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A comparison of beef, pork, and whale liver meals as enrichment sources in the ration of growing rats Forrest, Robert James 1955

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A COMPARISON OF BEEF, PORK, AND WHALE LIVER MEALS AS ENRICHMENT SOURCES IN THE RATION OF GROWING RATS by ROBERT JAMES FORREST A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE in Animal Husbandry in the Division of Animal Science We accept this thesis as conforming to the standard required from candidates for the degree of MASTER OF SCIENCE IN AGRICULTURE. Members of the Division of Animal Science THE UNIVERSITY OF BRITISH COLUMBIA August 1955 ABSTRACT The primary purpose of this investigation has been to attempt to assess the relative value of beef, pork, and whale liver after each has been processed into a meal by a similar method. The secondary purpose has been to establish the relative value of whale liver when processed into a meal by several different methods. A l l liver preparations have been found to be relatively low in thiamine when this vitamin i s assessed by bioassay using the Wistar strain rat as an experimental animal. In almost a l l cases , further supplementation with cod liver o i l has produced a bene-f i c i a l effect with respect to the growth rate and feed consumption of rats. Many of the difficulties associated with bioassay have come to light in the course of this investigation. These difficulties have been examined in detail and some suggestions have been made as to how the detrimental effect of these difficulties may be minimized. An attempt has been made in this thesis to analyze a l l the pertinant data statistically. It was pointed out that when group differences in feed consumption and weight gain are large there i s not too much difficulty in the interpretation of the experiment. However, when group differences are small and the group consuming the most feed makes the largest weight gain, caoution must be used i f one states the differences between groups are due to the ration only. Under this set of circumstances, i f the data are subjected to an analysis of covariance no difference may be noted between groups even though when either feed consumption or weight gain are analyzed separately the difference between groups are signif-icant. In this case one must rely heavily on common sense before asserting any conclusions. ACKNOWLEDGEMENT The w r i t e r wishes to take t h i s o pportunity to. thank Dean B l y t h e Eagles, Chairman of the D i v i s i o n of Animal Science f o r p r o v i d i n g the f a c i l i t i e s i n order that t h i s work could be accomplished* Sincere thanks are expressed to Dr* A* J * Wood, P r o f e s s o r i n the Department of Animal Science f o r suggesting t h i s problem and a l s o f o r h i s d i r e c t i o n , a s s i s t a n c e , and c r i t i c i s m , d u r i n g the course of t h i s study* The w r i t e r a l s o wishes t o express h i s g r a t i t u d e to B r i t i s h Columbia Packers L i m i t e d f o r t h e i r f i n a n c i a l a s s i s t a n c e i n the course of t h i s work. TABLE OF CONTENTS Page A* INTRODUCTION . . . . . . 1 B. EXPERIMENTAL li-l t Preparation of Samples . k 2. Basis of Animal Assay £ 3. Nutritive Requirements of the Rat . . . IS 1J-* Experimental Diets . . * . . 22 5 * Experimental Bloaseays 2k C. SUMMARY jk BIBLIOGRAPHY . . . . . . 77 A t INTRODUCTION With the discovery of eaoh new vitamin the animal feeding industry i s able to take an additional step i n the direction of providing " idea l " rations for the do-mestic animals. In some instances, the new discovery mere-l y entails the adjustment of the natural components of the ration although i n many cases direct addition of the new vitamin in crystal l ine form or as a biological ooncentrate Is required* It has been customary to add some natural source of aooessory factors to rations to allow for the poss ib i l i ty that as yet unknown compounds inoluded i n the natural source are required by the particular animal. Table I i l lus t ra tes several complete rations i n which natural sources of vitamins are included. From this Table, i t may be noted that dehydrated grass, skim milk powder and l i v e r meal may be expected to serve as a source of such unknown factors. This situation has been well summarized by Kon and Porter (1) when they stated:. Though i t i s probable, that the vitamins well characterized are those of greatest nutri t ional and therapeutic importance, there i s no reason to. assume that the l i s t of B vitamins i s closed. Some natural materials, part icularly l i v e r , produce i n experimental animals growth responses, over and above those obtained with a l l known vitamins. - 2 -Table I Four Examples of Domestic Regions U t i l i z i n g N a t u r a l Vitamin Sources U.B.Q.. Rat i o n No. 10-52 ' Mink R a t i o n U.B.C. Rat i o n No. 102-54 Dog Ration Ground wheat Wheat bran Fishmeal (70%) Wheat germ meal Powdered Skim m i l k Dried yeast Apple pomace Beet pulp Ground oats Meat scraps Soybean meal L i v e r meal Bone meal Dried grass S a l t 450 % 50 75 50 10 30 75 75 50 10 5 1000 Ground ye l l o w corn 12.5 Ground b a r l e y 12.5 Ground wheat 12.5 Oat groats 12.5 H e r r i n g meal 21.0 Soybean meal 6.0 Wheat bran . 5.0 S t a b a l i z e d e d i b l e f a t 4.0 Steamed bone meal 1*0 Drie d brewers yeast 2.0 Dri e d fermentation s o l u b l e s 1.0 Molasses 5*0 Io d i z e d s a l t 0.5 Whale l i v e r meal 1.0 F i s h o i l 0.5 Skim m i l k powder 3.0 2250 A-400D o i l 100.0 U.B.O. Ration No. 22-52 Swine Grower, Ration B r o i l e r R a tion Ground oats Ground wheat Ground b a r l e y F i s h meal (70$) Meat scraps Linseed meal A l f a l f a l e a f meal Steamed bone meal S a l t 250 700 700 125 50 50, 100 20 5 2000 F i s h meal £.0 Soybean o i l meal §,0 Meat and bone soraps g.O L i v e r meal 3.0 R i b o f l a v i n supplement 2.0 ( b u t y l fermentation) A l f a l f a l e a f meal 1.0 Bone meal 1.5 S a l t 0.5 2000A - 400D o i l 0.3 Manganese s u l f a t e 0.025 N i c o t i n i c a c i d Trace Choline, c h l o r i d e Trace Corn 1 67.7 100.0 * 3 ~ Mammalian l i v e r has proven to he a r i c h source of the vitamins* This i s not too surprising i f one appreci-ates the metabolio role carried out by this organ* Vitamin B-12 (2). pantothenic acid (3), and f o l i c acid (4) may be cited as examples of a few of the known vitamins for which l i v e r has served as a source i n the course, of their isolation and identification* The. established role of l i v e r meals i n commercial animal rations places a definite strain on world sources of suoh meals* The Increasing consumption of fresh l i v e r by the human population and its. use. by the pharmaceutical industry has meant that only the poorer grades of l i v e r nave been available for dehydration and use i n animal feeds* In addition, there i s circumstantial evidence to indicate that many suoh commercial l i v e r meals are of lower quality than would be anticipated from the known inherent value of the l i v e r * The l i v e r of marine mammals has reoeived consider-able attention as commercial sources of vitamins A and D* With the advent of synthetic vitamin A at competitive prloee, some interest has been evoked In the possible use of such li v e r s for vitamin B complex: recovery (5)» The av a i l a b i l i t y of appreciable quantities of whale l i v e r i n Canada on both the Atlantic and Pacific seaboards suggests that this materi-a l should be examined as a possible supplement for the animal feeding industry* The present thesis has as i t s objeotive a preliminary study of whale l i v e r meal to deter-mine i f i t might prove of use for this purpose* B. EXPERIMENTAL 1, Preparation of Samples On the P a c i f i c coast, commercial whales can he divided i n t o two d l s t i n o t types, namely, "toothed" (Odontooetl) and "baleen" (Mystaooceti) whales* (6) Of the "toothed" whales, only one species, the Sperm Whale (Physeter catodon) i s commercially important* Of the "baleen" whales, there are four important species, namely, Finback (Balanoptera  physalus), Sel (Balanoptera b o r e a l i s ) . Humpbaok (Mega.ptera hodosa), and Blue (Balanoptera musouius). As might be ex* peoted there are many differences i n "toothed" and "baleen" whales but f o r present purposes the most important i s probably the marked d i f f e r e n c e - i n l i v e r vitamin A between the two groups* Sperm whale l i v e r i n general has a higher vitamin A content* Since sperm l i v e r i s l i k e l y to remain an important source of vitamin A, i t was decided to f i r s t examine "baleen" whale l i v e r f o r i t s n u t r i t i v e value* For t h i s purpose, one of the most p l e n t i f u l species, Finback, was chosen f o r the preliminary study* a* Whale l i v e r preparation The c o l l e c t i o n of samples was c a r r i e d out by the author at Coal Harbour B* C* i n the summer of 195^. Because of the si z e of t h i s organ, whole whale l i v e r s oould not be used* To ensure a representative sample, f i v e s l i c e s were removed from eaoh of thirty-five Finback whale livers* Each liver contributed approximately a four pound aliquot* The sampling was carried out approximately thirty hours post mortem* It would have been desirable, to obtain sam-ples immediately after death, but the size and nature of this, mammal precludes immediate sampling under B* C« whaling conditions* As soon as the samples were collected, they were placed In a freezer maintained at 0°F until the entire sample had been eolleoted. The complete sample was then delivered to this laboratory in the frozen state* After partial thawing, the liver was ground, mixed in a large Hobart mixer, distributed into quart or gallon cans, sealed, and stored in the frozen state until used* b* Beef and pork liver preparation The beef and pork liver samples were obtained immediately post mortem from a local slaughter house* The beef sample was obtained from two livers and the pork from ten livers* These samples were ground and prepared in the same manner as for the whale liver* o* Preparation of liver meals i * Vacuum dried.whale liver (VPWL), yaeuum dried  beef liver IVDBL), and vacuum dried pork liver. IVDPL) The vacuum dried beef, pork, and whale liver were a l l prepared in a similar manner* The ground liver, was distributed in approximately one pound portions between two, 2-litre bomb flasks. The liver was evenly distributed about the Inside surface by rotation of the flask* The two flasks were then attached to a vapour trap and a vacuum pump. The vapour trap consisted of a 2-litre suction flask immersed in a bath of dry ice and isopropyl alcohol. The system was then evacuated and maintained under a vacuum of less than one mm, of mercury for a period of 2k to k&. hours during whloh time 72 to 75 percent of the sample was removed as moisture, 'The dry samples were then taken from the flasks and stored in sealed containers at 0°F, When a sufficient number of batches had been prepared In this manner, the dried l i v e r from each sample was pooled and passed through a No* 12 sieve in a small Wiley m i l l and stored frozen un-t i l used, 11, Enzyme hydrolyzed whale l i v e r (EHWL) The enzyme hydrolyzed whale l i v e r was prepared by masoeratlng approximately 71 pounds of raw l i v e r and mixing i t with 210 pounds of water. This mixture was then heated to H5°F and 160 gm, of rhozyme B-6, a commercial proteo-l y t i c enzyme of fungal origin, was added and the mixture was agitated and maintained at constant temperature for two hours. This material was then screened and centrifuged to remove'approximately 19 pounds of undigested solids. The c l a r i f i e d liquor was then evaporated under 25 to 28* inches of vacuum to a final., consistency of 35,g percent solids. The material was then acidulated with HCL to a pH of lJ-,5 and stored frozen u n t i l used. Unfortunately, due to d i f f i -culties involved in the processing of this enzyme hydrolysste, no quantitative comparison can be made as to how much original whale l i v e r was represented i n the f i n a l product* This product was prepared by the laboratory staff of Norman L* Armstrong Ltd., Vancouver, B. 0. T" ..... i l l . Enzyme hydrolyzed a i r dried whale l i v e r (ADWL) The hydrolyzed a i r dried l i v e r was prepared by placing 1000 grams of the above hydrolysate i n a ftunnel a i r dryer? maintained at 105°G for seven hours. The re-sulting dried material was then passed through a Wiley m i l l and stored frozen u n t i l used* iy* Spray dried whale l i v e r (SDWL) The spray dried whale l i v e r was prepared from the enzyme hydrolysate* The spray drying was carried out by Bowen Engineering Inc. of New Jersey. The drying-was carried out In five different batches in which influent a i r temperatures of 300, 400, and 5 0 0° F and maximum feed rates of 90, 130, and 1$0 ml per minute respectively were used* The five batches of dried.material were later mixed and stored frozen u n t i l used. The f i n a l product was finely divided and tended to be somewhat hydroscopic as were the vacuum dried l i v e r samples, v. Drum dried whale l i v e r (DDWL) The drum dried whale l i v e r was prepared at B, 0. Packers Whaling Station at Coal Harbour B. C, during the summer of 1954, This l i v e r was processed on a commercial drum drier using the. standard conditions for its. operation. v i . 50/50 Drum dried whale l i v e r (50/50 DDWL) The 50/50 drum, dried whale l i v e r was prepared i n the same manner as the drum dried whale l i v e r . However, . prior to the drum drying, 5© percent by weight of whale solubles was added to the l i v e r * The whale solubles are the concentrated material obtained after removal of the o i l from the blubber and bone portion of the whale* This product i s generally u t i l i z e d as a protein source but also may have some value for i t s vitamin content ( 7 ) * v i l * Commercial l i v e r meal (CLM) The commercial l i v e r meal used in this project was a sample of a l i v e r meal that i s available tp the animal feeding industry i n considerable quantities* This meal i s composed of both f i s h and mammalian li v e r s * In Table II the proximate analysis, and chemical analysis of Vitamin A. for the nine samples l i s t e d above and raw whale l i v e r are given* These analyses were also carried out by the laboratory staff previously mentioned* 2* Basis of Animal Assay A number of methods for the assessment of the nutritive value of natural materials have been used* For example Gray et a l . (5) have recently carried out micro? biologloal. assays on livers from, several species of whales* Their results are reproduced In part in Table III* Much valuable information has been obtained by such assays but unfortunately, for a variety of reasons, the ultimate oriterion of nutritive value must be by means of animal bioassay. This i s borne out by the work of Unna et a l * (£>) In which i t was demonstrated that close agreement between a microbiological procedure and an animal assay, was not obtained. Such discrepancies are not uncommon.and must •d © a ^ «d © P> > *H 3 V i O 0) aS © , © (4 •H © Qa-3 © O- OS «d © © H O © W -H © g © ^ ^ tn m ni O OA tO • • • - • • ON UN OV VO U N in H . • KN <H CO C-^ ^ iso xo U N r-H -3" *0 KN O rH • • • • • - • « I— *H O VO O f"— -H C! H VO ^ ^ ^ ^ ON O VO ISO VO VO rH H IT\ H • • • • • • • • Tea rH VO ON Jd" LfN CS rH VO CM • • • • r-i VO 1 I UN H . rH CU O VO •sh-i n u © © rH S at BO - • r-i • « 01 © u 3 © «H O fo c •ri © o A © 5* 0 O T50 r — ON 0 CU cu -=fr ON iso H ON « 0 rH • • X a) © © a .Q 3 ' < +» aa c d ri 0 w- +>• +*• u •H 0 >• •H © _ © •d a © © a > © o 4-1 >>Oi4 rH O •d* © >»«d > §.&© «d U © © •H > U -H (=» J 3^ «d © a © ? > wH O © UN3E _ **' •d. © © > «H -H © a H ^ ^ \K O O -=h ON UN f>- KN H • • • • • • • • VO VO KN KN rH rH UN UN CU KN UN O KN UN TSO v o CU CU • • • • • • • .sa-.=r LfN UN i n r-i «H (3 ISO • • H ^ ^ KN rH BO .=*• J * O • •• • • • • t— UN VO UN O UN •60 VO H H UN ON KN O • '• « • • KN 150 «0 KN VO H •H G KN KN UN KN" KN UN KN UN • • • • ' • • • UN ON VO UN r -H v o -=*• ON 160 H • • at © < © » a 3 •ri © B © *» xi CD +» O 4» O U ca •H P* ON ISO a CQ a o o Table III Vitamin. Content of Four Species of Whale and Whole Beef Liver Milligrams per 100. grams Solids . Finback Sel Humpback Sperm Whole beef Thiamine 0.51 0.64 0.64 O..56 O.36 - O.39 Riboflavin 5.50 6.00 6.00 4.90 O.90 - 3.00 Niacin 31.00 33.00 140.20 44.20 S.OO -23.00 Pantothenic Pyridoxins Acid S.20 0.42 13.40 1.03 12.50 0.S5 12.50 O.SO 4*50 - 9.00 O.lK) - 1.00 Vitamin B 1 2 ° 0.06 O.13 0.26 0 , l 4 0.15 Vitamin B l g 0.06 O..09 0.12 0.11 0 Lactobacillus l a c t i s 0 0 Escherichia ooli -11 - 1 Inevitably, arise for a number of reasons* The a b i l i t y of microorganisms to synthesize a specific vitamin from i t s precursors may be cited as an example (9)* Mammalian cell s i n many cases lack this a b i l i t y * Under suoh circum-stanoes one i s not l i k e l y to. obtain good agreement between animal and microbiological results* Even with many of the speclfio ohemioal methods for vitamin determinations It has been found that discrepancies exist between results obtained with chemical methods and results obtained with animals* Perhaps the best example of this type of discrep-ancy i s provided by the work of Morton and Stubbs (10) on vitamin A* The d i f f i c u l t i e s referred to, above are magnified and confounded when one attempts'tp obtain an overall assessment on a natural product such as l i v e r meal* How-ever, for the present work, animal bipassay was selected* Such methods are not new, i n fact, one of the original bioassays may be cited as follows: And It came to pass at the end of forty days, that Noah opened the windows of the ark which he had made: And he sent forth a raven, which went to and fro, Until the waters were dried up from the earth* Also he sent forth a dove, from him, to see i f the waters were abated from off the face of the ground; But the dove found no rest for the sole of her foot, and she returned unto him into the ark, for the waters were on the face of the whole earth; then he put forth his hand, and took her, and pulled her in unto him into the ark* And he stayed yet other seven days; and again he sent forth the dove out of the ark; And the dove came i n to him i n the evening: and lo, i n her mouth was an Olive leaf pluokt off; so Noah knew that the waters were abated from the earth* (Genesis, g, vi-xi) ~ 12 -As Finney (11) has pointed out "The three essential constituents of an assay, stimulus (depth of water), subject (the dove)., and response (the plucking of an olive leaf), are present in this description* Knowledge of the response enabled Noah to estimate, or rather, in this instance, to place an upper limit to the size of the stimulus*. The limitations of his animal house made his replication less than would to-day be thought, adequate, but In other respects his experiment was admirable for' Its purpose*11. Just as discrepancies exist between types of assay so. do discrepancies exist within a particular type of assay* Bloassay is no exception* A single animal, on a specific ration, under a given set of conditions can only give one response and generally one interpretation provided the experiment Is properly designed* As the number of ani-mals on test is increased so the complexity of the experi-ment and the results obtained increases* Then one must re-ly on common sense to evoke the correct interpretation of the experimental results* At the same time, sight must not be lost of the most important feature of the experiment* This feature, is very aptly put by Carrol (12) when he states: "The value of any experiment lies not in the results ob-tained, but In the usefulness of those results for; pre-dicting what can be expected in the future*" There are various, ways of approaching the problems concerned with bloassay and various, procedures that can be followed in carrying i t out* One of the first choices that - 13 -presents i t s e l f Is group or individual feeding* About the only point i n favour of group feeding i s i t s economy* Some may hold that group feeding has an advantage i n that i t induces competitive feed intake and consequently higher feed consumption than Individually fed animals* This type of reasoning has l i t t l e s c i e n t i f i c basis (13) and no place of importance i n the. more desirable controlled experiment* Falatabillty on the other hand may be of importance i f the main concern of an experiment i s to assess two rations as a whole on. a qualitative basis or i f the toxicity of cer-tain ingredients i s of importance* However i f the main concern of the experiment i s a quantitative comparison of two rations to determine their relative nutritive value, palatability must be disregarded* I t would appear then that individual feeding i s vastly superior to group feeding* Individual feeding allows an interpretation of the response of eaoh animal as a unit rather than merely as a group* Consequently a more reliable mean value i s obtained and the data i s more applicable to s t a t i s t i c a l analysis* A clear picture may also be obtained of the specific feed consumption and weight gain relationships* The specific disadvantages of group feeding are discussed i n detail by Crampton ( 1 4 ) , A fundamental principle that should be adhered to not only in bloassay but In any type of experiment i s that only one variable be varied at a time (15). For instance, i f there are two variables i n an experiment, i n the f i n a l analysis i t cannot be ascertained whether the effect was due - 14 -to either variable singly or due to interaction of the two variables* If the two variables are affecting the experi-ment the case may arise where the effect of one i s n u l l i -f i e d by the other* This case does not often arise i n blo-assay but often when two variables are present, an addatlve effect w i l l be occluded i n the results* Another faotor that should be considered with animal experimentation i s environmental effect* Obviously suoh faotors as season, temperature, altitude, length of day, etc* w i l l exercise an effect on animals* An animal husbandman i s not too interested i n research projects carried out on a researoh station as such but he i s particu-l a r l y interested i n how a projeot w i l l effect his animals on his farm* For example, i f a comparison Is to be made of the relative value of two protein sources as a winter supplement on the range, one may assume that i t i s necessary to carry out the experiment under winter conditions on the range* This concept i s entirely fallacious* The primary Interest i s the comparison of protein sources and not the ab i l i t y of cattle to withstand winter conditions* Of course this second factor i s very Important but i t should be consi-dered in a Separate experiment* Sinoe the primary concern i s protein comparison the environmental effect should be eliminated as much as possible so that only one variant affects the results* This principle i s i n accordance with the above considerations* - 15 -Perhaps one of the most important considerations in bioassay is the method of feed allotment* Hopkins (16) in 191.2» and later Osborne and Mendel (17) were probably the first writers.to show a marked interest in the complex problem involving differences in feed consumption between experimental groups* Armsby (1*3) was probably the first; man to attempt an experiment involving equalized feed in-? take* Later Mitchell (IS) clarified and developed the paired feeding method* Much controversy has since ensued on whether to use ad libitum or controlled feeding practices* The ad libitum.feeding method has a very important limi-tation* If by ohance in a feeding t r i a l , two groups con-sume the same amount of feed or i f the weight gains, are identical, the interpretation of the results is straight-forward* However, as is usually the case, the feed con-sumption and weight gains of two groups will differ marked-ly and consequently no definite conclusions can be stated since the difference in weight gain may have been due to the nutritive content of the rations or merely due to differences in feed concumptlon* By using the paired feeding method, in which two (or more) genetically similar animals consume the same amount of food, governed by the member of the pair consuming the least food, this discrep-ancy is eliminated* The weight differences can then be assessed more accurately since now this difference can only be due to the superiority of one ration or theother. However the paired feeding method is not the final answer-16 ? i n animal experimentation s i n c e two d i f f i c u l t i e s can a r i s e . The member of the p a i r which has i t s f e e d consumption r e s t r i c t e d may be adversely a f f e c t e d i n that i t w i l l tend to be more a c t i v e than i t s mate* Consequently some of the food consumed w i l l be d i s s i p a t e d as " a c t i v i t y . 1 1 A l s o * as an animal gains weight, the composition of the g a i n changes from l a r g e l y p r o t e i n m a t e r i a l d u r i n g the f a s t growing phase to l a r g e l y f a t during the maturing p e r i o d . The f a s t e r growing animal w i l l a l s o have a h i g h e r maintenance cost but t h i s f a c t o r can be c o r r e c t e d without too much d i f f i c u l t y and w i t h f a i r accuracy (13), These d i f f i c u l t i e s can be overcome i n p a r t by e q u a l i z i n g the weight g a i n and v a r y i n g the feed consumption but f o r o r d i n a r y bloassay technique t h i s procedure would be f a r too l a b o r i o u s and time consuming. At any r a t e , the p a i r e d feeding method should be used wherever p o s s i b l e i n preference t o ad l i b i t u m feeding s i n c e i t has many d i s t i n c t advantages. A f u r t h e r p o i n t t h a t should be considered w i t h bloassay I s the method of experimental i n t e r p r e t a t i o n . The i n t e r p r e t a t i o n begins not at the end of the experimental p e r i o d but r a t h e r before any animals are p l a c e d on t e s t or before any r a t i o n s are formulated. An assay should not be designed i n such a manner that one i s dependent on the r e s u l t s to provide the answers. The experiment should be designed w i t h a preoonceiyed concept or hypothesis i n mind. The purpose of the t r i a l then, i s to prove or disprove t h i s hypothesis i n much the Same manner that the *?nul hypothesis" - 17 -i s used in s t a t i s t i c a l procedures* I t should not be con-strued that this preconceived hypothesis introduces any bias, for the experimenter i s merely concerned with whether the hypothesis i s right or wrong* Any " i f " or "but" Interpretations should be used with caution* I f one finds i t necessary to resort to "If" and "but" interpretations i t usually can be' inferred that the experiment was not designed with the proper amount of control* In any event, the most valuable tool in any interpretation i s common, sense (19). I f two groups of animals on test show a wide difference in weight gains and i f every member of one group was superior to the corresponding animal i n the other group, i t requires l i t t l e imagination to conclude that the results are due to experimental treatment* However, when mean values are not too different and i f individual re-sponse deviates from this mean value to a considerable extent, one cannot ascertain by observation whether the difference i s significant* In.this oase we must resort to st a t i s t i c a l analysis to assist i n the interpretation* This analysis takes into consideration not only- the mean differ-ences between groups but also the dispersion of the data* It provides a measure of the minimum amount that mean values must dif f e r before i t can be concluded that these, differences are due to treatment rather than chance* Assessing the significance of an experiment by mean values alone can often lead to erroneous conclusions since the IS* -mean value may fluctuate markedly due to. extreme values* Here again the importance of knowing Individual' response is illustrated* 3* Nutritive Requirements of the Rat The rat has long been used as an experimental animal and consequently its nutritive requirements are fairly well understood* Its nutritive requirements with respect to carbohydrate are easily met as are the fat re-r qulrements since the three essential fats linolenio, lino-leio, and arachldonic are widely distributed in natural feed constituents* The picture with respeot to protein and particularly amino acids has. been olarifled by the work of Rose (20) with his determinations of the essential and non essential amino acids* The rat requirements for inorganic elements are not too well understood partially because many elements are required in such minute quantities* However, their requirements for minerals Can be met. without d i f f i -culty. The f i f t h class of nutrients, the vitamins, have been, and are today the subject, of a vast amount of research. It should be noted that the term vitamin has no chemical significance (21) since the members of this group are not related chemically and are engaged in a wide variety of physiological processes;• However the members of this group have continued to grow over the years and there is no reason to suppose a limit has been reached as yet (1). The role of vitamins in animal nutrition is varied and oomplex* The picture is further confounded by the Interrelationship between the specific members, A shortage of one vitamin may lead to lack of u t i l i z a t i o n of a second vitamin even though the latter i s present i n adequate quantity. It i s also very d i f f i c u l t to ascribe speoifio symptoms to vitamin deficiencies, especially the B complex group. For instance lack of thiamin results i n anorexia and nervous disfunction. The f i n a l symptoms are a combination of thiamine deficiency and starvation. Loss of hair and the various forms of dermatitis are also associated with B.oomplex:. deficiency but are very d i f f i c u l t to assooiate with any specific vitamin. On the other hand, graying of the hair in black rats has been alleviated by pantothenic acid (8) but other factors such as biotin have also shown therapeutic value* One of the most important functions of the B complex vitamins i s their activity as coenzymes* Thiamine, as oocarboxylase, a thiamine phosphoric ester, catalyzes the decarboxylation of pyruvic acid. Riboflavin, or War-burg's yellow enzyme, serves a redox: function i n various forms as does nicotinamide. Further research w i l l probably bring forth evidence for the enzymatic activity of other vitamins. However, a l l vitamins appear to play intrloate roles i n cellular metabolism. The eluoidatlon of their metabolic function has been f a c i l i t a t e d by the discovery that certain of the.microorganisms require the specifio vitamins for their normal growth and development. It i s not an easy matter to designate the 20 quantitative vitamin requirements of the rat slnee a l l the required vitamins have not been identified* In ad-dition the vitamin requirements w i l l ohange with stage of growth* It i s now well established (22) that generally, the B vitamins are required i n direct proportion to total energy metabolism or total feed intake whereas the fat soluble vitamins, especially Vitamin A are required i n direct proportion to body weight* In the growing, animal, in the early stages of rapid growth the animal Is synthesizing tissue and vitamin requirements are high whereas i n the mature state the animal i s simply replacing tissue and the vitamin requirements are greatly reduced* When dealing with dietary requirements the picture can also be. greatly confused by the Intestinal microflora* The commensal microflora may synthesize vitamins and lower dietary requirement, or they may u t i l i z e vitamins and raise dietary requirements* In 194l Richardson et. a l * (23) indicated that sub optimal growth and reproduction could be induced In rats on a synthetic diet supplemented with Vita-mins. A, D, E, thiamine, riboflavin, calcium pantothenate and choline* It would appear that these rats were also u t i l i z i n g other vitamins in sub optimal amounts probably produced by intestinal microorganisms* A specific diet w i l l also tend to establish a characteristic f l o r a i n the digestive tract* Thus the carbohydrate source, be i t suorose, starch, or lactose, may tend to have a speolfio microflora associated with i t * It would seem reasonable to suppose then,. * 21 -t h a t the v i t a m i n requirements c o u l d change by simply a l t e r -i n g the r a t i o n c o n s t i t u e n t s * I n view of the above c o n s i d e r a t i o n s , the d l f f i -o u l t y Involved w i t h assessing q u a n t i t a t i v e v i t a m i n r e q u i r e -ments becomes obvious. Wide d i s c r e p a n c i e s w i l l ' a l s o be found i n the l i t e r a t u r e * I t would appear then that e n v i r o n -mental f a c t o r s , i n as much as they i n f l u e n c e metabolic r a t e , as w e l l as n u t r i t i o n a l f a c t o r s w i l l e f f e c t the s p e c i f i c v i t a m i n requirements* I n view of the present knowledge w i t h respect to vitamin s and I n s p i t e of the d i f f i c u l t i e s i n v o l v e d , i t i s h i g h l y d e s i r a b l e t h a t a base l i n e be e s t a b l i s h e d w i t h r e -spect to the minimum amount of vit a m i n s r e q u i r e d by the r a t * Table IV i l l u s t r a t e s the recommended in t a k e of the v a r i o u s known vitam i n s * Table IV The Recommended Vitamin Requirements of the Rat: Vitami n Requirements per Day 4 mgm* 30 USP u n i t s 1 mg* 10 mgm* Uo mgm* 10 mgm* 15 mg* A D Thiamine R i b o f l a v i n P y r i d o x i n s Choline - 22 -k. Experimental Diets In the bioassay portion of this thesis the experimental diet used is typical of those used in studies with the rat* The composition of this diet and the salt mixture used in i t are shown in Table V. To this basal diet, various levels of liver meals were added* The de-tailed preparation of these liver meals, nine in a l l , has been described previously* For simplicity* in the following disoussion these preparations will be designated by their oode letters* Table V Composition of Basal Ration and Salt Mixture Constituents Amount Sucrose 63 Vitamin Free Casein 18 Mazola Oil 10 Salt Mixture No* 2 U*S.P* XIII _J+ 100 Caloium Bl. phosphate 13*5$ Calcium Lactate 32*70 Ferric Citrate 2*97 Potassium Phosphate (Dibasic) 23*961 Sodium Blphosphate $*72 Sodium Chloride 4.35 Magnesium Sulphate 13*70 100.00 - 23 -Since the diet tabulated in Table VI i s a v i t a -men free diet, i t would be desirable to see what effect the addition of vitamins to this diet would have on growth* When this was done, the vitamin f o r t i f i c a t i o n mixture (VFM) of the composition shown i n Table VI was used. Table VI, Composition of Vitamin Fortification Mixture (VFM) (Titurated i n Dextrose to Make 1 Kilo) Vitamin gms. Vitamin A (200,000 units per gram), Vitamin D (400,000 units per gram) o .25 Alfa Tocopherol 5.Q Aosorblc acid 45.0 Inositol 5*0 Choline Chloride 75*0 Menadione 2.25 P Amlnobenzolo Acid 5*0 Niacin 4.5 Riboflavin 1.0 Pyridoxlne Hydrochloride 1.0 Thiamine Hydrochloride 1.0 Calcium Pantothenate 3.0 Biotin 0.20 Folio Aold 0.9 Vitamin B-12 0.QI35 - 24 -To return to the basal diet (Table V), some question may arise as to the adequacy of the constituents* There can be l i t t l e criticism as to the carbohydrate source* The protein souroe may be low In certain of the amino, acids but this has been compensated for i n part by raising the protein level above the recommended requirements* The selection of the salt or mineral mixture may cause concern i n view of the work of Lyman and Elvehjem (24), Kandutsoh and Bauman (25) and others i l l u s t r a t i n g the l a b i l i t y of thiamine in purified rations* However the work of Rambouts (26) indicates that not a l l salt mixtures exert a detrimen-t a l effect on B complex vitamins* Thus there i s good reason to suppose that the drastic reduction i n thiamine reported by Kandutsoh and Baumann need not apply i n the case of the Simplified salt mixture chosen for this investigation* This w i l l be substantiated later In the experimental portion of this thesis* 5* Experimental Bloassays The primary purpose of this investigation has been to attempt to assess the supplementary value of whale l i v e r when added to a synthetic diet devoid.of vitamins* The work has been considered i n two phases: (1) to determine the nutritive value of whale l i v e r with respect to the vltar mine by a direct comparison to beef and pork l i v e r , the values of which have been well established by the literature, and (2) to determine the relative value of whale liver, meal after various methods of preparation* - 25 -The second phase of the work Is very Important sinoe to make use of l i v e r In the animal feeding Industry i t i s desirable to< f i r s t convert i t to a dehydrated form to f a c i l i t a t e storage and handling* From this It, i s essential to know what effect the desiccation process has on the nutritive value of the product i n question* The assay portion of this investigation consisted of a number of biological assays using the Wistar strain of rats as experimental animals* Weanling rats were placed on test at the age of 20.to 24 days* The majority of the ani-mals weighed from 40 to 50 grams at this age* The experi-mental groups i n most oases were made up of five rats con-sisting of three females and two males* In. general, the assay period was 42 days* In many of the assays, thiamine and)or cod l i v e r o i l (3000A - 400D) was added to the ration* When this was the oase, thiamine was added at a level of 0*025 grams per kilogram of ration and cod l i v e r o i l was added at the one percent level* a* Assay 1 The objective of this assay was to compare beef, pork, and whale l i v e r when added to the basal diet at the three, six, and nine percent levels* To accomplish this, nine rations having the compositions shown i n Table VII were prepared* An attempt was made to follow Mitchell ls -* 26 -Table VII Composition of Rations Used In Assay 1. In Prams Levels Ration No, Basal Ration VDPL VDBL VDWL Total 150Q15QQ 1500 1500. 1500 1500 1500 1500 I50.O 1socaloric feeding method (IS)* A total of 5^ weanling rats were chosen i n groups of three l i t t e r mates of the same weight ( 3 gm.). Thus a total of six rats were alloted to each ration and the three Corresponding rats on a specific level were l i t t e r mates* The rats, on different levels were not necessarily related* It should be noted that the total weight of the three groups on a specific level were the same* Feed was weighed out daily and body weights were reoorded every three days* Table VIII shows the i n i t i a l and f i n a l weights of the rats on a 27 day experimental run* Since the I n i t i a l group weights, within levels at least, were almost the same, i t was decided to test the f i n a l weights only for significant differences, between species (type of l i v e r supplement) and levels* The analysis of variance (Table IX) indicates that there i s a • # W W 1 2 3 » .5 6 7 * Q 1^55 1455 1455 1 4 1 0 l4 io . l4 io . 1365 1365 1365 ^ 90 _ 135 _ _ " _ ^5 ^ ^ 90 _ _ 135 _ — — ^5 _ . ' 90 _ 135 * 27 Table, VIII I n i t i a l and Final Weights In drams, of Rats Used In Assay 1 Level Sex- VDPL VDBL VDWL I n i t i a l Final I n i t i a l Final I n i t i a l Final M RI 76 52 71 51 7* M % 69 W 70 44 69 3# F 51 68 52 66 49 74 F 51 72: 52 67 52? 61 M 49 72 49 71 % 64 F kj 53 43 50 42 58 F 48 81 48 82 47 84 M 44 96 44 96 44 98 F 38 87 ft 92 39 9^  F 43 84 44 91 43 82 M 41 107 40 109 40 111 F 39 so 39 82 39 85 F 43 103 41 113 42 112 M 50 125 49 135 *9 129 F 41 104 41 101 42 102 F 49 120 48, 116 47 109 M 13 98 32 95 30 97 F 41 108 39 114 39 118 Table. IX Sum of Degrees of Mean Variance Source of Variation Squares Freedom Square Ratio Species (of liver) 13,5 2 6*8 0.6 Levels 17»^77»5 Z- 8,238*7 710*2 Sex 1 7 2 , 5 1 172*5 Species x levels interaction 63*0 4 1 5 * 7 1*4 Species x sex " 9,0 2 4 , 6 0 . 4 Levels x Sex • 1,327*5 2 663.J 57.2 Sex x level x species M 2,763*0 4 11,6 5 9 , 6 Error 4 1 7 . 7 36 Total 22 , 2 4 6 . 7 53 - 28 -significant difference between Increasing levels of supple-* mentation but no signifleant difference between beef, pork, or whale l i v e r at either of the three levels used. The most tempting conclusion at this point Is to state that beef, pork, and whale l i v e r are equivalent with respect to enrichment value and that a l l three are low i n at least one vitamin at the three and six percent levels of supplemen-tation. However, there are two points that make one hesi-tate to draw any conclusions at this point. In Table X Is li s t e d the average weights of the various groups at three day intervals. They indicate that the rats on the three and six peroent levels were losing weight in the latter portion of the assay. The rats on the nine percent level were not making the rapid gains that would be expected at this stage of growth. Also, while the dally feed a l l o t -ments were being made, i t was noted that the rats being fed alration containing whale l i v e r almost invariably limited the feed consumption of their l i t t e r mates. It was decided that further investigation of the experimen-t a l method was essential* Table X Average Weights of Groups 1-9 at 3 Day Intervals In Assay 1 Days Group 0 3 6 9 12 15 18 21 2k 27 1 48 55 66 2 h 56 65 I & g £ 5 kz 51 61 6 k2 52 63 7 43 51 62 8 k2 50 62 9 k2 49 60 70 77 SO 78 7? 73 68 70 76 75 7J 7> 71 13 66 21 Z g 16 67 72 S3 89 §2 n 94 8$ 73 39 92 96 95 92 73 82 -S8" 92 96 95 92 74 6k 90 100 107 109 110 72 8-5 91 101 106 110 112 72 82 SS 95 101 108* 111 - 29 -b* Assay 2 Assay 2, c a r r i e d out i n three p a r t s , was designed to i n v e s t i g a t e f u r t h e r , the r e l a t i v e value of beef, pork, and whale l i v e r as enrichment souroes, and a l s o to examine the experimental method* A t o t a l of 15 weanling Wistar r a t s were chosen i n groups, of three l i t t e r mates, and set up i n the same manner as groups f o u r , f i v e , and s i x : of Assay 1* The r a t i o n s used i n the f i r s t p a r t of Assay 2 were the same as those used i n f o u r , f i v e , and s i x groups of of. Assay 1* I n p a r t s two and three Assay 2, the r a t i o n s were supplemented w i t h thiamine h y d r o c h l o r i d e ( i n c r y s t a l -l i n e form)* I t was decided that i t would be more d e s i r a b l e to t e s t the s i g n i f i c a n c e of weight g a i n as weight g a i n over a s p e o l f l o p e r i o d r a t h e r than u s i n g only the f i n a l weights* This w i l l tend to take care o f s m a l l d i f f e r e n c e s i n i n i t i a l weights* I t immediately beoomes obvious t h a t i n comparing i n d i v i d u a l s or groups i n t h i s manner no account i s taken of the f a c t t h a t growth and feed consumption are not l i n e a r w i t h respect t o time* I t i s w e l l known (22) t h a t percentage growth r a t e decreases w i t h time (although I t may be constant; over a smaller s p e c i f i c time i n t e r v a l ) and o b v i o u s l y i f an animal i s to grow feed consumption must inorease s i n c e the maintenance cost i s i n c r e a s i n g * However, t h i s assumption need not introduce any e r r o r since a l l groups are oonsidered on the same b a s i s * The weight gains and feed consumption of the r a t s over a s p e c i f i c time I n t e r v a l are t a b u l a t e d i n Table XI ( i n a l l three oases, the weight ga i n and feed consumption are c a l c u l a t e d from the beginning of the experiment although p a r t s two and three were a o t u a l l y only 12 days i n l e n g t h ) * - 30 -Table XI T o t a l Weight Gain and Feed Consumption I n grams of Rats i n Assay 2 Weight Gain R a t i o n Group Sex P a r t 1 6#VDBL 1 6#VDPL 2 6j£VDWL 3 F M F M t F M F M F F M F M F C o n t r o l l e d Feeding .27 days FO 157 163 166 158 12S l§9 1|5 165 158 128 *57 164 163 157 I25-WG 41 48 40 51 p 62 40 40 1 P a r t 2 C o n t r o l l e d Ad. l i b . Feeding (12 days)Feeding Thiamine added .(12 days) 39 clays Thiamine added 51 days PC WG 220 26S 241 232 196 224 267 240 232: 196 220 263 239 228 190 85 51 71 58 •8 §7 8 FC WG 345 375 308 340 425; 3Z§ 386 307 287 34| 318 30* 254 79 113 93 10" 14: 90 61 96 74 The weight g a i n data of Table XI were analysed s t a t i s t i c a l l y and the r e s u l t s are t a b u l a t e d i n Table X I I . Feed consumption data have been i n c l u d e d i n Table XI but were not analysed. I t was noted at t h i s stage of the i n v e s t i g a t i o n ; as one might: expect, that there appeared to be a c o r r e l a t i o n between f e e d consumption and weight g a i n , o r , s t a t e d i n other terms, the group that- made the l a r g e s t gains consumed the most f e e d . - 31 -Table X I I A n a l y s i s of Variance of Assay 2 V a r i a t i o n DP SS MS VR Sex; 1 6,ljk 6,134 Speoies 2 1,249 625 17*7 Time 2 16,979 8,475 Sex x Speoies 2 197 99 Sex x Time 2 1*279 6* Time x Speoies 4 2,088" 522: Sex x Time x Speoies 4 168 42 E r r o r 27 954 35,3 T o t a l 44 29,01S Mean d i f f e r e n c e r e q u i r e d f o r s i g n i f i c a n c e (at p a 0*01) = 13*9 gm Mean d i f f e r e n c e between groups 1 and 3, P a r t 3, Table XI= 17.2 gm Mean d i f f e r e n c e between groups 1 and 2, P a r t 3, Table XI= 13*4 gm Mean d i f f e r e n c e between groups 2 and 3* P a r t 3* Table XI= 35.6 gm A l l other species d i f f e r e n c e s are. n o n - s i g n i f i c a n t . The a n a l y s i s of variance i n Table X I I i n d i c a t e s t h a t i n P a r t 1 of Assay 2 the r e s u l t s of Assay 1 are confirmed* P a r t 2 i n d i c a t e d that thiamine may not be the l i m i t i n g f a o t o r In the l i v e r p r e p a r a t i o n s . The r e s u l t s of Part 3 i n d i c a t e d that the three l i v e r p r e p a r a t i o n s were indeed not equivalent when t e s t e d under c o n d i t i o n s of ad l i b i t u m f e e d i n g * There was a strong i n d i c a t i o n (Table X I I ) that pork l i v e r -was su p e r i o r to beef l i v e r which i n t u r n was sup e r i o r to whale l i v e r * Whether or not t h i s occured as a r e s u l t of thiamine a d d i t i o n c o u l d not be a s c e r t a i n e d from the above data* - 32 -c. Assay 3 Although the r e s u l t s of Assay 2 gave no i n d i c a t i o n that thiamine was a l i m i t i n g f a c t o r i n the whale l i v e r , the r e s u l t s of previous work (27) d i d i n d i c a t e t h a t thiamine was a l i m i t i n g f a o t o r i n whale l i v e r at l e a s t * Before examining the l i v e r p r e p a r a t i o n s f u r t h e r , i t was decided to examine thiamine d e f i c i e n c y i n r a t s and a l s o the e f f e o t of thiamine a d d i t i o n to the b a s a l r a t i o n used i n t h i s i n v e s t i g a t i o n * To accomplish t h i s , 20 weanling r a t s were chosen and d i v i d e d i n t o three groups r e c e i v i n g r e s p e c t i v e l y , a v i t a m i n f r e e b a s a l r a t i o n , a v i t a m i n f r e e b a s a l r a t i o n p l u s thiamine, and a v i t a m i n f o r t i f i e d d i e t l a o k i n g thiamine* Ad l i b i t u m feeding was c a r r i e d out and weights were reoorded every three days* I n Table X I I I are l i s t e d the weight data, at s i x day i n t e r v a l s * The data i n Table X I I I i n d i c a t e s the primary r o l e of thiamine i n the n u t r i t i o n of the r a t * Although the r a t s r e c e i v i n g the v i t a m i n f o r t i f i e d r a t i o n made gr e a t e r weight g a i n than the r a t s on the b a s a l r a t i o n , there was no a p p r e c i -able d i f f e r e n c e i n the s u r v i v a l time* However on the other hand, thiamine added to the b a s a l r a t i o n appeared to lengthen the s u r v i v a l time* At. t h i s p o i n t the q u e stion may a r i s e of whether o r not to use a d e p l e t i o n p e r i o d before p l a c i n g the r a t s on t e s t * I n view of the f a c t that none of the r a t s i n t h i s assay gained any weight a f t e r the 18th day I t was f e l t that, the added work of u s i n g a d e p l e t i o n p e r i o d was not warranted* T a b l e X I I I Weights o f I n d i v i d u a l R a t s I n A s s a y 3 ait 6 Pay . I n t e r v a l s Days R a t i o n . Sexc Q 6 12 l g 24 P 38 43 43 45 48 M 43 51 50 44 4o. V i t a m i n F 51 59 63 63 55 F r e e M 44 50 4g 4g 4g F 35 39 38 36 33 F 4o 46 4g 4g 51 V i t a m i n M 46 54 58 52 52 F r e e F 52 64 71 69 67 p l u s M 49 51 50 49 48 Thiamine F 53 59 66 65 6 l M 66. 82 9.3 94 &5 M 62 76 89 81 73 M 57 6g 79 go 79 V i t a m i n M 62 75 g4 g4 g4 F o r t i f i e d M Rg 69 91 97 g7 l a c k i n g M 42 62 65 57 45 Thiamine M 4 l 6l 69 63 56 M 40 60. 64 62 57. M 41 62 69 65 59 M 43 66g 87 100 91 30 ?6 42 44 37 45 MM* 11 t 44 — — 4g . 46 4g — . 62 62 63 H 46 43 58 57 59 68 MM 62 ___ 68 54 69 — 75 — 46 45 51 81 69 MMI. I t was a l s o - f e l t t h a t s i n c e t h i s i n v e s t i g a t i o n was d e s i g n e d a s a r e l a t i v e r a t h e r t h a n a q u a n t i t a t i v e c o m p a r i s o n , no s i g n i f i c a n c e o f the t e s t s would be- l o s t by not u s i n g a d e p l e t i o n p e r i o d p r o v i d e d t h e a s s a y p e r i o d was o f adequate l e n g t h * As a r e s u l t , i t was decided- t o use a 42 day a s s a y p e r i o d wherever p o s s i b l e . - 3 4 -d* Assay 4 Assay 4 continued the i n v e s t i g a t i o n of the t h i a -mine content of whale l i v e r * At t h i s time i t was a l s o f e l t f u r t h e r c o n s i d e r a t i o n should be gi v e n to i s o o a l o r i o f e e d i n g * Up to t h i s p o i n t , l i v e r p r e p a r a t i o n s had been assessed as a t o t a l v i t a m i n source w i t h no c o n s i d e r a t i o n being given to f a t and water s o l u b l e v i t a m i n s as separate e n t i t i e s * There-f o r e Assay 4. was designed to examine the e f f e c t of i s o o a l o r i o f e e d i n g , and the a d d i t i o n of thiamine and cod l i v e r o i l to a b a s a l r a t i o n supplemented w i t h f i v e percent VDWL* Four r a t i o n s of the composition l i s t e d i n Table XIV were prepared* A t o t a l of 20 weanling r a t s were chosen and maintained on Table XIV Composition o f Rations Used i n Assay 4 C o n s t i t u e n t B a s a l VDWf. Thiamine Cod l i v e r o i l T o t a l 2000 2000 2000 2000 t e s t f o r a p e r i o d of 30 days* These r a t s were chosen i n groups of f o u r l i t t e r mates of the same weight ( 2 gnu). The t o t a l feed consumption and weight g a i n of i n d i v i d u a l r a t s are recorded i n Table XV* Here again, as w i t h assay 1, i t would be tempting to conclude, even without s t a t i s t i c a l a n a l y s i s , that the whale l i v e r has adequate v i t a m i n s A, D, Rat i o n 1 2 3 4 •• 1900 I860 1900 I860 100 100 100; 100. 0.05 0*05 40 * ~ 40 - 35 -Table XV T o t a l Weight g a i n and Feed Consumption of Rats In Assay 4 Groups Sex B a s a l B a s a l p l u s B a s a l p l u s B a s a l p l u s Thiamine Cod l i v e r o i l Thiamine p l u s Cod l i v e r o i l FC m FO W G F C W G F C WJ$ F 199 §4 196 50 196 64 197 622 M 165 61 166 61 166 59 I69 62 F 181 61 1S2 64- 1S2 68 18b* 66 M 201 70 205 64 203 79 206 70 F. 167 49 165 39 16S - 51 167 56 and t h i a m i n e . However since the weight gains and feed consumptions of the r a t s i n t h i s essay d i d not seem to be as h i g h as one would normally expect, i t was decided to ca r r y out a s i m i l a r assay on an ad l i b i t u m f e e d i n g b a s i s r a t h e r than on an i s o c a l o r l o i n t a k e b a s i s as was done i n t h i s assay* e. Assay 5 In Assay 5, 20 weanlings were used. Four r a t i o n s were made up using the c o n s t i t u e n t s shown i n Table XVI. Feed was a l l o t e d ad llbutum to the fo u r groups- of r a t s and I n d i v i d u a l weights were recorded every three days. The weights are recorded at 6 day i n t e r v a l s I n Table X V I I . Table XVI Composition of Rations Used I n Assay 5 Con s t i t u e n t R a t i o n  x 2 3 "+ B a s a l l l 5 5 l4"4"o 1IE55 lW) VDWL 45 45 % 45 Thiamine — — 0.04 0.0% Cod l i v e r o i l — 15 — 15 T o t a l 1500 1500 l^OO 1500 - 36 • Table XVII Weight of Rats In Assay 5 at 6 Day Intervals Weight in grams  Ration Sex 0 6 12 l g 24 30 36. 42' p — 45 58 68 71 70 63 62-M 44 46 71 78 77 67 59 P 41 50 63 62 5^  54 48 40 H 46 63 77 79 78 72 60 P, — 47 62 68 61 55 45 37 p - - 4 1 47 67 76 78 76 72 M 47 63 84 87 85 84 78 72 P 41 50 64 76 71 72 66 64 M 45 56 74 76 72 72 65 56 P 35 48 6& 75 74 74 60 P 34 45 58 64 72 78 82 92 M 44 57 76 81 88 94 103 109 P 42 57 71*- 86 89 94 98 105 5 5^ 57 73 §3 91 97 98 107 P — 43 47 65 78 93 96 104 p 48 69 87 87 108 118 127 M 47 61 83 109 129 145 160 182 P 42 52 72 86 96 106 115 128 M 45 60 77 94 H I 125 I36 150 P 3o 44 57 72 85 99 110 123; These rats died due to failure to eat or drink and were replaced* Prom the results of data shown in Table XVII, i t becomes apparent that thiamine i s the limiting factor In dried whale l i v e r , at least at the three percent supplemen-tation level* It also became apparent that ood l i v e r o i l or probably vitamin A was also a limltlng>.' faotor* Before this, i t had been assumed that since whale liver, i s generally - 37 valued for Its vitamin A content, this would not be a limiting factor i n the l i v e r meals* However, the vitamin A analysis shown i n Table II lndioates that the raw l i v e r has adequate vitamin A but the dried l i v e r meals have not* Therefore, even the low temperature vacuum drying process reduces the. vitamin A content* This i s probably due to some oxidative process and i n fact may acoount i n some way for the low thiamine levels* It was decided that i n the future, consideration would have to be given to cod l i v e r o i l and thiamine supplementation before assaying the pre<* pared l i v e r meals* It should be noted that as these preceeding assays were i n progress, several other assays were being carried out. The results of these assays were so erratio i n many oases that they were deemed non-significant but they are mentioned here because their results aided in planning the design of future experiments. Two controlled intake assays were carried out comparing VDWL to DDWL and 5 0 / 5 0 DDWL. The results were similar to Assay 1 only i n this case rats on the drum dried l i v e r tended to. limit the feed consumption of the l i t t e r mate pairs. Supplementation with thiamine at 30 days appeared to have l i t t l e effect even when ad libitum feeding was allowed* This was probably due i n a large measure to the very poor condition of the rats at the time of thiamine supplementation. It was also possible that the long self Induced and foroed starvation period may have caused - 3« -Unrepairable p h y s i o l o g l o a l damage* I t was a l s o noted t h a t at l e a s t d u r i n g the e a r l y stages of the experiments the r a t s consuming VDWL almost without exoeption consumed a l l feed o f f e r e d and tended to be much more a c t i v e than t h e i r l i t t e r mates* The Inference drawn from these assays was that VDWL appeared to be su p e r i o r to the drum d r i e d l i v e r and a l s o that the p a i r e d feeding method used under the c o n d i t i o n s of the gssays tended to n u l l i f y any s u p e r i o r i t y i n one or the other of the l i v e r p r e p a r a t i o n s * Another l a r g e assay was a l s o i n progress i n which VDWL, DDWL, 50/50 DDWL, and GLM were being compared at the three, s i x , nine, and twelve percent l e v e l of a d d i t i o n to b a s a l r a t i o n by ad l i b i t u m feeding* Growth response and feed consumption were poor i n a l l cases even at the twelve percent l e v e l s and at the end of 3 ° days p r a c t i c a l l y a l l r a t s were l o s i n g weight* At 3 ° clays a l l r a t i o n s were supplemented w i t h thiamine* There was an immediate response to the thiamine a d d i t i o n even at the lower l e v e l s but i n most cases t h i s response was s h o r t - l i v e d * Only the nine and twelve percent l e v e l s of GLM and VDWL showed a l a s t i n g r e -sponse f o r a p e r i o d of 12 days, a f t e r which the assay was discon t i n u e d * The r e s u l t s of t h i s assay were very i n c o n -c l u s i v e but they d i d i n d i c a t e that i f t h i s type of assay was to be continued, supplementation w i t h thiamine at l e a s t would be r e q u i r e d from the beginning of the assay p e r i o d * This assay a l s o i n d i c a t e d that p o s s i b l y ; J r VDWL and CLM were s u p e r i o r to the drum d r i e d l i v e r meals* - 39 -Up to this point much Information had come to light regarding the d i f f i c u l t i e s involved in the ^ioassay type of experimentation. It was f e l t that the marked d i f f e r -ences exhibited by the various l i v e r preparations disallowed any further effective use of Mitchell?s paired feeding method and therefore further assays would involve ad libitum feeding. With this decision i t was'realized that further d i f f i c u l t i e s would be involved In experimental interpretation. Under conditions of ad libitum feeding, the various groups would rarely be expected to consume the same amounts of food. Thus some method would have to be devised to deter-mine whether differences, in feed consumption were due to experimental treatment or chance. The preceedlng assays indicated the close relationship between feed consumption and weight, gain and also indicated that there was good reason to suppose that improvement of the ration tended to increase the feed consumption. This f i r s t point i s quite evident but the seoond requires c l a r i f i c a t i o n . With respect to feed consumption three distinct conditions exist in bloassay: (1) a growing animal may con-sume food at or below the maintenance level and w i l l eventu-ally die, (2) an animal may consume slightly more than maintenance requirements and w i l l grow at a slow rate, or, (3) an animal may consume a "maximum'' amount of food and w i l l grow at a fast rate. A l l three of these conditions Can and do occur in feeding t r i a l s under conditions of ad libitum feeding. The f i r s t case may be dispensed with - 40 -slnoe l i t t l e Information can be gained from i t except that the r a t i o n i s unable to promote growth or sustain l i f e * The second two cases, however, are more complex since be-tween these two classes one can obtain a gradation of re-sponses* The point i n question i s best i l l u s t r a t e d with an example* Table XVIII Total Feed Consumption Weight Gain and E f f i c i e n c y of Grain of Rats on Two Separate Assays A* Basal Ration plus 5$ VDWL plus Three Levels of VFM F i r s t 21 Days Second 21 Days Total 42 days l e v e l Q*05# 0*15 0.25 0*05 0.15 0*25 0.05 0.15 0.25 FO 76T 810 ""§48 746 1172 1324 1452 1982 2172 m 263 31S 373 1* ,293 3^ 3 1^7 611 716 Eff 2.68 2.55 2.27 4.84 4.00 3*86 3*51 -3.24 3*03 B. Basal Ration plus Three Levels of EHWL F i r s t 21 Days Second 21 Days Total 42: days l e v e l _ 2 g _ 1 2 _ 1 5 _ l g -15 -2 12 15, FO 771 837 835 613 666 881 1384 1503 1716 WG 241 284 307 49 101 184 290 385 491 E f f 3*20 2.95 2.72. 12*27 6*59 4.79 $,77 3.90 3.49 Table XVIII shows the r e s u l t s of two separate assays, chosen f o r purposes of i l l u s t r a t i o n only* The t o t a l feed consumption, weight gain, and e f f i c i e n c y of gain has been tabulated i n two 21 day periods as well as for the t o t a l assay period. I t becomes evident from t h i s table that consider-able differences appear when the assay i s considered i n two, , sections. This marked difference Is probably due to r e s i d -u a l vitamin storage by the rats before being placed on test and indicates that a comparatively long assay period i s desirable i f a depletion period i s not used (that i s long with respect to the maturation time of the rat).. O r d i n a r i l y i f two rations are of oomparable n u t r i t i v e value except that one i s s l i g h t l y higher i n I n d i -gestible matter, I t would probably be found that the animals on the more i n d i g e s t i b l e r a t i o n would consume more feed to meet t h e i r requirements and would consequently show a higher e f f i c i e n c y of gain. This appears to be the case- i f one Compares the f i r s t 21 day periods of groups A and B. i n Table XVIII. There i s also a tendency f o r the rats consuming the most feed to gain the l e a s t . I t would appear, therefore, that at lea s t the r a t s gaining the most are consuming l e s s than t h e i r physical maximum (that allowed by the oapaolty of the digestive system) and consequently some other, factor i s governing feed consumption. Since i n a l l cases increasing supplementation brings about Increased feed consumption, I t would be reasonable to suppose that t h i s other factor Is the r a t i o n * To carry t h i s point farther i t would be reasonable to suppose that the n u t r i t i v e content of the r a t i o n governs the weight gains through feed consumption. This becomes more evident when the second 21 day period of part B Table XVIII i s considered. At t h i s stage, the 'iessentlal" stored nutrients have probably been metabolized and sinoe they are not present i n s u f f i c i e n t quantity i n the r a t i o n growth has been severely depressed. I t i s also quite evident at t h i s stage t h a t the r a t s are consuming l e s s than t h e i r p h y s i c a l maximum and t h i s can be a t t r i b u t e d to an e f f e c t of the r a t i o n . However, t h i s assumption must not be- c a r r i e d too f a r . When group d i f f e r e n c e s or p r e f e r a b l y i n d i v i d u a l d i f f e r e n c e s between d i f f e r e n t groups are l a r g e , l i t t l e d i f f i c u l t y i s i n v o l v e d i n the experimental i n t e r p r e t a t i o n . However, when group d i f f e r e n c e s are small c a u t i o n must be used i n the i n t e r p r e t a t i o n of r e s u l t s e s p e c i a l l y i f the group making the l e a s t g a i n consumes the l e a s t feed since many f a c t o r s may govern small d i f f e r e n c e s I n feed consumption* To. a v o i d r e p e t i t i o n t h i s p o i n t w i l l not be considered f u r t h e r here but w i l l be di s c u s s e d w i t h a o t u a l examples i n the f o l l o w i n g assays. I t w i l l s u f f i c e , to s t a t e t h a t many authors a v o i d this, problem by simply u s i n g weight g a i n data and make no mention whatsoever of feed consumption o r perhaps only mention i t obscurely as e f f i c i e n c y of g a i n . Another problem th a t appears from the r e s u l t s o f Table XVTII i s whether or not one can compare d i f f e r e n t l e v e l s i n d i f f e r e n t assays. I n other words whether one can s t a t e that a 15 percent IHWL r a t i o n i s s u p e r i o r to a 0.05 percent VFM p l u s three percent; VDWL r a t i o n . I f i t were c e r t a i n that the same f a c t o r was l i m i t i n g i n both r a t i o n s , t h i s would be a v a l i d Comparison,. However, when growth enhancing f a c t o r s i n the r a t i o n s d i f f e r markedly, care must be taken I n making such comparisons. I n s p i t e of t h i s , p r ovided the materials, compared I n d i f f e r e n t r a t i o n s are of s i m i l a r composition,, there i s some j u s t i f i c a t i o n i n making comparisons. This p o i n t i s o f t e n overlooked when two fee d i n g . 43 -s t u f f s are compared as p r o t e i n sources* One p r o t e i n source may be s e r i o u s l y p e n a l i z e d when In e f f e c t i t may be an e x c e l l e n t p r o t e i n Save f o r only one e s s e n t i a l amino a c i d * ( I t i s a l s o d i s h e a r t e n i n g to r e a l i z e that thousands of tons of feeding s t u f f s are bought and s o l d on,the b a s i s of n i t r o -gen content.) At any r a t e the best r e s u l t s are obtained i n a fe e d i n g t r i a l when a s i n g l e component such as an amino a c i d or a v i t a m i n I s compared i n d i f f e r e n t feed s t u f f s by b i o l o g i c a l assay. As mentioned before, i f s e v e r a l components are compared at the same time,, d i f f i c u l t i e s w i l l be encountered i n determining which i s the l i m i t i n g f a c t o r and whether i t i s the on l y a f f e c t i n g v a r i a t e * I t should now be evident that many of the problems d e a l i n g w i t h d i f f e r e n c e s i n feed consumption are e l i m i n a t e d by c o n t r o l l e d i s o c a l o r i c feed i n t a k e . However, when feed consumption i s l i m i t e d to a l e v e l c l o s e to maintenance, there w i l l be l i t t l e i f any growth e x h i b i t e d . I n t h i s case, the animal on the b e t t e r r a t i o n may l o o s e i t s advantage e n t i r e l y due to excess a c t i v i t y , i h i s i s probably one of the few cases I n which ad l i b i t u m feeding i s e s s e n t i a l to a l l o w one to"make v a l i d comparisons* f* Assay 6 At t h i s stage the p a t t e r n f o r the f o l l o w i n g assays had been e s t a b l i s h e d . Feeding was to be ad l i b i t u m and experimental groups would c o n s i s t of three females and two males. This p o i n t may be c r i t i c i z e d since "sex d i f f e r e n c e " was not a primary oonoern of t h i s t h e s i s * However i t was * 44 declded to continue using the two sexes *so that i n the f i n a l analysis a comparison could be made throughout the entire groups of assays* From the analysis of variance i n Table XI I t can be seen that there i s a s i g n i f i c a n t difference i n sex re-sponse and sex and l e v e l i n t e r a c t i o n * This i s what one would expect since males and females were placed on test at the same weight and consequently the males were physiologi-c a l l y younger, ThuSj, on a low plane of n u t r i t i o n the f e -males made better responses than the males* On the other, hand, on a high plane of n u t r i t i o n the males made larger gains than the females over the 42 day assay period* Shis i s the reason f o r the marked significance of the sex: l e v e l i n t e r a c t i o n * However t h i s difference i n sex response to treatment i s taken into account by the analysis of variance and simply means that, s l i g h t l y larger group mean values w i l l be required f o r significance* Assay 6 was designed to ascertain whether the protein rather than the vitamin content of the l i v e r preparations was responsible f o r the Increased growth r e -sponse with increasing supplementation l e v e l s * Four rations of the oomposltion shown i n Table XIX were prepared. The protein content of the four rations was equalized using vitamin free casein. The casein was assessed as 100 percent protein and the VDWL as 70 percent* The t o t a l weight gain and feed consumption of the ra t s used i n Assay 6 are recorded i n Table XX* I t should be noted that the rats on the three Table XIX Composition of Rations Used In Assay 5 VDWL Basal Vitamin Free Al fa VDWL Total l eve l Ration Casein c e l l  3# 1760 126 54 60 2000 6i 1760 84 36 120 2000 c$ 176O 42 18 180. 2000 12# 1760 — — 240 2D00 Table XX Total Feed Consumption and Weight Gain of Rats in Assay 6 level Sex 6% _lgg FC WQ> FO WG- FC WGr F 200 47 271 78 247 70 M 272 69 293 io4 352 128 F 206 37 250 62? 280 84 M 295 89 - - -- 350 133 F 243 58 278 87 278 66 (rats on 3% l eve l fa i led to gain weight after 21 days on test). percent l eve l have not been recorded. It was f e l t that since growth was to be assessed as total weight gain over a speoiflo assay period that a primary requisite should be that the rats gain weight over the entire assay period* If the growth curve for a speoiflo group drops i n the la t ter part of the assay, one i s measuring l i t t l e more than vitamin storage i n the weanling ra t . This point was discussed i n detai l In - 46 ~ connection w i t h Table X I I I and need not be elaborated f u r t h e r . From Table XX i t can be seen that an enrichment f a c t o r (or f a c t o r s ) other than p r o t e i n i s causing the added growth response w i t h i n c r e a s i n g supplementation l e v e l s . These data were not analysed s t a t i s t i c a l l y because one male r a t on the nine percent l e v e l d i e d f o r unknown reasons. How-ever, the marked growth d i f f e r e n c e s noted between the s i x , nine and twelve percent l e v e l s are evidence enough to mer i t the c o n c l u s i o n s t a t e d above. g- Assay 7 1. Experimental r e s u l t s Assay 1 was c a r r i e d out i n f o u r p a r t s to assess by r a t growth the r e l a t i v e value of ( l ) s i s , nine, and twelve percent VDWL, CLM, DDWL, 50/50 DDWL, and ADWL added to b a s a l r a t i o n w i t h thiamine added and s i x percent beef and pork l i v e r added to b a s a l r a t i o n w i t h thiamine added, (2) s i x , nine, and twelve percent SDWL added to b a s a l r a t i o n and s i x percent SDWL added to b a s a l r a t i o n w i t h thiamine added, (3) nine, twelve, and f i f t e e n percent EHWL added to fthecbasal r a t i o n w i t h thiamine added, and (4) s i x percent VDWL, VDBL, VDPL, CLM, SDWL, DDWL, and 50/50 DDWL added to b a s a l r a t i o n w i t h thiamine and cod l i v e r o i l added. The r e s u l t s of t h i s assay are l i s t e d i n Tables XXI to XXIV showing t o t a l weight g a i n and feed consumption over a 42 day assay p e r i o d . - 47 * Table XXI T o t a l Feed Consumption and Weight G-aln of Rats I n Assay Seven Part One l e v e l M _2& 12* group Sex: FC. W& FC WGr FC WjJ 63 351 73 266 S5j 113 315 102 357 127 VDW1 F 302 96 295 86 323 92i 48 309 99 339 116 77 269 87 266 74 47 274 79 : 297 94 102 335 S3 313 112 CLM F 231 59 332 105 340 96 " " ~ 77 354 124 428 146 79 357 101 271 92 43 240 65 247 59 - ^ 52 235 54 250 67 50/50 F 200 29 232 40 266 70 72 309 87 286 89 45 300 64 228 43 : £0 F 240 M 339 M F 285 F 264 M 369 M 261F 303 F 224 M 234 M 288 F 241 F 199 M 241 F 342 M 315 F 310 F 253 M 186 F 228 M 223 F 243 F 320 M 377 s 289 371 F 370 F 8|6 M F 1"53 M 411 F 295 4o 282 66 259 48 58 196 33 277 50 DDWL 111 205 45 224 54 " $6 271 71 297 69 70 13S£ 29 320 58 61 270 8 5 250 70 45 294 91 267 89 AD.WL S 44 ' 221 62 257 56 " -54 354 112 252? 85 57 266 81 305 87 VDBL E  88. 147 116 50 127 VDPL F 533 110 147 78 - 48 ~ Table XXII Total Feed Consumption and Weight Gain In Assay 7 Part 2 Level Ration Sex: 6% 3% 12# 6% (wltmhlamlne PC WG PC PC § PC WG F 301 83 266 71 296 89 267 75 M 342 86 294 112 294 111 307 98 SDWL F 275 69 253 78 25? 71 279 77 M 276 94 324 115 324 123 327 108 P 283 78 277 85 226 67 287 78 Table XXIII Total Feed Consumption of Rats In Assay 7 Part 2 Level Ration Sex. 3% 12% 15% FO PC WG PC WG F 251 40 29I 66 336 94 M 207 48 280 82 383 119 EHWL F 306 54 278 51 311 79 M 286 66 321 90 340 113 F 334 82? 333 96 346 86 49 -Table XXIV T o t a l Feed Consumption and Weight Gain of Rats I n Assay 7 P a r t 4 Rat i o n L e v e l Sex SDWL VDPL 6% F M F M F F M F M F FO 335 392 32 9 228 286 2Q3 298 342 WG FO l H H 9 98 362 DDWL WG l U 152 160 113 VDBL 3 ft 444 182 324 98 50/50. DDWL; CLM 70 204 91 247 66 222= 92 243 107 239 61 3 9 197 249 232 242 273 52 74 84 FO 1*5 400 4 l 0 439 WG 1?2*? 202 118 166 142 11• S t a t i s t i c a l r e s u l t s This assay has been considered s t a t i s t i c a l l y i n f i v e separate s e c t i o n s since the data and o b j e c t i v e s are too complex to consider under a s i n g l e a n a l y s i s of va r i a n c e . Considering the data i n t h i s manner a l s o tends to inorease the s i g n i f i c a n c e of the s p e c i f i c s e o t l o n s . A l s o c e r t a i n seotlons of Table XXI were h i g h l y e r r a t i c i n I n d i v i d u a l response to treatment and were deemed unworthy of consider-a t i o n under the c o n d i t i o n s of t h i s assay* aa) A n a l y s i s of variance of the weight gains of groups r e c e i v i n g s i x , nine and twelve percent VDWL, 50/50 DDWL and CLM added to b a s a l r a t i o n s supplemented w i t h thiamine* Table XXV  A n a l y s i s of Variance of P a r t aa V a r i a t i o n DP SS MS. VR Sex 1 4,547 4,547 L e v e l 2 4,480 2,240 8.3 Species 2 10,519 5,260 I 9 . 6 Sexrx l e v e l 2 4j6 234 Sex x Species 2 bb 33 Speoies x< l e v e l 4 574 W Sex xr. species x; l e v e l 4 S58 215 E r r o r 27 7,270 269 T o t a l 44 28,761 Mean d i f f e r e n c e r e q u i r e d for. s i g n i f i c a n c e (at p = 0,05) = 21.3 gms. Mean d i f f e r e n c e r e q u i r e d f o r s i g n i f i c a n c e (at p ** 0,10) * 17.7 gms. Mean d i f f e r e n c e f o r l e v e l s (gm) Mean d i f f e r e n c e f o r speoies Speoies 6-9* 9-12* 6-12* 50/50DDWL 13.8 4,6 18.4 VDWL 9,0 9?4 18.4 CLM 25*2? 10.0 35*2 Speoies 6* 9* 12* 50/50 DDWL&VDWL 32,2: 27.4 32.2: VDWL and CLM ' 7,6 8.6 9.2? 50/50 DDWL&CLM 24.6 36.0 4l,4 The r e s u l t s of t h i s preceedlng t a b l e i n d i c a t e t h a t not too much improvement i s obtained by doubling the l e v e l of supplementation of the l i v e r meal except I n the case of the CLM, On the other hand, the r e s u l t s i n d i c a t e that VDWL and CLM are of s i m i l a r q u a l i t y at e i t h e r of the three l e v e l s of supplementation w i t h thiamine added* and that both of these samples are s u p e r i o r to 5°/50 DDWL when assayed under the same c o n d i t i o n s . I t may be r e c a l l e d that s i m i l a r r e s u l t s were obtained, but weight gains were lower, when these preparations were assayed w i t h no thiamine added. However, even at the 12 percent supplementation l e v e l none of the weight gains of the r a t s analysed are what would be considered normal* For purposes of comparison, a group of r a t s were placed on U*B*C* stock r a t i o n No* 10-54, The r a t colony at the Laboratory of Animal N u t r i t i o n at U.B.C. i s maintained almost s o l e l y on t h i s r a t i o n * The growth response and feed consumption of t h i s group on a 42 day assay p e r i o d i s shown i n Table XXVI, Table XXVI T o t a l Weight Gain and Feed Consumption of Rats  Maintained on U*B,C* Stock R a t i o n 10-54 f o r 42 Pays Sex FC WG F 513: 131 M 671 201 F 448 94 M 662. 192 F 456 108 bb) A n a l y s i s of variance of the weight gal'rls of the groups r e c e i v i n g nine percent DDWL, 50/50 DDWL, ADWL, VDWL, and CLM, added to b a s a l r a t i o n w i t h thiamine added; and SDWL added to b a s a l r a t i o n w i t h no thiamine added. - 52- * Table XXVII  A n a l y s i s of Variance of Part bb SS MS VR 2,330 2,330 9,475 1,895 7*93 750 150 4,302 239 16,857 Mean d i f f e r e n c e r e q u i r e d f o r s i g n i f i c a n c e (at p = 0,05) * 20.6 gms. Mean D i f f e r e n c e s (gm)  DDW1 5Q/5QDDWL . ADWL VDWL SDWL CLM Group Means 48,8 62,0 86,2: 89,4 92.2 98,0 Mean D i f f e r e n c e 13,2 24,2 3*2 3.8 4.8 V a r i a t i o n DF Sex 1 L e v e l s 5 Sex x l e v e l s 5 E r r o r 18 a b t a l 29 The r e s u l t s of Table XXVII i n d i c a t e that ADWL, VDWL, and CLM are of equivalent enrichment value and s i g -n i f i c a n t l y s u p e r i o r to DDWL and 50/5O DDWL when added to a vi t a m i n f r e e b a s a l r a t i o n at the 9 percent l e v e l w i t h thiamine supplementation. A l s o , SDWL w i t h no thiamine added i s e quivalent to the former three mentioned p r e p a r a t i o n s * However, here again the growth response i n a l l cases I s muoh lower than that obtained w i t h r a t i o n 10 - 54, 0 0 ) A n a l y s i s of variance of the feed consumption and weight g a i n of the groups r e c e i v i n g s i x percent 50/50DDWL, DDWL and CLM when added to b a s a l r a t i o n supplemented w i t h ood l i v e r o i l and thiamine* - 5 J -Table XXVIII  A n a l y s i s of Variance of P a r t oo SS MS VR V a r i a t i o n DF FO WO- FO W FG WO-Speoies 2 4,610 1,112 2,305 556 1*42 3.05 Sex: 1 1,913 208 1,913 20S 1»18 1.14 Sex: x species 2 314 17 157 8.5 0.10 O.05 E r r o r 9 14,591 1,642 1,621 182 T o t a l 1% 21,428 2,979 No s i g n i f i c a n t d i f f e r e n c e at the 5* l e v e l . The r e s u l t s of t h i s assay i n d i c a t e t h a t the a d d i t i o n of cod l i v e r o i l and thiamine to the two drum d r i e d l i v e r samples b r i n g s them up to the same q u a l i t y as the commercial l i v e r meal. However these three samples w i l l s t i l l r e q u i r e f u r t h e r supplementation before they w i l l equal the stock r a t i o n w i t h respect to growth a t t a i n e d . I t may a l s o be noted t h a t the a d d i t i o n of thiamine to the CLM enhanced growth but f u r t h e r supplementation w i t h cod l i v e r o i l d i d not. I t would appear then that the CLM may have adequate amounts of A and D but i t i s d e f i c i e n t i n at l e a s t one f a c t o r , probably a v i t a m i n , other than thiamine* dd) A n a l y s i s of variance and oovarianoe of the feed consumption, weight g a i n , and e f f i c i e n c y of g a i n of the groups r e c e i v i n g s i x percent SDWL, VDPL, VDWL, and VDBL added to b a s a l r a t i o n supplemented w i t h thiamine and cod l i v e r o i l . - 54 -The s t a t i s t i c a l a n a l y s i s I n t h i s s e c t i o n has been c a r r i e d out i n d e t a i l to i l l u s t r a t e the method used. The a n a l y s i s i s based on a p r e l i m i n a r y sample (Table IX) c a r r i e d out w i t h the a i d of Dr. V. C. B r i n k of> the D i v i s i o n of Pl a n t Science at the U n i v e r s i t y of B r i t i s h Columbia, The a n a l y s i s i s a l s o based; on. samples and methods I l l u s t r a t e d by Dr. J , Wishart (28), (29). Table XXIX A n a l y s i s of Variance and Covarlance of P a r t dd A. Feed Consumption, Weight "lain and E f f i c i e n c y of Grain Sub T o t a l s and To t a l s For a 42 Day Assay SDWL VDPL VDWL VDBL F ~~ M F M F " M F M FC 330 449 352 409 329 503 345 512 W& 97 166 111 152 116 196 122 202 E f f 3.40 2,70 347 2.69 2*84 2.57 2.83 2.53: 335 392 345 414 404 444 400 410 99 156 113 160 l 4 l 182 118 166 3.38 2.51 3.05: 2.59 2.87 2.44 3.39 2.47 329 362. 324 4?9 93 113 98 142: 4294 3533 994 136S 138O 294 37.71 20.50 10.14 841 322 5.21 -M .F-*. 1059 823 337 312 9.42 5.28 ... X057 947 -3SS 3 8 5 7 8.84 5.01 1184 922 382 368 9.31 5.00 7S27 1835 2748 616 58.21 15.35 1882 649 14,70 2004 1*5.85 2106 750 14.31 ~ 55 ~ Table XXIX continued  B. Calculated Sum of Squares and Products F.C. S.S. W.fl. S.S. FCxWG- S.P. E f f i c i e n c y SS Correction Factor 3,063,096 377,575 1,075,430 169.4202 Total 3,125,649 ?|063,0?6 -399,062 577,575 -1,109,862 1,075,430 -171•6085 169.4202 62,753 21,507 34,432 2.1883 Species mm 3,072,080 3.0o3.096 -380,089 277,575 -1,080,042 1.075.430 -I69.6622 169.4202 8,984 2,514 4,612 0.2420 Sex 1,536,536 1.560.261 155,952 238,050 489,516 609.^43 118.5307 52.5313 mm 3,096,797 3,063,096 * 394,002 ?77i575 -1,098,959 1,075,430 -I7I.O35O 169.4202 33,701: 16,427 23,529 1.6148 Sex x Species 1,5^2,674 ii565,752 239,668 492,214 612,420 H8.7926 52.5613 - 3,108,626 3,105,781 - 369,986 396,516 - 1,104,634 1,103.571 - 171.3539 171.2770 2^ 84 5 470 1,063 O.Q769 - 56 ~ Sroup FC WC E f f , Table XXIX continued  C. Tabulated A n a l y s i s of Varlanoe V a r i a t i o n DF SB MS VR Species Sex Sex x speoies E r r o r 3 1 3 12 33,7?1 17,223 2,9-82,7 33,701.0 ?48,3 1,435.3 2.09 23.4S 0.66 Total t 62,753 Species Sex: Sex x species E r r o r 3 , l 3 12: 2,514 16,427 470 2,096 838.0 16,427*0' 15657 17M 4. So 94.03 T o t a l 19 21,507 Speoies Ser. Sex x speoies E r r o r 3 • 1 3 121 0.2420 1.6l4s 0.0769 0.2546 O.O.807 1.6148 0,0256 0.0212 . 3.81 76.12 1.21 T o t a l 19 2V1883 Mean d i f f e r e n c e r e q u i r e d f o r s i g n i f i c a n c e at p « 0,05 f o r Wa sj sq. r t . 174.2 x: 2 x 2.179 - 13.2 5 f o r E f f s sq. r t . 0.0212 x< 2 x 2.179 * 0.201 5 Table XXIX continued D. Mean D i f f e r e n c e Table Group SDWL VPPL VDWL VDBL WG Mean 123.2 129.8 146.6 150.0 Mean D i f f . 6.6 16,8 3.4 E f f Mean 3,07 2.94 2,77 2.86 Mean D i f f . 0.13 0.08 0.09 E, A n a l y s i s of Oovarlance V a r i a t i o n DF, FO, Species 3 8,984 Sex: 1 33,701 Sex x species 3 2,845 E r r o r . 12 17,223 T o t a l 19 62,753 Species + e r r o r 15 26,204 Sex + e r r o r 13 50,924 I n t e r a c t i o n * e r r o r 15 20,068 SP 4,612 23,529 1,063 5,228 34,432 9,8* WG 2,514 16,427 470 2,096 21,507 4,610 b f D i f f PF 406 3 1,775 l 85 3 1,587 509 11 ?'695 915,, 28,757 18,523 16,239 2,284 6,291 2,566 1,972 594 Species Sex: Sex x species E r r o r MS VR 1 11 1 1 • 135,3 2,92 1775.0 38.36 26,3 0.61 46.27 0 Wishart 1950 page 17. - 58 -The r e s u l t s of t h i s t a b l e are elaborated on I n d e t a i l under the s e c t i o n d e a l i n g w i t h d i s c u s s i o n of r e s u l t s * ee) A n a l y s i s of varianoe and oovarianoe of the feed consumption and weight g a i n of the groups r e o e i v l n g nine, twelve and f i f t e e n peroent EHWL added to b a s a l r a t i o n supplemented w i t h thiamine* Table XXX A n a l y s i s of Varianoe and Oovarianoe of Pa r t ee V a r i a t i o n Df PC SP WG_ b f d£ D i f f DP Sex: 1 163 -347 846 1,099 1 L e v e l 2 11,317 6,707 4,0W 394 2 Sex x l e v e l 2> 3,014 1,531 484 2 E r r o r 9 11,754 4,174 2,289 1,482 807 8 T o t a l 14 26,248 12,065 7,663 Sex * e r r o r 10 11,917 3,827 3,135 1,229 1,906 L e v e l + e r r o r 11 23,071 10,881 6,333 5,132 1,201 I n t e r a c t i o n • 11 14,768 5,705 2,773 2,204 569 e r r o r V a r i a t i o n Mean Square VR PC —m r — n f f , P U — m — s i f f Sex: lbT 84"cT lTol^ 0*1? 3*33 loTsT* L e v e l 5,659 2,022 197 4*33 7*96 1*95 Sex x l e v e l 1,507 242 — 1.15 0*95 — E r r o r 1,306 254 101 111* D i s c u s s i o n of R e s u l t s The r e s u l t s of Tables XXV, XXVII, and XXVIII r e -q u i r e l i t t l e e x p l a n a t i o n since they are s e l f evident* I n summary they i n d i c a t e that DDWL and 50/5Q DDWL are not very - 59 -promising as enrichment sources when added to a v i t a m i n f r e e b a s a l r a t i o n even when t h i s b a s a l r a t i o n i s supplemented w i t h thiamine. The CLM and VDWL samples are b e t t e r , but s t i l l not passable when assayed under the same circumstances* With the a d d i t i o n of thiamine and cod l i v e r o i l to the b a s a l r a t i o n , DDWL and 50/50 DDWL Improve, and are equiva-l e n t to CLM as enrichment sources* Also CLM i s equivalent to VDWL when they are both assesed w i t h thiamine added* Further a d d i t i o n of cod l i v e r o i l to the CLM does not Im-prove t h i s product* I t should a l s o be noted (Table XXII) that the same reasoning can be a p p l i e d to SDWL w i t h r e s p e c t to thiamine r a t h e r than cod l i v e r o i l * I f one compares the r e s u l t s t a b u l a t e d i n Tables XXVIII and XXIX without r e s o r t i n g to s t a t i s t i c a l analyses, i t becomes, evident that VDWL, SDWL, VDBL, and VDPL are v a s t l y s u p e r i o r to DDWL, 50/50 DDWL, and CLM when a l l have been supplemented w i t h thiamine and cod l i v e r o i l * A l s o , a l l seven of these samples except CLM are improved by the a d d i t i o n of cod l i v e r o i l * The ones most markedly e f f e o t e d are SDWL, VDPL, VDWL, and VDBL* This p o i n t can be v e r i f i e d by oomparing the group means as has been done i n Table XXXI* I t does not r e q u i r e too much imagination to see that 50/50 DDWL, CLM, and DDWL are r e l a t i v e l y poor enrichment sources and of about equal value even when supplemented w i t h cod l i v e r o i l and thiamine. The a n a l y s i s of vari a n c e shown I n Table XXVIII i n d i c a t e s that there, i s no s i g n i f i c a n t d i f f e r e n c e - 60 -Table XXXI Group Mean Weight Gains of Rats R e c e i v i n g 6% DDWL, 50/50DDWL VDPL, VDWL, VDBL, o r SDWL w i t h Thiamine Added and With and Without bod L i v e r O i l Added  50/50 R a t i o n DDWL CLM DDWL SDWL VDPL VDWL VDBL p l u s Thiamine 48.2 72#6 p l u s Thiamine and CLO 65.2 69.4 Dlf f e r e n c e 17.0 -3*4 75.0 87.2 102,4 80.4 119.4 85.2: 123.2 129.8 146.6 150.O 10*2 36.O 27*4 66*2 3O.6 i n e i t h e r the feed consumption or weight g a i n of these three groups. Here one does not even have the problem of d i f f e r -ences i n feed consumption and weight ga i n to consider* How-ever the s i t u a t i o n i s not as c l e a r when we consider the other f o u r l i v e r p r e p a r a t i o n s l i s t e d i n Table XXXI. (SDWL, VDPL, VDWL, and VDBL). There can be no question t h a t a l l f o u r samples are markedly improved by the a d d i t i o n of cod l i v e r o i l . There i s a l s o some evidence to suggest that the o r i g i n a l whale l i v e r meal contains l e s s V i t a m i n A and/or D since i t i s the most markedly improved by the a d d i t i o n of cod l i v e r o i l . There i s a l s o some evidence to suggest that beef l i v e r meal i s s u p e r i o r to whale l i v e r meal when both are supplemented w i t h thiamine. This may be deduced from the a n a l y s i s of variance g i v e n i n Table XXXII. At t h i s p o i n t i t would be advis a b l e to s c r u t i n i z e some of the data and i t s a n a l y s i s c a r e f u l l y . Table XXIX I l l u s t r a t e s I n d e t a i l an a n a l y s i s of variance on the feed - 61 -Table XXXII A n a l y s i s of Varianoe of Growth Response of Groups Receiving VDWL, VDBL, VDPL, SDWL added to B a s a l R a t i o n Supplemented w i t h Thiamine V a r i a t i o n DF Sex 1 Species 3 Sex x species 3 E r r o r 12 T o t a l 19 SS 4,826 Km 2,181 16 Mi MS 4,826 1,503 727 415 Mean d i f f e r e n c e r e q u i r e d f o r s i g n i f i c a n c e (at p * ,05) = 28*2: gm* VR 3.62 Group Mean Mean D i f f . Mean Differen c e s . VDWL SDWL VDPL VDBL 80*4 87*2 102*4 119*4 6.8 18*0 17*0 consumption, weight g a i n , and e f f i c i e n c y of g a i n and an a n a l y s i s of oovarianoe on. the feed consumption and weight g a i n * I n the a n a l y s i s of varianoe, a s i g n i f i c a n t d i f f e r e n c e between samples was noted f o r the weight g a i n and e f f i c i e n c y of g a i n data hut d i f f e r e n c e i n feed consumption were found to be non s i g n i f i c a n t . The s i g n i f i c a n c e noted f o r e f f i c i e n c y of g a i n need not be considered f u r t h e r since i t merely p a r a l l e l s the weight g a i n data. At f i r s t appearance, s i n c e the d i f f e r e n c e i n weight ga i n are s i g n i f i c a n t and d i f f e r e n c e s i n feed consumption are not s i g n i f i c a n t , there i s a strong i n d i c a t i o n to suggest th a t group d i f f e r e n c e s are due to treatment whloh i n t h i s case i s d i f f e r e n t l i v e r samples. (SDWL, VDPL, VDWL and VDBL w i t h thiamine and cod l i v e r o i l added). However t h i s type of a n a l y s i s con-s i d e r s the feed consumption and weight ga i n as separate e n t i t l e s . I t has been shown that these two are i n f a c t c l o s e l y c o r r e l a t e d and i t would t h e r e f o r e be more de-s i r a b l e to consider t h e i r e f f e c t j o i n t l y . This has been attempted i n the a n a l y s i s of oovarianoe* (Table XXIX, part E ) . The feed consumptions and weight gains of the groups of r a t s i n t h i s assay (Table XXIX p a r t A) are a case i n p o i n t f o r the s i t u a t i o n mentioned e a r l i e r i n whloh group d i f f e r e n c e s are not l a r g e and the group making the l a r g e s t g a i n d i d indeed consume the most feed. Thus, when the feed consumptions and weight gains of the v a r i o u s groups are considered J o i n t l y , as i s done i n the a n a l y s i s of oovarianoe, the s i g n i f i c a n c e of group d i f f e r -ences disappears. Thus one would h e s i t a t e to a s c r i b e any s u p e r i o r i t y of one of these samples over any of the other three at l e a s t under the c o n d i t i o n s of the assay. I t would a l s o appear probable that had t h i s assay been con-ducted on an l s o o a l o r l o feed i n t a k e b a s i s there would have been no s i g n i f i c a n t d i f f e r e n c e I n the group weight gains* However, although the data i n the a n a l y s i s of oovarianoe does not a l l o w one to a s c r i b e s u p e r i o r i t y to any of the samples the problem may be considered from-a d i f f e r e n t - 63 *• aspect. The a n a l y s i s of variance d e f i n i t e l y i n d i c a t e s the VDBL and VDWL are equivalent as enrichment sources under the c o n d i t i o n s of the assay. This c o n c l u s i o n i s su b s t a n t i a t e d by the a c t u a l data f o r feed consumption and weight g a i n of the r a t s i n these groups* For s i m i l a r 1 reasons one would f e e l j u s t i f i e d i n s t a t i n g that SDWL and VDPL are eq u i v a l e n t * The a n a l y s i s of oovarianoe t a b u l a t e d i n Table XXX i s an example of the improper a p p l i c a t i o n of t h i s type of a n a l y s i s * I n t h i s oase both feed consumption and weight gains of the r a t s i n the three groups are s i g n i f i -c a n t l y d i f f e r e n t when considered s e p a r a t e l y and non s i g n i f i c a n t l y d i f f e r e n t when considered j o i n t l y * However the a c t u a l group d i f f e r e n c e s i n feed consumption and weight g a i n (Table XXIII) are enough to warrent concluding that the treatment i s governing the weight g a i n through feed consumption since the same supplement i s used i n a l l three r a t i o n s * T h i s i s t y p i c a l of the response obtained from i n c r e a s i n g l e v e l s of supplementation as i n a b i o l o g i c a l assay (or m i c r o b i o l o g i c a l a s s a y ) . Since group d i f f e r e n c e s are r e l a t i v e l y l a r g e , one i s J u s t i f i e d i n t e s t i n g the s i g n i f i c a n c e of the data from weight response only* As w e l l as the above mentioned f a c t o r s , the r e -s u l t s of Assay 1 i n d i c a t e d s e v e r a l other p o i n t s that should be noted* « 64 -I n essence, the b a s a l r a t i o n used i n t h i s assay was complete exeept f o r v i t a m i n s . I t seems reasonable to suppose that the l i v e r i s a l s o supplying other f a c t o r s that may or may not be vitamins ( l ) . For t h i s reason the term enrichment source has been used throughout t h i s t h e s i s i n preference to v i t a m i n content when d e s c r i b i n g the e f f e o t of adding l i v e r p r e p a r a t i o n s to the b a s a l r a t i o n . I t may be seen from the r e s u l t s of t h i s assay that s i x percent VDBL or VDWL, supplemented w i t h thiamine and cod l i v e r o i l and added to b a s a l r a t i o n c o n s t i t u t e a. d i e t comparable to r a t i o n 10-54. Comparable i n that both r a t i o n s produce as good growth i n r a t s on a 42 day assay p e r i o d . This i n i t s e l f i s e x o e l l e n t evidence that a p r o p e r l y prepared l i v e r meal i s h i g h l y d e s i r a b l e as an enrichment, souroe i n animal r a t i o n s * D i f f i c u l t i e s i n v o l v e d i n c o r r e l a t i n g b i o l o g i c a l and ohemioal data are a l s o i n d i c a t e d by t h i s assay. The r e s u l t s of the chemical a n a l y s i s f o r v i t a m i n A (Table I I ) i n d i c a t e that the l i v e r p r e p a r a t i o n s are devoid of t h i s v i t a m i n * However, although the samples d i d appear to be low I n t h i s v i t a m i n they at l e a s t possessed some sub-stance w i t h v i t a m i n A a c t i v i t y . Otherwise the r a t s on r a t i o n s that were not supplemented w i t h cod l i v e r o i l would not l i k e l y have continued to g a i n weight f o r a 42 day assay p e r i o d . A l s o , no previous mention has been made - 65 -of the fact that i n whale l i v e r , vitamin A i a stored as k i t o l , a. complex dimer of vitamin A ( 3 0 ) • The r e l a t i o n -ship of t h i s dimer to vitamin A has not been considered i n t h i s i n v e s t i g a t i o n , A recent review by Morton ( 30 ) gives good evidence of the i n t r i c a t e i n t e r r e l a t i o n s h i p between the f a t soluble vitamins which may further compli-cate the picture* Another recent a r t i c l e ( 3 1 ) indicates that the methods used i n B*C* to obtain concentrates of vitamin A.may markedly effect the s t a b i l i t y of t h i s v i t a -min* Because of the Importance of thiamine i n re-l a t i o n to t h i s investigation, i t . was f e l t necessary to determine with certainty whether or not i t was stable when supplemental to the rat i o n * I t had been noted i n e a r l i e r assays that when thiamine was the l i m i t i n g factor In the r a t i o n , an immediate Increase i n feed consumption and weight gain resulted from i t s addition to the ra t i o n * However there was no evidence that the thiamine was necessarily stable i n the ra t i o n * To c l a r i f y t h i s point, several groups of r a t s i n Assay 1 which, had been main-tained on rations previously supplemented with thiamine were kept a f t e r the regular 42 day assay period* Their rations were supplemented a second time with thiamine and the assay continued f o r 12 days* This procedure d i d not produce any marked change i n either the growth rate o r feed consumption of any of the groups tested* I t was f e l t that t h i s was good evidence to indicate that thiamine was stable i n the p u r i f i e d diet used In t h i s investigation* , Up t i l l now l i t t l e has been s a i d regarding the var i o u s l i v e r p r e p arations used I n t h i s assay exoept f o r g i v i n g a d e t a i l e d account of t h e i r p r e p a r a t i o n * I n re? gard to the vacuum d r i e d samples, no s p e c i f i c c o n t r o l was maintained on temperature d u r i n g the d r y i n g time* However, duri n g the i n i t i a l stages of the d r y i n g p e r i o d the temperature was spontaneously maintained at app r o x i -mately f i v e degrees centigrade due t o the l a t e n t heat of vapour!zation requirements* A f t e r a few hours, the d r y i n g process slowed down and the temperature of the sample g r a d u a l l y rose t i l l I t reached room temperature where i t remained t i l l the d r y i n g process was disco n t i n u e d * Thus the f i n a l product was the r e s u l t of vacuum d r y i n g at r e l a t i v e l y low temperature which i n t u r n would l e a d one to expeot a minimum amount of d e s t r u c t i o n of the b i o l o g i -c a l l y v a l u b l e compounds i n the l i v e r * The importance of temperature i n d r y i n g b i o l o g i -c a l m a t e r i a l s cannot be overemphasized i n view of the Vant HoffiAprhennlus temperature c o e f f i c i e n t concept whloh estimates that a r i s e of 1°C in c r e a s e s the ohemioal r e a c t i o n r a t e by 20 to 30 percent (22)* Thus the l e n g t h of d r y i n g time, the temperature, and the "type" of heat used i n the d r y i n g process would a l l be. expected to have a marked e f f e c t on the c o n d i t i o n of the f i n a l product* Three other samples used i n t h i s assay, ADWL, SDWL, and DDWL are the product of three d i f f e r e n t d r y i n g - 67 ~ processes* The ADWL was prepared hy passing a current of warm a i r (106°) oyer the prooessed enzyme, hydrolyzed l i v e r f o r a p e r i o d of ten hours* The SDWL was prepared by s u b j e c t i n g t h i s same m a t e r i a l to a higher a i r temper-ature f o r a much shor t e r p e r i o d of time* The DDWL was prepared by s u b j e c t i n g the raw l i v e r to a pre h e a t i n g p e r i o d and d r y i n g was e f f e c t e d by d i r e c t oontact of the l i v e r w i t h steam heated metal* An I n s p e c t i o n of the weight responses made by the r a t s on r a t i o n s c o n t a i n i n g ADWL, SDWL, and EHWL (Tables XXI, XXII, and XXIII) w i l l r e v e a l t h a t both d r y i n g processes r e s u l t e d I n d e s t r u c t i o n of some of. the n u t r i t i v e q u a l i t y of the l i v e r * However, there, i s a very good i n d i c a t i o n t h a t the A i r d r y i n g process i s considerably more severe than the Spray d r y i n g process* This r e s u l t i s not s u p r i s i n g i n view of the proceeding c o n s i d e r a t i o n s . No d i r e o t comparison can be made as to the r e l a t i v e e f f e c t of drum d r y i n g on whale l i v e r since the drum d r i e d samples were de r i v e d from d i f f e r e n t basio m a t e r i a l s * However the r e s u l t s of t h i s assay s t r o n g l y euggest t h a t the drum d r y i n g i s the severest of the three d r y i n g processes* No data i s a v a i l a b l e on the p r e p a r a t i o n of the CLM but i t was purported to be the product of a vacuum - 68 drying process. The low protein content of this product would lead one to believe that i t is not solely the combination of fish and mammalian liver, h. Assay 8 The effect of increasing levels of vitamin fortification mixture added to a vitamin free basal ration with and without 3 percent VDWL added. Table XXXIII Total Feed Consumption and Weight Gain of Rats on Assay 7 A. Without 3* VDWL Added V,F»M. Levels  0,15* 0*25 0.25 Sex FC WG FC WG FC. WG F 329 57 285 69 208 M 292 5.6 277 52 221 41 F 266 51 254 41 252: 48 M 226 45 282 53 210 45 F 249 40 187 18 246 52 0.5.0 0*75 1.00 F 218 40 384 115 389 110 M 231 57 445 136 540 163 F 20.8 38 382: 95 408 115 M 230 37 377 122 441 139 F 270 65 464 125 432 119 1*50 2*00 3*00 F 442 . 112 339 :;96 440 150 H 439 149 376 128 473 148 F 400 110 408 121 375 95 M 466 145 479 164 389 116 F 364 105 421 113 457 121 * 69 -Table XXXIII continued  B* With "5% VDWL Added V.F.M. L e v e l s  0.025$ 0.05 0.10 Sex: Fc WO; FO WG FC W& F 265 52 319 64 — M 216 40 261 83 390 135 F 277 63 239 53 365 i n M 190 44 316 105 435 164 F 194 46 32f 92 285 89 0.15 0»25 0.50 p M M i M mmmmwmmmmm HMIMW F 338 89 393 M 504 166 566 F 339 94 416 M 432 163 464 F 369 99 333 1.00 F 364 125 x 452 158 F 399 130 M 449 165 F 367 124 105 380 118 204 471 161 123 380 112 185 *59 I65 99 338 119 The purpose of t h i s assay was to determine i f whale l i v e r would g i v e a growth response over and above that obtained by maximum amounts of VFM. A p r e l i m i n a r y assay was c a r r i e d out i n which the b a s a l r a t i o n was supplemented w i t h two percent VFM. Three r a t i o n s were prepared from t h i s m a t e r i a l , two of whioh were supplemen-ted w i t h one and two percent VDWL r e s p e c t i v e l y . The t h i r d - 70 -r a t i o n had only VFM as a supplement. An assay was c a r r i e d out using the p a i r e d feeding method and no s i g n i f i c a n t d i f f e r e n c e was obtained between the three groups. I t was decided to reverse the assay tedhnique, vary the VFM and keep the VDWL constant, and ca r r y out another assay on an ad l i b i t u m feeding b a s i s . The r e s u l t s of t h i s assay are t a b u l a t e d i n Table X X X I I I , I t may be noted from t h i s t a b l e that when VDWL was present i n the r a t i o n , maximum growth was obtained w i t h 0,25 percent VFM, When VDWL was not present i n the r a t i o n , i t r e q u i r e d one percent VFM t o a t t a i n maximum growth. I n n e i t h e r case d i d h i g h e r VFM supplementation produce any added growth e f f e c t s . However, at the higher l e v e l s of, supplementation, the groups, r e -c e i v i n g VDWL made c o n s i s t a n t l y g r e a t e r weight gains than the groups not r e c e i v i n g VDWL, V i s u a l observations a l s o i n d i c a t e d that the r a t s r e c e i v i n g VDWL were i n a t h r i f t i e r and h e a l t h i e r c o n d i t i o n * Thus, l i v e r supplementation does indeed produce a b e t t e r growth response than that obtained w i t h a r a t i o n supplemented w i t h a l l the known vitamins o n l y . I t would be very i n t e r e s t i n g to c a r r y t h i s l i n e of i n v e s t i g a t i o n f u r t h e r but t h a t , of course, would c o n s t i t u t e a t h e s i s i n i t s e l f * I t was a l s o noted from t h i s assay t h a t there was a marked d i f f e r e n c e i n the feed e f f i c i e n c i e s of the two main groups, A c l o s e r examination of the data revealed t h a t f i v e male r a t s i n each of the two groups consumed almost the same amount of food. The feed consumption and - 71 * weight gains of these ten rats were appropriately paired and analysed using Students paired data method. (Table XXXEV) Table XXXIV Analysis of Data Using Students Paired Data Method Basal + VFM Ration Basal +3$ VDWL + VFM Ration F£ m FO WG 473 148 471 181 466 145 464 18" 439 149 435 16 445 136 449 165 441 139 439 165 SS of weight differences = 341.20 SD s 0,236 S.E. difference « 4.131 t 0 28.6 * 6.923 4.131 S i g n i f i c a n t at p = 0.01 I t Is quite evident from t h i s data that the group d i f f e r -ence i s highly s i g n i f i c a n t . Since the feed consumption of the two groups are I d e n t i c a l , the growth difference must have been due to treatment, or, i n t h i s case, to the b e n i f l o i a l effects of VDWL. This demonstrates not only the value of the paired feeding method i n operation but also the advantages of including a high q u a l i t y l i v e r meal i n an animal r a t i o n . The section of t h i s assay i n which the basal - 72: -r a t i o n i s supplemented with. VDWL and VFM a l s o c l e a r l y demonstrates s e v e r a l p o i n t s discussed e a r l i e r * The weight, gains of the groups which r e c e i v e d 0*025, 0*05, 0*15, and 0*25 percent VEM (Table XXXIII F a r t B) were subjected to an a n a l y s i s of variance (Table XXXV)* The r e s u l t s Table XXXV An A n a l y s i s of Variance on a P o r t i o n of. the Data, of Assay 7 V a r i a t i o n DF SS MS VR L e v e l s 3 26,127 8 7^24 60.8 Sex 1 7,874 7,874 55.9 Sex x l e v e l s 3 7,446 2,4S2' 17*3 E r r o r 12 1,722 143*5 T o t a l 19 43,214 Mean d i f f e r e n c e r e q u i r e d f o r s i g n i f i c a n c e . (at p # 0*02) m 20*3 gm* Mean D i f f e r e n c e s L e v e l 0*025 0.05 0*15 0.25 Mean 4 9 . 0 llT? 122*2 11JT2 Mean D l f f * 34*4 38*8 21.0 l n d l o a t e a s i g n i f i c a n t d i f f e r e n c e (p - o.Ol) f o r edx, l e v e l s , and sex l e v e l i n t e r a c t i o n . The data f o r feed consumption a l s o i n d i c a t e s a pr o g r e s s i v e i n c r e a s e i n feed consumption w i t h i n c r e a s i n g l e v e l s of supplementation. This substan-tiates!) the statement made e a r l i e r that the plane of n u t r i t i o n governs the weight g a i n through feed consumption and weight gains may be used to i l l u s t r a t e group d i f f e r e n c e s provided these d i f f e r e n c e s are l a r g e * The d i f f e r e n c e i n sex response to a given r a t i o n i s also w e l l i l l u s t r a t e d here. This difference i s due to differences i n physio-l o g i c a l age and the fact that at maturity the male i s heavier than the female* Also, the marked difference in: sex l e v e l i n t e r a c t i o n may he attri b u t e d to differences i n p hysiological age, as mentioned e a r l i e r , and the fact that the male rat grows at a fa s t e r rate than the female and w i l l consequently be more adversely affected by a lower plane of n u t r i t i o n since i t s requirements for s p e c i f i c growth essentials w i l l be higher* 0. SUMMARY An attempt has been made to assess the n u t r i t i v e value of whale l i v e r by d i r e c t comparison to beef and pork l i v e r * An attempt has a l s o been made to assess the r e l a -t i v e value of whale l i v e r when subjected to va r i o u s d r y i n g methods* I n c a r r y i n g out these two o b j e c t i v e s , many of the problems Involved i n bioassay have been encountered and discussed i n d e t a i l * The s a l i e n t f e a t u r e s of t h i s i n v e s t i g a t i o n have been i r e c o r d e d below* 1* When vacuum d r i e d whale l i v e r , commercial l i v e r meal, drum d r i e d whale l i v e r , and 50/50 drum d r i e d whale l i v e r and s o l u b l e s are supplemented to a v i t a m i n f r e e b a s a l r a t i o n at the 12 percent l e v e l and t h e i r value as enrichment sources t e s t e d by r a t growth, there i s some i n d i c a t i o n that the former two samples are of equivalent value and s u p e r i o r to the l a t t e r two* I n no case, however, was the growth response anywhere near what would be c a l l e d " normal " f o r t h i s species of animal* 2* When commercial l i v e r meal, spray d r i e d whale l i v e r , vacuum d r i e d whale l i v e r , enzyme hydrolyzed a i r d r i e d whale l i v e r , 50/50 drum d r i e d whale l i v e r and s o l u b l e s , and drum d r i e d whale l i v e r are added at the nine peroent l e v e l to a v i t a m i n f r e e b a s a l r a t i o n supplemented w i t h thiamine and t h e i r r e l a t i v e value as enrichment sources are assessed by r a t growth, the former f o u r samples are of equivalent value and a l l f o u r are s i g n i f i c a n t l y s u p e r i o r to the l a t t e r two samples. The a d d i t i o n of thiamine im-proved a l l samples t e s t e d except the spray d r i e d whale l i v e r but the growth response obtained i n a l l oases was not yet l a r g e enough to be c a l l e d "normal". Vacuum d r i e d beef l i v e r when assayed under the same c o n d i t i o n s et the si x ; peroent supplementation l e v e l produced a growth r e -sponse s i g n i f i c a n t l y s u p e r i o r to t h a t obtained from any of the p r e v i o u s l y mentioned samples. 3* When Vacuum d r i e d beef l i v e r , vaouum d r i e d whale l i v e r , vacuum d r i e d pork l i v e r , spray d r i e d whale l i v e r , commercial l i v e r meal, drum.dried, whale l i v e r , and BO/50 drum d r i e d whale l i v e r and s o l u b l e s are added at the s i x percent l e v e l to a v i t a m i n f r e e b a s a l r a t i o n supplemented w i t h thiamine and cod l i v e r o i l and t h e i r r e l a t i v e value as enrichment sources are assessed by r a t growth, the former four samples are s i g n i f i c a n t l y s u p e r i o r to the l a t t e r t h r e e . The growth response obtained from the former f o u r samples was comparable to that obtained from U.B.Q. stock r a t i o n No. 10-54. Under the c o n d i t i o n s mentioned, no s i g n i f i c a n t d i f f e r e n c e was obtained i n growth response of the groups r e c e i v i n g : (a), drum d r i e d whale - 76 -liver, 50/50 drum dried whale liver and solubles, or commercial liver meal, (b) spray dried whale liver or vacuum dried pork liver, or, (c) vacuum dried whale liver or vacuum dried beef liver. In this instance, a l l seven samples except commercial liver meal were improved by the addition of cod liver o i l . 4. Some investigation jets been carried out in this thesis concerning the severity of four different drying processes on the biological value of liver. There is good evidence to indicate that drum drying is the most severe process followed by drying by air, spray drying, and vacuum drying respectively in a decreasing order of severity. 5. It has been demonstrated that a carefully prepared sample of beef, pork, or whale liver meal has excellent supplementary value when added to a purified diet devoid of al l vitamins except thiamine and cod liver o i l . 6 . When vacuum dried whale liver is added to a purified diet supplemented with a l l the known vitamins) growth response in rats may be improved. BIBLIOGRAPHY 1* Bourne, G.H., and Kidder, G.W,, Ed., "Biochemistry of Physiology and N u t r i t i o n " , Academic Press I n c . , New York, 1953* 2* Shenoy, K.G., and Ramasarma, G.B., I n d i a n J o u r n a l of Pharmacy XVI, p. 11,. 1954. 3. W i l l i a m s , R.J., Truesdale, J.H., Weinstook, H.H., J r . , Rohrman, E., Lyman, CM., and MoBurney, C.H., J . Am. Chem. Soc.,. 60, 2719, 1938* 4. West, E .3 . , and Todd, W.R., "Textbook of Biochemistry," The MacMUlan Co., New York, 1951* 5* Gray, D.J.S., N o v e l l l e , L., Sephton, H.H., and Shuttleworth, R.G., J.Sc, Pood A g r i o . , 2, p. 450, 1951. 6. B a i l e y , B.E., (ED.) B u l l e t i n No. 89, F i s h e r i e s Research Board of Canada, 1952* 7* Gray, P.J.S., Hougen, F.W., N o v e l l l e , L», Sephton, H.H*, . S p r u i t , C.J.P., T a r i o a , V.. and Volckman, O.B., J . S o l * Pood A g r i o * 2, p. 451, 1951. 8. Unna, XT., Riohards, G.V., and Sampson, W.L., J * Nutr,, 22, p. 553, 1941* 9. Baldwin, E«, "Dynamic Aspects of Biochemistry," Cambridge U n i v e r s i t y P r e s s , 1952* 10* K o r r , L», Chemist-Analyst, k2t p. 15, 1953* 11* Plnney, D.J., " S t a t i s t i c a l Methods i n B i o l o g i c a l Assay," Charles G r i f f i n & Co, L t d . , London, 1952* 12. C a r r o l l , W.E,, Proc. Am. Soo* Animal Prod., p. 34, 1930, 13. Dunlop, C , J . A g r i c . S c i . , 23., p. 58O, 1933, 14* Crampton, E.W., Proo* Am. Soc, Animal Prod., p. 56, 1930. - 78 -1 5 * M i t c h e l l , H*H., P r o c Am. Soc. Animal Prod., p. 6 3 , 1 9 3 0 * 1 6 * Hopkins, F.G., J . P h y s i o l . 4 4 , p. "+25, 1 9 1 2 . 1 7 . Osborne, T.B. and Mendel, L.B., J . B i o l . Chem*, 2 6 , p. 1 , 1 9 1 6 . 18* M i t c h e l l , H.H., and Beadles, J*R», J . Nutr., 2 p. 2 2 5 , 1 9 2 9 . 1 9 * Lush, J.L., Proc* Am. Soc. Animal Prod., p. 4 4 , 1 9 3 0 . 2 0 . Rose, W.C., P h y s i o l * Revs., 18, p. 1 0 9 , I 9 3 S . 2 1 . Maynard, L.A., "Animal N u t r i t i o n " , McGraw-Hill Book Co. Inc., 1 9 5 1 . 2 2 * »Brody, S., "Bioenergetics and Growth", Reinhold P u b l i s h i n g Corporation, New York, 1 9 4 - 5 * 2 3 * Richardson, L.R., P r o c Soc* Exp. B i o l . Med., 4 -6 , p. 5 3 0 , 1 9 ^ 1 * — 24-* Lyman, R.L., and Elvengem, P r o c S o c Exp* B i o l * Med., 2 1 , P* 8 1 3 , 1 9 5 1 . 2 5 . Kandutsoh, A.A., and Baumann, C.A., J . Nutr., 4 -9 , p. 2 0 9 , 1 9 5 3 . 2 6 . Rambouts, J*E*, J . Nutr., ^ p* 2 5 5 , 1 9 5 3 * 2 7 * F o r r e s t , R.J., "WhaleLiver As a B-complex: Source I n the Ration of Growing Rats", U n i v e r s i t y of B r i t i s h Columbia, Ondergraduate Essay, 1 9 5 4 . 2 8 . Wishart, J«, C.A.B., Teoh. Comm. No* 1 5 , 1 9 5 0 * 2 9 * Wishart, J . , C.A.B., " F i e l d T r i a l s : . T h e i r Layout and S t a t i s t i c a l A n a l y s i s , " 1 9 4 0 . 3 0 . Morton, R.A., J . S o l * Food A g r i c , _6, p. 3 4 - 9 , 1 9 5 5 * 3 1 * Swain, L.A., and. I d l e r , D.R., Progress Reports No* 1 0 3 , F i s h e r i e s Researoh Board of Canada, p* 18, 1 9 5 5 . 

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