0.6927#* These differences were not significant*

xi

The calcium and the phosphorus content of the milk samples of the different groups were very similar. Cal­cium values ranged from 96*35 to 103.60 mg.$, and the phosphorus values from 101*65 to 103.44 mg*$.

The bacterial count of the milk samples showed no significant difference among the groups, the values being 9*400, 9,000, 12,200, and 12,700 per c.c. for the control, tyrothricin, aureomyein, and vitamin B12 groups, respec­tively* The results show that the feeding of antibiotics or vitamin has no effect on the bacterial flora of the milk* The samples produced normal acid curd on incu­bation at 35° C, indicating that the antibiotics were not being secreted into the milk at the level fed.

The vitamin content of the milk samples, assayed microbiologically, averaged 1.777 mpg. per ml. for the control group and 1.697 mpg. for the vitamin coirs,

Thus it is concluded that the feeding of aureomyein or tyrothricin or vitamin supplement at a 1% level had no effect on the milk production, percentages of fat, ash, tltratable acidity, calcium, phosphorus, and vitamin Bjjj. The supplementation of tyrothricin at a level of 1% did increase the total solids, solids-not- fat, protein and casein values of the milk. The vitamin B^2 content of cow's milk was not increased by feeding vitamin B12 supplement at a 1% level.

adi

B* Experiment II involved supplementing the basal ration with higher levels of vitamin B*^ determine whttthsr an inemaa. in vitamin B12 value of the milk would occur* Group 1 served as the control, groups 11,III, and IV received a 2, 3# or 4# vitamin supple* meat, respectively, for a period of 15 days* This amounted to 1*66, 2*49, or 3*32 mg, of vitamin B^2 per cow daily. All other treatments were similar to the previous trial*

The milk production for the different groups shewed no signifleant difference*

Butterfat production was significantly depressed when the higher levels of vitamin supplement was fed*

The vitamin B ^ content of the milk for the differ*” ent groups was similar* The vitamin content varied from 3*90 to 4*12 apg per ml* of milk* The actual values being 4*06f 4*10, 3*90 and 4*12 mpg per ml* for groups I, II, III, and IV, respectively*

Thus it appears that the vitamin content of cow’s milk can not be increased by feeding as much as 3*32 mg* of vitamin dally* More than 0*63 mg* of vlta~ mln depressed the percentage of fat in the milk*

xiii

I* INTRODUCTION

Numerous studies hsve been made on the growth promot­ing effect of antibiotics and vitamin • These investi-12gations were conducted on poultry, hogs, rats and young dairy calves, and showed that the antibiotics have a bene­ficial effect on most of the animals so far studied* Vitamin B was also found to stimulate the growth of ntt. «m £., bo,.. nt»a» hu iU. boon ob..rydto promote the hatchability of eggs and the lactation of rats and carried over to the young animals from the preg­nant mothers* Until now no work has been reported in the literature about the effect of vitamin or the anti­biotics on laetating dairy cows*

Milk is the vital complete food of the human being, and the production of milk has been greatly encouraged* When milk production is low and demand is too high any attempt to increase milk production is highly welcomed*In many areas milk production per cow is very low, which might be due to malnutrition of the animals* Any method to increase the production of milk in any area by addi­tion of any kind of supplement will be beneficial* Since the antibiotics and vitamin have been found to have a beneficial effect on other animals it was thought that these supplements might be beneficial to the mature dairy cow*

1

2

fhe object of this study m s to find out whether or not supplementation of the vitamin or the antibiotics (aureomyein or tyrothricin) in the feed would8 1* Affect the milk production, fat production, and/or

any one of the following constituents, namely) total solids, solida-not-fat, ash, titratable acidity, pH, calcium, phosphorus, bacterial count, and vitamin**12 c°2l ezi #

2m Have any effect on the quality of milk,3# Increase the vitamin content of milk when the

ration is supplemented with vitamin B •

II* mam of literatureA* Antibiotic®

!• Origin and history* The discovery of antibiotics may be traced back as early as 1377, with the information that some airborne organisms had an inhibiting effect on the anthrax bacillus* Pyocyanase was isolated from Pseudomonas -aeruginosa in 1399, which inhibited the growth of diph- there, cholera, typhoid and plague cultures*

Antibiotics may be defined as substances which are produced by microorganisms and which have the ability to inhibit the growth or to destroy the other microorganisms* They usually are considered drugs rather than nutrients (50).

a)* Aureomyein* Duggar (33) in 1943 isolated aureo- mycln* The substance was produced in liquid corn steep liquor medium in the fermentation tank*

The new antibiotic principle was active against certain viruses and rickettsla and against both gram positive and gram negative microorganisms* The antibiotic has been named aureomyein from the yellow color of the parent actinomycete and the golden color of the crystalline antibiotic (16).

b)* Tyrothricin* Duboe (37) in 1939 isolated tyro­thricin from a soil sample* Tyrothricin is a spore bearing bacillus capable of causing the lysis of living positive cocci*

3

4

2* Chemistry#&)» Aureomyein# Aureomyein is a weakly basic com**

pound which contains both nitrogen and nonionic chlorine* Aureomyein when treated with alcoholic ferric chloride gives a greenish color by reflected light and reddish color by transmitted light# The crystalline free base has a melting point of 16S-1690 C (16) * The crystalline hydrochloride is soluble in distilled water and to a lesser extent in saline* Solution of this salt in water had a pH 4*5# and when made alkaline rapidly lost anti­bacterial activity ($3)#

b}* Trrothrlc^Ln# Tyrothricin is a dual antibiotic composed of the cheaioally and baeteriologically dissi­milar antibiotics gramicidin and Tyrocidine# Tyrocidine hydrochloride was originally referred to as graminic acid; and a crystalline mixture of tyrooi din© and its hydro­chloride as graaidinic acid (5)#

Gramicidin is a colorless platelet with pointed ends with a melting point of 230-231° C* It is soluble in lower alcohol, acetic acid and pyridine#

Tyrocidine is a colorless needle or rod truncated at angle of 32° £ 3° with parallel extinction# It has a melt­ing point of 237-239° C* It is moderately soluble in methanol, ethanol, acetic acid and pyridine#

3# Mechanism of nctlon, It Is not yet clear how anti­biotics function to promote growth in animals# Certain

5

theories have been proposed which state that feeding anti­biotics does ndt change significantly the total number of bacteria and/or microorganisms in the alimentary tract, but changes the type of organisms present* So it is quite logical to think that they produce favorable microorganic flora in the alimentary tract* It is possible that they synthesise some unknown nutrients which stimulate growth and well being of animals* Some workers prefer the theory that antibiotics lower the toxic organisms which are either competing for nutrients or inhibiting the proper function of tissues (7)*

4* Mastitis and antibiotics* Cows infected with streptococcal mastitis were treated with 150 mg, of tyro­thricin per quarter and 93 % of the animals recovered (17)* Bryan et al. (IB) observed better results with 150 mg* per quarter or 600 mg* per cow* This level of dosage was found to be least irritating but effective to cure Streptococcus agalaetlae with fewest treatments* They infused the drug by gravity and allowed the drug to remain in the udder for 6 hours* Tyrothricin did not have any effect in chronic mastitis and where the induration of the udder had taken place* They have also stated that the number of treatments has no correlation with the length of time during which the cow has been infected* Wine years of Infection was cleared up by one treatment, while 2 months infection in another case required 3 treatments* The cows returned to

6

normal milk within 3 weeks of treatment* Milk from the tow treated with antibiotics remained unfit for human consumption for about a week* Similar results have been reported by Bean and hie coworkers (6)*

Little £& £&*(56) treated infected cows with grami­cidin* Gramicidin reduced the number of streptococci but did not eliminate them* Gramicidin gave better results when the animals were in early stage of lactation than later stage of lactation* The dose of gramicidin consisted of 60-240 mg* in ltQQ0 c*c* of distilled water* injected and allowed to remain in the udder until the next milking* The same authors (59) in another study reported that grami- cidln suspension in water caused swelling and mild fever in cows* With mineral oil emulsion there was little or no reaction* Gramicidin emulsion in heavy mineral oil gave better results than water suspension of gramieidin* Tripp ££,£^*(105) reported similar results and stated that olive oil emulsion was less irritating than mineral oil for emulsifying tyrothricin*

Packer (74) used aureomyein hydrochloride for the treat­ment of mastitis* Two hundred mg* of aureomyein in 7*5 grams of ointment was injected from the tube into the udder through the teat canal* He reported that 200 mg* dose gave 34*3$ suecess in Staphylococcic aurens Infection* With two injections the recovery was 63*5$* Aureomyein did

7

not give any result in two eases of E. coll infection* Bell and Jordan (10) reported the same results.

Eaaterbrooks (39) obtained better results when 2.5 grams of aureomyein in 250 ml. of distilled water was injected intravenously. A total of 27.5 grama in 5 days completely recovered the animal.

5. Animal nutrition. Since the discovery of anti­biotics numerous studies have been made to find out their beneficial effects. These experiments were con­ducted on dairy calves (50f 60, 61, 87* 113) 5 on swine (7, 21, 23, 32, 47, 107, 112, 113); and on chickens (4, 13, 42, 43, 71, 102, 110). The results showed bene­ficial effect to these animals,

a). Dairy cattle. Rusoff (8b) reported that when 2# aureomyein supplement was fed along with a simple plant protein ration to 14*week-old dairy calves the growth rate increased by 35# over the control group.In a subsequent study he and his coworkers (87) reported that aureomyein supplement at 2% level in an all plant protein ration of young calves stimulated the growth of Jersey calves by 20% over the control group through 16 weeks of age. Pure aureomyein (75 mg. daily) was effec­tive in preventing the onset of scours.

Williams (113), in a review of the effect of aureo- mycin on calf growth, reported 20-70# Increase in gain in weight when different levels of aureofac were fed to

young dairy calves. In milk replacement experiments aure-* ofac gave a 20$ increase in the rate of gain in weight over the control group• Marked reduction in incidence of scours was observed in all experiments.

Murley (66) reported that when 60 mg. of aureomyein was fed daily to calves from 4 to 60 days of age the growth rate and the physical appearance were superior to the control group. The incidence of secure was much reduced.

Loosli e£ &1. (60) reported that 2% antibiotics in a milk replacement diet or in whole milk diet stimulated the gain in weight over the control group. The difference in gain in weight was statistically significant. They also reported less incidence if scours.

Jacobson (50) has summarised the effect of feeding antibiotics, especially aureomyein, to dairy calves.The effects most commonly observed are 2

(1) Increase in growth rate (gain in weight).(2) Better or improved appetite.(3) Greater feed intake.(4) Less incidence of scours.(5) Possibly some increase in efficiency of feed

Intake.However the initial thriftiness and breed of calf play

a greater part in the degree of response.Though there has been much work on the effect of anti-

9

bioties on dairy calves* the following still remain un­solved s

(1) The exact amount of antibiotics that will pro­duce the maximum effect*

(2) The comparative effects of different antibiotics*(3) Long-time effect of feeding*(4) The way in which the antibiotics exert their

effect on growth*b)* Swine* Carpenter (21) stated that when 0*5$

aureomyein feed supplement was fed 49-93 days prior to parturition and during lactation period the litters were 44% heavier than the litters of the control group* Pigs from the litters fed rolled oats containing 2 #4 and 6 grams of aureomyein per 100 pounds during the last 26 days of a 56-day lactation period averaged 33*1* 31*3 and 31*5 pounds at the weaning compared to 21*9 pounds for pigs in control litters*

Catron et (23) reported similar results* Aureomyein in all levels gave better results than the control groups*

Cunha (32) reported that supplementation of aureomyein to a eorn-peanut meal ration containing all known vitamins and minerals for pigs growth gave the following results:

(1) Increased rate of gain from 26 to 135% averaging 100*2% in 12 trials*

(2) Decreased feed requirement per 100 lbs* gain from 5 to 44%* averaging 26% in 12 trials*

ID

Heiderbrecht (4-7) observed that pigs suckling sows Ted the aureomyein fortified diet were heavier and more thrifty at 56 days of age than pigs suckling sows fed no aureomyein*

Wahlatrom and Johnson (107) found significant increase in average daily gain over the basal group when pigs consumed 100 mg* of aureomyein per kilogram of dry matter* They also reported that aureomyein, streptomycin and penicillin had no effect on coliform, lactobacillus or yeast cell counts of the feces* Williams (112, 114) reported the same.

Luecke £& aj, (61) did not observe any beneficial effect when pigs were fed 100 mg* streptomycin per pound of corn* soy oil meal basal ration* However, addition of 15 mg* of niacin, 10 mg* of Ca-pantothenate and 2 mg* of riboflavin per pound of basal ration plus the streptomycin stimulated the gain in weight about 50$ over the control* One mg* of penicillin per pound of ration was as effective as 10 mg* of aureomyein in one pound of ration in promoting increase in growth*

Beeson (7) has summarised the beneficial effect of anti­biotics for hogs as follows!

(1) Feeding adequate quantities of antibiotics even in the presence of known nutrients increases the growth rate on an average by 20$*

(2) On a well-balanced ration antibiotics reduce the

11

feed requirements by 5 to 10$*(3) Favorable response 1® obtained from antibiotics

fed to hogs either on pasture or on dry lot#(4) Figs healthy or unhealthy will respond signifi­

cantly to antibiotics*(5) Biological response is obtained by supplementing

5 to 7*5 mg# of antibiotics per pound of ration*(6) Antibiotic is not stored and there is no carry­

over effect of the same#(7) Antibiotics have protein sparing action*

c)* Poultry* Briggs (13) reported 10 to 15$ increase in the rate of gain when antibiotics were fed to poultry, either in pure form or in certain crude antibiotic sources* He did not observe 50$ increase in the rate of gain as previously reported*

Groschke and Evans (42) observed a favorable effect on th. growth or chleks -hen wnttatl. ritmbm. vif-la streptomycin or aureomyein was fed* The greatest response was observed from vitamin

Ole son gt (71) observed no beneficial effect when aureomyein alone was fed to chicks# However when aureomyein was fed along with vitamin a definite growth increase was observed* They found that a level between 2*1 micrograms and 4*2 micro grams of vitamin B^2 per kilogram of ration was needed to get the growth stimulating effect of aureo- myciri* With this level aureomyein in as small amount as

12

5 mg* per kilogram of diet will show beneficial effect*Nhitehill and Ole son (110) observed similar results*Stokstad (101) obtained a 40$ increase in rate of gainin weight* He also reported that aureomyein is notcarried over from hens to chicks as is vitamin B_*12Growth response was also obtained with streptomycin, terramycin, penicillin, certain sulfa drugs and pheny* larsonic acids*

Atkinson and Couch (4) reported that streptomycin, terramycin, and penicillin alone did not give any benefi- clal effect, but exerted growth response when added to the b m l ration to which vitamin Bu had boon added. Halick et £l„(43), Stokstad (102), and Cravens (31) re~ ported similar results*

d)* Other animals* Bau and Colby (7$) reported that lambs receiving aureomyein went almost entirely off feed* However Jordan and Bell (51) in 1951 observed higher rate in gain in weight when 5 mg* of aureomyein was fed to suckling lambs for a period of 6 weeks*

Bell e£ a^ (3) in 1950 reported that steers produced anorexia and diarrhea when 0*6 gram of aureomyein was fed daily* The symptoms were observed within 2 to 3 days* However Neumann ££ al- (6£) in 1951 did not observe any harmful effect when 2 mg* of aureomyein was fed to beef cattle*

13

B. Vitamin B12

1* Hjatorvand isolation* Biochemists and haemotolo- gists were concerned with the problem of antipernicious anemia factor of liver and were studying its chemical nature and mode of action for the last quarter of the century* In 1926 for the first time the so-called addi- sonian or pernicious anemia of dog responded dramat­ically to oral treatment with large amounts of liver.Since then several methods have been developed to fractionate the antiperaicious anemia factor. Precipi­tation of impurities with alcoholic calcium acetate and the precipitation of the active principle with Reinecke acid or uranium acetate was also developed. Scandinavian workers (96) made the most important contribution In this respect. They extracted the active portion from aqueous solution with liquefied phenol and found that it could be adsorbed on to relatively small portions of activated charcoal and eluted again with liquefied phenol. Norwegian workers (96) were the first to prepare concentrates that gave a classical response in doses of only few milligram. In 1946 an active concentrate was prepared which was active in a single dose of 1 mg., but that as we know now contained only 1% of vitamin (96).

Since then no significant publication occurred until Eickes and his eo-woxkers (79) announced the Isolation and

14

purification of antipernicious factor in 1946* *Ehis active principle of liver was isolated in the form of a crysta­lline substance red in color# for which they suggested the name vitamin B12* A few weeks later Smith (94)# a British scientist, also reported the isolation and puri­fication of antipernicious factor from liver* His isolation was controlled by the clinical test whereas the works of Rickea e£ &L. {79) were controlled in part by microbio­logical tests.2* and physical properties. The constitution

of vitamin is not yet fully known. Smith (95) in 1946 gave the first report that vitamin contained one atom of cobalt and phosphorus* Brink (15) reported the empiri­cal formula for vitamin as c6l-64%6-92 % 4 ®13^

From the results of different methods of fragmentation it is now possible to write the following partial formula of vitamin (96).

15

o— OH

Co

CN

The first indication of the multiple nature of vita­min was the appearance of a second red sone contain­ing chemically active material* Fierce et al. (77) separated the second red substance and named it vitamin B12* Later work proved that vitamin is identicalwith vitamin B^ga F^spsred by treatment of vitamin with hydrogen and » platinum oxide catalyst. Vitamin B12b is identical with vitamin except for the replacement of cyan group with hydroxyl* It can be obtained also by treating vitamin with bisulphite or by exposure to light* Vitamin B ^ or vitamin 0-^ can be converted to

vitamin by treating with cyanide* Vitamin mole* eule without cyan group has bean named oobalamin, and with cyan group vitamin *12 is named cyanocobalamin and vitamin is named hydroxocobalamln (96)*

An®low jgt al, (1) isolated two other factors of vitamin 3^2 from the fermentation liquors of Streetomvees grisens and named them vitamin B ^ 0 and vitamin B12d* Vitamin B12c differs from vitamin B^2 iu that it contains nitrite in place of cyan group. Vitamin behaves similarly tovitamin and arises on removal of the nitrite fromvitamin B12c.

The molecular weight of vitamin B is about 1300*Ji AVitamin oan be heated at 100^ C for a long period*Vitamin darkens from red to black at 210 to 220® Cand does not melt below 300® C* Vitamin in aqueoussolution at pH 4 to 7 can be autoclaved without losingmore than a small part of its activity (96)*3* Mode of action* Vitamin and folio acid were

found to be concerned in transmethylation reactions inrats and chicks* Methionine could be synthesised byliver slices from rats given vitamin from homocystinetogether with choline or betaine but not by liver slicesfrom rats deficient in vitamin In rats vitamin12 12appears to have a lipotropic effect and have a protective effect of liver Injury caused by carbon tetrachloride* Vitamin increases the biological value of casein* In

17*

stimulating the growth improvement vitamin is prob- ably related to the metabolism of carbohydrate and fat (96)#*.*■«», tf.ntitt Em. TWWU, 1»». w

used to obtain a common unit of measurement for the acti­vity of vitamin B^2 for the antipernicious effect and for animal growth, so that the unit can be used inter­nationally* Thus several methods have been developed for the assay of vitamin Among them chicks (57, 72), rats (12, 45) and microbiological assay methods (20, 91,94) are best known*

a)* Microbiological assay of vitamin . Shorb (91) reported that Lactobacillus lactis Corner was found in liver extracts in concentration bearing an almost linear relationship to the unit potency of the extract used In the pernicious anemia* Vitamin was assayed for L* ifgfcig Dorner activity and the growth promoting effect was compared with that of a liver concentrate used as an arbitrary standard for L* lactis Domer* The standard was assigned a potency of 1,000 units /mg. According to this basis the potency of vitamin was found to be 11,000,000 units/mg when a 23-hour growth period was employed*

Capps jg£ jQ,* (20) reported that Isicfaaannii 4797 required a similar nutrient as that required by L. lactis Domer. Thus, they were able to utilise leichmannii in the assay of vitamin They used crystalline vitamin

B-jj2 •* standard ranging from 0*01 to 0*1 mg* per tube* level. The results of the assay were evaluated turbid- imetrically after Id hows growth at 37° C. They re­ported that Jj. leichmannii did not grow at all in absence of vitamin B^ .

Stokstad et al. (100) reported that &.313 (ATCC 7930} responded to the crystalline antiper-nicious anemia factor (vitamin B^)* Further, theyreported that thiogly colic acid increased the growthresponse produced by vitamin Thioglycolic acid wasfound to protect the destruction of vitamin duringautoelavinge Protection was also accomplished by theaddition to the medium of other reducing agents suchas ascorbic acid or certain natural supplements thathad been freed of thymidine and vitamin B •12

Thompson £L, (10k) reported the usage of 1* leichmannii in the assay of vitamin B^* They prepared the standard solution by diluting the vitamin in sterile 0*G2M HaHPO^ - KHgPO^ buffer pH 6*£. A standard curve was pre­pared by adding sufficient stock solution to a series of tubes giving 0*025, 0*05# 0*075# 0*1, 0*25# and 0*75 mp& of vitamin per tube* Distilled water and double strength medium were added to bring the volume to 10 ml.(5 c*c* medium)* The tubes were autoclaved for 3 minutes at 121° C* Then in a steamed room 1 drop of inoculum was added to each of the tubes except the blank* The assays

19

were red turbidimetrically after 15 hours to 18 hoursincubation in a constant temperature of 37° C*

Skeggs ^t jfci. (93) were able to develop a medium forthe assay of vitamin B^2 with L* leichmannii* They usedcasein hydrolysate which was of great importance* Thepreparation of casein hydrolysate had a great import onceon the growth of the bacteria* Repeated adsorption ofthe add hydrolysate casein on carbon resulted a poorresponse of the organism to the vitamin and increased12the requirement of £* leichmannii for the growth factor*

5# Kutrltive values inia)* Dairy calves* Anthony and eoworkers (2) in 1950,

reported beneficial effect with vitamin when dairycalves were fed 25 pg* of vitamin B12 in capsule daily* Vitamin fed calves gained 23 pounds more than the control group at 4 months of age. The vitamin fed calves had exceptionally sleek and glossy coats*

Busoff and Haq (88) in 1950 did not observe any bene­ficial effect in calves when 0*5 mg* of vitamin was supplemented in 100 lb* of an all plant protein calf starter ration up to 13 weeks of age* Williams and Knodt (111) also reported the eame* They did not find any value in supplementing A*P.F* containing vitamin to milk replacement diet*

In a recent study MaeKay ©£, &L. (62) reported that vitamin and aureomyein enhanced the growth of the

20

dairy calves up to 12 weeks of age*jEMSklBft* ott his coworkers (72) in 1948

reported that crystalline vitamin as small as 6 y per kilo of the diet stimulated the growth of chicks fed a diet low in A*P*F* They stated that under the experimental condition the optimum growth requirement appeared to be less than 30 y per kilo of the diet*

Lillie si (57) in 1949 observed superior gain in weight in chicks fed vitamin as compared to control group.

Oleeae at al. (70) in 1950 reported that the egg pro­duction decreased to sere in from 3 to 6 weeks when hen. were fed « diet lew in vitamin The additionof A«P*F* concentrates improved the egg production and hatchability of eggs from hens fed the purified diet* They suggested that starch promotes the intestinal synthesis of vitamin and possibly some unknown fac­tor required for embryonic development in fowl*

Oleson and Hutchings (71) in 1950 obtained an increase in gain in weight when vitamin was fed along with aureomyein to chickens* Groschke and Evans (42) in 1950 reported similar results*

Richardson and Blaylock (B0) in 1950 stated that chicks can be raised on soybean oil meal supplemented with a vitamin concentrate supplement equivalent to 10 to 20 of crystalline vitamin Per kilo of the

21

diet*Schaefer £t (89) in 1950 reported that vitamin B12

and folacin are required for maximum chick growth* When both are preeent in the diet the resulting gain is an additive effect* Vitamin B ^ could replace folacin for hemoglobin production in chicks* Both vitamin andJLiCfolacin in addition to choline are concerned in the pre-vention of perosis in chicks*

Gillis and Norris (41) in 1951 reported that chickshaving sub normal storage of vitamin B utilised betaine12more efficiently than choline* They suggested that chicksutilise choline when vitamin B__ is deficient in the diet*12

Jukes and Stokstad (52) in 1951 stated that the choline requirement for the maximum growth of chicks was higher in absence of vitamin B^« However* the amount of choline requirement for the prevention of perosis was not de­creased by supplying vitamin B •

J m

Thus* vitamin supplementation in poultry rations stimulates the growth of chicks and increases the hatch- ability of eggs* Vitamin B ^ along with aureomyein gives better results in promoting the growth of chicks# It re­duces the choline requirement for growth but does not reduce the amount necessary for the prevention of perosis in chicks*

c)* Bats* Information is accumulating that vitamin stimulates the growth of rats* Hat assay methods have

been developed for assaying the vitamin B activity of12different feed and feed stuffs*

Bosshardt j& (12) reported that the new«*bom micedied when they received milk from their mothers whichwere depleted animal protein factor during pregnancy*

Hartman (45) observed that rats receiving 10% driedskim milk to supply the nutrient X averaged 204 gramsin weight whereas the control group averaged 116 gramsfor the same period*

Dinning e£ (34) reported that rats receiving amethionine deficient purified diet developed leneopenia*The lsucopenia was prevented by the supplementation ofmethionine with or without folie acid and vitamin B.12or by choline or betaine with the supplements of folic•eld u d vitamin 8^. Supplement, of choline or betainein the absence of added folic acid and vitamin B failed12to prevent lsucopenia*

Dryden §t jijL. (35) stated that female rats fed vitamin" w * — «»««•»»*«• «*«<»>• -ppi.— «««th

ail other known nutrients did not show greater number of nonpregnancy or resorption than the stock ration*The else of the litters was smaller and the youngs generally had lower average birth weights* Supplementation of the vitamin deficient rations with crystalline vitamin ranging from Q«& mg* to 3*2 mg* per 100 grams of the ration* had no effect on the else of the

23

litters produced by the rats, but the average birth weights did definitely increase.

Meyer £& j&. (1*3) reported that oral administration of crystalline vitamin at the rate of 0.1 pg* per day did not overcome the lactation failure frequently seen in rate fed roast pork as the sole source of pro** tein, but helped in overcoming the conception lag. The failure of vitamin B^2 was not due to poor absorption,as injected Vitamin 33 did not show any improvement.12

Strengh eit £l. (103) In 1951 stated that the choline requirement of rats for the maintenance of normal liver fat was greatly reduced by supplementing the diet with vitamin and folacin. Vitamin B^2 and folacin appeared to be essential for the maximum utilisation of methionine or betaine for the biological synthesis of choline from aminoethanol or methyl amino ethanol.

Drydsn et £1.(36) in 1952 reported early death of new-born rats from mothers fed a vitamin B10 deficientJLsSeroln diet. Supplmaant.tlon of vitamin B12 improved the survival rate of the young rats. Schaefer apU (S9) in 1950 reported that vitamin B12 and folacin protected the kidney hemorrhage.

In summary, rats require vitamin in their rations for normal growth. Vitamin B12 also reduces the choline requirement • The vitamin and folacin appear to be essen­tial for the maximum utilisation of methionine and betaine

In the biological synthesis of choline from aminoethanol end methyl amine ethanol*

d). Boses* timorous studies have been made on the effect of vitamin on hogs*

Catron and Culbertson (22) in 1949 reported faster gain of hogs with A*P.P. They have euimaarised the bene­ficial off acta aa followss

(1) A 19 to 20% faster gain than is obtained from all-plant basal ration.

(2) Finished pigs ready for market up to 20 days earlier.

(3) In a properly balanced ration A.P*F« produced 100 pounds of pork with 7 to 10# leas feed*

(4) Proa a nutritional standpoint, concentrate A*P.P. can replace the factor commonly found In such animal proteins as condensed fish solubles, fish meal, meat scraps or tankage.

(5) Only 10 micrograms of vitamin was needed per pound of total ration to improve the dally gain by 2456# and feed efficiency by 10%.

They also reported that adding 0*5% vitamin con­centrate to the basal ration produced an average dally gain of l«6l pounds compared to 1*46 pounds for the basal ration alone, 1*54 pounds for the basal ration plus 6% meat scrape, and 1*69 pounds for supplemented 0*556 A*P*F* concentrate* Catron j&i* (23) 1950 in

25

another study reported that pig© receiving 10 pg vitamin BI2 per &oymtSi ration and different level© of aureo­myein gained significantly more than those receiving no vitamin B^*

Richardson §&. (dl) in 1951 reported that pigs re­ceiving different level© of vitamin (5, 10 and 20 pg* of crystalline vitamin per pound of ration) did not show any difference in gain in weight* Vitamin B^2 alone produced only slight increase in daily gain in weight, hut in the presence of antibiotic© the increase was highly significant*

Blight £& jy.# (11) in 1952 observed no significant effect in gain in weight of unthrifty pigs when vitamin

was added to the basal ration* Briggs and Beeson (14) in 1952 reported similar results* Addition of aureomyein along with vitamin stimulated the growth. Sheffy At M-(92) in 1952 reported that streptomycin and vitamin

gave better results than streptomycin alone*Thus, vitamin supplements in the rations of pigs

produce a growth stimulating effect and increased feed efficiency* Vitamin B with antibiotics produces betterXwresults in promoting the growth of pigs*

e)* Human* Vest (106) in 194$ reported that 3 patients with pernicious anemia responded following a single intra­muscular injection of 3, 6, and 150 micrgrams respec- tively* Four similar patients given single injection

26

of amorphous liver concentrates containing 20,GOO to 40,000 LU) units of the compound showed strong hema­tologic response, while three patients given 10,000 LLD units or less manifested little or no response*He also reported that the red blood cell count in­creased considerably when crystalline vitamin was injected*

Spies J&&L«(99) in 1946 stated that 2 patients with tropical sprue in relapse responded clinically and hems- tologically with a single intramuscular injection of 6 mierograas of crystalline vitamin B^. By the sixth day soreness of the mouth and tongue had completely dis­appeared and appetite had returned* The patients showed a rise in reticulocytes which reached a peak on the eighth day* This was followed by an increase in red blood cells*

Hall and Campbell (44) in 1946 treated 6 patients suffer­ing from pernicious anemia with vitamin B^. Boses of 25 mierograas given intramuscularly at weekly Intervals pro­duced excellent hematologic responses* When the interval was more than a week the rise in erythrocytes was not always so great*

Campbell gj* (19) in 1949 in another study treated11 cases of pernicious anemia with crystalline vitaminB10* Weekly doses of 25 micrograms or less manifested x*hematologic improvement, with blood counts rising to normal levels* The minimum effective dose has been found

27

to be 1 otlcrogran daily intramuscularly. Vitamin B,„12appeared to bo the erythrocyte maturation factor of liver*

Vitamin <nu*a pernicious anemia in humane. Boses of 25 mlerogrems or less manifest hematologic improve­ment, with blood counts rising to normal levels# Vlta- min appears to be the erythrocyte maturing factor in the liver#

6# Occurrence of vitamin B ^ . Vitamin seems to eeeupy a unique position among B vitamins. Its biologi­cal synthesis appears to be a function of micro-organisms, especially bacteria and molds f not shared by higher plants and animals# One part of fresh soil and two partsof water contain the sane amounts of vitamin B_ found in12cow*a milk. Vitamin B-^ found in soil is evidently the results of extracellular synthesis by soil bacteria*Boots of the majority of garden plants contain vitamin

amounting to from 0*0002 to 0*01 micrograms per gram of fresh material but it is not found In the leaves or fruits (&2)* Fish solubles, various fermentation broths, glandular meats, milk, eggs and milk products contain vitamin B^*

a)* Milk* Very little information is available re­garding the vitamin content of milk*

Collins &t gl (27) in 1950 reported that rata fed goat9s milk mineralised with iron, copper and manganese

2$

grew poorer as compared to rats fed mineralised cow’s milk* They reported that goat’s milk contained very little vita­min They also reported that the addition of vitamin C to the cow’s milk diet produced a large increase it the vitamin content of the liver# whereas no such activityoccurred when vitamin C was added to the goat’s milk diet# In their further studies (2d) in 1951 they reported the vitamin content and folic acid content of the milkof different species of animals* They collected milk samples of cow’s milk and goat’a milk from the herd of Wisconsin University and from the hospital# The milk sam­ples from the cows and goats were taken from the mixed milk of a single milking and the samples of other species were collected while the youngs were being nursed* Most of the samples were assayed within two hours of collection according to the method used by Skeggs et a^. (93) r table 1* In order to find out whether or not storage would in­crease the vitamin content of zailk, they stored goat’s milk for about 4 weeks and assayed it weekly* They reported that the vitamin B content of the goat’s milk Increased considerably on storage t table 2* Colostrum contained more vitamin than the normal milk* The cow’s milk was 2 to 50 times higher than the goat’s milk, depending upon the stage of lactation* The vitamin content of human milk was very low and the vitamin content of non-ruminants studied was highly variable* One important factor affecting

the vitamin content of milk appeared to them to he thedietary intake of vitamin The lower value® presentedfor the aow, rat, and dog represent milk from the animalsmaintained on all-plant rations which were believed to below in vitamin , while the higher values of these12ranges represent milk from the animals receiving supple­ments containing vitamin B •12

TABLE 1lltmla B eeoto.t of .ilk of different .pool...

Bays'postSpeciesVitamin B

Mean 1,2*.m i .. .... W T .. -

Cow 90-150 6*6 3*2 *• 12*4Sheep ao-ioo 1*4 1.0 - 2.0Goat 30- 60 0.12 0.07 * 0.18Human 3- 21 0.41 0.10 ■» 0.15Sow otH 1.05 0.03*- 2.7Bat 3- 21 11.0* - 95.0Dog 3- 6 — — 0.70*- 13.0Horse 10 0.02 .»***!>Commercial raw milk 3.4 2*4 — 4*6Commercial pasteurised milk 4*0 1*6 — 6*5

30

TABLE 2nt«d. Bl2 « m « t of "lit.

Days of storage Mean JBJWBttftAa-r___Rangeps/i JUg/l

0 0.11 0.07 - 0.157 0,08 0.05 * 0.1514 0.25 0.10 - 0.5021 0.21 0.12 * 0.4023 0.27 0.22 - 0.40

Collin et al*{2£) also made a comparative study of the vitamin B*^ content of milk of different breeds of cows (Holstein, Guernsey, and Jersy cows), table 3* Cows from which milk samples were collected received a grain mixture containing 1% of traced mineral salt- There was no differ* ence in vitamin content among the milks of different breeds, though appreciable differences were found among the groups*

Anthony jg£ (3) in 1951 assayed the vitamin con­tent of colostrum and normal milk of Jersey and Holstein cows* They collected samples at an interval of 24, 43,72, 144, and 192 hours and 15, 30, and 60 days after calving to determine the vitamin content* The initial concentration of vitamin in colostrum from Holstein

31

cows was greater in most Instances than the concentra­tion round in Jersey colostrum, table 4*

TABLE 3Vitamin B 2 content of milk from the

Holstein, Guernsey and Jersey breeds of cattle#

Week Holst* In Mean RangeVitamin

J m a m xMean Range ifi£9SX Mean Harngeu s A ) «A pi/l tig/l ]«g/l jag/1

1 St* 7.4 4.9-9.2 8.7 7.0-11.0 8.7 7.0*11.02 nd* 9.2 7.0-12.0 5.8 4.4—6.8 6.3 3.2*9.03 rd* 3*d 3*2—4*6 5.6 4.2-7.4 4.5 4.1-5.0

b)* Blood* Couch gt pi* (29) in 1950 reported on the rltamin content of blood from different species ofanimals* They studied the blood of human, dog, rat, cow, sheep, goat, pig, horse, rabbit, turtle, chicken and turkey* The average value varied from 0*5 to 10*10 mjjtg* per ml* of whole blood, table 5#

Anthony g£ j&» (3) in 1951 studied the blood samples of newborn calves and colostrum-fed calves* The blood samples were analyzed microblologically* No increase in vitamin in bhe blood of the calves after ingestingeolostrum was apparent, table 6*

32

TABLE 4Vitamin in colostrum and normal milk

from Holstein and Jersey cows*

Tina after SaAftSiftift ..ftffltgcalving Bang* Mean Range Meanapg/ii mpg/ml rajig/ml mjug/ml

£ 6 hra. 28*78 49 4 5.4 3*56 21 4 4.624 " 15-40 25 4 2.3 2*35 18 j 2.748 " 12-37 24 4 2,2 4*40 17 4 2.972 • 13-45 27 ^ 3.0 6*27 16 ^ 1.38 days 11-38 22 ^ 2.5 3*25 10 4 1.615 • 5-36 16 jjf 4.4 3*15 7 ^ 1.730 « 3-24 10 4 1.9 2*14 7 jf 1.445 • 3*15 9 i 1.0 2*11 7 ^ 0.960 • 2-9 5.9 4 0.6 2*13 5.6 4 0.8

*}« feed atnffs. Hartmen et al. (46) obtained vitamin activity for the normal maftmaia in some of the food

and feed staff*, table 7*

33

TABLE 5Vitamin Bi, content of whole blood from various spades of animals

Source of sample Rangecontent

Averagempg/ml ^ mpg/rai

Hasan 0*6 - 1*4 0*3Dog 0*5 - 1.1 0.9Cotton rat 3.3 - 3*3 3.6Calf (dry lot) 0.3 - 1.0 0*9Cow (pasture) 0.4 - 0.5 0*5Sheep 0*6 - 0.9 0.7Goat 0*5 - 0*9 0.7Pig 0.3 - 1.3 1.3Horse 1*2 - 3.4 2.1Babbit 6.4 -15.0 10*1Turtle 5.9 - 6.9 6*6Chick 3*3 *» 6*6 5.6Turkey 4* 5 — 6*0 5.3

34

table; 6Vitamin Bj, content of the blood of dairy calves before and following ingestion of colostrum.

Age in hrs« j&Mft&LiiaimBangs Mean Jersey calves Bangs Meanmpg/na rapg/mi mpg/ml mug/ml

z * 1.0 - 1.2 1.1 0.7 - 1.3 1.024 hrs. 0.9 - 1.7 1.2 0*9 • 1.4 1.14& * 0.6 - 1.2 1.0 0.6 — 1.6 1.172 • 0.6 - 1.9 1.1 0.7 - 1.5 1.196 » 0.7 - 1.4 1.0 0.7 - 1.1 0.8144 ■ 0.8 - 1.6 1.2 0.4 - 1.7 1.1192 * 0.7 - 1.3 1.1 0.5 - 1.3 0.9

Peeler et al. (75) reported that fish meal and related products and liver products contained appreciable quanti­ties of vitamin B-j,, while cereals and their products were found to contain very little, if any, vitamin B12.

d). Tissues. Couch et gj.. (29) reported the vitamin Bj, content of the different tissues of chicks when vita­min B12 was supplemented to their rations and when no vitamin B12 was supplemented. The chicks were fed differ­ent levels of vitamin B ^ up to 10 weeks of age. Their

35

TABLE 7Food and food stuffs containing vitamin B12 activity

for normal mammals*

Vitamin B^o activityHovitamin B,~ activity

Milk White flourSkim milk Enriched wheat flourChaese Whole wheat flourCasein, crude Wheat branLiver extrace YeastBeef muscle Com mealPork mssele Soybean mealEgg yolk Linseed oil mealAlfalfa or alfalfa hay Com silageLettuce Distiller*© dried solubleTimothy hay CarrotsKentucky blue grass TomatoesRice polishing concentrate Egg white

Cottonseed flour (deglanded or defatted)Butterfat

36

data are shown In table 6*

TABUS 6Vitamin content of the tissues of chicks 12fad various levels of vitamin 12

Diet Liver Kidney Pancreas Spleenpg/llMg pg/i65g pg/I06gpg/156g

Control20 ug vitamin B ^ A e of diet10# alfalfa1056 alfalfa pins 20 ug vitamin per Kg of diet “10JJ alfalfa plus 135 ug vitamin B.0 per Kg of diet

3.6 4.4 5.7 10.6

id«6 4.3 7.3 12.42.9 3.2 3.4 5.2

4.2 10.2 6,2 6.6

21.4 21.4 11.1 20.6

Control 4*1 3*9 2*5 5*41 ug of vitamin Bn? injected per birdper week 11*6 9*7 3*2 7*9

Smith (96), reviewing the sources of vitamin B^, re­ported that cooked beef contained 3 to 6 pg of vitamin B12 per 100 gram, fresh beef kidney and liver contained 42 to 47 ug/100 grams, veal and lamb contained 2 to 6 pg/lQO grams and cooked pork contained 0*1 to 5 pg« of

37

vitamin par 100 grams of the sampls*

III. EXPERIMENTAL METHODS A. Experiment I.

1. Statement of the problem, Thie investigation was conducted to determine whether or not vitamin B£2# aureomycln or tyrothrlcin feed supplement in the regular herd ration would affect the milk produo-* tion, fat production and/or any other milk constituents namely, total solids, solida-not-fat, ash, calcium, phosphorus and vitamin content* A study was also made to determine whether or not vitamin supple-* mentation would increase the vitamin content of milk*

2# Experimental design* A comparative group feeding trial was conducted* The design of the experiment is given in table 9*

TABLE 9 Experimental design*

Groups No* of animals Breed Ration fedI 5 Holstein Basal (L* S* B* dairy herd ration}*II 5 w Basal / 1% tyrothrlcin supplement*III 5 It Basal / 1% aureomycin supplement*IV 5 «» Basal / 1% vitamin supplement*

3B

39

3* Experimental animals. Twenty lact&ting Holstein cows were obtained from the L» S. U. herd in February 1951* These 20 cows were divided into 4 comparable groups on the basis of lactation and gestation. Each group contained 5 animals. The age and the lactation period are shown in table la in the Appendix.4-* Bations fed. All the cows received a 15*5$ di­

gestible protein basal grain mixture for the entire period of the experiment. The composition and the in­gredients of the basal ration are given in table 10.Group I (control) received the basal ration, group XI received the basal ration plus 1% tyrothrlcin supple­ment^ containing l.d grams of tyrothrlcin per pound ofthe supplement , group III received the basal ration plus

2a Ifo aureomycln supplement containing 2 grams of aureo-mycin per pound of the supplement , and group IV received

3the basal ration plus a 1% vitamin supplement which contained 12.5 mg. of vitamin per pound of the supple­ment.

^Courtesy of 3.B. Penick and Co., New York.^Courtesy of Lederle Laboratories Division,American Cyanamide Co., Pearl River, N.Y.3Courtesy of Merk and Co., Inc., Rahway, N.J.

40

The sows were fed 1 pound of grain mixture for each 4 pounds of milk produced* They were in pasture throughout the experimental period of 60 days* The supplements were fed to the cows daily in one feeding and the supplements Wire given directly in the grain mixture at the time of feeding*

5. Collection of aaraolaB. Milk sample a were collected from the individual cows separately at the beginning and at 15-day intervals over a period of 60 days* Samples collected in the evening were stored in the cool room (40° F) and the morning samples were also collected in the same battle* The composite samples were analysed as soon as possible*

TABLE 10 _ composition of the ration# — -

Digestible Total digestible Crude Ingredients Quantity proteins nutrients* fiber*Yellow corn ..(XETl— ' iTbVr-" '---7IE7J----— nsnmeal 300 21 243.6 1.8Crimped oats 300 36 259.6 9.9Cottonseed meallOO Soybean oil 33 70.6 10.4meal 100 37 76.4 5.7Wheat branOyster shellpowderDeflorinatedphosphateSalt

2001000301020

26155

134.4782.8 19.25?Co

Percent . TO'B ".. 4.70

"calculated from Morrison (65)•

41

6. Anllr... af allk sample. Samples wara analysed for the following constituents!

a* Per cent of butterfat* b. Specific gravity,e. Ash* d* Total solids*e. Solids~not-fat. f* Calcium*g. Phosphorus h* Protein.

1. Casein.i* Bacterial count* j* Titratable acidity.

1. pH*The methods used in analysing the milk samples are

described in the Appendix, pages 90-92.Percentage of butterfat was determined for individual

samples and for the group as a wholeJ the rest of the analyses were made on a composite sample of the group as a whole*

Vitamin B. content of the milk samples of the con**4 Atrol group and the vitamin f*d group were determined* Vitamin B ^ was assayed microbiologically as per the method of Skeggs et §X (93).

Standard bacterial plate counts were used in counting the bacteria (54)*

B* Experiment II.1. Statement of the problem. The second experiment

was conducted to find out whether higher levels of vita­min Bjjg supplement would increase the vitamin con** tent of the milk, since the 1j£ level of feeding did notshow any increase in vitamin B^2 in the milk, arid also

6 %• i v\,j~r)

42

to determine the effect of vitamin supplement on milk and fat production.

2* Jtoftrittental deaim. The experimental design is presented in table 11. The age, number of lactations, and stage of lactation of each cow are given in table lb in the Appendix.

TABLE 11Level of vitamin supplement in the rations.

Groups Ho. of animals Breed Ration fedI 5 Holstein Basaln 5 ft Basal / 2% vitamin supplement.

i n 5 9 Basal / 3% vitamin B12 supplement.IV 5 9 Basal / 4$ vitamin B-« supplement*

3. R-rpftrimental animals. Twenty lactating Holstein cows obtained from the L. 3. B. herd were divided into 4 comparable groups. Each group contained 5 animals. Animals were fed the same basal ration as in experiment I (table 10). Group I served as the control, groups II, III, and IV were fed a vitamin eupplement containing 12.5 mg. vitamin B^2 P®r pound, at a 2, 3# and 4$ level, respectively.

43

4* Collection ofsamolea* Milk samples were collected from individual cows at the beginning of the experiment and at the end of the 15-day experimental period* Samples were collected in the same manner as in experiment 1*

5m Analyses of the samples* Samples were analysed for the percentage of butterfat and for the vitamin in the same way as was done in experiment no. 1*

Statistical analyses were made according to the method of Snedeeor (9#)*

I?., RESULTS AND DISCUSSION A* Experiment I

1* Milk production* The necessity of more milk for human consumption has led to the development of differ­ent methods of feeding and management practices (40)*, Milk production also is dependent on the climate and season of the year (40, 65)*

In the present experiment the cows were under normal feeding and management practices* They received a grain mixture containing 15 • 5% digestible protein as shown in table 10* Group I served as the control, groups II, III, and 17 were fed supplemental tyrothrlcin, aureo- myeln or vitamin B^, respectively, at a 1% level of the grain mixture for a period of 60 days* The dally amount of F*C«M* (f at-corrected milk) produced by each group is presented in table 12*

This table shows that the control group (group I) averaged 103*61 pounds of 4# P.C.M. daily at the be- ginning of the experiment and 145*66 pounds of F*C*M* dally for the experimental period* Thus there was an increase of an average of 41*65 pounds of k% F.C.M* daily during the experimental period* The tyro- thriein-fed cows (group II) showed an increase of 40*02 pounds of k% 7*C*M* daily, the aureomycIn-fed cows (group III) 39*72 pounds of k% F*C*M* and the vitamin Bj^-fed cows (group IV) 40*04 pounds of k% F*C*M* over

44

45

TABLE 12Average daily pounds of k% fat-corrected milk* (F.C.M.}•

ExperimentalGroup and Differenceration fed Initial Final Difference per cow* ■‘I K ----

I Control 103*31 145.63 41*35 3.37II Basal ration / 1$ tyrothrlcin supple- asnt 77*97 U7.99 40.02 3.00III tanl ration / l£ aureomycln supple­ment 92.03 131.30 39.72 7.9417 Basal ration / 1% vitamin Bio supple­ment 90.55

.130.59 40*04 3.00

the amount in the initial period* The differences in milk production among the groups of cows were very * small. The control group increased the most in milk production and the aureoaycin-fed group the least* the difference be­tween these groups being 2.13 pounds of 4$ F.C.M. daily. The statistical analysis by the method of Snedeoor (96) (analysis of variance* Appendix table 9a) showed that the differences in milk production among the group© of cows were not significant. Thus* it is concluded that feeding of tyrothrlcin, aureomycln or vitamin supplement did not increase milk production.

46

2* gat production* The production of butterfat in the milk of Mire depends not only on the breed of the animal but also on the season and stage of lactation# Overman (73) reported that Holstein cows produced on the average 3*6$ butterfat , whereas Jacob (49) obtained 3 *4$ butterfat in Holstein cows* Davis et al. (33) observed under Arisona condition, that the Holstein cows produced butterfat ranging from 3*0# to 4#4$, with an average of 3#5$ butterfat* The results of butterfat production in this experiment are given in table 13* The values of 3 *24 to 3*54 are very close to those obtained by the above investigators# The overall average of all groups (1, II, III, and IV) at the beginning of the experiment was 2#975$ butter­fat and at the end of the experiment the overfall average was 3 #3 55$ butterfat* The data reveal that the percentage of the butterfat did not increase considera­bly during the experimental period of 60 days* The con­trol group increased 0*46$, while the tyrothrlcin group, aureeaycln group and vitamin B^^fed group increased 0.44$# 0.2$$ and 0.34$ butterfat respectively, over the initial period* This may be explained by the fact that the cows were on pasture throughout the experi­mental period of 60 days, which influenced milk produc­tion, thus tending to keep the butterfat percentage very close to the initial percentage#

The differences in the percentage of the butterfat

47

production among the groups of the cows wore not sig­nificant* (Appendix table 9b) and therefore the small differences in butterfat production are of no practical ▼slue*

TABLE 13Butterfat produced by the animals before the experimental feeding and during the experimental period*

GroupPerseaS*ge pf M i t e ri!St urodiiciiInitial ExperimentalBangs Average

I (Control) 2.9 3.2 - 3.4 3.36IX (Tyrothrlcin) 3 .1 3*4 • 3>8 3.54III (Aureomycln) 3.0 3.2 • 3.4 3.26IV (Vitamin B^) 2.9 2.9 m 3.4 3.24

3# Titratablc acidity. Table 14 shows the percentage of titratable acidity of the milk samples of the differ­ent groups' on the experiment* The control group averaged 0.161$ at the beginning of the experiment and 0.156$ at the completion of the experiment while groups IX*III* and I? averaged 0.160* 0.157, and 0.160$,respec­tively at the beginning and 0.15#* 0.152* and 0.155$ respectively during the experimental period* Thus the titratable acidity of the milk was not affected by feeding the supplements.

TABU 14 Titratable acidity of the milk*

Group AverageInitialI 0*161 .0*135-0*165 0.156

i nII 0.160 0.130^0.175 0.156

0.157 0.135*0.160 0.1520.160 0.135-0.160 0.155

4* The pH. pH values of the milks were made only during the experimental period. No effect on pH values were observed by feeding the supplements; the value for the control group being 6.61. the tyrothrlcin group 6.62. the aureomycln group 6.60 and the vitamin B^-fed group

5. Total solids. Davis gt &1. (33) reported that the percentage of total solids in the milk of the Holstein cows varied from 10.5# to 12.26#. However. Overman (73) found from 12.055# to 12.701# total solids in Holstein milk. The values on total solids of the present study are shown in table 15» The over—all average of all the groups was 10.625# at the beginning of the experiment and 11.532# at the completion of the experiment. These results are close to the findings of the other investi-

6.60.

49

gators* During the experimental period the control group increased in total solids by 0*63#* the tyro- thricin group by 0*94/£, the aureomyein group by 0*35$ and the vitamin group by 0«4$^» these differences might be due to the monthly fluctuation as has been suggested by Overman (73) and Davis (33 )• theaverage values of total solide of the different groups sere 10*93, 10*72, 10*62, and 10*93# for the control, tyrothrlcin,aureomycln and vitamin B^-fed groups, respectively, prior to the experimental feeding and during the experimental feeding averaged 11*61, 11*66, 11*47 and 11*46 respectively* The tyrothrlcin group was higher than the other groups*

TABLE 15Total solids present in the milk*

.. Fereentaee iota,! aolida...EscBfrJjaftntaGroup Initial Range AverageI (Control) 10.98 11.37-11.89 11.61IX (Tyrothrlcin) 10.72 11.24-11.96 U.66III (Auroemyeln) 10*62 11.23-11.59 11.47IT (Vitamin B^) 10.98 11.01-11.96 11.46

50

6* Jacob (49) reported that Holsteincows produced 3*63# solids-not-fat in their milk, whereas Overman (73) observed from 3*77# to 9*079#* Davis et ftl* (33) in 1947 reported that Holstein cows under Arizona conditions produced aolids-not-fat varying from 3*1 to 3*5# with an average of 3*3#* table Id presents the data of soUda^not-fat of the present study* The milk from the control group averaged 3*03#, the tyrothricin group 7*62#, aureomycln group 7*62#, and the vitamin B 3*03# prior to experimental feeding* The values during the experimental period of 60 days milk Increased for the control group to 3*25#, the tyrothricin group 3*16#, the aureomycln group 3*19# and the vitamin group 3*22#* The tyrothricin and aureomycln groups produced more solids-not-fat than the other groups| however, they did not exceed the limit of monthly fluctuation observed by other investigators (40, 33, 73)* These differences were found to be significant*

7* Protein* Jacob (49) found 3*32# protein in Holstein cows milk, and other workers (33, 73) reported 3*20 to 3*432#, and 2*2 to 3*2# protein* In the present study, the percentage of protein varied from 2*69 to 3*26#, as is seen in table 17* The control group averaged 2*69# protein, the tyrothricin group 2*47#, the aureo­mycln group 2*76# and the vitamin group 2*77# before the experimental period; and during the experimental

51

period the average* were 2*96* 3«10, 2*97 and 3«13$ protein* respectively*

TABUS 16 Solid-not-fat in the milk*

Pereentage *ollds-not-fat..Exoerimeni;*1...Group Initial Range AverageI (Control) 4,08 4.17*4.49 4.25II (TJrrothrieln) 7.62 7.94-4.46 4.16III (Aureomycln) 7.62 8.13-8*22 4.19IV (Vitamin B^) 4.04 7.44-4.54 4.22

The percentage of protein in the milk samples of the cows receiving tyrothrlcin was higher than that of the milk samples from the other groups* This was found to be signlfleant*

a)* Casein* Table 17 also gives the casein values*The control group averaged 2*03% casein before the experl~ mental feeding and 2*16# at the end of the experiment*The tyrothricin group increased from 1*72 to 2*26$; the aureomycln group from 1*91# casein to 2*15# and the vita­min B 2 group from 1*92 to 2*22# after the experimental feeding* The tyrothricin group was also highest in per­centage casein*

Thus it appears that tyrothrlcin supplementation increases the protein content of the milk*

TABLE 17Percentage of protein and casein In the milk from different groups of animals

Grouprercen

InitiaPercent pageinsals&satjsszs.Initi

I 2.69 2.83-3.09 2.96 2.03 2.03—2.32 2.16II 2.47 2.86-3.26 3.10 1.72 2.23-2.46 2.26III 2.76 2.97-3.16 2.97 1.91 2.02-2.26 2.15IV 2.77 3.02-3.26 3.13 1.92 2.07-2.37 2.22

VnK)

53

Asrtju Jacob (49) in 1944 reported that the ash content of Holstein cow* s milk varied from 0*560 to 0*364$, while Overman (73) in 1945 found 0*66 to 0.692$ ash in the milk. Espe (40) in 19^3 reported that milk from Holstein cows contained on the average 0.63$ ash. The ash content of milk of the present study is shown in table 13. The ash content varied from 0.6524$ to 0.7023$* These figures are in accordance with the data presented by the above investi­gators. The ash content of the milk during the pre-experi- mental period ranged from 0.6524$ to 0.6703$. During the experimental period the control group averaged 0.6913$ ash, the tyrothricin group 0.7023$* the aureomycln group0.6357$ and the vitamin 0.6927$ ash. The differences among the group of the cows in ash content were not signi­ficant.

TABLE 13 Percentage of ash in the milk*

Percentof ashGroup Initial ExperimentalRange Average

I (Control) 0.6524 0.6757*0.7048 0.6913II (Tyrothricin) 0.6745 0.6929-0*7119 0.7023III (Aureomycln) 0.6682 0.6796-0.7116 0.685717 (Vitamin B^) 0.6708 0.6747-0.7201 0.6927

54

9* Bata on milk calcium arc shown in tabic 19*The calcium content of the milk of various groups varied from 91.57 mg. # to 107*61 mg. #. Espe (40) reported that Holstein cow's milk contained 120 mg. % of calcium. In the present study the control group averaged 96.43 mg* % cal­cium for the pre-experimental period and 103.20 mg. % dur­ing the experiment. The tyrothriein-fed-cows increased from 96.90 *mg. % to 101.24 mg* %, the aureomyeln-fed-covrs from 93.33 mg. % to 96.65 mg. %, and the vitamin B^-fed-cows from 95.37 mg. % to 101.29 mg. % calcium. The differences in calcium content of milk among the groups of cows are not significant since the increase in values from the pre- experimental and the experimental period are very close.

TABLE 19 Calcium values of the milk.

me. % of daleitsnExDerlmentulGroup Initial sang. AverageI (Control)II (Tyrothrlcin) III (Auroomycin) IT (Vitamin B12)

98.4396.9093.3395.37

91.59-107.6197.86-105.7891.57-100.6295.62-105.78

103.20101.2496.85101.29

10. Phosphorus. Table 20 gives the average phosphorus values in the milk of each group of cows on the various

55

rations* The control group averaged 92*05 mg % at the be­ginning of the experiment and 102*90 mg* # daring the experimental period* The tyrothricin group increased from 94*77 mg* % to 101*93 mg* the aureomycln group from92*05 mg* % to 101*65 mg* %, and the vitamin groupfrom 94*77 mg* % to 103*44 mg*

The control group increased at a higher rate during the experimental period than the other groups* The differ** ences between the groups in phosphorus values of milk were not significant*

TABLE 20 Phosphorus values of the milk*

me. S PhoBohorusGroup BxperimoaMJ, Initial sange AverageI (Control)II (Tyrothriein) III (Aureomycln) IV (Vitamin Bu )

92.0594.7792.0594.77

96.90-117.2496.90-117.24 92.05-113.65 94.77-117.24

102.90101.93101.65103.44

11. Bacterial count* Table 21 presents the number of bacteria per e*c* of milk for the different groups of animals* On the average the vitamin group contained the maximum number of bacteria per c*c* (12,700/c*c*) of milk, the aureomycln was the next highest (12,200/c.c*)

56

followed by the control group (9,400/c*c*), wad the tyro­thricin group (9,0QQ/c*c*)*

TABLE 21Humber of bacteria per e«c« of milk*

Av, mwtar at ..'saaiaria. D«r s.c.Group Initial SSBS£3aSB&&.Hangs AverageI (Control) 8,600 1,050-22,000 9,400II (Tyrothrlcin) 6,000 1,050-19,000 9,000III (Auroomyein) 7,400 2,500-21,000 12,200IT (Yitaain B^) 6,000 1,000-26,000 12,700

Krlenke (53) reported that aureomycln therapy In mastitis reduced the quality of milk* Milk from the treated cows was not suitable for cheese culture* Bell and his coworkers (9) also found higher concentrations of aureomycln in the milk of cows when aureomycln was administered by lntramammary infusion* In the present study aureomycln or tyrothricin supplement (1*6 gram to 2*0 gram per pound) was fed orally at a level of 1% of the grain mixture which supplied 130 mg* of the aureomycln or tyrothricin daily per animal* To ascertain whether or not aureomycln or tyrothricin affected the bacteria, concerned with cheese culture milk samples were Incubated* These developed lactic acid on incubation and produced a normal acid curd* This Indicated that the

57

antibiotics wars not being secreted into the milk, or if present at all* were of each low concentration as not to interfere with the curd formation*12* HSfiBtiL5i2e Vitamin has been reported to pro*

mete the growth of animals* However* vitamin did not influence the growth of the young calves (88, 111}* It is possible that ruminants synthesise this vitamin and do not require supplemental B10 in their ration* Collins al*JLd*(2B) reported that oowvs milk contains an average of 6*6 ug* vitamin per liter of milk* The amount varied from 3*2 pg* to 12*4 fig* per liter milk* Anthony && pi* (3) obtained lower values for the vitamin content of milk* Their figures varied from 3*2 jag* to 4*6 pg* of vitamin ®12 ?&ble 22 gives the amount of vitamin B ^present in the milk of the various groups in the present experiment.

TABLE 22 Vitamin content of milk*

l»erloGroup and No* of ___ration fed______ samples Initial _ J&n&e Averagem p g . / c . c . m u g * / c * c * I Basal ration 4 1*35 1*76*1*89 1*77II Basal ration /

vitamin 4 1*33 1*50-1*83 1*69

56

The average vitamin content of the milk of Holstein coirs ranged from 1*33 ®pg* per c*c* to 1*77 mpg* per c«c« which are much lower than the values reported by other investigators (2, 2ft)* The control group averaged 1*35 mpg* per c.e* of the milk at the beginning of the experi- ment, and the vitamin B^-fed cows averaged 1*33 ®pg* per e*e* for the same period* At the completion of the eaqaerl- mental period the control group averaged 1*77 mpg* of vita- min per c*c* of milk and the vitamin B^-fed cows averaged 1*697 mpg* These differences among the groups in vitamin content were not significant* The vitamin content of the milk of Holstein cows can not be increased by feeding a vitamin supplement (12*5 Big* per lb*) at a 1% level in the ration*

B* Experiment II

1. Milk production. Table 23 presents the data of aver­age daily k% F*C*M. of each group of animals that received the various levels of vitamin B^2 for the experimental period of 15 days* The control group averaged 66.90 pounds F.C.M. daily prior to eaqjeriaental feeding and averaged 61*20 pounds daily during the experimental period* Group II receiving 1*66 Big. 3 ^ daily averaged 65*70 pounds F*0*M* daily prior to experimental feeding and 60.10 pounds daily during the experimental period* Group III receiving 2*49 mg. Bj2 daily averaged 73*20 pounds F.C.M* daily before and

59

67*50 pounds daily during the experimental period. Group IT receiving 3*32 mg. daily averaged 71*10 pounds F.C.M. dally prior to and 65.50 pounds daily during the experimental periof. Animals in this experiment were in a late stage of lactation, hence the milk production at the completion of the experiment was less than the milk produced prior to the experimental feeding. The difference in the milk production among the groups were vey small and were not statistically significant.

TABLE 23Average amount of F.C.M. of the various groups of cows*

InitialGroup and ration fed

I Basal 66.9011 Basal / 1.66 mg. vita­min B|? daily 65.70111 Basal / 2.49 mg. vita­min Bjjj daily 73.20IT Basal / 3.32 mg. vita­min Bi? dally 71.10

61.20

60.10

67.50

65.50

DifferenceIF-5.70

—5«6o

-5.70

—5.60

2. Fat production. The average percentage of butterfat produced by the different groups of animals prior to the experiment, and the range and average for the experimental period of 15 days are shown in table 24. Prior to experl-

mental feeding the control group averaged 3*S$ butterfat , group II averaged 3*54 %, group III averaged 3*54 %$ and group IV averaged 3 .52 %* During the experimental period the value# for butterfat increased to 4.12* 3*70* 3*70, and 3*70 %9 respectively.

TABLE 24Percentage of butterfat in the milk.

Average nairesnfi or tuiterfatGroup and ration Tad Initial Experimental---Bangs AverageI Basal 3.60 3.6-4*4 4.12II Basal / 1*66 mg*. vita­min B12 P*r cow daily 3.54 3-4-3.9 3.70

I H Basal / 2.49 og. vita- ain Bjjj par cow daily 3.54 3.1—4.2 3.70IT Basal / 3.32 ng. vita­min B-jjg par cow dally 3.52 3.1-3.9 3.70

The differences between the average initial and the aver** age final percentagee of butterfat produced by the different groups of cows were statistically significant. The control group had the highest percentage of butterfat production indicating that the vitamin supplement at a level above 1% slightly depress butterfat production.3. y^fc^in b<i? content* In the first experiment no in­

crease in vitamin content of milk was obtained whether

61

or not & vitamin supplement was fed to tho cows* InJLmthis phase higher levels of vitamin B 2 worn fed to tho cows to find whether this would have any effect on the vitamin content of the milk* Table 25 prosente the vitamin B^g content of the milk of the different groups of cows prior to experimental feeding and after the 15 day experimental period* Group I (basal) averaged 1*71 |ig* of vitamin par Uter of milk prior to the experimental feeding and 4*06 pg* per liter of milk at the end of the experiment; group II averaged 1*75 pg* prior to and 4*10 pg* at the completion of the experiment; group III averaged 1*60 pg* per liter of milk prior to and 3*90 pg* at the end of the experiment; and group IV averaged 1*75 Ug* prior to and 4*12 pg* B12 per Uter at the completion of the experiment* These increase in vitamin B«|g content of milk from the initial to the end of the experimental period is difficult to explain * Various fac­tors may be involved, i*e* pasture fed, stage of lactation or environment• In any ease, all groups showed the same trend* The average values of 3*90 pg* to 4*12 pg* of per liter of milk for the different groups are also lower than the average value of 6*6 pg* reported by Collins al» (2d) and is in the range of 3*2 to 4*6 pg* reported by Anthony et al* (3) •

Anthony sk cl* (3) stated that the vitamin B12 content of the blood of dairy calves was not Influenced by feeding

62

colostrum which contained higher amounts of vitamin B -g* Chow and his coworker® (24) reported a higher level of vitamin is the urine of rats when vitamin Bjjj> was injected subcutaneously or was fed orally* Their results showed that feeding or injecting Vitamin did in** finance the vitamin content of blood* Collins sk si- (2d) reported that when rats, sovs, and dogs were fed an all-plant ration the vitamin content of their milk was lower than when their rations were supplemented with vitamin However they did not mention any Influence or ration on the vitamin content of the milk of other animals* Couch &£ (30) reported higher concentrationof vitamin in tissues of chicks when vitaminwas supplemented in their rations* It is known9 that chicks need vitamin for efficient growth* However, no beneficial effects were observed In feeding vitamin supplements to dairy calves (£6, 111)*

In this study a vitamin supplement was fed to a mgYimmn level of k$ in the grain mixture which supplied approximately 3*32 mg* of vitamin dally per cow* This high amount of vitamin B ^ did not influence the vitamin B ^ content of the milk as seen in table 23* This clearly shows that the vitamin content of the Holstein cow* a milk can not be Influenced by feeding large amounts of vitamin B ^ supplement* It might be that the ruminant® synthesise this vitamin in adequate amounts and do not

63

need any external supply of this vitamin* It is possible that massive doses of vitamin might affect the vitamin B 2 content of the milk* However, this would not be prac­tical*

TABLE 25Amount of vitamin in the milk*

Group and Ho* ofration fed sample* ExperimentalInitial Hang© AverageJW./I1.71

)»«•/!3*64-4.44

ug*/I4.06

1.75 3 * 96-4.44 4*10

1.80 3*20-4*44 3*90

1.75 3 *96-4*42 4*12

I Basal 4II Basal / 1*66 mg* vitamin B 2 daily 4III Basal / 2*49 mg* vitamin B3.2 daily 4IT Basal / 9*32 mg* vitamin B ^ daily 4

V. GENERAL DISCUSSION

Since the isolation end purification of vitamin much work has been conducted on its effect on different animals# Vitamin has been reported to be beneficial in stimulating the growth of chicken, rat, swine, and in curing the antipernicious anemia of man# However, it has no beneficial effect on cattle#

Anthony fi& fii# f3) in 1951 assayed the vitamin content of the blood of young calves fed colostrum and before they received colostrum# They could not find any difference in the vitamin Bio content of the blood be-Jkmfore and after ingestion of colostrum* Colostrum contained higher amount of vitamin than the normal milk# Colo­strum contained on the average 49 mug of vitamin Per ml#, whereas normal milk contained only 25 mug vitamin B22 par ml# Though the calves consumed higher amounts of vitamin B^, the blood picture was the same# The higher amounts of vitamin did not influence the vitamin B^2 content of the blood#

Collins fi£ al# (2£J reported that the vitamin B^2 content of milk samples from sows, rats, and dogs was influenced by feeding animal protein in the ration#When these animals were receiving an all-plant protein ration the vitamin B ^ content of the milk was very low# Vitamin also has growth stimulating effect on these

64

65

animals*Chow e£ al. (24) in 1950 observed no vitamin

activity in the urine of rata until a dosage of at least 270 ug of vitamin P®r 300 grams rat was admin±stered# However, they found that the subcutaneous injection of crystalline vitamin to rats caused appearance of microbiological activity of this vitamin in the urine in amounts essentially equal to the amount injected.They concluded that rats can absorb and utilize vitamin

for normal physiological function* Tamamoto &t al. (115) in 1951 also reported the same.

In the case of dairy cattle any supplementary vita­min in their ration is uneconomical since the present study showed that the added vitamin B_ did not influence the vitamin B12 content of the milk. The nonnal dairy herd ration and pasture are quite sufficient to meet the need of vitamin B-- for the physiological or biochemical functions of dairy cattle.

VI. SUMMARY AMD CONCLUSIONS.

This investigation was conducted to determine the effect of tyrothricin, aureomycin or vitamin supple­ment on the milk production, fat production, total solids, solids-not-f at, ash, tit rat able acidity, pH, calcium, phosphorus, bacterial count, and the vitamin content of milk. Also, to determine whether the vitamin con­tent of milk could be increased by feeding high levels of vitamin

Experiment 1.Twenty lactating Holstein cows obtained from the

Louisiana State University herd were divided Into four comparable groups. Group I served as the control and was fed a basal ration containing 15.5# digestible protein.Group II received the basal ration plus tyrothricin supple­ment at a 1# level (1.& grams of tyrothricin per pound of the supplement), Group III received the same basal ration plus aureomycin supplement at a 1% level (2.0 grams of aureomycin per pound of the supplement), Group IV received the basal ration plus vitamin supplement at a 1% level (12.5 mg. of vitamin P*r pound of the supplement). All groups consumed pasture throughout the experimental period of 60 days. The supplements were fed once daily and were placed directly on the grain mixture at the time of feeding. The cows received 1 pound of grain mixture for each 4 pounds of milk produced. Milk samples were collected from each animal separately prior to the experi-

66

67

mental feeding and a 15-*day interval for analyses* Except for the percentage of butterfat all analyses were conducted on the group basis. The percentage of butterfat was deter­mined for the individual sample and for the group*

Experiment II.Twenty laetating Holstein cows obtained from the L*S*U*

herd were divided into 4 comparable groups. Each group con­sisted of five eows. Group I served as the control and re­ceived a 15.5$ digestible protein grain mixture* Group II received the same basal ration supplemented with 2% levelof vitamin Bn 0 supplement (1.66 mg. vitamin Bx2 per cowdaily); Group III received the basal ration plus vitaminB|3 supplement at the level of 3# (2.49 mg* vitaminper cow daily); Group IV received the same basal ration plus vitamin B12 supplement at the level of k% (3*32 mg* vitamin per cow daily), for a period of 15 days* Milk samples were collected from individual cows prior to the experimental feeding and at the completion of the experiment* Samples were analysed for butterfat and for the vitamin content of the milk*

Results of experiment I showed that;1. The supplementation of tyrothricin, aureomycin or

vitamin at a l/£ level to the basal ration of lactating dairy cows was of no value* The supple­mentation did not influence the milk production over a period of 60 days*

6g

2* The percentages of butterfat production of the groups were similar* showing that fat production can net be influenced by feeding the supplement at a 1# level#

3* The percentage of total solids of the milk samples from cows fed tyrothricin was higher than In the milk of the other groups• Tyrothricin might have in­fluenced the total solids of the milk#

4# The percentage of solida-not-fat in the milk samples of the cows fed tyrothricin was also higher than in the milk of the other groups# Tyrothricin may have influence on the solida-not- fat of the milk#

5# The titratahle acidity and pH values of the milk samples of all groups were similar#

6# The ash content of the samples of all the groups was similar# The supplements did not influence the ash content of the milk.

7# The percentage of total protein and casein in the milk samples of the cows fed tyrothricin was defi­nitely higher than in the milk of the other groups# Tyrothricin influenced the protein and casein con­tent of the milk#

£# Calcium and phosphorus values of the milk of all the groups were similar# Supplementation of the antibio­tics or vitamin B^ n0 influence the calcium or

69

phosphorus value of the milk*9* Standard bacterial plate counts showed no differ*

eace in the bacterial content of the milk* Milk samples developed lactic acid on incubation, and produced a normal acid curd* The results indicated that the antibiotics wears not being secreted into the milk or, if present at all, were of such a lew concentration as net to interfere with the curd formation*

10. Microbiological assay of vitamin showed that the feeding of 0*&3 mg* vitamin B.0 did not in**JUCfinance the vitamin B*^ content of the milk*Milk is sold on the basis of butterfat present

in the milk* From the present experiment it can be said that the feeding of antibiotics or vitamin 3 ^ supplement is of no value for the practical dairyman* The dairyman may feed aureomycin or tyrothricin at a 1% level if oral administration of these antibiotics prevent or cure mas­titis* Oral administration of these antibiotics at a X% level did not interfere with the curd formation for the cheese starter*

Experiment II*1. The feeding of higher levels of vitamin did

not Influence milk production* Feeding as much as 3*32 mg* of vitamin Per cow d&i-ly was of no value in increasing the production of milk.

70

2* The percentage of butterfat was depressed by feed*ing the levels of vitamin fL higher than 0*03 mg*12daily*

3* The vitamin B^2 content of the milk can not be in*creased by feeding vitamin as high as 3*32 mg*of vitamin per cow daily*

For the practical dairyman# feeding dairy cows supple* ments of vitamin is harmful as the data indicates thatJmSthe fat content of the milk is depressed*

VII., REFERENCES

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72

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79

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100* Stokstad, E# L*R*, Dombush, A* C., Franklin,A* L*, Hoffmann, C. E*, Hutching, B* L*, and dukes, T# H* Microbiological Assay of Vitamin ky Lactobacillus lactis* Fed* roc# 6: 257# 1949#

60

101. Stokstad s S. L* R. Effect of Aureomycin on Animal Nutrition. Feed Stuffs 22} 17*16. 1950.102* Stokstad, E. L. R. Recent Advances in the Use of Aurofac In the Nutrition of Poultry. Nutrition Symposium. N. Y* Dot* 31* 1951*103. Strength, D. R., Schaefer, £. A., and Salmon, W* B. The Relation of Vitamin Bi$ and Folaeln to the Utilisation of Choline and its Precursors for Lipotropism and Renal Protection in Rats. «J* Nutrition 451 329-344. 1951.104. Thompson, H. T., Deitrich, L. S., and Elvehjem,C. A. The use of Lactobacillus leichmannii in the Estimation of B10 Activity. J. Biol. Chem.164: 175-160. 1950.105. Tripp, L. H., and Laurence, C. A. Clinical Obser- vation on the Use of Gramicidin in the Treatment of Bovine Mastis. Cornell Vet. 321 90-95. 1942*106. Ungly, C. C. Vitamin Big. A Review of the Clinical Aspect. Nutrition Abstr. & Rev. 21} 1-26. 1951.107. Vahlstrom, R. C. and Johnson, B. C. Growth Effect of Various Antibiotics on Baby Pigs fed Synthetic Rations. Fed. Proc. 10s 397. 1951.106. Vest, R. Activity of Vitamin B19 in Addisonian Pernicious Anemia. Science 107? 396. 1946.109. Vest, R., and Reisner, E. H., Jr. Treatment of Pernicious Anemia with Crystalline Vitamin Bio.Am. J. Med. 6: 643-650. 1949.110. Whitehill, A. R«, Ole son, J. J., and Hutchings,B. L. Stimulating Effect of Aureomycin on the Growth of Chicks. Proc. Soc. Exp. Biol. & Med*

74t 11-13. 1950.HI. Williams, J. B., and Knodt, C. B. A.P.F. Supple­ments in Milk Replacements for Dairy Calves. J. Dairy Sci. 33t 360. 1950.112. Williams, L. V. Recent Results with Aurofac in Hogs Diets. Nutrition Symposium. N.I. 1951.113. Williams, L. V. The Use of Aurofac in Calf Feeding. Nutrition Symposium, N.Y. Oct. 31. 1951.

114.

115.

81

Williams. L. W. Aureomycin and othar Antibioties in Swine nutrition. Lederle Lab. Pearl River.N.T. 1951.Yamamoto. R. , Barrows. C. Jr.. Lang. 0.. and Chow.B. F. Further Studies of the Absorption of Vitamin B-.~ following Oral and Parenteral Administration.J. nutrition. 45t 507-520. 1951.

m i JLPPSKSZX

iz

83

TABLE laThe data of birth, ago, number of lactation, and the stage of lactation of eaeh eow on experiment I*

cow ' sate of 1' too, ot' " totage ofS£2H2 fl£s--kiHkt_Age lactation...... lactation---y r s . m o a . d a r e

1 10.1.39 11-5 S> i n

2 11.21.43 7-3 3 205I 3 1.17.40 11-1 7 267

4 1.25.48 3-1 1 104_________ 2___12*2«A6 4-3_______ 2....... -334.....

6 6.26.44 6-8 4 1167 12.21.44 6-2 3 291

U 8 9.25.46 4-5 2 2029 12.18.46 4—2 2 166

10 12.25.47 3-3 1_________ 13&..11 9.1.46 4-3 2 17812 7.14.45 5-7 3 133

III 13 3.19.42 8-11 6 15014 10.27.38 12-4 9 137

15 2.19.48_______ ________i--------- m -----16 9.17.43 7-5 4 28917 12.31.45 5-2 2 156

IV 18 9*6.41 9-5 7 10919 10. Id. 46 4—4 2 4420 2.13.48 3-0 1 141

64

TABLE lbThe date of birth, age. mother of lactation, and the stage of laetatien of eaeh co«r on experiment IX*

Got*... XXOm tiaie of birth ..Me... TBrar—.lactation lactation1 6*26*44

yX*ft«£ri0ji»6-11 4days226

2 12.21*44 6-5 3 411I 3 9.25*46 4-6 3 312

4 9*6*46 2-9 1 159...5 12.25.47 3-6 ....1_____ _ ___246_____

6 10.1*39 11-6 9 3067 11.21.43 7*6 3 316

U 6 1.17*40 11*4 7 3779 1.25.46 3-4 1 214

- -rl- -- - .. 4-6 ... 2...... 24411 9*1.46 4-6 2 26612 7*14.45 5-10 3 243

i n 13 3.19.42 9-2 6 26014 10.27.36 12-7 9 24715 2.19.46 3-3 1 _ 27916 9.17*43 7-6 4 39917 12.31*45 5-5 2 266

IV 16 9.6*41 9-6 7 21919 10.16.46 4-7 2 15420 2.13.46 3-3 1 251

TABUS 2ftThe amount of 4# F.C.K. for each animal before the experimental feeding and during the experimental parted of 60 days.

Group and ration fed Cow Initial no. daily

Wdaytotal It.

daily

I Control (&tad.)

1 31.662 17.213 16.914 17.755 20.06

600.7334.6 3#.?340.6 326.9

685.6

364.2406.0

709.5367.1432.9374.4380,0

705.2338.9453.1362.5369.7

2701.01393.91656.71461.71506.6

45.0223.2327.6124^9

Total 103.61 1941.9 2264.7 2263.9 2269.4 6739.9 145.66

II Basal / Tyrothricin

6 16*947 16.40 6 15.40 9 13.3610 33.35

369.9294*1339.2299.0276.6

385.9341*0406.0346.5400.8

366.6331.5 400*8 326*8396.6

364.5316.9396.7301.0409.2

1488.91285.51542.71277.31465.2

24.6221.4225.7121*2924,75

Total 77.97 1560.6 1332.2 1626.3 1790.3 7079.6 317.99

&\Tt

TABLE 2a (CONTINUED)

Group and Coir Initialration fed No. dailyX 50“day

total11 16.72 359.4 394.2 393.4 410.0 1557.0 25.9512 26.42 406.1 417.6 413.4 402.6 1641.7 27.36

III Basal / 13 16.40 307.2 390.9 364.0 392.9 1455.0 24.25Aureomycin 14 18,62 473.7 525.6 566.3 527.9 2113.5 35.2315 _13-»92 274.5 271.0 272.2 . 323.0 2140,7 19.01

Total 92.06 1622.9 1999.3 2029.3 2056.4 7907.9 131.60

16 12.72 260.5 302.9 297.0 293 i6 1154^ 19.2319.29 297.1 346.1 321.1 310.5 1274.6 21.25I? Basal / IB 19.36 336;9 466.3 424.7 426.9 1656.8 27.61Yitamin 19 24.73 501.7 574.1 567.3 637.6 2300.9 38.3520 14.45 313.0 390,7 375.7 _ 369.3 1446.7 24,15Total 90.55 1709.2 2060.1 2005.6 2040.1 7635.2 13Q.59

07

TABLE 3»Average daily 4$ F.C.M. of each animal prior to and at the completion of the experimental period of 60 days.

Group anci Cow — u a s n r - "AVeragojatian fed _ no* daily Difference— ..1 31.00 45.02 13.142 17.21 23.23 6.021 Control (Basal) 3 16.91 27.61 10.704 17.75 6.945 20.06 25.11 5.05

Total 103.01 145.66 41.05

6 10.94 24.62 5.007 16.40 21.42 5.02II Basal / 0 15.40 25.71 10.31Tyrothriein 9 13.30 21.29 7.9110 13.05 ...24.75.. 10.90Total 77*97 117.99 40.02

11 16.72 25.95 9.2312 26.42 27.36 0.94III Basal / 13 16.40 24.25 7.05Aureomycin 14 10.62 35.23 16.61_15_ _...13.92.. . 19.01._ __5_.09Total 92.00 131.00 39.72

16 12.71 19.23 5.5217 19.2? 21.25 1.96IT Basal / 10 19.36 27.61 0.25Vitamin B12 19 24.73 30.35 13.6220 14.45. _ 24.13 , . 9.70Total 90.54 130.59 40.05

TABLE 4aThe average daily Wf> F.C.M. of oaeh aninal on axperlnaat XI.

1 2 3 4 ju aiujuyvxiUiDifference

Group and Cow Total of Avorage betweenration fed No* Initial 15 days daily cols. 3 A 5

W j — n s r r ’ "Tibrj'..1 13.1 182.2 12.1 -1.02 10.0 121.4 8.1 -1.9I Control (Basal) 3 134 190.4 12.7 -0.74 17.6 244*4 16,3 -1.3A ___ li.d 180.7 12,0 —0.8

Total 66.9 61.2 -5.7

II Basal / 1*66 6 2$*1 397.3 26.5 —1.6yitamin B12 d&ily 7 9.5 132.4 8,8 -0.7per cow* £ 11.8 199*2 13.3 A. 5

9 21.0 259.9 17.3 -2.710 15.3 _ 212.8 14.2... ___r —1.1Total 85.7 80.1 -5.6

TABLE 4a (CONTINUED)

1 2 3 4lriaMmimmT.

5 6uirxerenceGroup and Cow Total of Average betweenration fed No. Initial 15 days dally cols. 3 & 5

(lb.) (lb.) (lb.) (lb.)11 14.9 203.7 13.9 -1.0III Basal / 2*49 og» 12 11.3 156.7 10.4 —0.9vitamin B-»0 daily 13 14.7 197.7 13.2 *•1.5per cow 14 13.4 249.9 16.7 -1.715 13.9 193.9. 13.3 . —0*6Total 73.2 67.5 -5.7

16 9.5 105.4 7.0 -2.5I? Basal J 3*32 mb. 17 11.9 169.3 11.3 -0.6

vitamin Bio daily X& 16.7 247.2 16.5 —0.2per cow 19 21.9 302.9 20.2 -1.7

20 11.1 157.1 10.5 -0.6Total 71.1 65.5 —5.6

uaed in analysing thr M lk asgplaa for tb« lag conai-.?.t;Uam ta.1* Butterfat— -Babcock method (54) •2# Spec i n c gravity— Lactometer (54)*3« Total solids-— Calculated aa per method of the

Association of Official Agricultural Chemists, (A«OiA»C»)t (69)*

4* Ash— A*0*A*C* method (69)*5* Protein-— A*0#A#G# method (69)*6* Calcium and phosphorus-— Method of Morris al*

(64) •7* Bacterial counts— Plate counts (54)*&* Vitamin Method of Skeggs £& j|i* (93 )•Bgtai.la._of, yitamin Ba Aaeay, Bacto-vitamin B12

Assay medium as described by Capps et jgi* (20) was used in this experiment* The prepared medium was obtained from the Difco Laboratories, Detroit, Michigan* Seventy-six grass of Bacto-vitamin Assay medium were dissolved in 1000 ml* of distilled water* The slight precipitate which formed was distributed by shaking# Five ml# of this medium was added to each tube and plugged with cotton#

Vitamin standard solution was prepared by dls* solving 0*1 gram vitamin with sodium chloride con­taining 1 mg* of crystalline vitamin Bi# per gram of vitamin Bi2 with sodium chloride, to 100 e*e# of distilled water* One c*e* of this solution was diluted to 100 e*e*

91

with distilled water, one c*c* Of this dilution was fur* ther diluted to 100 c*e* with distilled water* Thus the final dilution contained 0*1 mug of vitamin Per c*c* of the solution*

Duplicate tubes were used for each concentration of the standard vitamin solutions* Tube no# 1 did not contain any vitamin tubes no* 2 to 9 contained vita* nin *t the rate of 0.01, 0*03, 0*05, O.Od, 0*1, 0.2, 0*3, and 0*5 mpg* of vitamin B^2 respectively* The volume .in each tube was made to 10 e.c* by adding didtilled water* The tubes with the container were sterilised by awtoclavlng for 5 minutes at 15 lb# pressure (121° C).

Preparation of inoculum. Inoculum for the assay was prepared by subculturlng from a stock culture of

leichmannii no* 4797 to tube containing 10 c*c* of Bacto-Micro inoculum broth# After 24 hours incubation at 37° C the cells were centrifuged under asceptic conditions and the supernatant liquid was des­canted* The cells were washed by suspending in 10 ml* of sterile physiological saline solution and cetrifuging* The washing was repeated a total of 4 times* The fifth sue* pension was prepared by diluting the cells to 20 ml* with sterile physiological saline solution*

After the tubes had been autoclaved and cooled, one drop of the prepared inoculum was added to the content of

the tubes and the tubes were Incubated for 72 hours at 37° C* At the end of the incubation period the content© of the tubes were chilled to stop further growth of bacteria and then were titrated with 0+1 N NeOH solution until a light blue color was obtained using brom thymol blue as Indicator

The standard curves were drawn from the results eh* tained* The milk samples were treated in a similar way* Known amounts of milk were added instead of vitamin standard solution in the tubes* The vitamin ®12 content of the milk was read and calculated from the standard curve

93

bactoVITAMIN ASSAY MEDIUM DEHYDRATED

Baeto-Vitamin Free Casamin> Acide 12 g«Bacto-Dextrose 40 g*Sodium Acetate 20 g.1-Cystine, Difeo 0*2 g*dl-Tryptophane 0.2 g.Adenine 0*02 g.Guanine 0*02 g*Uracil 0*02 g*Xanthine 0*001 g.Thiamine Hydrochloride 0*002 g*Riboflarln 0*002 g*Niacin 0*002 g*Calcium Pantothenate 0*0002 g*Pyrldoxine Hydrochloride 0*004 g*p—Aminobenselc Acid, Dtfco 0*0002 g*Blotin 0*00001 g*Folic Add 0.0001 g.Tfcreen BO 2*0 g*Dipotasalum Phosphate 1*0 g.Monopotassium Phosphate 1*0 g*Magnesium Sulfate 0*4 g •Sodium Chloride 0*02 g*Ferrous Sulfate 0*02 g.Manganese Sulfate 0*02 g*

TABLE 5aThe number of c.c. of *1 N NaOH Solution required to trltnte

the acidity produced In eaoh tube*

ho* of tube*

c.c. ormediumusedc.c. of testmaterial

Amount of vitamin Bi? Standard c.c. of

water addedTotalC«6e

e*e« ofNaOHseeded

1 5.0 0.0apg

0.0 5.0 10*0 1.5**5.0 0.0 0.0 5.0 10*0 1.32 5.0 0.1 0.01 4.9 10*0 2.35.0 0.1 0.01 4.9 10*0 3.03 5.0 0.3 0.03 4.7 10*0 3*25.0 0.3 0.03 4.7 10*0 3.64 5.0 0.5 0.05 4.5 10*0 5.05.0 0.5 0.05 4.5 10*0 5.45 5.0 0.3 0.03 4.2 10*0 6.35.0 0.3 0.03 4.2 10*0 7.06 5.0 1.0 0.1 4.0 10*0 3.65.0 1.0 0.1 4*0 10*0 3.97 5.0 2.0 0.2 3.0 10;0 10.05.0 2.0 0.2 3.0 10.0 9.33 5.0 3.0 0.3 2.0 10.0 ruo5.0 3.0 0.3 2.0 10*0 11.39 5.0 5.0 ' 0.5 0.0 10*0 13.55.0 5.0 ...0.5 0.0 10.0 13.3^Duplicate tubes were innoeulated and incubated.

ocd

i— i

oo

□71mjig. vitamin B^*-

Fig, 1. Response of L, leichmannii 4797 to vitamin (table 5a),

TABU 5bNumber of c.c. of 0.1 N NaOH solution required to titrate the acidity

produced in each tube of the milk samples.

_ Control• OfExperimental

CeCo OfNaOH Vitamin needed B 2Tube CeCe Of CoCe ofno* medium milk

c.c* ofwateraddedTotalCoC*

c.c NaOH needed Vitamin

B12

3

4

5

6

7

5.05.05.05.05.05.05.0 5*05.05.05.05.05*05.05.0_______ 5.0*This amount o?

0.00.00.10.10.30.30*50.50.30.31.01*01.51*52.02.0

5.05.04*94.94.74*74*54.54.24.24.04.03.53.53.0 O

10.010.010.010.010.010.010.010.010.010.010.010.010.010.010.010.0

1.31.39.0 9*312.012.414.013*715*215.516.216.016.6 16*517.3

mpg.

0.1290.1520.3730.412

3.99.2

12.134*113*6

15.2

16.016.416*636.7

mpg,

0.1200.1430.410.339

was subtractedin drawing 4she curve and the vitamin 8 2 content.inavAluating

97

TABLE 6aBunber of c.c. of 0*1 N NaOH solution required to titrate the acidity.

Ho* ortubeC.C* Oftestmateriel

Amount of vitamin B19 Standard

1 CaC. 0#NaOHneeded(Averagemug

1 0.0 0.0 1.22 0.1 0.01 2.23 0.3 0.03 3.04 0.5 0.05 4.75 o.a 0.0$6 1.0 0.10 9.77 2.0 0*20 10.9a 3.0 0.30 12.09 5.0 0.50 14.3

N NaOH

l—1 *o

oo

0.50.30.1mpg. vitamin B^. ^

Fig. 2. Response of L. leichmannii 4797 to vitamin B ^ (table 6a).

TABLE 6bNumber of e*c» of 0*1 N NaOH solution required to titrate the acidityproduced in each tube of the milk samples*

Tubeno* c.c. of medium c.c. of»nirc.c. ofwateradded Totalc.c.

Contror c.c. o£NaOH Vitamin needed

izDe&mental c.c. oxNaOH Vitaaii needed

npg. mpg.1 5.0 0.0 5.0 10.0 1.2 1.22 5.0 0.1 4.9 10.0 10.6 0.176 10*5 0.1665.0 0.1 4.9 10.0 10.5 0*166 10.3 0.1503 5.0 0.2 4*3 10.0 12.7 0,360 12*3 0.366

5.0 0.2 4.3 10.0 12.9 0.373 12.7 0.3604 5.0 0.3 4.7 10.0 15.7 15.65.0 0.3 4.7 10.0 15.6 15*5

TABLE 7«Number of c.c* of 0.1 N NaOH solution required to titrate the aeidity*

Tubeno.c.c. ofmediumused

e.c. of testmaterial

Amount of vitamin Bin Standard e.c. of water added Total6»6»

e.e. ofNaOHneeded* mjig.1 5.0 0.0 0.0 5.0 10.0 3.0

5.0 0.0 0.0 5.0 10.0 3.32 5.0 0.1 0.01 4.9 10.0 4.0

5.0 0.1 0.01 4.9 10.0 4.23 5.0 0.3 0.03 4.7 10,0 4.55.0 0.3 0.03 4.7 10.0 4.64 5.0 0.5 0.05 4.5 10.0 6.25.0 0.5 0.05 4.5 10.0 6.55 5.0 0.3 0.08 4.2 10.0 7.55.0 0.8 0.03 4.2 10.0 7.96 5.0 1.0 0.1 4.0 10.0 9*55*0 1.0 0.1 4.0 10.0 9.37 5.0 2.0 0.2 3.0 10.0 11.55*0 2.0 0.2 3*0 10.0 11*73 5.0 3.0 g;3 2.0 10*0 13*55.0 3*0 0.3 2.0 10.0 13.49 5.0 5.0 0.5 0.0 lo;o 14*75.0 5*0 0.5 0.0 20.0 14.9*The msnber of c.c. required to titrate the blank tube was subtracted in drawing tbs normal curve.

11 ’

oa

oo■a

o0.3 0.40.1 0.2 • s

mpg. vitamin B^

Fig. 3. Response of L. leichmannii 4797 to vitamin B-^ (table 7a).

101

TABLE 7bNumber of c.c. of 0*1 B NaOH solution required to titrate the acidity

produced in each tube containing milk*

Tubeno.6«Ceofmedium

GeCeofmilk*

e.c.ofwateradded Total

e.c. HaOH reouired litpio Bi?I.

Group II III nr I Grownn m i?

apg.1 5.0 0.0 5.0 10.0 2.1 2.1 2.1 2*12 5.0 1*0 4.0 10.0 7.0 7.0 7.5 7.5 .033 .086 .039 .0915.0 1.0 4.0 10.0 7.3 7.2 7.0 7.33 5.0 2.0 3.0 10.0 10.5 9.5 10.5 10.5 .173 .153 .13 .1345.0 2.0 3.0 10.0 9.7 10.0 9.3 10.04 5.0 3.0 2.0 10.0 11.5 11.5 11.0 11.5 .234 .25 .23 .225.0 3.0 2.0 10.0 11.0 11.7 11.4 10.35 5.0 4.0 1.0 10.0 12.5 13.0 33.5 13.0 .335 .412 ,42 .376

OAlb T»_

5.0 4.0 1.0 10.0 13.2 13.3 13.0 13.4♦liilk was diluted to 1:20 (1 c.c. milk plus 19 c.c. of distilled Water).

TABLE £aNumber of c.c* of 0*1 N NaOH solution required to titrate the acidity*

c.c. of c.c. of Aooimt of c.c* ofTube medium test vitamin B^2 CmC* Total NaOHno. used medium Standard water added c.c. needed

1 5.0 0.0BQlg.0.0 5.0 10.0 1.25.0 0.0 0.0 5.0 10.0 1.4

2 5.0 0.1 0.01 4.9 10.0 2.55.0 0.1 0.01 4.9 10.0 2.73 5.0 0.3 0.03 4.7 10.0 3.05.0 0.3 0.03 4.7 10.0 3.34 5.0 0.5 0.05 4.5 10.0 3.95.0 0.5 0.05 4.5 10.0 4.05 5.0 0.3 0.03 4.2 10.0 4.55.0 0.3 0.03 4.2 10.0 4.76 5.0 1*0 0.1 4.0 10.0 6.55.0 1.0 0.1 4.0 10.0 6.37 5.0 2.0 0.2 3.0 10.0 3.15.0 2.0 0.2 3.0 10.0 3.53 5.0 3.0 0.3 2.0 10.0 11.35.0 3.0 0.3 2.0 10.0 11.59 5.0 5.0 0.5 0.0 10.0 13.75.0 5.0 0.0 0.0 10.0 13.5

0.1 0.2 0.3nqig. vitamin B^2

Fig. Response of L. leichmannii 4797 to vitamin B 2 ( table 8a ).

TABLE 8bNumber of c.c. of 0*1 N BaOB required to titrate the acidity produced in each tube containing milk.

Fubeao.c.c.ofmedium

e.c.ofmilk

c.c.ofwateradded Totalc.c.

. HaOH reauired Titamia B12I IT I GroroII III IT

ops.1 5.0 0.0 5.0 10.0 1.0 0.9 1.0 0.82 5.0 0.05 4.95 10.0 7.7 7.8 7.4 7.5 .182 .198 *16 .1985.0 0.05 4.95 10.0 3.1 8.0 7.8 7.9 .199 .199 .18 .1993 5.0 0.1 4.9 10.0 12.6 12.2 12.5 12.2 .42 .404 .436 .4125.0 0.1 4.9 10.0 12.7 12.6 12.7 12.4 .444 .444 *444 .4424 5.0 0.2 4.$ 10.0 14.0 13.8 13.4 13.35.0 0.2 4.8 10.0 14.0 14.2 14.2 13.7

TABI.E 9aAnalyses of variance* milk production (Experiment I)«

Sources of variance d.f. Sum o f squaresg IillI*#; f

Between the groups 3 Q.57 0,19 0.033.Within the individuals 16 2®1#0? 17.57Total 19 2&1.64

** Hot significant#

TABLE 9bAnalyses of variance * fat production (Experiment I)#

Sources of variance d#f# Sum of squaresSwan sum ofscraares F

Between the groups 3 0*142 0.04733 2.2948**Among the records 16 0*330 0.020625Total 19 0*472

* Calculated as per Snedecor (94)#** Hot significant#

TABLE 10aAnalyses or variance* milk production (Experiment II)**

Sources of variance d.f. Sum of squares sum ofsquares FBatmen the groups 3 *005 .00166 0.002**Among the individuals 16 12*90 .606Totals 19 12*905

me Hot significant*

TABU 10bAnalyses of variance9 fat production (Experiment 11)*

Seurees of variance d.f. Sum of squaresMean sum of squares f

Between the groups 3 .0395 .0296 5.96***Among the individuals 16 .060 .005Total .1695

* Calculated aa per Snedecor (9$) •*** Highly significant.

AUTOBIOGRAPHY

Muhammad Obayedule Haq was b o m In the village of Santanpara of the district Dacca, East Bengal, Pakistan, on January 1, 1920* Be received his primary education from his father, Mv. Abdul Maquit Khan, He passed the matriculation examination of Calcutta University in 1936 and was placed in the first division, where he obtained distinction in mathematics* He passed the Intermediate examination in Science in 193d from the Jagannath Inter* mediate College of the Board of Intermediate and Secondary Education, Dacca* He studied toward the degree of Bachelor of Science (in the Faculty of Science) in Physics, Mathe­matics, and Chemistry for about two years at which time he changed the course of his study and obtained the degree of Bachelor of Science (in the Faculty of Agriculture) from the Sail mullah Muslim Hall of the Dacca University, Pakistan, in 1942* He obtained the degree of Bachelor of Agriculture from the Bengal Agricultural Institute of the Dacca University in 1944* He was appointed and joined as District Agricultural Officer under the Bengal Governmenton May 20, 1944# He held positions in the different branches

*

of this department, both in administration and research* During his service he was selected by the Government of East-Bengal for further studies in Dairying in September 1947. He left his country on May 31, 1949 to do graduate

lOS

109

work at Louisiana State University in Dairying. June 9, 1949* he registered in the Summer school as a graduate student. He obtained the degree of Master of Science in Dairying in August, 1950. He was elected as a member of the honor society of Phi Kappa Phi in 1951# and also was elected a member of Sigma Xi in the same year. He is availing study leave as granted by his Government, and is a candidate for the degree of Doctor of Philosophy majoring in Dairy Nutrition.

EXAMINATION AND THESIS REPORT

Candidate: M* 0. Haq

M ajor F ie ld : D a iry in g

Title of Thesis: Effect of Antibiotics and Vitamin B-^ Feed Supplements on Lactating Dairy Cows

Approved:

M ajor Professor and Cxi airman

Acting Dean of the Graduate s c h o o l \

E X A M IN IN G C O M M ITTEE:

Date of Examination: