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The nutritive value of fish meal and condensed fish solubles as supplements in poultry rations March, Beryl Elizabeth 1962

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THE NUTRITIVE VALUE OF FISH MEAL AND CONDENSED FISH SOLUBLES AS SUPPLEMENTS IN POULTRY RATIONS by Beryl Elizabeth March, B.A.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE in the Department of Poultry Science  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA, April, 1962 •  In presenting  t h i s thesis i n p a r t i a l f u l f i l m e n t of  the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available f o r reference and study.  I further agree that permission  f o r extensive copying of t h i s thesis f o r scholarly purposes may granted by the Head of my Department or by his  be  representatives.  It i s understood that copying or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission.  Department The University of B r i t i s h Columbia, Vancouver 8 , Canada. D a t e ^ ^  J  /fC  ^  ABSTRACT THE NUTRITIVE VALUE OF FISH MEAL AND CONDENSED FISH SOLUBLES AS SUPPLEMENTS IN POULTRY RATIONS Fish meals, particularly British Columbia herring meals, and condensed herring solubles have been studied for their nutritive propertie as supplements in poultry rations. Both commercially and experimentally prepared herring meals were used in the study. Protein quality in fish meals was investigated to ascertain what degree of variability exists in commercial samples and to obtain information regarding the effects of raw material, processing temperature and  storage. On the basis of the Protein Index Values commercial fish meals  appeared to vary in the quality of their protein content.  Much of the  variability amongst different meals could be ascribed to the nature of the raw material. Experiments were carried out in which herring meals prepared under controlled conditions from material of known origin were compared. It was shown that the value as a protein source for the growing chick of meals produced under a considerable range of drying temperatures was similar. Differences in the vitamin content of fish meal were found to be responsible for differences in the rates of growth of chicks fed herring meals which had been dried at high and low temperatures.  With the basal  rations employed, f o l i c acid was the limiting nutrient when herring meal was used as the sole source of supplementary protein.  The folic acid  content of meals prepared from similar raw material was found to be  ii.  markedly affected by the temperature at which the meals were dried. Chicks and poults fed rations formulated to be complete i n the known nutrients grew at a faster rate when 2.5 to 5.0 percent of herring meal was added to the rations.  It was concluded that herring meal contains  a factor(s) not identified with any of the known vitamins.  Condensed  herring solubles was shown to be an effective vitamin supplement to practical type poult rations whether or not the ration contained herring meal. Extraction of the o i l from herring meals did not improve the growth response of chicks to the use of the meals as protein supplements. Normal and extracted commercial flame-dried meal and experimentally prepared low-temperature-dried meal were compared i n this regard. Storage of herring meals for one year at -25°, 21° and 37°, respectively, did not appear to affect the nutritive value of the meals as protein supplements for chick rations. Chemical changes did occur i n the fat present i n herring meals during storage.  The amount of ether-soluble material decreased with the  length of the storage period and the iodine value of the ether extract decreased.  The addition to the meal of 0.15 percent butylated hydroxy-  toluene before storage prevented any decrease i n ether extractability of the fat during a 9-month storage period and considerably reduced the drop in iodine value of the extract.  iii. TABLE OF CONTENTS Page Abstract  i  Table of Contents  i i i  Acknowledgment  v  Introduction.  1  Review of Literature...  4  Experimental:  20  A.  The variability in the composition of fish meals from different sources  B.  A comparison of the nutritive value of herring meals dried at different temperatures..  C.  Unidentified growth factors in herring meal  D. The use of condensed herring solubles as a vitamin supplement in turkey poult rations E. The effect of heat treatment and storage on the nutritive value for the chick of herring meals of various o i l contents. F.  20  21 26 31 33  The effect of storage temperature and antioxidant treatment on the chemical and nutritive characteristics of herring meal  40  Summary and Conclusions  50  Literature Cited  55  Tables: A-1  Chemical composition of twelve samples of commercial fish meal and meat meal A-2 Protein Index Values of twelve samples of commercial fish meal and meat meal  B-l  60 6l  Growth response of chicks to herring meals dried at different temperatures (Experiment l )  62  B-2 Growth response of chicks to herring meals dried at different temperatures (Experiments 2 and 3)  63  B-3 Effect of single vitamins and a mixture of vitamins of the "B complex" in improving the nutritive value of commercial herring meal (Experiment 4)  64  B-4 Folic acid activity of low-temperature and commercially processed herring meal (Experiment 5)  65  iv. TABLE OF CONTENTS (Continued) Page_ 66  B-5  Composition of basal diet used in Experiment 5  C-l  Growth response of chicks in Experiment.1  67  C-2  Growth response of chicks in Experiment 2  68  C-3  Growth response of chicks in Experiment 3  C-4  Composition of synthetic basal diet  C-5  Growth response of chicks in Experiment 4  C-6  Growth response of poults i n Experiments 5 and 6  72  D-l  Composition of basal diets  73  D-2  Average final weights of poults in Experiments 1, 2 and 3 . .  74  E-l  Average weights of chicks fed unheated and heated, fatextracted or unextracted herring meals as protein supplement in Experiment 1 . .  75  E-2  Average weights of chicks fed extracted and unextracted herring meals in Experiment 2  76  E-3  Average weights of chicks fed various extracted and unextracted herring meals in Experiment 3  77  E-4  Stability of f o l i c acid in different samples of herring  «  69 70  •  71  meals stored forayear  78  F-l  Composition of diets  79  F-2  Chemical analysis of stored herring meals  80  F-3  Ranking of meals according to growth rate of chicks i n the various biological tests  81  I.  Extract ability of fat from herring meal  82  II.  Iodine values of ether-extractable fat in herring meal  83  Figures:  V.  ACKNOWLEDGMENT The author wishes to thank Professor Jacob Biely for introducing her to the f i e l d of nutritional research.  Professor Biely  pioneered in studies of the nutritive properties of British Columbia fishery by-products for poultry feeding and the present study was initiated by him and was carried out under his supervision. Most of the research was conducted as part of a cooperative project with the Pacific Fisheries Experimental Station and with the financial assistance of the Fisheries Research Board of Canada. Throughout the programme Dr. H.L.A. Tarr, director of the Pacific Fisheries Experimental Station, assisted in the planning of the experiments. Thanks are due also to members of the staff of the Fisheries Technological Station for the preparation of experimental herring meals, and to technicians in the Department of Poultry Science for assistance i n the execution of the experiments.  -1-  INTRODUCTION Excess fish and fish offal in different forms have been used from earliest times i n livestock feeding.  It i s surprising, therefore, that when  the f i r s t reduction plants went into operation the potentialities of fish meal as a feedstuff were largely disregarded.  For many years fish meal was  used principally as f e r t i l i z e r and was usually referred to as fish guano. The f i r s t large scale feeding t r i a l s with fish meal were carried out in Norway in 1892 (Woodman, 1937) to ascertain i f fish meal were a suitable feed for farm animals.  At that time there was also considerable interest in the feed-  ing of fish meal in Germany. The success of the Norwegian and the German experiments resulted i n the general use on the continent of fish meal as an animal feed.  In England and i n America, i n spite of the European findings  and the growing use in Europe of fish meal for animal feeding, fish meal continued to be used, as f e r t i l i z e r for some years.  It was not u n t i l after  the First World War that the use of fish meal in feeding became an accepted practice. Early records of fish as feed for livestock dealt mainly with cattle, sheep and pigs, and similarly the f i r s t research into the nutritive properties of fish meal was conducted using these animals.  The greater part of fisheries  products used in feeds today, however, goes into poultry rations and most of the studies carried out in recent years on the nutritive properties of fish meals and other fisheries products have been in connection with poultry. Much of the earlier research on the nutritive value of fish meal was carried out to assess only what may now be termed the "over-all" value of the product as a feed supplement.  It i s d i f f i c u l t to evaluate results based  on experiments conducted at a time when nutrition was only a rudimentary science and before the discovery of most of the vitamins.  This i s particularly true of  -2studies on the feeding of fish meal to poultry prior to the synthesis of riboflavin and the subsequent availability of the vitamin for use in feeding t r i a l s . Today, with a detailed knowledge of nutrition available and an appreciation of the number of essential nutrients, there can hardly be any general statement of the value of any natural product where a variety of nutrients i s involved.  The amount and availability of protein, energy,  vitamins and minerals present in the product, as well as the composition of the remainder of the diet, of which the product i s to be a part, a l l have to be taken into consideration in any evaluation of nutritive worth. A distinction used to be made between animal and vegetable protein with the former assumed to be of higher nutritional value.  The emphasis on the  importance of animal protein as an essential dietary constituent grew out of the fact that, before the development of adequate processing methods for vegetable protein concentrates, i t was extremely d i f f i c u l t , i f not impossible, to compensate for the amino acid deficiencies in the cereal portion on the ration without recourse to some protein derived from animal sources.  Furthermore, animal protein concentrates supplied vitamins which  were deficient in the vegetable protein concentrates.  Today, with the  detailed knowledge of nutrient requirements, the formulation of feeds i s based more and more on the analysis of feedstuffs for a l l the nutrients. The factors determining whether or not a given product is used are not whether i t happens to be of plant or animal origin, but rather the,  "*  particular nutrients supplied by the product and their relative cost from this source. Actually, the use of fish meal and condensed fish solubles constitutes one of the few exceptions to this practice because of evidence for the presence of substances necessary for maximum growth rate and hatchability i n poultry which have as yet to be termed unidentified factors.  In view of the above considerations a programme of research has been carried out on the nutritive value for poultry of fish meal, particularly British Columbia herring meal, and of condensed herring solubles. Variability i n nutritive value and factors affecting nutritive quality have been studied.  Principal attention has been given i n the programme to protein  quality, vitamin content and the nutritive properties of the fat content of the products.  - 4  -  REVIEW OF LITERATURE Fish meal i s purchased on the basis of i t s protein content.  The  amount of protein present i s calculated from the total percentage of nitrogen on the assumption that the protein contains 16 percent nitrogen.  Assurance  is given only of the stated minimum level of crude protein i n the meal with no account being taken of the amino acid composition of the protein, i t s digestib i l i t y or of the amount of nitrogen present i n compounds other than proteins. Over the years many testing methods have been proposed i n an attempt to increase the information available beyond the bare guarantee of minimum protein level. Biological tests provide the best measure of protein quality but tend to be too expensive and time-consuming for routine testing.  Furthermore they have  to be designed to give a specific answer to a clearly formulated question. It i s accordingly not possible to devise a single test which w i l l indicate the performance of a protein supplement under a l l conditions. From time to time various chemical procedures are suggested fot giving a rapid estimate of protein quality.  Procedures based on the percentage  of protein hydrolyzed i n vitro digestion with pepsin, i.e., the percentage of digestible protein, the amount of non-protein nitrogen and the amount of copperprecipitable protein have been worked out.  Almquist (1941) developed a procedure  to measure a Protein Quality Index which was used by several investigators. This method involves the determination of the non-protein nitrogen, the indigestible protein, the copper and the phosphotungstic acid precipitable fractions, and the hot-water-soluble fraction of the protein.  The method  showed quite good correlation with the results of chick growth tests i n assessing the the relative value of animal protein concentrates of the f i s h meal and meat meal type.  As yet there i s no satisfactory chemical procedure  that has general application for routine testing of a l l types of protein concentrates for nutritive quality.  - 5 -  More recently there has been interest i n the development of chemical assays for the measurement of individual available amino acids. The composition of the raw material, for each kind of fish at least, i s quite constant (as opposed to f i s h scrap and offal).  Differences i n the nutritive quality of the  resultant meals therefore most probably arise as the result of variations i n processing procedure.  Overheating during drying and, i n certain types of fish  meal, during the early stages of storage, may result i n reduced biological availability of certain amino acids. Lysine i s particularly l i k e l y to be affected i n this regard.  Since lysine i s a limiting amino acid i n cereal  protein the amount and availability of lysine i n a supplementary protein i s of primary importance.  A method for the determination of "available" lysine  in animal protein concentrates has been devised by Carpenter ( i 9 6 0 ) . Results obtained with this chemical method have given good correlation with chick growth response i n several laboratories. Microbiological analyses for the essential amino acids present d i f f i c u l t i e s for routine work.  They have to be carried out on hydrolysates  of the protein material and the method of hydrolysis has a definite bearing upon the results obtained.  Unless the hydrolysis simulates that occurring  in vivo there i s ano assurance that amounts of amino acids found to be present in a given protein concentrate are available to the animal.  A microbiological  procedure for the evaluation of the protein as a whole has been proposed by Ford (I960). As already stated, biological tests which should give the most accurate appraisal of quality, have to be designed to give an answer to a specific question.  Many of the discrepancies noted between the results of the  biological tests carried out i n one laboratory and another are referable to  -6-  differences in assay procedure and particularly to differences in the basal diets used.  Even when nitrogen balance studies are undertaken to measure  the quality of the protein fed, the results do not necessarily provide an estimate of the quality of the meals as protein supplements. From what i s known today i t i s possible that diets used i n earlier experiments have not always been balanced as to energy content and vitamin level.  Since both the  energy and the vitamin content have to be so balanced in the diet as to obviate the effect of energy and vitamins supplied by the fish meal in question, i t i s d i f f i c u l t to be sure that differences obtained i n many of the experiments reported in the literature have been due to differences in the protein quality and not to some other nutrients supplied by the f i s h meal. Heiman, Carver and Cook (1939) evaluated protein concentrates on the basis of the response of chicks to diets containing 8 percent protein from cereal sources and 3 percent from the protein concentrate in question. The results of this assay procedure are expressed as "Gross Protein Value" relative to the response obtained with casein within the same experiment. Grau and Williams (1955), in an investigation of the quality of commercially produced fish meals from the standpoint of their value as amino acid sources for the chick, used the meals in the test diets to supply 20 percent of crude protein.  In this assay procedure the vitamins, minerals  and carbohydrate were supplied in purified forms so that the diet was dependent upon the fish meals only as sources of amino acids, and no data on the values of the meals as vitamin and mineral sources were obtained. The method does not differentiate among various meals as sources of amino acids for the supplementation of other proteins. In estimating the value of fish meals as supplements in rations, i t would seem that a more c r i t i c a l and meaningful test would be one in which  - 7 -  the fish meal i s used to supplement a cereal or cereal mixture i n accord with actual practice i n feed formulation.  An efficient balance of amino  acids i n a protein to be used as the sole source of protein i n a diet w i l l not be the same as i n a protein used to supplement other proteins. Nor are the needs of the animal necessarily satisfied by supplying the minimum requirements for each of the essential amino acids i n the ration. Harper (1959) has reviewed some of the papers that have appeared regarding the effects of imbalances and excesses i n the amino acid composition of the diet.  It may be remarked that the effects of imbalances of amino acids are  much more apparent when the diet fed i s low i n protein content than when i t contains a high level relative to the requirements of the animal. Differences among species i n the composition of the flesh i t s e l f are not responsible for any appreciable differences i n the protein quality of meals processed from different kinds of f i s h .  Nilson et al.(1947) fed  acetone-extracted fish flesh from 17 species of fish to rats i n a basal diet containing some supplementary protein.  From the growth rates of the  rats i t was concluded that the proteins were about equal i n nutritive value. Konosu et a l . (1956) found that the amino acid composition of fish muscle proteins was relatively constant without distinction among species.  Differences  among meals from different sources would seem to be influenced more by the nature of the method of manufacture and whether or not whole fish or fish offal i s the raw material. Fish meals made from some kinds of fish are more variable than others i n nutritive quality.  Generally speaking, herring and menhaden meals,  which are prepared from whole fish, are more uniform than meals made from the scrap and trimmings of fish processed for human consumption.  For example,  whitefish meal, as the meal made from any of the white fish such as cod, haddock, halibut, etc. i s called, may vary i n quality, not  -8-  because of any species differences in the amino acid composition of the body proteins, but because of differences i n the relative amounts of muscle, heads, skins and viscera that may provide the raw material for a given batch of meal.  Scrap meals, when made from more than one kind of f i s h , may also  vary from one lot to another because of processing d i f f i c u l t i e s associated with material of this nature. There i s l i t t l e information regarding the nutritive value of fish meal prepared from fresh fish as opposed to fish which had undergone various degrees of putrefaction.  What l i t t l e data there are regarding the effect of  the condition of the fish used in making meal are contradictory. (1951)  Lassen et al  reported that spoilage of raw fish before processing had l i t t l e effect  on the nutritive value of the resultant meal. When spoiled fish are processed there i s a much greater loss i n the stickwater so that the yield of meal i s reduced.  Likewise in the case of mackerel meals, a meal processed from fish  that were soft and broken with obvious spoilage was equal in protein value for chicks to one made from firm fleshed mackerel suitable for canning according to the data of Grau and Williams  (1955).  .Tuna meal differs from  the meals tested above in being made from cooked fish.  It was found that  meal made from tuna that has undergone spoilage i s of poor quality as compared to meal produced from unspoiled cooked fish and does not support as rapid chick growth (Anon,  1958).  Whole or f u l l meal are the names given to fish meal which has had presswater added back.  On the basis of Almquist's Protein Index analysis,  dried fish presswater would not appear to be of high protein value because of the high content of hot-water-soluble protein (Almquist,  1941).  This  was borne out by the growth response of chicks to blends of dried presswater and ordinary fish meal. I t should be mentioned that this finding i s contrary to the results of earlier experiments (Wilgus, 1 9 3 3 ) which showed protein  -9-  efficiency to be superior in fish meals containing the presswater.  In  view of the data obtained in the latter study i t seems likely that the whole meal preparation supplied water-soluble vitamins which were a factor in the efficient utilization of the protein.  For valid appraisals of whole  meal from the standpoint of protein quality, only the results of more recent experiments can be taken into consideration. Laksesvela (1958) has reported the results of an extensive study of the nutritive value of condensed herring solubles and herring meal.  He  found that, when fed in the diet as the only source of protein, herring solubles appeared to be of l i t t l e nutritive value and did not sustain l i f e for more than 2 to k weeks when fed to day-old chicks.  Certain mixtures of  herring solubles with meal, however, were superior to a protein source to the meal alone. solubles.  Tryptophane was the limiting amino acid in the herring  Nevertheless, although the solubles do not provide a balanced  source of amino acids for the chick, they may be used to advantage to supplement presscake protein. Of the various aspects of fish meal manufacture the one which has perhaps received the most widespread attention i s that of drying temperature. This i s understandable i n view of the vast literature dealing with the destruction of vitamins by heat, the impairment of protein nutritive quality by heat and the lowering of the digestibility of fats and even the production of toxic products resulting from heat treatment of fats. Early work on fish meal processing (ingvaldsen, 1929) showed that high drying temperatures increased humin and volatile basic nitrogen and reduced the arginine and cystine content of the meal. Much of the experimental work on the effects of drying temperature was in relation to the type of drying equipment, i.e., steam or vacuum dryer.  In feeding tests with  different animals, vacuum and steam dried meals were generally reported to be  -10better supplements than flame dried meals (Maynard and Tunison, 1932; Record et al, 1934; Harrison et al, 1935; Oshima and Itaya, 1938; Daniel and McCollum, 1931; Schneider, 1932; Wilder et al, 1934). More recent studies under carefully controlled conditions show that meals of equal nutritive value may be produced by either the vacuum or the flame methods of drying, provided care is taken that the meals are not overheated (Clandinin, 1949; Grau and Williams, 1955). In meals scorched during drying the availability of the amino acids to the animal is reduced.  The  liberation of lysine, arginine, and possibly threonine was found by Clandinin (1949) to be affected by overheating even when tested by in vitro acid hydrolysis, and the liberation of a l l the essential amino acids was greatly depressed. Clandinin further reported and concluded on the basis of feeding experiments with chicks involving diets in which fish meals were used to supplement cereal protein, that "over-heating fish meal during drying depressed availability of the amino acids to such an extent that the damage done is irreparable by simple supplementation with the essential amino acids which acid hydrolysis values suggest as limiting".  A combination of lysine,  arginine, threonine, glycine, and methionine improved growth over that obtained with lysine alone, but growth rate was s t i l l not comparable to that obtained with fish meal that had been dried at a lower temperature. It seems probable, however, that although the amino acids may be rendered unavailable when fish meals are subjected to heat treatment as evidenced by the above reports, a rather severe heating is required to render the fish meal protein resistant to the action of the digestive enzymes. Under normal conditions the temperatures used in food manufacturing procedure are not sufficiently high, nor is heating sufficiently prolonged, to result in protein damage. Comparative microbiological assays (Bissett and Tarr, 1954) of chemical and enzymatic hydrolysates of herring meal show that the availability of the  -lies sent i a l amino a c i d s was commercial p r o c e d u r e one  hour a t 159°C.  impaired.  I t was  n o t e d t h a t when h e a t i n g was  C a r p e n t e r e t a l (1954) found no d i f f e r e n c e s i n t h e p r o t e i n by d i f f e r e n t  d r y i n g methods.  More r i b o -  d e s t r o y e d i n the flame d r y i n g t h a n i n o t h e r p r o c e s s e s , but  p r o t e i n q u a l i t y and v i t a m i n methods.  continued f o r three  the a v a i l a b i l i t y o f a l l t h e e s s e n t i a l amino a c i d s  q u a l i t y o f w h i t e f i s h meals produced f l a v i n was  i n normal  nor by h e a t i n g meal under e x p e r i m e n t a l c o n d i t i o n s f o r  h o u r s a t t h i s temperature was  not a l t e r e d by f l a m e - d r y i n g as performed  The  B 2/|  w e r e  ±  a  s  high  a  s  with the lower  the  temperature  s l i g h t l y d a r k e r c o l o u r o f t h e f l a m e - d r i e d meals was  not  n e c e s s a r i l y an i n d i c a t i o n o f p o o r e r q u a l i t y i n t h e s e e x p e r i m e n t s .  Laksesvela  (1958), however, found t h a t t h e e x t e n t to which meals were d i s c o l o u r e d by spontaneous h e a t i n g a f t e r manufacture i n d i c a t e d t h e e x t e n t t o which n u t r i t i v e v a l u e o f the meals had been a f f e c t e d . M i l l e r (1955) c o n s i d e r s t h a t the impairment i n p r o t e i n q u a l i t y o f commercial f i s h meals d u r i n g d r y i n g i s due t o the a c t i o n o f h e a t i n the presence  o f m o i s t u r e and  i s the r e s u l t o f the M a i l l a r d r e a c t i o n .  Miller  r e p o r t e d t h a t commercial f i s h meals from v a r i o u s s o u r c e s had p r o t e i n  utili-  z a t i o n v a l u e s (body weight X d i g e s t i b i l i t y ) o f 60 as compared w i t h 80 f o r fresh f i s h . is  The e f f e c t o f o v e r - h e a t i n g on the p r o t e i n q u a l i t y o f f i s h meals  d i f f e r e n t t o t h a t o c c u r r i n g w i t h p r o t e i n supplements o f h i g h  content.  With p r o t e i n meals which c o n t a i n l i t t l e  o r no f r e e  carbohydrate  carbohydrate,  o v e r - h e a t i n g r e s u l t s i n l y s i n e and p o s s i b l y a r g i n i n e and h i s t i d i n e new  forming  l i n k a g e s t o o t h e r amino a c i d s which a r e r e s i s t a n t t o enzyme a c t i o n  c o n s e q u e n t l y not a v a i l a b l e t o t h e animal a c c o r d i n g t o Almquist L y s i n e i s one  and  (1951).  o f t h e amino a c i d s most apt t o be r e n d e r e d u n a v a i l a b l e i n f i s h  meals t h a t have been o v e r - h e a t e d .  S i n c e l y s i n e i s the p r i m a r y l i m i t i n g amino  a c i d when c e r e a l s a r e f e d as t h e o n l y source o f p r o t e i n , any p r o t e i n must f i r s t o f a l l compensate f o r the l y s i n e  supplementary  deficiency.  -12Th e e f f e c t s o f s t o r a g e on n u t r i t i v e v a l u e o f f i s h meal p r o t e i n have n o t been c o n c l u s i v e l y e s t a b l i s h e d . Evans, C a r v e r and Hairm (1944) r e p o r t e d t h a t t h e r e was  no l o s s i n the  supplementary  p i l c h a r d meals a f t e r s t o r a g e f o r 10 months i n paper, Storage  a t room temperature  protein value of c o t t o n o r b u r l a p bags.  f o r 3 months d i d not a f f e c t the p r o t e i n n u t r i t i v e  v a l u e o f d r y cod p r o t e i n s a c c o r d i n g t o M i l l e r  (1955).  Almquist  (1956) on  t h e o t h e r hand, on the b a s i s o f i n v i t r o t e s t s , found t h e d i g e s t i b i l i t y o f p r o t e i n by p e p s i n t o be d e c r e a s e d upon s t o r a g e . t u b e s , hoxvever, showed l i t t l e  alteration.  Samples s e a l e d i n g l a s s  L e a , P a r r , and  Carpenter  (1958)  r e p o r t e d t h a t whereas h e r r i n g meals s t o r e d i n a i r d e c r e a s e d by 9 p e r c e n t i n t h e i r a v a i l a b l e l y s i n e c o n t e n t , meals which were s t o r e d i n n i t r o g e n , o r w h i c h were d e f a t t e d and then s t o r e d i n a i r , d i d not decrease i n a v a i l a b l e  lysine  content. Lanham and N i l s o n (1942, 1947) of and  s a r d i n e meal w i t h heat and m o i s t u r e i n which b o t h c h e m i c a l  decomposition  c h e m i c a l h y d r o l y s i s took p l a c e d i d not a d v e r s e l y a f f e c t t h e n u t r i t i v e  value o f the p r o t e i n . conducted  Subsequent s t u d i e s (1947) i n which f e e d i n g t e s t s were  on a v a r i e t y o f commercial meals p r e p a r e d from p i l c h a r d , menhaden  and l e a n marine ground f i s h f i l l e t of  found t h a t e x p e r i m e n t a l s p o i l a g e  s c r a p , showed t h a t e x p e r i m e n t a l s p o i l a g e  t h e s e meals under c o n d i t i o n s o f h i g h heat and h u m i d i t y and under a e r o b i c  c o n d i t i o n s , d i d n o t produce any t o x i c compounds. Vitamin  Content I n t e r e s t i n t h e v i t a m i n c o n t e n t o f f i s h meals was  at f i r s t  princi-  p a l l y i n c o n n e c t i o n w i t h t h e r i b o f l a v i n c o n t e n t , t h e n v i t a m i n B 2 » and more x  r e c e n t l y w i t h r e g a r d t o the e x i s t e n c e o f some u n i d e n t i f i e d f a c t o r o r Little  factors.  c o n s i d e r a t i o n i s g i v e n a t the p r e s e n t t i m e t o t h e content o f t h e f a t -  soluble vitamins.  The l e v e l o f t h e v a r i o u s v i t a m i n s i n f i s h meals may  vary  c o n s i d e r a b l y a c c o r d i n g t o b o t h the raw m a t e r i a l and t h e p r o c e s s i n g p r o c e d u r e .  -13Because many of the vitamins are heat-labile, over-heating may seriously reduce the nutritive value of the fish meal through destruction of the vitamin content.  For example, flame-drying reduces the amount of  riboflavin according to the data published by Harrison et a l , 1935;  Record  et a l , 1934; and Carpenter et a l , 1954). Klungs^yr et al (1953) reported the decrease i n the content of riboflavin, vitamin B3.2 and pantothenic acid in herring presscake to vary according to the drying temperature.  Carpenter  et a l (1954) compared the vitamin B 2 content of meals produced by different ±  commercial drying procedures and found the vitamin  content of flame-  dried meals to be as high as that of meals dried at lower temperatures. Southcott and Tarr (1953) likewise do not consider there to be a significant difference in the vitamin B]_2 content of experimentally produced low temperature air-flow dried meal and commercial herring meal. Karrick and Stansby (1954) analyzed samples of presscake and of meals prepared from the presscake in different processing plants for riboflavin, vitamin B 2 and niacin. x  There was no significant loss of vitamins upon drying the presscake except in the case of the niacin when the meal was dried in a direct flame-dryer.  The  vitamin B 1 2 content of the presscake was affected when the presscake was dried at 80 - 100°C. for 5 hours. Most of the early studies on the nutritive value of fish meal favoured the meals prepared by the dry-rendering process over wet-rendered meals. In view of the vitamin deficient diets employed at that time, this conclusion was probably due to the fact that the water-soluble vitamins were retained in the dry-rendered meals.  In 1931 Harrison drew attention to the  considerable losses of material that occur in the wet reduction of menhaden. Likewise Beall in 1933 reported the nutrient losses in the effluent of pilchard reduction plants in British Columbia.  During the last war a consider-  able production of condensed fish solubles developed.  The use of condensed  -14-  s o l u b l e s was p a r t i c u l a r l y p o p u l a r  i n those  areas  i n t h e U n i t e d S t a t e s where  c o r n and soybean o i l meal a r e the p r i n c i p a l components o f p o u l t r y f e e d s . A f t e r pure r i b o f l a v i n began t o be produced commercially, continued  fish  solubles  t o be used, a g a i n p a r t i c u l a r l y i n t h e mid-western U n i t e d  States,  f o r what came t o be w i d e l y r e f e r r e d t o as t h e "animal p r o t e i n f a c t o r " . With t h e d i s c o v e r y o f v i t a m i n B  x  2 the 'fenimal p r o t e i n f a c t o r " was i d e n t i f i e d  w i t h t h i s l a t e s t member o f t h e B-complex.  As i n t h e case o f r i b o f l a v i n , i t  was n o t l o n g b e f o r e i t was p o s s i b l e t o produce v i t a m i n B]_2 ^ t r a t i o n as a p r o d u c t  o f fermentation.  h i g h concen-  The use o f condensed f i s h  solubles  as a f e e d supplement c o n t i n u e d d e s p i t e the a v a i l a b i l i t y o f r i b o f l a v i n and v i t a m i n B 1 2 from o t h e r s o u r c e s .  Condensed f i s h s o l u b l e s was c o n s i d e r e d t o  be a v a l u a b l e n a t u r a l source o f t h e v i t a m i n B-complex and, i n a d d i t i o n , t h e r e was evidence  f o r t h e p r e s e n c e o f an u n i d e n t i f i e d f a c t o r / s i n t h e  product. Hill  (1948) r e p o r t e d t h a t f i s h meal and condensed f i s h  were c a r r i e r s o f a n u n i d e n t i f i e d f a c t o r . p u r i f i e d d i e t , found t h a t 3 p e r c e n t weeks i n c h i c k s and p r e v e n t e d diet.  soluble  C a r l s o n e t a l (1949), u s i n g a  f i s h meal g r e a t l y improved growth t o 4  t h e m o r t a l i t y which o c c u r r e d w i t h t h e b a s a l  Sunde e t a l (1950) found evidence  that chicks f e d p r a c t i c a l rations  r e q u i r e d an u n i d e n t i f i e d f a c t o r i n a d d i t i o n t o v i t a m i n BT_2 and o t h e r known v i t a m i n s f o r maximum growth.  Combs and S h a f f n e r  (1950) r e p o r t e d t h a t  fish  meal s u p p l i e d an u n i d e n t i f i e d f a c t o r which s t i m u l a t e d growth i n t u r k e y p o u l t s . Heuser and N o r r i s (1951), working w i t h f i s h meal, condensed f i s h homogenized f i s h , concluded factor.  t h a t they c o n t a i n e d  McGinnis e t a l (1952) a l s o o b t a i n e d  f i s h s o l u b l e s w i t h -both p o u l t s and c h i c k s .  s o l u b l e and  some as y e t u n i d e n t i f i e d  a growth response t o condensed P a t r i c k (1951) r e p o r t e d t h a t  p o u l t s f e d h i g h soybean o i l meal r a t i o n s responded t o supplements o f f i s h meal, f i s h s o l u b l e s and  a methanol s o l u b l e f r a c t i o n o f f i s h s o l u b l e s .  There  -15was  no d i f f e r e n c e i n the e f f e c t o f f u l l meal and The.  required  question  o f whether t h e r e may  a l l - v e g e t a b l e d i e t and  condensed f i s h s o l u b l e s . f o u r weeks t h e was  no  change i n the  retarded  diet.  on t h e The  same d i e t w i t h t h e  a d d i t i o n of 13  hens were kept on b u i l t - u p l i t t e r .  Within there  condensed  also i n d i c a t e d a decreased hatching  growth i n the c h i c k produced by t h e hens on the  on  percent  dropped 15 p e r c e n t but  h a t c h a b i l i t y o f eggs from the hens f e d t h e  Progeny s t u d i e s  A r s c o t t and  has  Stephenson and Glower (1952) kept b r e e d i n g hens  h a t c h a b i l i t y o f f e r t i l e eggs had  f i s h solubles. and  a l s o be u n i d e n t i f i e d f a c t o r s  f o r maximum h a t c h a b i l i t y as w e l l as f o r maximum r a t e o f growth  a l s o been i n v e s t i g a t e d . an  solubles.  weight  all-vegetable  Combs (1953) found t h a t f i s h s o l u b l e s gave a more pronounced  improvement i n the h a t c h a b i l i t y o f eggs from b i r d s housed on w i r e f l o o r s t h a n those on l i t t e r .  There was  a l s o evidence o f a c a r r y o v e r  u n i d e n t i f i e d f a c t o r s u p p l i e d by condensed f i s h s o l u b l e s .  t o the c h i c k o f  the  Ferguson e t a l (1956)  f e d t u r k e y hens on an a l l - v e g e t a b l e b a s a l r a t i o n ( f o r t i f i e d w i t h v i t a m i n and  supplemented i t w i t h v a r i o u s  condensed f i s h weeks t h e r e was mented d i e t s .  s o l u b l e s and  BTJ-J)  combinations o f dehydrated a l f a l f a meal,  vitamin  E f o r a p e r i o d o f 20 weeks.  From  1-8  l i t t l e d i f f e r e n c e i n h a t c h a b i l i t y from t h e d i f f e r e n t l y s u p p l e From 9 - 20 weeks t h e r e was  a drop i n h a t c h a b i l i t y w i t h a l l  d i e t s , except t h a t supplemented w i t h the combination o f dehydrated meal, f i s h meal and  vitamin  E.  The  d a t a suggest the p o s s i b i l i t y  alfalfa  t h a t condensed  f i s h s o l u b l e s c o n t a i n an u n i d e n t i f i e d f a c t o r which reduces embryonic m o r t a l i t y during  the l a s t week o f i n c u b a t i o n .  p u r i f i e d d i e t and from a h i g h bation.  The  K u r n i c k e t a l (1956) f e d b r e e d e r hens a  found t h a t the d i e t r e s u l t e d i n poor h a t c h a b i l i t y r e s u l t i n g  incidence  o f embryonic m o r t a l i t y d u r i n g  the  f i r s t week o f  u n i d e n t i f i e d f a c t o r , t h e d e f i c i e n c y o f which was  found i n l i v e r p r e p a r a t i o n s ,  f e r m e n t a t i o n p r o d u c t s and  Supplementation o f the b a s a l d i e t w i t h f i s h  incu-  responsible,  condensed f i s h  was  solubles.  solubles increased h a t c h a b i l i t y  -16from 28 to 84 percent.  In addition to the above reports indicating the  presence of an unidentified factor promoting high hatchability, there have also been instances reported where no effect on hatchability could be demonstrated.  Whether or not the differences noted i n the response to the inclu-  sion of condensed fish solubles i n the diet with regard to hatchability are due to some management factor influencing the amount of the factor available to the hens through coprophagy or to strain or breed differences in the requirement of birds for the factors has yet to be elucidated. Attempts have been made to factionate fish solubles i n order to concentrate the factor.  Menge et a l (1953) showed that the factor was soluble  in water, phenol, 50, 60 and 80 percent ethanol and i n 70 percent methanol, but insoluble in ether.  It was also found to be dialyzable and stable to  autoclaving at pH 2.0 to 11.0.  According to Taminie (1955) the growth stimulus  resulting from the addition of fish meal to a purified diet i s due to at least two unknown factors.  One factor i s extractable from the meal with water and i s  acetone soluble and the second factor i s present in the ash.  Taminie considers  the fish factor to be identical with the liver factor but distinct from the whey factor, the alfalfa factor and the factor present i n d i s t i l l e r s ' dried solubles.  Ritchey et a l (1956) found that intact fish meal always increased  growth rate significantly but that the response obtained to the ash from fish meal was variable.  s  Assay methods for the unidentified^factors have been described by L i l l i e et a l (1953) and by Barnett and Bird (1956). The latter investigators observed that the response to a growth factor i n fish solubles apparently depends upon the stock used and upon the presence of a particular microorganism or group of micro-organisms in the environment.  -17Fat  Content Most o f t h e i n v e s t i g a t i o n s i n t o t h e n u t r i t i v e v a l u e o f f i s h meals  have been concerned w i t h p r o t e i n q u a l i t y o r w i t h v i t a m i n c o n t e n t .  Little  a t t e n t i o n has been g i v e n t o t h e e f f e c t o f o i l c o n t e n t on the n u t r i t i v e o f these meals. oil.  Commercial f i s h meals n o r m a l l y c o n t a i n from 5 t o 10  value  percent  T h i s o i l , because of. the temperatures reached i n p r o c e s s i n g and the  l a r g e s u r f a c e a r e a exposed i n the f i n a l p r o d u c t , may be s u b j e c t t o c o n s i d e r a b l e a l t e r a t i o n t h r o u g h p o l y m e r i z a t i o n and o x i d a t i o n . The d a r k e r  c o l o u r n o t e d i n some samples o f f i s h meal i s a p p a r e n t l y  due, i n p a r t , t o r e a c t i o n s between the p r o t e i n and t h e o i l a t the c a r b o x y l and amino groups, o x y p o l y m e r i z a t i o n  and o x i d a t i o n o f u n s a t u r a t e d  a c c o r d i n g t o V e n o l i a e t a l (1957).  L e a e t a l (1958) found t h a t d e f a t t e d meals  did  oils,  n o t darken upon s t o r a g e n o r d i d i n t a c t meals when t h e y were s t o r e d i n an  atmosphere o f n i t r o g e n .  V e n o l i a e t a l (1957) showed t h a t emulsions o f menhaden  o i l and p r o t e i n r e a c t t o form a deep brown c o l o u r . that f i s h o i l s  (1938) showed  adsorbed on s a r d i n e meal and o t h e r p r o t e i n f o r 3 0 days and then  removed were darkened by c o n t a c t w i t h the p r o t e i n . on n o n - p r o t e i n  Oya and Nonaka  They n o t e d t h a t  m a t e r i a l d i d n o t g i v e t h e same d a r k e n i n g .  adsorption  That t h e d a r k e n i n g  o f t h e f i s h meal i s an i n d i c a t i o n o f a r e d u c t i o n i n t h e amount o f  utilizable  l y s i n e i n t h e f i s h meal has been shown i n s t u d i e s conducted by L e a e t a l (1958). The chemical is readily oxidized.  c o n s t i t u t i o n o f t h e o i l i n f i s h meals i s such t h a t i t Aure (1957) g i v e s the i o d i n e v a l u e o f o i l e x t r a c t e d from  h e r r i n g meal as 136 as compared w i t h a v a l u e o f 1 2 3 from t h e h e r r i n g o i l produced i n the r e d u c t i o n o f h e r r i n g .  Aure e x p l a i n s t h e d i f f e r e n c e between  the v a l u e s as b e i n g due t o a c o n c e n t r a t i o n o f p h o s p h o l i p i d s the  f a t t y a c i d s o f which a r e more u n s a t u r a t e d  i n the meal f a t ,  than h e r r i n g o i l f a t t y a c i d s .  I n a d d i t i o n t o the s u s c e p t i b i l i t y o f t h e o i l c o n t e n t  o f t h e meal t o o x i d a t i o n ,  t h e p r e s e n c e o f hematin i n the meal c a t a l y z e s t h e p r o c e s s .  Banks (1939) showed  -18that hematin accelerates rancidity in the o i l content of fish tissue and more recently i t was demonstrated (Anon., 1 9 5 8 ) that the content of hematin i s a major factor in determining the rate at which fish meals deteriorate. The question as to whether oxidized oils are deleterious i n feeding has occasioned a great deal of research. Studies with rats have shown that oxidized and polymerized oils seem to contain toxic factors as well as being poorly digested (Lassen et a l , 1 9 4 9 ; Fraser, et a l , 1 9 4 9 ; Crampton et a l , 1951).  The exact nature of the apparent toxicity i s not altogether clear.  Crampton and his associates, have made an extensive study of the effects of heat treatment of various o i l s on the nutritive qualities of the o i l s . It was concluded  (1951)  that,in the case of linseed o i l , peroxidation is not  concerned with the development of any toxic factor and that the damaging factor i n heated o i l i s not produced oxidatively.  Further studies  (1953)  showed that the toxicity of heat-polymerized linseed o i l was associated with the presence of polyene acids i n the original o i l . Johnson et al  (1957)  found that thermally oxidized corn o i l inhibited growth when fed to rats whereas margarine base stock and butter which had been heated under similar conditions gave only a slight growth depression, or did not depress growth. Again i t appeared that products resulting from the thermal treatment were related to the unsaturated fractions of the o i l . Witting et a l  (1957)  found  that the toxicity of oxidized and oxidatively polymerized fish o i l produced by blowing with a i r for three days at room temperature was less than that of o i l thermally polymerized without oxidation. When the oils were fed to rats i t was noted that the harmful effects of the oxidized o i l could be more easily offset than those of the thermally polymerized o i l by an increased intake of pyridoxine, riboflavin or protein. Old samples of fish meal contain as much as 4 0 percent of free fatty acids i n the o i l according to Moen  (1933).  It is doubtful i f a high free  -19-  fatty acid content in fish meal i s , in i t s e l f , harmful since pure fatty acids may be fed to chicks without depressing growth (Sunde, 1956).  With meat  scraps i t has been found that no i l l effect results from feeding samples high in free  fatty acids (Gray and Robinson, 1941).  Siedler et a l , 1955)  concluded that animal fats that vary in free fatty acid content are utilized equally well by the chick.  It seems probable that the fat content o f the  fish meal may exert indirect effect on the nutritive value o f the meal through the activity of peroxides i n destroying vitamins i n the f i s h meal i t s e l f or in mixed rations to which the meal may be added.  -20EXPERIMENTAL A.  The variability in the chemical composition of fish meals from different sources The f i r s t experiment in the study was carried out to ascertain the  variability i n the chemical composition of fish meals from different sources. Several samples of meat meal were included i n the test for comparison.  The  meals tested are listed together with their gross analyses in Table A-1.  The  protein nutritive values of the respective meals were assessed by the Protein Quality Index method of Almquist (1941).  The values are shown in Table A-2.  The Protein Index Values of the fish meals ranged from 65 to 82. As can be seen from Table A-2, the amounts of the total nitrogen content of the meals present in the indigestible fraction and i n the non-protein fraction, as well as the proportion of the protein which was hot water soluble, differed considerably among the meals analyzed. The meat meals gave lower Protein Index Values than did the fish meals with a range of 48 to 72.  The lower values were largely the result of  a higher percentage of hot water soluble protein.  In addition, the phospho-  tungstic acid precipitable fractions indicated a greater degree of protein decomposition. Both fish meals and meat meals normally contain varying proportions of blood, bone, cartilage, connective tissue, muscle and viscera.  Different  methods of processing may result i n meals of different composition from essentially the same type of material.  The Protein Index Values w i l l be seen  to reflect both the nature of the raw material used and the method of processing.  Thus, of the fish meals, the sample designated as "scrap" meal had a  higher percentage of hot water soluble protein.  This i s doublLess the result  of the naturally high collagen content of the raw fish scrap, since skin and bone constitute a large proportion of fish trimmings.  The meat meals gave  -21lower Protein Index Values (48-72) than the fish meals, largely because the percentage of hot water soluble protein was greater.  In addition, phospho-  tungstic acid precipitable fractions indicated a greater degree of protein decomposition. B.  A comparison of the nutritive value of herring meals dried at different temperatures Previous experiments (Tarr et a l , 1951) showed that herring meals  prepared by air-flow drying at 100-110° F. had, for the chick, a higher nutritive value than commercial meals.  The herring meals used in these exper-  iments were not, however, s t r i c t l y comparable since they were not prepared from identical press cake.  In the following experiments, herring meals were  compared which were made from the same batches of press cake dried at different temperatures, thus eliminating any variations due to the raw material. Fresh whole herring were used within one day of capture to make the following meals: Meal 1.—A  typical light-coloured product prepared in November 1951 by  direct flame drying of the press cake under the following conditions:  Fire  box temperature (range) 1000-1150°F.; stack temperature (range) 227-257°F.; meal temperature as discharged from dryer (range) 125-130°F. Meal 2.—A  second portion of the same batch of press cake was dried at a  much higher temperature than that usually employed: Fire box temperature (range) 1210-l600°F.; stack temperature (range) 315-340°F.; meal temperature as discharged from dryer (range) 175-200°F. Meal 3.—A  low-temperature-dried meal was produced by drying i n an air  current at 100-110°F. a third portion of the same herring press cake as was used in the production of Meals 1 and 2. Meal 4.—A  typical light-coloured commercial meal was prepared by normal  direct flame drying of herring press cake in early January 1952.  -22Meal 5.—A low-temperature-dried meal dried similarly to Meal 3 and made from a sample of herring press cake taken i n the same plant and on the same day as that for Meal 4. Each of the herring meals was used as the sole source of supplementary protein in chick starting rations formulated to contain 17$ protein in Experiment 2 and 21$ protein in Experiments 1 and 3. Ground yellow corn comprised the cereal portion of the rations.  The amounts of corn meal and  herring meal in each ration were adjusted according to the protein content of the herring meal and the protein level desired in the experimental ration. Water was added to the rations to make up for any differences in the moisture content of the herring meals. Limestone and bone meal were added to the rations to adjust the calcium and phosphorus content to 1.0 and 0.6$ respectively. The following supplements were added per 100 l b . of ration: iodized salt, 0.5; feeding o i l (2250 A - 300 D3), 0.25; choline chloride (25$), 0.25 lb.; riboflavin, 0.16; calcium pantothenate, 0.5; niacin, 0.8; manganese sulphate, 10.0 gm. Duplicate lots of 20 one-day-old White Leghorn cockerel chicks were fed each ration in Experiments 1 and 3, and duplicate lots of one-day-old White Leghorn pullet chicks were fed each ration in Experiment 2. In these, and in the other chick biological tests in the study, the chicks were kept i n the compartments of battery brooders with wire screen floors.  Water and the  experimental diets were given ad l i b . Experiment 1.—The average weights of the chicks at 3 weeks are given in Table B - l . It w i l l be seen from the data presented that the two herring meals which viere dried at low temperatures promoted a faster rate of growth than the meals prepared commercially.  The meal which was dried conmer-  c i a l l y at higher temperatures than usual supported a rate of growth equal to that with the normal commercial meal.  Since the meals compared were a l l  -23prepared from similar raw material, the data of this experiment show conclusively that some nutritive factor (or factors) i s destroyed or i s rendered unavailable to the chick by the commercial drying process. Experiments 2 and 3.—  These were designed to ascertain i f the  limiting growth factor(s) in the commercially dried herring meals was one of the known vitamins. Accordingly, Meals 1, 2, and 3 were again used as the only protein supplements in rations similar to those fed in Experiment  1,  with and without the addition of the following vitamins per 100 l b . of rations: Thiamin hydrochloride Pyridoxine hydrochloride Folic acid 2-Methyl naphthoquinone Inositol Alpha-tocopherol Para-aminobenzoic acid Biotin Vitamin B 2 ±  0.050 0.160 0.035 0.018 5.0 0.15 4.5 0.0045 0.0005  gm. gm. gm. gm. gm. gm. gm. gm. gm.  The average weights of the chicks in Experiments 2 and 3 are given in Table B-2.  With the basal diet the low-temperature-dried meal, as in  Experiment 1, was superior in nutritive value to the commercially dried meals. The difference i n the nutritive value of the meals was apparent with both the 17 and the 21% protein ration. When the vitamin mixture was added to the rations containing a lowtemperature-dried meal, there was no improvement in growth rate of the chicks. T h e r e was, however, a marked improvement with the commercially dried meals. The chicks fed the normal commercial meal supplemented with vitamins were similar i n weight to those fed the low-temperature-dried meal. The chicks fed the overheated commercial meal with the vitamin supplement were heavier than those fed either the low temperature of the normal commercial meal.  The  differences in the nutritive value of the various herring meals were apparent with both the 17 and 21% protein rations.  The data are interpreted as indicating  that the sample of herring meal dried at a low temperature contained adequate  -24quantities of one or more vitamins which were markedly deficient in the commercially dried meals. Presumably some heat labile vitamin(s) were destroyed or rendered unavailable by the normal flame-drying procedure employed i n the manufacture of commercial herring meal. Experiment 4.—The next step was to determine whether any single vitamin was responsible for the growth stimulus obtained with the mixture of vitamins in the previous tests.  The basal diet used was similar in compo-  sition to the 21$ diet used above and a sample of commercially prepared herring meal constituted the protein supplement. This basal diet was supplemented with the respective vitamins of the mixture and with the entire mixture. The average weights of the chicks at 4 weeks of age are shown in Table B-3. Folic acid was the only single vitamin producing a growth response approaching that obtained with the vitamin mixture.  Data i n the literature  likewise suggested that folic acid might be a limiting factor i n chick rations unless care i s taken to provide a source of this vitamin. Biely et a l (1951) showed that when fish meal alone replaced the combination of soybean o i l meal, fish meal, and meat meal ordinarily used in the Connecticut high energy broiler ration, the growth rate of the chicks fed the ration was lowered.  It was  suggested that the elimination of soybean o i l meal from the ration lowered the f o l i c acid content of the ration below the optimum requirement of the chick. L i l l l e and Briggs (1947), in a study of the f o l i c acid content of different feedstuffs by the chick assay method, found that menhaden meal showed no folic acid activity.  Cheldelin, Woods, and Williams (1943) reported that cooking  destroyed or rendered unavailable as much as 74$ of the folic acid content of halibut and salmon flesh. Experiment 5.—This experiment was designed to study specifically the effect of processing temperature on the f o l i c acid content of herring meals.  -25Two sets of meals were tested.  The f i r s t set was made from herring caught  in November 1951, and the second from herring caught i n January 1952.  Each  set comprised a low-temperature meal and a normal meal made from the same lot of herring press cake. The low-temperature meals were made by drying the press cake in a current of a i r maintained at 100°-110°F.  The normal  meals were dried by the commercial flame-drying method. The f o l i c acid content of the herring meals was determined microbiologically'", using Streptococcus faecalis as test organism.  The herring  meals were prepared for analysis by digestion of oil-free samples with hog kidney conjugase as recommended by Bird et a l (1946). The results of the assay are given i n Table B-4.  It w i l l be seen  that there was a marked difference in the folic acid content of the herring meals prepared from the same raw material, depending on the temperature at which the meals were dried.  The two meals that were dried at 100°-110° F.  contained 4.1 and 4.7 V folic acid/g.  The commercial herring meals, although  processed from the same lots of herring press cake as the corresponding lowtemperature meals, contained only 0.29 and 0,27-/ f o l i c acid/g. In addition to the microbiological assays, a chick biological experiment was carried out with the various herring meals.  Each of the herring  meals was added as the sole protein supplement to the chick ration shown in Table B-5.  The experimental rations were fed with and without supplementary  folic acid.  Each ration was fed ad l i b to duplicate lots of 20 day-old White  Leghorn cockerels. From the average weights of the chicks on the different experimental rations as shown in Table B-4, i t w i l l be seen that, with the basal ration employed, both the meals dried at low temperatures promoted a faster rate of ''"Difco f o l i c acid assay medium was used.  -26growth than did the corresponding commercially dried meals.  The addition of  0,5 mg folic acid/lb of diet was without effect on the growth rate of chicks fed the diets containing the low-temperature dried meals. When, on the other hand, this amount of folic acid was added to the rations containing the commercially dried herring meals, the growth rate of the chicks fed these rations was stimulated to equal that of the chicks on the diets containing the meals dried at low temperatures. It is concluded from the data obtained in the microbiological assays and the chick biological test that the high temperatures employed i n the flame-drying process in the commercial manufacture of herring meals are responsible for the low f o l i c acid content of these meals. C.  Unidentified growth factors i n herring meal Experiments 1. 2 and 3 The composition of the basal diet employed in Experiment 1 is  listed in the second footnote to Table C-l. To this basal diet were added soyabean-oil meal and herring meal in varying proportions.  Protein, calcium  and phosphorus were adjusted to the same levels in a l l diets.  The resulting  average weights of the chicks fed the various rations are given in Table C-l. It w i l l be noted that the inclusion of 5$ to 10$ herring meal in the basal ration slightly increased the growth rate of the chicks fed the ration. It i s also interesting to note that the rations in which both soyabean-oil meal and herring meal were included promoted a faster rate of growth than the rations in which either of these protein supplements was used alone. In Experiment 2, wheat and corn again comprised the cereal portion of the basal diet.  In addition to the supplements added to the ration in  Experiment 1, 3% liver meal and 2% dried d i s t i l l e r s ' solubles were added to each of the diets i n Experiment 2.  Soyabean-oil meal and fish meal were  added to the diets i n various proportions.  The results of Experiment 2 are  -27given i n Table C-2. The addition of fish meal to the diets resulted i n better growth than was obtained with the soyabean-oil meal ration, indicating that fish meal supplied some factor or factors not present in the samples of l i v e r meal or dried d i s t i l l e r s  1  solubles used i n the basal diet.  Although the results of Experiments 1 and 2 indicated the presence in f i s h meal of some factor(s) absent (or present only i n small amounts) i n the natural ingredients of the control diets, there was the possibility that this factor(s) might be one of the known vitamins.  Accordingly a third exper-  iment was designed in which optimum amounts of a l l the known vitamins were added to the rations. Corn and soyabean-oil meal were the principal ingredients. One-tenth of 1% of dl-methionine was included in a l l rations. In addition to the vitamin and mineral supplements incorporated i n amounts similar to those given in the footnote to Table C-l, the following supplements were also added: thiamin hydrochloride, pyridoxine hydrochloride, folic acid, menadione, i-inositol, a-tocopherol, g-aminobenzoic acid, biotin, ferric citrate, cupric sulphate, nickel carbonate, cobalt acetate, aluminium sulphate, and zinc acetate. Bone meal was added i n varying amounts depending on the fish meal content of the ration. Two samples of herring;meal were used as supplements at 2,5$ and 5$ levels.  One herring meal was a commercial sample and the other a specially  prepared, low-temperature (100° to 110° F.) dried meal. Both meals were prepared from whole herring caught in January 1952. Adjustments were made to maintain the same levels of protein, calcium and phosphorus in each of the diets.  The basal ration (containing no fish meal) and the rations containing  the fish meal at the 5$ level were fed both with and without the addition of 15 mg. procaine p e n i c i l l i n per pound. From the resulting average weights of the chicks in Experiment 3, given i n Table C-3, i t w i l l be seen that a slight improvement in growth rate  -28-  was obtained when either fish meal or the penicillin was added to the basal diet.  However, statistical analysis of the data showed that the increase i n  growth rate was not significant with either the fish meal or the penicillin When, on the other hand, penicillin and 5% of herring meal were both  alone.  included i n the ration, there was a statistically significant increase i n the rate of growth of the chicks as compared with those fed the basal diet. It may therefore be concluded that the fish meal contributed a factor(s) which was not supplied either by the basal diet or by the addition of the antibiotic p e n i c i l l i n . Experiments 4 and 5 Several groups of investigators, working independently, have established that fish meal contains an unidentified factor which stimulates chick growth.  Similarly i t has been reported that dehydrated green feed (DGF)  contains an unidentified growth factor, distinct from that present i n fish meal.  In the present experiments British Columbia herring meal and DGF  (two samples, "A" and "3") were added to a basal synthetic diet as sources of unidentified factors to study possible interaction between the two factors. The composition of the basal diet is given in Table C-4. The basal diet contained adequate levels of a l l the known vitamin and mineral supplements which are required by the chick.  There i s , at present, some uncertainty  whether vitamin C (ascorbic acid) promotes growth of young chicks when i t i s added to synthetic diets.  The effect of vitamin C in the proportion of 45  grams per 100 pounds of ration when added alone, with herring meal or DGF or both, was therefore investigated. Herring meal was added at a level of 2% and DGF at the 5% level.  Adjustments were made in the levels of soya protein,  dextrose and cellulose to maintain the protein energy and fibre at constant levels i n a l l diets.  -29In Experiment 4 , New Hampshire pullet chicks were fed the basal diet for one week. At one week they were distributed on a weight basis into lots of seventeen birds each.  Similarly in Experiment 5, in which New Hampshire  cockerels were used, the chicks were fed the basal diet for ten days before they were distributed into standardized lots of fifteen birds each. The average weights of the chicks at the termination of the tests are presented in the accompanying table. In Experiment 4 , neither DGF "A" nor herring meal used alone stimulated growth.  There was a slight response in growth rate when DGF "B" was  added to the basal diet.  When herring meal was added to the diets supplemented  with either of the dehydrated green feeds, the chicks were heavier at 33 days than those fed the corresponding diet with the DGF alone.  In Experiment 5  there was a response to both the dehydrated green feeds and to herring meal when these supplements were added singly.  When herring meal was added to the  diets already supplemented with DGF the rate of growth was substantially greater than with herring meal or DGF alone. Vitamin C added to the diets without herring meal i n Experiment 4 depressed the growth of chicks.  The addition of herring meal to the diets  supplemented with vitamin C stimulated growth. In the presence of vitamin C a response of the chicks to DGF was not apparent whether or not the diet also contained herring meal. Further evidence has been presented that herring meal and DGF contain separate factors, both of which stimulate growth rate in chicks.  In addition,  i t has been shown that the response obtained to each of these factors i s affected by the presence of the other in the diet.  -30Experiments 6 and 7 Since turkey poults grow at a faster rate and have higher vitamin requirements than do chicks, i t was consequently considered that they might be used to advantage i n biological tests to demonstrate the presence of any unidentified growth factors in fish meal. In Experiments 6 and 7 wherein turkey poults were fed a ration of the corn—soyabean meal type, the response to addition of fish meal and of dried brewers' yeast was measured. The vitamins indicated in Table C-6 were added to the diets.  Five percent herring meal and 5% dried brewers' yeast  were each added to the control ration, adjustment being made to maintain a similar protein level in a l l rations.  In Experiment 6 the control ration and  the ration supplemented with fish meal and dried brewers' yeast were each fed with and without chlortetracycline and i n Experiment 7 a l l of the experimental rations were fed with and without chlortetracycline. tests are given i n Table C-6.  The results of these  It i s evident that 5% of herring meal included  in the corn—soyabean meal ration stimulated the growth rate of the turkey poults fed the ration in spite of the fact that the ration was supplemented with the vitamins shown i n Table C-6.  The inclusion of dried brewers' yeast  either i n the basal ration or i n the ration supplemented with herring meal did not e l i c i t any additional growth response.  When the diets were supplemented  with the antibiotic chlortetracycline, the addition of 5% of fish meal stimulated the growth rate over that obtained with chlortetracycline alone. It w i l l be seen from a comparison of the results obtained above that fish meal had a growth-stimulating effect for both the chick and the poult when included in an all-vegetable ration supplemented with a l l the known vitamins.  Furthermore, an added growth response was obtained with fish meal  even when the rations were supplemented with an antibiotic.  It i s interesting  to note that while these studies were in progress i t was announced by the U.S.  -31-  Department of Agriculture Research Centre at Beltsville, Maryland, that 5% fish meal added to a broiler ration containing a l l the known nutrients, plus an antibiotic, resulted in an increase in growth rate of chicks fed the ration. The effect of the new factor was more pronounced on the growth of males than that of females and more pronounced on the growth of the fast-growing strain of chickens than that of a slow-growing strain.  Although the effect of the  unknown factor can be observed in four-week-old chickens, i t is s t i l l greater in ten-week-old birds." It i s concluded from the data presented above that the chick and the poult require for maximum growth rate a factor(s) which is present in herring meal but which i s not present, to any appreciable extent at least, in liver meal, dried distillers* solubles, or dried brewers' yeast. factor(s) has not been identified with any of the known vitamins.  The The require-  ment of the chick and the poult for the factor(s) does not appear to be modified by the inclusion of an antibiotic in the ration. D.  The use of condensed herring solubles as a vitamin supplement i n turkey poult rations Turkey poults, because of their rapid rate of growth, have high and  c r i t i c a l nutritional requirements.  The following tests were conducted to  determine i f condensed herring solubles might not be equally as efficacious as dried brewers' yeast as a natural supplement supplying B-complex vitamins in poultry rations.  Since condensed herring solubles i s essentially an evaporated  water-extract of whole herring, i t contains high levels of B-complex vitamins-. It .therefore seemed possible that the product might, in addition to supplying vitamin B 2 x  and unidentified factors, reduce the need for products commonly  used as vitamin B-complex supplements. Three different basal diets were employed in the study (Table D-l). In the f i r s t experiment the diet contained 10% herring meal.  In the second  and third experiments no fish meal was included in the diets but the two diets fed differed in that they contained feed screenings and corn respectively as the principal cereal component. Riboflavin, niacin, and calcium pantothenate are readily available in high potency, commercial vitamin concentrates.  Since i t is not a general practice to rely on natural products,  these vitamins were added in purified form to the experimental diets employed here, thus removing them from the possible factors involved in any response to the two products being compared. Vitamin B 2 was added to Diets 2 and 3 x  but not to Diet 1 which contained 10% herring meal. In Experiment 1 the growth responses of Broad Breasted Bronze poults to condensed herring solubles and to dried brewers' yeast were compared when the products were used to supplement Diet 1 at levels of 1.5 and 3.0$.  Each  dietary treatment was imposed on 20 male and 20 female poults from 0-6 weeks of age. In Experiment 2 the poults employed were Beltsville Small White poults Condensed herring solubles and dried brewers' yeast were added to the basal diets 2 and 3 at 3%. level.  Triplicate lots of 13-14  poults of mixed sexes were  fed each of the experimental diets until they were 5 weeks of age. In Experiment 3> Broad Breasted Bronze poults were fed Diets 2 and 3 with and without 3% of condensed herring solubles and dried brewers' yeast. Each diet was fed to 19 male and 19 female poults for almost 9 weeks  (f-\\6l  days). The  average weights of the poults at the termination of each exper-  iment are shown in Table D-2.  With the three different control diets (with  and without fishmeal and containing wheat, com  and feed screenings as the  principal cereal component) employed, condensed herring solubles stimulated growth to a greater extent than did dried brewers' yeast.  Although for the  -33purposes o f e x p e r i m e n t a l d e s i g n , v i t a m i n B 2  w  a  s  x  f e d i n Experiments 2 and  i n c l u d e d i n a l l the  3, i t i s most p r o b a b l e t h a t the p o u l t s  3% o f condensed h e r r i n g s o l u b l e s r e q u i r e d no a d d i t i o n a l B 2 x  diet.  The  observed r e l a t i v e l y low  compared w i t h t h a t to the  stock.  The  iments and  receiving  supplement i n t h e  response t o the d r i e d brewers' y e a s t  condensed h e r r i n g s o l u b l e s cannot be  d i f f e r e n c e s i n the c a r r y o v e r  diets  as  a t t r i b u t e d to  o f known o r unknown f a c t o r s from t h e b r e e d e r  f a c t t h a t s i m i l a r r e s u l t s were o b t a i n e d  i n three  d i f f e r e n t exper-  t h a t the p o u l t s were f r o m d i f f e r e n t b r e e d e r f l o c k s i n each  instance  would make t h i s p o s s i b i l i t y u n l i k e l y . I t i s c o n c l u d e d t h a t , w i t h the t y p e o f p r a c t i c a l t u r k e y s t a r t i n g d i e t used, condensed h e r r i n g s o l u b l e s i s an e f f e c t i v e supplement.  Poults  from t h r e e d i f f e r e n t s o u r c e s showed a b e t t e r response to condensed h e r r i n g s o l u b l e s t h a n t o d r i e d brewers' y e a s t . t o condensed h e r r i n g s o l u b l e s and  The  d i f f e r e n c e between t h e  t o d r i e d brewers' y e a s t o c c u r r e d  response with a  d i e t c o n t a i n i n g h e r r i n g meal and w i t h d i e t s c o n t a i n i n g no h e r r i n g meal but supplemented w i t h v i t a m i n E.  The  B 2. x  e f f e c t o f heat treatment and  storage  on the n u t r i t i v e v a l u e  f o r the  c h i c k o f h e r r i n g meals o f v a r i o u s o i l c o n t e n t s Experiment 1 Low-temperature h e r r i n g meal was at 37-43°C. i n an a i r c u r r e n t .  made by d r y i n g h e r r i n g p r e s s  cake  In o r d e r t o t e s t a l o w - f a t meal, samples o f  t h i s s p e c i a l l y p r e p a r e d meal were e x t r a c t e d w i t h hexane a t low temperature t h e e x c e s s s o l v e n t was  removed i n vacuo.  Samples of b o t h the normal and  and  the  e x t r a c t e d meals were h e a t e d i n 1 0 - l b .  l o t s i n a r o t a t i n g s t a i n l e s s s t e e l drum  a t 149°C. f o r p e r i o d s o f 60, 120,  180  and  n o n - e x t r a c t e d meals ranged from 7.30 e x t r a c t e d meals from 0.15 porated  t o 0.28  t o 7.55  percent.  i n chick starting rations.  minutes.  The  percent, The  f a t content o f  and t h a t o f the  the hexane-  e x p e r i m e n t a l meals were i n c o r -  In a d d i t i o n t o the r a t i o n s c o n t a i n i n g  the  -34normal and extracted herring meals, a third set of rations was formulated which contained the extracted meals and fresh herring o i l .  The amount of  herring o i l added corresponded to that contributed by the unextracted meals. Ration 1 was composed as follows: ground yellow corn, 78.0; herring meal, 18.0; bone meal, 0.5; limestone, 1.0; iodized salt, 0.5; dehydrated cereal grass, 1.0; choline chloride (25$), 0.4; manganese sulphate, 0.0|)5 lb. per 100 l b . plus the following expressed as grams per 100 lb. nicotinic acid, 0.8; calcium pantothenate, 0.5; riboflavin, 0,2; fish o i l (35,000 A), 6.0 gm.; and dry vitamin D^, 13,000 units per 100 l b . Rations 2 to 12 were supplemented with the various herring meals as indicated in Table 1. Adjustments were made so that the relative amounts of protein contributed by the corn meal and the herring meals did not vary throughout the experiment.  Sucrose and water were  added to compensate for the fat and water lost during the extraction and the heating.  The rations were fed to day-old New Hampshire cockerel chicks kept  in electrically heated battery brooders and given free access to feed and water. The experimental data are shown in Table E - l .  When the chicks were  ten days old, the experiment was terminated because the differences i n growth of the chicks fed the various rations were obvious at this time.  From the  average weights of the chicks i t appears that the o i l content did not affect the nutritive value of the low-temperature meals or of those heated for 60 minutes.  Heating at 149°C. for 120 minutes, however, impaired the nutritive  value of the original meal but was without effect on the hexane-extracted meal. Heating for 180 minutes markedly reduced the nutritive value of both the original and the extracted meals.  The addition of fresh herring o i l to rations  containing the extracted meals had no effect on the growth rate of the chicks fed these rations for the 10-day eaqjjerimental period.  -35-  Experiment 2 It has been shown that the temperature employed in commercial processing of herring meal resulted in considerable destruction of the folic acid content.  It was therefore considered possible that the heated meals  retarded growth because of the destruction of folic acid rather than because of any direct effect of the o i l i t s e l f .  It w i l l be noted that the rations  fed contained only 1% of dehydrated green feed.  This quantity would hardly  meet the chick's need for f o l i c acid i f the particular sample of herring meal used did not contain a considerable amount. An experiment was accordingly designed to determine whether the quality of the o i l i s important i n i t s e l f or mainly by way of some indirect effect on the vitamins in the ration. tested for their effect.  Three samples of herring o i l were  The f i r s t was a sample of fresh herring o i l , the  second a sample of the same o i l that had been held at a heat of 110°C. for 30 hours and the third another sample of the same o i l heated for 105 hours. To accelerate oxidation, a i r was bubbled continuously through the o i l during the heating period. The basal ration used was similar i n composition to the diet i n the previous experiment except that the dehydrated cereal grass was omitted.  The  herring meal used in the ration was a low-temperature meal which had been extracted with hexane. The oils were added to the ration at a level of 2 percent.  The o i l content of the rations containing the added o i l was therefore  comparable to the o i l content of the ration containing the original herring meal. Each ration was fed with and without the following vitamins: f o l i c acid, 0.035; thiamine HC1, 0.050; pyridoxine HC1, 0.160; menadione, 0.018; inositol,  5.0; alpha-tocopherol, 0.150; para-aminobenzoic acid, 4.5; biotin,  0.0045 and vitamin B , 0.0005 gm. per 100 l b . Except i n rations 6 and 7, l2  herring o i l when added,was mixed into the whole ration.  In rations 6 and 7,  -36-  the o i l was mixed with the herring-meal content and allowed to stand overnight at a temperature of 21° C.  The o i l was absorbed by the herring meal  and i t was hoped that the mixture approximated the original herring meal. The rations were fed to day-old White Leghorn pullet chicks under conditions similar to those of the chicks in the previous experiment. weights of the chicks at 4 weeks of age are given i n Table E-2.  The average The results  show that when sufficient vitamins were added to the diet, growth was not retarded by the  addition of any of the o i l s , whether fresh or heated.  At  the levels fed, therefore, the oils were not in themselves toxic. When, however, the basal ration was deficient i n the vitamins, the addition of o i l retarded growth. Heat treatment of the oils used in the present experiment did not seem to increase their growth-depressant  action.  When the growth rates of the chicks fed either the original meal or the extracted meal were compared, no difference i n nutritive value appeared as a result of extraction.  This was the case whether the rations were  fortified with the additional vitamin supplement or not.  The vitamin supple-  ment stimulated growth to the same extent with both meals. When added to the rations without the supplementary vitamins noted above, a l l of the oils retarded growth of the chicks. The method by which the o i l was added to the rations did not affect the nutritive value of the rations.  Growth was retarded whether the  o i l was added directly to the mixed diet or blended previously with the herring meal. Experiment 3 The third experiment i n this series concerned three herring meals a l l prepared from the same sample of herring press cake.  Meal 1 was a low-temperature  dried meal prepared according to the procedure already outlined.  Meal 2 was a  typical light-coloured meal prepared commercially by direct flame-drying of press cake.  Meal 3 was a press cake dried at a much higher temperature than usual.  -37Samples of the three meals were extracted with hexane.  Three growth tests  with chicks were carried out to compare the nutritive values of the various meals. The f i r s t test was carried out using a basal diet of composition similar to that in Experiment 2. in Table E-3.  The average weights of the chicks are given  The basal diet supplemented with the low-temperature dried meal  was superior i n nutritive value to the commercially dried meals.  It w i l l be  seen that in the commercially dried meals, overheating caused no greater deterioration than did the normal heating procedure. Samples of the three meals described above were used in a further test to determine the effect of storage on the relative nutritive value of herring meals processed under different conditions. As part of this test, samples of the meals were extracted with hexane to ascertain i f fat-free meals kept better than normal meals. TKe  meals were stored at three temperatures,  -25°, 21° and 37° C , for one year.  The chick starting rations in which the  various samples of meal were tested were similar to those used previously and were supplemented by the vitamins indicated for Experiment 2.  In addition, the  rations contained 0.25$ of an antibiotic supplement.* The data given i n Table E-3 show that no marked differences occurred in growth rate.  Even at a storage temperature of 37°C. there did not appear to  be any loss i n the quality of the meals. The presence or absence of o i l in a meal did not, in general, result in any difference in nutritive value before or after storage.  Meals dried at normal or excessively high temperatures kept as  well as meals dried at a low temperature. Since i t had already been shown that f o l i c acid i s particularly subject to destruction during the processing of herring meal, i n the present *Aurofac-A which contains at least 1.8 grams of chlortetracycline per l b .  -38-  test the f o l i c acid content of the meals was determined microbiologically at intervals during the storage period.  The folic acid content of the meals  as shown in Table E-4 dropped considerably during the year.  The meals listed  in this table as stored at 0°C. are samples which were kept in the laboratory refrigerator for use as reference standards with each assay.  It will be seen  that there was l i t t l e difference between the f o l i c acid content of these meals and that of the meals stored at 21°C.  Extraction of o i l from the meals before  storage did not increase the stability of the folic acid in the meals, and, i n fact, accelerated destruction of the vitamin. DISCUSSION The data obtained in the above experiments indicate that the o i l content does not have an important effect on the nutritive value of herring meal used as a supplement in chick rations, provided the rations are adequately supplemented with vitamins.  In Experiment 1 i t was observed that heating more  quickly reduced the nutritive value of a herring meal i f the meal contained the normal amount of fat than i f the meal was fat-free.  The basal ration fed to  the chicks in this instance was marginal in folic acid. When the basal ration in which the various herring meals and oils were- tested was supplemented by folic acid and other vitamins, no adverse effect was noted from the addition of herring o i l to the rations, regardless of the quality of the o i l added. In Experiment 2 a sample of herring meal was tested in the original state and after hexane extraction. No difference i n the nutritive value was noted, even when the basal ration was deficient in members of the vitamin B-complex.  The  addition of fresh or heat-treated herring o i l to the ration containing the extracted herring meal, but not supplemented with the vitamin mixture, retarded growth of the chicks fed the ration.  The adverse effect upon growth was  completely overcome by the addition of the vitamins.  Since there was no differ-  ence in growth whether the o i l present in the herring meal was extracted or not,  -39-  the o i l naturally present i n the meal evidently did not promote oxidation of vitamins in the ration.  The sample of fresh herring o i l and the two samples  of heated o i l s t i l l contained peroxides which destroyed some vitamin or vitamins present in suboptimal amounts in the ration and consequently lowered the growth rate of the chicks fed these rations.  Possibly the o i l in the  herring meal was oxidized to the extent that peroxides were destroyed.  Lease  et a l (1938) reported that heating of rancid fats lowered both their peroxide value and their a b i l i t y to destroy vitamin A. In the present experiment a sufficient excess of the deficient vitamins completely overcame the growthdepressant action of the added herring o i l . The samples of herring meal which were tested for storage stability did not deteriorate in nutritive value as protein supplements during a period of one year.  The. basal ration in which the various meals were tested was well  supplemented with vitamins. Extraction of the fat from the different samples of herring meal did not change their nutritive value as supplements to the basal ration employed, nor was the nutritive value of the extracted meals after storage different from that of those not extracted. It i s particularly interesting to note that storage at a temperature of 37°C. had apparently no effect on the value of herring meals as protein supplements. As was expected, the f o l i c acid content of the meals dropped during storage.  The stability of folic acid in the meals decreased with increasing  storage temperature.  In meals extracted with hexane before storage, folic acid  was less stable than in the original meals.  The folic acid content of the normal  commercial and the overheated commercial meals was so low that the difference observed between stability in the extracted and unextracted meals was insignificant.  In the low-temperature dried meals held at 21°C, however, there was  a difference of 1,3 micrograms of f o l i c acid per gram i n favour of the unextracted meal,  ^ t is possible that some natural antioxidants were removed from the meals  -40with hexane. Vitamin deficiencies encountered as the result of oxidation of vitamins present at marginal levels in mixed rations appear to be the basis for the alleged toxicity of rancid fish o i l s .  The data obtained in the  present study do not indicate that the quality of o i l present in commercial fish meal is a factor in the nutritive value of fish meal even when fed at levels above those normally used in chick rations. F.  The? effect of storage temperature and antioxidant treatment on the chemical and nutritive characteristics of herring meal The following experiments were designed to determine and follow  the changes which occur in herring meal stored under different conditions, and to ascertain what effect antioxidant treatment might have upon any changes that normally take place in meals kept under ordinary conditions of storage. PROCEDURES The herring meals for the experiment were prepared on December 9, 1958, at a commercial reduction plant.  The meal required for the experiment  was taken directly from the bagging operation and portions were treated as follows: A.  Stored at 25.5°C. This meal w i l l be referred to henceforth as the "normal" meal.  B.  Stored at -20°C.  C.  Spread out on trays to f a c i l i t a t e cooling in a freezer room at -20°C. overnight and then bagged. Stored at 25.5°C.  D.  Spread out in a freezer room at -20°IC. overnight and then bagged. Stored at -20°C.  E.  Stored at 25.5°C. for one week. Butylated hydroxytoluol* was then added at a level of 0.15$. Stored at 25.5°C.  F.  BHT added immediately to the hot meal at a level of 0.15$. Stored at 25.5 C.  *A finely divided product: 90$ < 100 mesh, 70$ ^ 200 mesh.  (BHT)  -41The meals were a l l s t o r e d i n p o l y e t h y l e n e - l i n e d m u l t i w a l l paper bags. Samples o f meals A and D were t a k e n f o r immediate c h e m i c a l and  samples o f t h e r e m a i n i n g meals were withdrawn f o r a n a l y s i s t e n days a f t e r  preparation. 24,  analysis  A l l the meals were sampled on January 16, March 16 and September  1959, f o r chemical  a n a l y s i s and b i o l o g i c a l t e s t i n g w i t h  chicks.  Chemical T e s t s . — W e i g h e d samples o f the meals were d r i e d i n vacuo at  99-100°C. f o r f i v e h o u r s .  d i e t h y l ether i n a Soxhlet  They were t h e n e x t r a c t e d w i t h  apparatus f o r 20 h o u r s .  were determined on t h e e t h e r e x t r a c t s . e x t r a c t e d with  acetone.  Iodine numbers  The e t h e r - e x t r a c t e d  The a c e t o n e - e x t r a c t e d  HC1 and e x t r a c t e d a g a i n w i t h acetone a c c o r d i n g  peroxide-free (Hanus)  samples were t h e n  samples were next d i g e s t e d t o t h e AOAC procedure  with  (1955).  The two s u c c e s s i v e acetone e x t r a c t s were weighed s e p a r a t e l y . Samples o f t h e whole meals and o f t h e e t h e r - e x t r a c t e d meals were subjected to pepsin h y d r o l y s i s (Almquist, digestibility  o f the p r o t e i n c o n t e n t  1941) i n o r d e r t o determine t h e  o f the meals.  B i o l o g i c a l T e s t s . — T h e comparative p r o t e i n v a l u e s o f t h e meals were determined i n growth s t u d i e s w i t h  chicks.  The meals were used t o supplement  the c e r e a l p r o t e i n i n a b a s a l d i e t composed o f n a t u r a l i n g r e d i e n t s and a l s o as t h e s o l e sources o f p r o t e i n i n a p u r i f i e d d i e t . o f b o t h t h e n a t u r a l and the p u r i f i e d d i e t s was 20$. conducted w i t h meals t h a t had been i n s t o r a g e  The t o t a l p r o t e i n  content  In the f i n a l t e s t  f o r n i n e months, an a d d i t i o n a l  and more c r i t i c a l a s s a y o f the r e l a t i v e p r o t e i n supplementary v a l u e o f t h e meals was c a r r i e d o u t .  I n t h i s f i n a l t e s t t h e meals were f e d i n n a t u r a l  d i e t s c o n t a i n i n g o n l y 14$ t o t a l p r o t e i n .  The v i t a m i n B-complex content  the meals was compared when t h e meals were i n c o r p o r a t e d m a r g i n a l i n t h e v i t a m i n B-complex. i n Table F - l .  of  i n a basal diet  The formulae o f t h e b a s a l d i e t s a r e g i v e n  -42The chicks used in a l l experiments were 10-14 days old and were standardized as to weight. a l l the experiments.  They were New Hampshires of the same strain in  The parent stock was maintained on a ration which was  marginal i n vitamin content i n order that the carryover to the chicks should be as constant as possible and not so high as to preclude the possibility of response to vitamin supplements. Prior to standardization and the feeding of the experimental diets, the chicks were given a chick starting diet which permitted a moderate rate of growth. The chicks were reared i n battery brooders and were given the experimental diets and water ad libitum. RESULTS Chemical Tests.—The normal meal A contained 73.2 percent crude protein, 10.7 percent ash, 9.5 percent ether-extractable fat and 7.0 percent moisture when analyzed on the day after manufacture.  A l l the meals were  analyzed for moisture content at the time of each set of analyses.  The results  of the various analyses expressed on the basis of the moisture-free meals are shown in Table F-2.  A l l the values given below are likewise i n terms of the  moisture-free meals. The percentage of ether-extractable material in the dried fish meal was i n i t i a l l y 10.2 percent.  In the case of the two meals treated with BHT  (meals E and F) there was no decrease in the amount of ether extract even after storage at 25.5°C. for nine months. Meal D, which was spread out to cool at -20°c. overnight, and then rebagged and held at -20°C, showed the most rapid drop i n ether-extractable material. Meal B which was stored at -20°C, but not spread out for rapid chilling, showed a similar content of ether-extractable material (8.7$) at the end of nine months storage at -20°C. In the case of the latter meal, however, the decrease in ether-extract was gradual rather than rapid during the f i r s t six weeks after processing as in  -43the c a s e o f meal D. 25.5°C. a f t e r b e i n g the f i r s t  Meal C was rebagged.  c h i l l e d after processing The  D.  the percentage of ether-extractable  The  iodine values  intermediate  From the  meal B (-20°C.) t h a n i n meal C  between the  The  m a t e r i a l decreased at a f a s t e r r a t e i n  ( F i g . 2) o f the e t h e r  e x t r a c t s showed a The  a decrease i n the  128,  no  r e s p e c t i v e l y ) o f the e t h e r  p r o c e s s i n g w i t h BHT There was, the  t h e r e was  I.V.  change.  and t h a t o f the meal t r e a t e d one  decreased during  The  meal which was  showed a h i g h e r u n s a t u r a t i o n normal meal on the day  storage.  and  After (126  and  processing.  meals i n t h e r a t e a t which  I n the meal k e p t w i t h o u t BHT  f i r s t week, but  t h e r e a f t e r there  t r e a t e d w h i l e s t i l l hot from t h e  (I.V. 143)  i n the  a f t e r manufacture.  cooled  material.  week a f t e r  The  iodine values  f o r the The  r a p i d l y t o -20°C. immediately a f t e r  the was  drier  ether e x t r a c t than d i d  meals became s i m i l a r sometime a f t e r t h r e e months a t 25.5°C. and meal C, which was  considerable  e x t r a c t s o f the meal t r e a t e d immediately a f t e r  d e c r e a s e d more r a p i d l y d u r i n g the  little  134T135.  d i f f e r e n c e between the i o d i n e v a l u e s  however, a d i f f e r e n c e between the two  iodine value  value  ether-  s o l u b i l i t y o f the f a t i n the o r i g i n a l meal, a l s o p r e v e n t e d to a degree t h e drop i n the i o d i n e v a l u e o f the e t h e r - e x t r a c t a b l e  general  iodine  f r e s h l y p r o c e s s e d h e r r i n g meal was  (BHT), as w e l l as p r e v e n t i n g  n i n e months s t o r a g e  storage  (25.5°C).  e x t r a c t s from the  antioxidant  at  decrease  s i x t h t o the f o u r t e e n t h week o f  r e l a t i o n s h i p t o the amount o f e t h e r - e x t r a c t a b l e m a t e r i a l . o f the ether  stored  decrease i n e t h e r - s o l u b l e m a t e r i a l i n  s i x weeks f o r t h i s meal was  o c c u r r i n g i n meals B and  but was  the two  normal meal A processing  s u b s e q u e n t l y s t o r e d a t 25.5°C, showed s i m i l a r r a t e s o f d e c r e a s e i n the  i o d i n e value  o f the e t h e r  e x t r a c t s a f t e r t h e f i r s t week.  the r a p i d l y c h i l l e d meal D which was t o 79  during  the  The  iodine value  s t o r e d a t - 2 0 ° C , however, f e l l  f i r s t month o f s t o r a g e  of  sharply  as compared t o meal B which was  stored  -44-  at-20°C. but not chilled quickly to that temperature. By the end of the storage period the iodine values of the extracts from meals B and D were very similar (66 and 52, respectively). The drop in the total fat content of the meals (Fig. 1) from 14.3 to 13.0$  in the f i r s t month after processing was due as much to a decrease  in the amount of the f i r s t acetone extract as to a decline in the ether solubility of the fat in the meal. Acetone extraction of the normal meal and the meal chilled overnight which had been extracted with ether on the day after the meals were prepared showed higher values (1.49 and 2.30$, respectively) than any of the meals sampled after one week. After three and nine months of storage the acetone extract was considerably lower and there was not a great deal of difference among the percentages found in the various samples.  The material soluble in acetone only after acid treatment  varied comparatively l i t t l e during the f i r s t week after preparation of the meals. During the f i r s t six weeks this fraction of acetone soluble material decreased i n the two meals to which BHT had been added. Thereafter the amount of this material decreased in a l l of these meals. From three to nine months the percentage of the HCI - acetone soluble fraction diminished rapidly so that the average of a l l the meals had dropped from an original value of 2.26 to 1.77$  at the time of the final analysis. The variation i n  the total acetone (acetone plus HCI - acetone) extract over the nine-month storage period i s shown in Fig. 1.  The amount of l i p i d in the meals that  was not ether soluble but which was extractable with acetone, either before or after treatment with HCI, showed marked changes up to three months but relatively l i t t l e change during the next six months of storage. On the basis of pepsin hydrolysis of ether-extracted samples the original digestibility of the protein of the meals was 96 percent. storage this dropped to 94-95 percent.  After  Digestibility of the protein content  -45-  of the meals, as judged by the results of in vitro pepsin hydrolysis, demonstrated that antioxidant treatment somewhat inhibited the formation of indigestible nitrogenous compounds. Immediate application of BHT gave consistently better results than application of the antioxidant one week after processing.  This was true whether pepsin digestibility of the protein  was determined on the whole meal or on ether-extracted samples. Meals B and D, which were kept at -20°C, showed the most rapid formation of undigestible nitrogenous compounds. Meals A and C were intermediate in the drop in digest i b i l i t y of the protein. Biological Tests.— The growth response of the chicks to the herring meals under the different dietary regimens imposed are given i n Table F-3. The chicks were not standardized at the same weight i n a l l tests and the duration of the experiments varied somewhat. It i s , therefore, not possible to make comparisons among the absolute weights of the chicks in experiments conducted on different dates nor among the different experiments on a given date.  Within each experiment the final weights of the chicks were subjected  to statistical analysis and significance tested according to Duncan's multiple range test (1955).  Table F-3 gives the average weights of the chicks within  each test expressed relative to the average weights of the chicks receiving the normal meal which was assigned for purposes of comparison a value of 100. When the meals were tested in diets composed of natural ingredients there were no statistically significant differences in the responses obtained to the various meals. This was true whether the meals were considered as a source of supplementary protein only or were considered as a vitamin B-complex supplement as well.  In the f i n a l test on the meals after storage for nine  months, there were no differences in the protein supplementary value of the meals, either as supplements to a practical type diet containing a total of 20 percent protein or when used to supply only 3 percent of protein to give  -46a total protein level of 14 percent. In contrast to the results of the chick assays for supplementary protein quality, there were differences i n the response obtained with the different meals when they were fed as the sole source of protein in a purified diet.  In none of the tests conducted over the storage period was  any of the meals s t a t i s t i c a l l y superior to the normal meal A.  Except i n  the test on the meals after storage for three months when there were no significant differences in the responses to the meals, the chicks fed meal D (the chilled and frozen meal) were significantly lighter than the chicks fed the normally handled meal. DISCUSSION The most striking alterations in the herring meals were those occurring in the meals subjected to a temperature of -20°C. An i n i t i a l low temperature appeared to favour the development, during the f i r s t month of storage, of compounds which were no longer split by HCI, or of compounds which did not yield acetone soluble products after HCI treatment.  The meals  treated with BHT showed no increase in amount of material which was acetoneextractable only after HCI digestion.  The effect would seem to have been  against formation of co-polymerization products from which the l i p i d fraction could be extracted with acetone after splitting with HCI rather than through the blocking of further change in such products. The effects noted in the meals which were spread out to promote rapid chilling after processing were not due merely to the exposure to air during the procedure.  Comparison of  the progressive decrease in iodine values of the ether-extracts of the different meals shows a more rapid decline in meal D, which was chilled and then stored at -20°C, than in any of the other meals.  In meal B, which was not  chilled but which was stored at -20°C, the I.V. had not decreased appreciably after six weeks of storage, but thereafter dropped rapidly until after nine  -47months i t was similar to that of the meal which had been chilled before being put into low temperature storage. Furthermore, the meal which was chilled and then kept at 25.5°C. showed an i n i t i a l drop in iodine value, but after one week the rate of decline was similar to that occurring in the normal meal. It may accordingly be concluded that the lower temperature favoured rapid oxidation of the unsaturated fatty components of the meal. In this connection Astrup (1958), using oxygen absorption as a measure of oxidation in herring meals, noted that the progress of oxidation differs, depending on the temperature.  Astrup suggests that the minimum range which he observed  indicates the formation of antioxidants i n the meal with elevated temperature. Lea, Parr and Carpenter (1958) likewise reported a decrease i n the rate of oxidation i n herring meal with higher storage temperatures and suggested an accumulation of oxidation inhibitors.  They consider i t possible that "whereas  oxidation of the l i p i d probably proceeds further at lower temperatures, coupling of the oxidized o i l to the protein (or i t s destruction) might be greater at higher temperature". Whatever the nature of the reactions favoured at the different temperatures, i t i s clear that so-called accelerated tests employing higher temperatures than those normally encountered i n the storage of herring meal are not applicable for a study of storage stability of fish meal. Moreover the rapidity with which oxidative changes occurred when the meals were subjected to a temperature of -20°C. renders i t d i f f i c u l t to establish a control for the study of changes taking place during the storage of fish meal. In earlier experiments described above the biological value of experimentally prepared herring meal dried at low temperature (37-43°C.) was compared with that of a normal commercial meal and a commercial meal that was overheated.  The meals were stored at -25°C, 21°C. and 37°C. for a year and  -48-  tested for their nutritive value as protein supplements in chick diets at the end of that time.  Samples of the meals were extracted with hexane prior  to storage to determine what changes in nutritive value may occur in f a t free meals during storage. By comparison of the nutritive value after storage of the meals of normal fat content with that of the extracted meals, the role of fat in any changes in nutritive value could be assessed. The only conclusion drawn at that time from the results obtained was that storage at a temperature as high as 37°C. had no effect on the nutritive value of herring meals as protein supplements. Reconsideration of the data in view of the results of the present investigation enables some additional observations to be made. After storage at 21°C. the extracted low temperature meal did not support as rapid growth as did the extracted meals dried at higher temperature.  When the meals were stored at -25°C. the extracted low temper-  ature meals again did not promote as rapid growth as did the extracted meals that had been dried at higher temperatures.  Of the unextracted meals, the  overheated commercial meal gave the fastest rate of growth. It was also observed that controlled heating of herring meals dried at a low temperature did not lower the nutritive value of the meals and that in many instances chicks grew better with the heated meals. From the results of these various experiments i t appears that certain changes in herring meal occur at a faster rate, or take a different course, when the processing and storage temperatures are low.  The absence, i n the present experiment, of significant d i f f e r -  ences in the biological responses obtained to the meals subjected to different temperatures i s probably accounted for by the fact that, i n contrast to the previous experiments, none of the meals was processed at low temperature. Antioxidant treatment of the meal prevented a decrease in etherextractability of the fat and to a large extent prevented a decrease in iodine  -49-  value of the ether-extractable f a t . The increase noted with some of the meals in the HCl-acetone extract during the f i r s t six weeks of storage appears to represent the formation of l i p i d complexes with protein which are split by HC1 treatment to yield an ether soluble l i p i d component. BHT  apparently  prevented the formation of these complexes. By inhibiting oxidation of the unsaturated l i p i d in the meal, BHT may have retarded the various types of polymerization that have been postulated for systems coritaining fat and protein and subjected to oxidative conditions (Tappel, 1955J Venolia and Tappel, 195&J Narayan and Kummerow, 1958).  It may be noted that postponing  BHT treatment of the meals until one week after manufacture permitted oxidative reactions in the fat that were, in this short time, apparently accompanied by polymerization with protein.  Consequently the i n vitro digestibility of the  meal treated immediately with BHT was better throughout the entire storage period than that of the meal treated one week after manufacture.  It i s inter-  esting that the advantage obtained with BHT treatment was constant at each sampling date.  Although the amount of ether-extractable l i p i d remained  similar for the two antioxidant-treated meals during storage, the iodine value of the meal stored without BHT for the f i r s t week dropped in the course of that week, but did not change appreciably thereafter.  Immediate addition of  BHT to the hot meal, on the other hand, inhibited oxidation so that after one week the iodine value was higher than that of the normal meal on the day after manufacture. In so far as the biological tests are concerned, i t should be noted that the diets fed were formulated so that in no instance was the available energy content of the f i s h meal a factor in the response of the chicks.  The  extent to which the fat content of the meals was utilized by the chick did not, therefore, affect the response of the chicks.  With the purified diets, however,  in which a relatively high level of fish meal was fed, i t i s possible that the  -50d e p r e s s i o n i n growth noted w i t h meal D c o u l d have r e s u l t e d from  destruction  o f v i t a m i n s i n t h e d i e t by t h e o x i d i z i n g f a t p r e s e n t i n t h e meal.  Although  a l l o f t h e v i t a m i n s were p r e s e n t i n c o n s i d e r a b l e e x c e s s , t h e y a r e e s p e c i a l l y labile  i n t h i s type o f d i e t .  SUMMARY AND CONCLUSIONS A study has been made o f t h e n u t r i t i v e v a l u e f o r p o u l t r y o f f i s h meal, p a r t i c u l a r l y B r i t i s h solubles.  A.  Columbia  h e r r i n g meal, and o f condensed  herring  The f i n d i n g s a r e summarized as f o l l o w s :  The v a r i a b i l i t y  i n t h e c h e m i c a l c o m p o s i t i o n o f f i s h meals from  different  sources E i g h t e e n samples o f f i s h meal and meat meal were a n a l y z e d f o r t h e i r P r o t e i n Index V a l u e s a c c o r d i n g t o t h e method o f A l m q u i s t (1941)•  The f i s h  meals ranged i n v a l u e from 65 t o 82; t h e meat meals from 48 t o 75. for  The v a l u e s  t h e meat meals were lower, p r i n c i p a l l y because t h e p e r c e n t a g e s o f h o t  water s o l u b l e p r o t e i n were h i g h e r .  I n a d d i t i o n , the phosphotungstic  acid  p r e c i p i t a b l e f r a c t i o n s i n d i c a t e d a g r e a t e r degree o f p r o t e i n decomposition i n t h e meat meals t h a n i n the f i s h  B.  meals.  A comparison o f t h e n u t r i t i v e v a l u e o f h e r r i n g meals d r i e d a t d i f f e r e n t temperatures The n u t r i t i v e v a l u e s f o r t h e c h i c k o f h e r r i n g meals made from t h e  same batches o f p r e s s cake d r i e d a t d i f f e r e n t temperatures were compared. When t h e meals were used as t h e s o l e s o u r c e o f supplementary  protein  i n c h i c k r a t i o n s c o n t a i n i n g e i t h e r 17 o r 21$ p r o t e i n , t h e meals which were d r i e d a t l o w temperatures higher temperatures.  supported f a s t e r growth t h a n d i d t h e meals d r i e d a t  S i n c e t h e meals compared were p r e p a r e d from s i m i l a r raw  m a t e r i a l , i t was apparent t h a t some e s s e n t i a l n u t r i e n t ( s ) was d e s t r o y e d o r was  - 51 -  rendered unavailable to the chick at the higher drying temperature. The next step was to ascertain i f the limiting factor i n the meals dried at the higher temperatures was one of the known vitamins.  When a  vitamin mixture consisting of thiamin, pyridoxine, f o l i c acid, inositol, p-amino benzoic acid, biotin, vitamin B-^, vitamin K and vitamin E was added to a ration containing a meal dried at high temperature, there was an improvement i n the growth rate of the chicks. When, however, the vitamins were added to rations containing meals dried at a low temperature, there was no improvement i n the rate of growth. It was therefore concluded that the meal dried at a low temperature contained adequate quantities of one or more vitamins which were deficient i n the meals dried at higher temperatures.  Presumably at the higher drying  temperature some heat-labile vitamin was destroyed or rendered unavailable to the chick. The results of the next experiment i n the series showed that f o l i c acid was the only single vitamin producing a growth response approaching that obtained with the entire vitamin mixture. A further experiment was designed to study specifically the effect of processing temperature on the f o l i c acid content of herring meals.  The  f o l i c acid content of herring meals prepared from the same raw material was found to be markedly affected by the temperature at which the meals were dried.  Experimentally prepared meals dried at a low temperature contained  4.1 and 4 . 7 ^ f o l i c acid per gram. Commercially dried meals contained only 0.29 and 0 . 2 7 r P  er  gram.  The above data regarding the f o l i c acid content of meals were obtained from microbiological assay of the meals.  Chick biological tests confirmed  the fact that the high temperatures employed i n the commercial manufacture of meals are destructive to the f o l i c acid content.  -52C.  Unidentified growth factors in herring meal The addition of herring meal to a corn-soybean meal ration supple-  mented with synthetic vitamins and with trace minerals stimulated growth of chicks slightly when the ration did not contain an antibiotic.  Supplementa-  tion of the basal diet with penicillin likewise resulted in a slight improvement in growth rate.  In neither case, however, was the improvement in  growth rate significant.  The addition of both herring meal and penicillin  resulted in a statistically significant increase in the rate of growth of the chicks as compared with those fed the basal diet.  It is therefore concluded  that the fish meal contributed a factor(s) which was not supplied either by the basal diet or by the addition of the antibiotic p e n i c i l l i n . Further experiments were conducted to study the effects on chick growth of herring meal and dehydrated green feed added singly and together to a synthetic basal diet. The results provided further evidence that herring meal and dehydrated green feed contained separate chick growth factors.  In  addition, the response obtained to each of these factors appeared to be affected by the presence of the other in the diet. Two experiments were carried out with turkey poults to determine i f the unidentified chick growth factor in herring meal stimulated growth of turkey poults.  The addition of herring meal to a corn-soybean ration was  shown to stimulate growth of poults.  When the basal ration was supplemented  with chlortetracycline the addition of herring meal to the ration resulted i n a greater stimulus to growth than was obtained with the antibiotic alone. D.  The use of condensed herring solubles as a vitamin supplement in turkey poult rations Condensed herring solubles was shown to be an effective vitamin  supplement to a practical type poult ration. A better response to herring solubles than to dried brewers' yeast was obtained with a diet containing  -53herring meal and with diets supplemented with vitamin B 2 but containing no x  herring meal. E.  The effect of heat treatment and storage on the nutritive value for the chick of herring meals of various o i l contents The. nutritive value for the chick of herring meal was not improved  when the o i l content was removed by extraction with hexane. This was true of commercial flame-dried meal as well as of an experimental low temperature dried meal. When a low temperature dried herring meal was extracted with hexane, the f o l i c acid in the extracted meal was less stable than that in the original meal. Storage of herring meals for one year at -25°, 21° and 37° respectively did not appear to affect the nutritive value.  It should be mentioned  that the basal diet in which the meals were fed was well fortified with vitamins and with f a t . F.  The effect of storage treatment and antioxidant treatment on the chemical and nutritive properties of herring meal Low temperature (-20°C.) storage resulted in a slightly greater drop  in ether extractability than did storage at 25.5°C. Low temperature storage greatly accelerated the decrease in iodine value of the ether-extract. Antioxidant treatment prevented any decrease in ether extractability of fat and, to a large extent, prevented any drop i n iodine value of the extract. The meals showing the most rapid decline i n iodine value of the etherextract during storage contained the greatest amounts of l i p i d material not extractable by ether and only extractable by acetone after HC1 treatment. Binding of the l i p i d into a complex from which the l i p i d was extractable  -54with acetone only after HCI treatment occurred early in the storage period. After six weeks, the amount of l i p i d represented i n this fraction decreased, presumably as a result of further oxidation and polymerization into more refractory compounds. In vitro digestibility studies of the meals xirith pepsin showed that meals treated with BHT contained the least amount of nitrogen bound i n compounds that were not soluble after the enzyme digestion. The meals that had been subjected to -20°C. either by chilling after processing or during storage, showed the highest content of undigestible nitrogen.  The normal  meal held at 25.5°C. contained an amount intermediate between these two groups. The meals snowed no differences in relative nutritive value as protein or vitamin B supplements i n chick diets composed of natural ingredients.  When the meals were fed to chicks as the sole sources of protein in  a purified diet, some differences i n growth response were noted. Growth of the chicks fed the meal which had been subjected to a temperature of -20°C. from the time i t was processed was slower i n the tests conducted at the beginning as well as at the termination of the storage period.  -55LITERATURE CITED Almquist, H.J., 1941. Chemical estimation of quality of animal protein concentrates. J. Nutrition 21: 347-50. Almquist, H.J., 1951. Nutritional applications of the amino acids. In "Amino Acids and Proteins" (Grunberg, D.M. ed.) Springfield, 111, Almquist, H.J., 1956. Changes i n fat extractability and protein digestib i l i t y i n f i s h meal during storage. J. Agr. Food Chem. 4: 638-639. Anon,, 1958. Oxidation research shows reasons for quality deterioration of certain foods. Com. Fisheries Rev. 20, no. 5: 18. Arscott, G.H, and G.F. Combs, 1953. Further evidence for an unidentified hatchability factor in condensed fish solubles. Poultry Sci. 32: 730-733. Astrup, H., 1958. Oxidation and self-heating in herring meal. scand. No. 242. 58-62.  Acta polyt.  Aure, L., 1957. Oxidation stabilization of herring meal by BHT on a technical scale. Arsberetning vedkommende Norges fiskerier 3:17-24. F.R.B. (Canada) Translation Series no. 214. Banks, A., 1939. Rancidity in fats. In "Food Investigational Report", page 49, H.M. Stat. Office 1949. Barnett, B.D. and H.R. Bird, 1956. Standardization of assay for unidentified growth factors. Poultry Sci. 35: 705-710. Beall, D., 1933. Loss in the effluent of pilchard reduction plants in British Columbia. Biological Board of Canada, Bull. 35: 1-U. Biely, J., B.E. March, G . L . Inkin and G.T. Eedy, 1951. Wheat vs. corn in the Connecticut broiler ration. Poultry Sci. 30: 593-598. Bird, O.D., M. Robbins, J.M. Vandenbelt and J.J. Pfiffner, 1946. Observations on conjugase from hog kidney. J . Biol. Chem. 163: 649-659. Bissett, H.M. and H.L.A. Tarr, 1954. The nutritive value of herring meals. 2. Effect of heat on availability of essential amino acids. Poultry Sci. 33: 250-54. Carlson, C.W., R.F. Miller, H.T. Puler, L.C. Norris and G.F. Heuser, 1949. The complex nature of the animal protein factor. Poultry Sci. 28: 750-752. Carpenter, K.J., I960. The estimation of the available lysine in animalprotein foods. Biochem. J . 77: 604-610. Carpenter, K.J., G.M. Ellinger and D.H. Shrimpton, 1954. The nutritive value of six white fish meals of known origin. Proc. Nutrition Soc. 13: 20-21,  -56Ch&ldelin, V.A., A.M. Woods and R.J. Williams, 1943. Losses of B-vitamins due to cooking of foods. J . Nutrition 26: 477-485. Clandinin, D.R., 1949. The- effects of methods of processing on the nutritive value of herring meals. Poultry Sci. 28: 128-133. Combs, G.F. and C.S. Shaffner, 1950. Further evidence for an unidentified growth factor required by the chick. Poultry Sci. 29: 623-624. Crampton, E.W., R.H. Common, F.A. Farmer, A.F. Wells and D. Crawford, 1953. Studies to determine the nature of the damage to the nutritive value of some vegetable o i l s from heat treatment. I I I . The segregation of toxic and non-toxic material from the esters of heat polymerized linseed o i l by d i s t i l l a t i o n and by urea adduct formation. J . Nutrition 49: 333-346. Crampton, E.W., F.A. Farmer and F.M. Berryhill, 1951. The effect of heat treatment on the nutritional value of some vegetable o i l s . J. Nutrition 43: 431-440.  Daniel, E.P. and E.V. McCollum, 1931. Studies on the nutritive value of fish meals. U.S. Bureau of Fisheries Investigation Rep. 2: 1-19. Duncan, D.B., 1955. Multiple range and multiple F tests.  Biometrics 11: 1-  Evans, R.J., J.S. Carver and W.S. Hamm, 1944. The influence of storage on the supplementary protein nutritive value of certain fish meals. Poultry Sci.  23: 491-94.  Ferguson, T.M., H.P. Vaught, B.L. Reid and J.R. Couch, 1956. The effect of vitamin E, dehydrated alfalfa meal and condensed fish solubles upon hatchability of eggs from Broad Breasted Bronze hens maintained on l i t t e r . Poultry S c i . 35: 872-875.  Ford, J.E., I960. A microbiological method for assessing the nutritional value of proteins. B r i t . J . Nutrition 14: 485-497. Fraser, A.C, J.M. French and H.G. Sammons, 1949. The relationship of the chemical structure of dietary triglycerides to their absorption in the small intestine. Proc. First International Congress Biochem. pp. 12-13. Grau, C.R. and M.A. Williams, 1955. Fish meals as amino acid sources i n chick rations. Poultry Sci. 34: 810-817. Gray, R.E. and H.E. Robinson, 1941. Free fatty acids and rancidity i n relation to animal by-product protein concentrates. Poultry Sci. 20: 36-41. Harper, A.E., 1959. Amino acid balance and imbalance. I. Dietary level of protein and amino acid imbalance. J. Nutrition 68: 415-418. Harrison, R.W., 1931. The menhaden industry. gation Rep. 1: 1-113.  U.S. Bureau Fisheries Investi-  Harrison, R.W., A.W. Anderson and S.R. Pottinger, 1935. Effect of manufacture on the quality of non-oily fish meals. U.S. Bureau Fisheries Investigation Rep. 30: 1-30.  -57-  Heiman, V., J.S. Carver and J.W. Cook, 1939. A method for determining the gross value of protein concentrates. Poultry Sci. 18: 464-474. Heuser, G .F. and L.C. Norris, 1951. An unknown nutritive factor in feeds of animal origin. Poultry Sci. 30: 470-471. H i l l , F.W., 1948. The multiple nature of the deficiency of unidentified nutrients in crude all-vegetable protein chick starter rations. Poultry Sci. 27: 536-541.  Ingvaldsen, T., 1929. Fish meals. I. Effect of high temperatures employed for drying on nitrogen partition in f i s h meals. Can. Chem. Met. 13: 97-99. Johnson, O.C., E. Perkins, M. Sugai and F.A. Kummerow, 1957. Studies on the nutritional and physiological effects of thermally oxidized o i l s . J . Am. Oil Chemists' Soc. 34: 594-597. Karrick, N.L. and M.E. Stansby, 1954. Vitamin content of fishery by-products. I. Effect of processing methods on riboflavin, nicotinic and vitamin B ± 2 content of solubles and meal. Com. Fisheries Rev. 16, no. 2: 7-10. KLungs^yr, M., G. Boge and T. Sparre, 1953. Relation of drying method to meal quality. Meldinger f r a SSF No. 2, 22-30. Chem. Abstr. 47: 11581f. Kurnick, A.A., R.L. Svacha, B.L. Reid and J.R. Couch, 1956. Further evidence for an unidentified factor required i n a purified diet for normal hatchabili t y . Poultry Sci. 35: 658-662, Konosu, S., S. Katori, R. Ota, S. Eguchi and T. Mori, 1956. Amino acid composition of fish muscle protein. Bull. Jap. Soc. Sci. Fisheries 21, no. 11: 1163-1166.  Laksesvela, B., 1958. Protein value and amino-acid balance of condensed herring solubles and spontaneously heated herring meal. Chick experiments. J. Agr. Sci. 51: 164-176.  Lanham, W.B. J r . and H.W. Nilson, 1942. Effect of heat and moisture on the feeding value of pilchard meal. U.S. Fish and Wildlife Service Rep. 3: 1-10. Lanham, W.B. J r . and H.W. Nilson, 1947. Some studies on the feeding value of fish meals. Com. Fisheries Rev. 9, no. 8: 1-2. Lassen, S., E.K. Bacon and H.J. Dunn, 1949. The digestibility of polymerized o i l s . Arch. Biochem. 23: 1 - 7 . Lassen, S., E. Kyle and H.J. Dunn, 1951. Fish reduction process. Chem. 43: 2082-2087.  Ind. Eng.  Lea, C.H., L.J. Parr and K.J. Carpenter, 1958. Chemical and nutritional changes in stored herring meals. B r i t . J. Nutrition 12: 297-312. Lease, E.J., J.G. Lease, J. Weber and H.S. Steenbock, 1938. Destruction of vitamin A by rancid fats. J. Nutrition 16: 571-583.  -58-  L i l l i e , R.J. and G.M. Briggs, 1947. Biological assay of folic acid activity in common feedstuffs. Poultry Sci. 26: 289-294. L i l l i e , R.J., J.R. Sizemore and H.R. Bird, 1953. Unidentified factors in poultry nutrition. Poultry Sci. 32: 855-862. McGinnis, J., L.R. Berg, J.R. Stern, M.E. Starr, R.A. Wilcox and J.S. Carver, 1952. Additional evidence for an unidentified growth promoting factor for turkeys and chickens. Poultry Sci. 31: 100-106, Maynard, L,A., and A. Tunison, 1932. Influence of drying temperature upon digestibility and biological value of fish protein. Ind. Eng. Chem. 24: 1168-1171.  Menge, H., C.A. Denton, J.R. Sizemore, R.J. L i l l i e and H.R. Bird, 1953. Unidentified factors in poultry nutrition. 2. Properties and preliminary fractionation of a growth factor i n condensed fish solubles. Poultry S c i . 32: 863-867.  Miller, D.S., 1955. 18:  The; nutritive value of fish proteins.  Chem. and Ind.  501-502.  Moen, A., 1933.  Tids, Norske Landbruk 40: 153-157.  Chem. Abstr. 30: 3669,  1936.  Narayan, K.A. and F.A. Kummerow, 1958. Oxidized fatty acid protein complexes. J. American O i l Chem. Soc. 35: 52-56. Nilson, J.W., W.A. Martinek and B, Jacobs, 1947. Nutritive value for growth of some fish proteins. Com.Fisheries Rev. 9, no. 7' 1-7. Oshima, K. and S. Itaya, 1938. Relation between quality and method of manufacture of f i s h meal and scrap. J. Agr. Chem. Soc. Japan, 14: 512-517. Oya, T. and Z. Nonaka, 1938.  Bull. Jap. Soc. Sci. Fisheries 7: 27-28.  Patrick, M., 1951. Studies on supplements for high soyabean o i l meal rations for early poult growth. Poultry S c i . 30: 70-72. Record, P.R., R.M. Bethke and O.H.M. Wilder, 1934. Effect of method of manufacture on the nutritive value of fish meals as determined by growth studies with chicks. J . Agr. Res, 49: 15-22. Ritchey, S.J., H.M. Scott, B.C. Johnson, 1956. Adenosine and ash as unidentified chick growth factors i n fish meal. Proc. Soc. Exp. Biol. Med. 93: 326-328. Schneider, B.H,, 1932.  N-balance studies with various fish meals.  J . Agr. Res,  44: 723-32.  Silkier, A.J., H.E. Scheid and B.S. Schweigert, 1955. Effect of different grades of animal fats on the performance of chicks. Poultry Sci. 34: 411-414. Southcott, B.A. and H.L.A. Tarr, 1953. The vitamin content of certain fishery products. J. Fisheries Research Board Can. 10: 64-68.  -59Stephenson, E.L. and B.B. Clower, 1952. The influence of an unidentified factor found i n fish solubles on hatchability. Poultry Sci. 31: 936-937. Sunde, M.L., 1956. Effect of fats and fatty acids i n chick rations.  Sci. 35: 362-368.  Poultry  Sunde, M.L., W.W. Cravens, C.A. Elvehjem, C.A. and J.G. Halpin, 1950. An unidentified factor required by chicks fed practical rations. Poultry  Sci. 29: 204-207.  Taminie, H.S., 1955. Bull. Japan. Soc. Sci. Fisheries 6: 201-208. Tappel, A.L., 1955. Studies of the mechanism of vitamin E action. I I I . vitro copolymerization of oxidized fats with protein. Arch. Biochem. Biophys. 54: 266-280.  In  Tarr, H.L.A., H.M. Bissett, B.A. Southcott, J. Biely and B.E. March, 1951. The nutritive value of fish meal and condensed fish solubles, I I . The effect of heat on herring meal. Fisheries Research Board, Canada. Progress Rep. Pac. Coast Stat. 87: 42-46. Venolia, A.W. and A.L. Tappel, 1958. Brown-colored oxypolymers of unsaturated fats. J . American Oil Chem. Soc. 35: 135-139. Venolia, A.W. A.L. Tappel and M.E. Stansby, 1957. Oxidative deterioration in fish and fishery products. III. Progress investigations concerning reactions resulting i n brown discoloration. Com. Fisheries Rev. 19, no.5A: 32-34. Wilder, O.H.M., R.M. Bethke and P.R. Record, 1934. The effect of manufacturing method on the nutritive value of fish meals as determined by nitrogen balance studies with rats. J. Agr. Res. 49: 723-30. Wilgus, H.S. Jr., R.C. Ringrose and L.C. Norris, 1933. Studies of the essential nutritive properties of some common protein supplements used i n poultry rations. Proc. 5th World's Poultry Congress. Witting, L.A., T. Nishida, 0,C. Johnson and F.A. Kummerow, 1957. Relationship of pyridoxine and riboflavin to the nutritional value of polymerized fats. J. Am. O i l Chemists' Soc. 34: 421-424. Woodman, H.E., 1937. White fish meal as food for livestock. Ministry of Agriculture and Fisheries Bull. 63, Great Britain.  -60-  T a b l e A - l C h e m i c a l c o m p o s i t i o n o f Twelve samples o f commercial f i s h meal and meat meal. Description  o f Meal:  %  % FFA  %  moist.  ash  %  in o i l  %  oil  protein  1.  whole h e r r i n g meal, flame d r i e d  6.2  7.1  6.3  11.9  70.4  2.  whole h e r r i n g meal, flame d r i e d  8.2  5.2  8.1  10.9  71.9  3.  h e r r i n g o f f a l meal, flame d r i e d  10.8  7.8  9.5  14.4  62.9  4.  h e r r i n g o f f a l meal, vacuum d r i e d  10.0  6.1  5.2  16.3  64.9  5.  h e r r i n g o f f a l meal, flame d r i e d herring spoiled  8.9  9.4  7.4  18.6  59.8  6.  herring  6.6  8.1  7.1  22.2  58.9  7.  whole p i l c h a r d meal, flame d r i e d  3.6  6.8  8.1  14.8  63.5  8.  p i l c h a r d meal, flame d r i e d ,  3.6  5.7 7.8  7.1 7.4  12.1 14.5  65.1 63.8  o f f a l meal, flame d r i e d  scorched  4.8 9.  p i l c h a r d meal, flame d r i e d  10.  mixed h e r r i n g - s a l m o n  (60:40) meal vacuum d r i e d  13.3  4.4  6.3  13.6  63.9  11.  mixed h e r r i n g - s a l m o n  (10:90) meal 15.5 vacuum d r i e d  4.2  3.5  17.6  58.7  21.2  5.6  6.9  28.6  41.1  8.0  6.0  33.3  48.0  12. f i s h  s c r a p meal  13. meat s c r a p meal  7.8  14. meat s c r a p meal  10.5  2.6  6.0  27.6  50.9  15. meat s c r a p meal  13.7  5.3  3.7  36.6  41.7  16. meat s c r a p meal  7.5  2.4  6.9  29.5  52.3  17. meat s c r a p meal  10.0  3.2  7.1  29.6  51.9  6.6  8.1  23.0  56.3  18. meat s c r a p meal  9.0  TABLE A-2 - Protein Index Values of twelve samples of commercial fish meal and meat meal  Description of Meal  Percent Crude Protein  Percent Crude Protein as: Copper precipitible  Indigestible  Hot Water Soluble  Phosphotungstic Acid ppt.  Nonprotein  Protein Index  1. Whole herring meal 2.  70.4 71.9  95.1  10.6 13.7  7.2 6.5  0.6 1.6  4.3 7.6  80  90.8  3. Herring offal meal 4. 5. 6.  62.9 64.9 59.8 58.9  90.7 92.1 93.0 97.8  11.6 9.5 12.4 9.2  6.3 13.2 8.7 10.9  2.8 2.5 1.3 1.0  6.5 5.4 5.7 1.2  77 76 76  82  7. Pilchard meal 8. 9.  63.5 65.1 63.8  92.8 95.0 93.0  12.4 14.3 11.2  5.6 5.8 6.5  1.6 0.3 1.8  5.6 4.7 5.2  78 77 79  10. Mixed fish meal 11.  63.9 58.7  91.9 94.3  11.7 11.9  10.3 9.5  5.2 0.4  2.1 5.3  76 78  12. Fish scrap meal  41.1  87.1  14.8  18.7  8.1  4.8  65  13. Meat scrap meal  48.0 50.9 41.7 52.3  84.3 90.4 65.3 87.8 90.0  8.8 9.5 9.2 10.2 9.2 :-9.8  20.8 22.2 29.8 22.0 17.9  6.8 5.1 24.7 5.2 1.3  8.9 4.5 10.0 7.0 8.7 6.4  66 70 48 67 70 72  14.  15. 16. 17. 18.  51.9  56.3  90.4  16.1  3.2  74  -62-  TABLE B-1.  -  Growth Response of chicks to herring meals dried at different temperatures (Experiment l )  of chicks  Meal Meal Meal Meal Meal  1 2 3 4 5  (commercial) (commercial, overheated) (low-temperature-dried) (commercial) (low-temperature-dried)  Lot A  Lot B  165 171 213 144 214  l6l 155 221 166 205  Average 163 163 217 155 210  -63-  TABLE B-2 - Growth response of chicks to herring meals dried at d i f f e r e n t temperatures (Experiments 2 and 3)  Herring meal supplement  Average weight (grams) o f chicks at 4 weeks o f age: Basal d i e t plus vitamin mixture  Basal diet  Experiment 2 (17$ protein) Meal 1 (commercial)  Lot A Lot B Av.  169 161 166  198 200  Lot A Lot B Av.  174 160 168  221 232  Lot A Lot B Av.  208  207 211 209  Lot A Lot B Av.  188 194 191  273  Meal 2 (overheated)  Lot A Lot B Av.  185 208 198  315 316 316  Meal 3 (low temp.)  Lot A Lot B Av.  272  280  281 289 285  Meal 2 (overheated)  Meal 3 (low temp.)  207  208  199  226  Experiment 7 (21$ protein) Meal 1 (commercial)  276  287 280  -64-  TABLE B-3 - Effect of single vitamins and a mixture of vitamins of the "B complex" in improving the nutritive value of commercial herring meal (Experiment 4 ) . Av. weight of chicks after 4 weeks (gms.) Basal ration  270  Basal ration + vitamin mixture  316  Basal ration + folic acid  291  Basal ration + thiamine HC1  274  Basal ration + pyridoxine HC1  269  Basal ration + menadione  264  Basal ration + ino s i t o l  259  Basal ration + GJ -tocopherol  246  Basal ration + p_-aminobenzoic acid  274  * 20 White Leghorn cockerel chicks i n each l o t .  -65TABLE B-4  - F o l i c a c i d a c t i v i t y o f low-temperature and c o m m e r c i a l l y p r o c e s s e d h e r r i n g meal (Experiment 5 ) .  Description of herring meal added to b a s a l ration^  Folic acid content of herring meal  Av. wt. (g) o f chicks fed basal rations  (Vg)  Low  temperature (Nov. 1951)  4.1  Av. wt. (g) o f chicks fed basal ration supplemented w i t h f o l i c a c i d (0.35 mg./lb.)  Lot A Lot B Av.  213 221 217  219  212  226  Commercial (Nov. 1951)  0.29  Lot A Lot B Av.  165 161 163  219 222 220  Low temperature (Jan. 1952)  4.7  Lot A Lot B Av.  214 205 210  203 218 210  Commercial (Jan. 1952)  0.27  Lot A Lot B Av.  144 166 155  223 214 218  -66-  TABLE B-5 - Composition o f b a s a l d i e t used i n Experiment 5  Ingredient  /IOO  Ground y e l l o w c o r n  lb.  78.16  lb.  0.5  "  Feeding o i l (2250A-300 D3)  0.25  "  Limestone  1.25  "  0.25  "  Iodized  Choline  salt  chloride  (25$)  Manganese s u l p h a t e  10.0  gm.  Niacin  0.8  »  Calcium pantothenate  0.5  "  Riboflavin  0.16  "  The h e r r i n g meal i n q u e s t i o n was added t o t h e above b a s a l d i e t t o g i v e a t o t a l p r o t e i n c o n t e n t o f 21$. Adjustment was made f o r t h e s l i g h t d i f f e r e n c e s i n t h e m o i s t u r e c o n t e n t o f t h e meals.  -67-  TABLE C-l - Growth response of chicks in Experiment 1 Herring meal added to diet (percent)  Average weight of chicks* at 5§ weeks of age (grams)  Ot  352  5.0  364  7.5  364  10. C  372  19.5+  354  * 40 mixed White Leghorn chicks were used for each l o t . + Composition of control diet per 100 lb.: ground wheat, 32.5; • ground corn, 32.5; soyabean-oil meal, 30.0; limestone, 1.0; bone meal, 2.0; dehydrated cereal grass, 1.0; feeding o i l (2250 A, 300 D), 0.25; iodized salt, 0.5; and choline chloride (25$), 0.44 lb.—Manganese sulphate, 6.0; riboflavin, 0.16; calcium pantothenate, 0.5; nicotinic acid, 0.8; and vitamin B , 0.0005 gm. 12  +  Herring meal constituted the sole source of supplementary protein.  - 6 8 -  TABLE C-2 - Growth response of chicks in Experiment 2  1  Protean supplement  Average weight of chicks* at 4 weeks of age (grams)  Soyabean o i l meal alone Soya—herring meal proportion  3 0 6  2:1  3 4 6  Soya—herring meal proportion 1 : 1  3 4 3  Soya—herring meal proportion 1 : 2  3 3 9  Herring meal alone  3 3 4  * 2 6 to 3 3 New Hampshire cockerel chicks per l o t .  -69TABLE 0-3 - Growth response of chicks in Experiment 3  Amount of herring meal added to diet  Average weight (in grams) of chicks* at 8 weeks of age Procaine penicillin not added  Procaine penicillint added  735  760  None  745  -  5.0$ Commercial herring meal  737  788  5.0$ Low-temperature herring meal  755  766  2.5$  Commercial  2.5% Low-temperature  744  * 40 White Leghorn pullet chicks per l o t . t Courtesy Merck and Co.  -70TABLE C-4 - Composition of synthetic basal diet lb./100 l b . Dextrose Isolated soya protein dl-methionine Bonemeal * Manganese sulphate Feeding o i l (2250 l.U. vitamin A 300 I.C.U. vitamin D^) Com o i l Ground cellulose Iodized salt Potassium chloride. Choline chloride  63.89 25.0 0.4 4.0 0.0125 0.25 3.0 2.0 0.5 0.8 0.15 gm./lOO l b .  .Niacin Calcium pentothenate Riboflavin Thiamin HCI Pyridoxine HCI Inositol £-amino benzoic acid Folacin Menadione Vitamin B Biotin «6-tocopheryl acetate....... Ferric citrate Copper sulphate Nickel carbonate Cobalt acetate Zinc acetate 1 2  2.27 0.908 0.363 0.454 0.227 45.4 4.54 0.0908 0.0908 0.0023 0.0091 0.15 5.0 0.5 0.025 0.05 0.25  -71TABLE C-5 - Growth response o f c h i c k s i n Experiment 4 and 5 Average Weights o f C h i c k s i n Grams Basal r a t i o n  alone  Supplement  T e s t 4 (17 c h i c k s per l o t ) None  7 days*  33 days  Basal ration+herring meal-  7 days*  33  % Increase i n weight due to h e r r i n g meal  days  85  424  85  412  -3  86  437  84  484  11  85  418  85  439  5  86  414  84  437  6  Ascorbic acid + DGF " A "  84  403  84  432  7  Ascorbic acid + DGF "B"  84  406  85  447  10  '.DGF "A" DGF  "B"  Ascorbic  acid  T e s t 5 (15 per l o t )  chicks  10 days  31 days  10 days*  31 days  None  64  311  63  326  5  DGF " A "  63  354  64  375  6  DGF »B»  63  338  63  374  11  S t a n d a r d i z e d on a weight  basis.  -72TABLE C-6 - Growth response of poults i n Experiments 5 and 6  Supplement to basal diet*  Test 4 Average weight of poultst at 6 weeks of age (grams)  Test £ Average weight of poultst at 5 weeks of age (grams)  870  872  971  1023  868  877  970  906  969  990  Herring meal + dried brewers'  Dried brewers* yeast + aureomycin Herring meal + dried brewers'  *  —  1088  —  1024  1061  1036  Composition of basal diet per 100 lb.: Ground yellow corn, 45.0; soyabean-oil meal, 48.0; dl-methionine, 0.1; iodized salt, 0.5; limestone, 1.5; bone meal, 4.0; choline chloride (25$), 0.5; manganese sulphate, 0.04 lb.—Riboflavin, 0.2; calcium pantothenate, 0.6; nicotinic acid, 2.25; thiamin hydrochloride, 0.05; pyridoxine hydrochloride, 0.15; menadione, 0.2; biotin, 0,01; folic acid, 0.05; vitamin BTJ2J 0.0001; i - i n o s i t o l , 45.0; p_-aminobenzoic acid, 45.0; a-tocopherol, 1.0 gm. Vitamin A, 400,000 units; Vitamin D3, 60,000 units.  t  15 Jersey Buff male poults per l o t .  %  12 to 13 Broad Breasted Bronze male poults per l o t . Supplied as 0,5$ "Aurofac" (Courtesy Lederle Laboratories)  -73-  TABLE D-l - Composition of basal diets Diet 1 lb./lOO l b . Ground wheat  25.3  Ground corn  15.0  Diet 2  Diet 3  lb./lOO l b . lb./ICQ l b .  31*9  — 36.3  Ground feed screenings  —  Ground oats  5.0  5.0  5.0  Dehydrated cereal grass...........  3.0  3.0  3.0  5.0  5.0  5.0  29.5  43.0  Limestone  2.0  2.0  2.0  Bonemeal  1.0  1.5  1.5  Iodized salt  0.5  0.5  0.5  Manganese sulphate  0.0125  0.0125  0.0125  Tallow  3.0  3.0  3.0  Feeding o i l (2250A - 300D)  0.5  0.5  0.5  Choline chloride 25$.  0.2  0.2  0.2  dl-Methionine  0.025  0.025  0.025  grams  grams  grams  Riboflavin  0.15  0.2  0.2  Niacin  1.0  1.5  1.5  Calcium pantothenate  0.5  0.5  0.5  Alpha-tocopheryl acetate  0.25  0.25  0.25  mg.  mg.  mg.  ~  °» 5  0.25  Herring meal (70$) Meatmeal (50$) Soybean o i l meal (44$)  Vitamin B  1 2  Procaine penicillin G  10.0  0.2  2  47.4  -74TABLE D-2 - Average f i n a l weights o f p o u l t s i n Experiments 1,2, and 3. Average weights (grams) Males  Mean  ( a t 6 weeks o f age)  Experiment 1 Control diet 1 + 1 . 5 $ condensed h e r r i n g s o l u b l e s + 3$ condensed h e r r i n g s o l u b l e s + 1.5$ d r i e d brewers' y e a s t £ 3$ d r i e d brewers' y e a s t  Females  1069  1236 1298 1334  1254 1282  Experiment 2  1087 1117 1058 1011  1152 1192 1226 1156 1146  ( a t 5 weeks o f age)  Control diet 2 + 3$ condensed h e r r i n g s o l u b l e s + 3$ d r i e d brewers' y e a s t  577 626 599  Control diet 3 + 3$ condensed h e r r i n g s o l u b l e s + 3$ d r i e d brewers' y e a s t  599 624 569  Experiment 3  ( a t 9 weeks o f age)  Control diet 2 + 3$ condensed h e r r i n g s o l u b l e s + 3$ d r i e d brewers' y e a s t  2206  2352 2356  1970 2048 2079  2088 2220 2218  Control diet 3 + 3$ condensed h e r r i n g s o l u b l e s + 3$ d r i e d brewers' y e a s t  2170 2402  1966  1993  2370  1943  2081 2184 2156  -75-  TABLE E - l - Average weights of chicks fed unheated and heated, fat-extracted or unextracted herring meals as protein supplement in Experiment 1.  Eation No  Treatment of meal  W  S ^ (16 chicks per lot) e  i  g  h  ^  raffiS  Lot A  Lot B  a t  Av.  1  Unextracted, unheated  86  89  38  2  Unextracted, heated 60 min.  86  90  88  3  Unextracted, heated 120 min.  69  73  71  4  Unextracted, heated 180 min.  54  57  56  5  Extracted, unheated  89  90  90  6  Extracted, heated 60 min.  88  94  91  7  Extracted, heated 120 min.  90  90  90  8  Extracted, heated 180 min.  56  57  54  9  No. 5, fresh herring o i l added  94  87  90  10  No. 6, fresh herring o i l added  91  84  88  11  No. 7, fresh herring o i l added  85  87  86  12  No. 8, fresh herring o i l added  57  58  58  -76-  TABLE E-2 - Average weights of chicks fed extracted and unextracted herring meals i n Experiment 2.  Supplement to basal ration  Weights in grams of chicks at 4 weeks (20 chicks per lot) Without vitamin supplement  With vitamin supplement  207  256  219  258  168  261  182  242  185  272  185  —  162  —  * Herring o i l mixed with herring meal and l e t stand at 21°C. overnight.  -77TABLE E-3 - Average weights of chicks* fed various extracted and unextracted herring meals in Experiment 3 . T es t  1 Weights i n grams of chicks+at 4 weeks  Description of herring meal supplement  Unextracted  (20) 163 (15) 193 (10)  Low temperature Normal commercial Overheated commercial  2 5 7  T es t  Extracted  258 (20) 178 (12) 167 (11)  2 Average weights i n grams at 25 days Stored at  21°C.  Unextracted low temperature Unextracted normal commercial Unextracted overheated commercial Extracted low temperature Extracted normal commercial Extracted overheated commercial  3 2 7  Stored at  37°C. 3 2 2  335 307  302  281  338  3 3 9  3 2 3  316  T e st3 Meals stored at -25°C,  Average weights i n grams at 5 weeks  Unextracted low temperature Unextracted normal commercial Unextracted overheated commercial  470 476 517  Extracted low temperature Extracted normal commercial Extracted overheated commercial  456 508 518  * Test 1: 2 0 White Leghorn pullet chicks per lot. 2 : 2 5 White Leghorn cockerel chicks per lot. 3 : New Hampshire cockerel chicks per l o t . t Figures in parentheses represent the number of survivors at 4 weeks.  -78-  TABLE E-4 - Stability of f o l i c acid in different samples of herring meal stored for a year.  Description of herring meal  Meg. folic acid per gram oil-free meal Storage Original temperature 0°C.  21°C.  37°C.  Extracted low temperature  9.6  5.4  5.4  3.8  Extracted normal commercial  0.72  0.44  0.40  0.32  Extracted overheated commercial  0.50  *  0.40  —  Unextracted low temperature  —  —  6.7  4.0  Unextracted normal commercial  —  —  0.42  0.36  Unextracted overheated commercial  —  —  0.42  * No analysis.  —  -79TABLE F - l - Composition o f d i e t s .  P u r i f i e d Diet 20$ Protein  /ICQ l b .  Herring meal Cornstarch. Hydrogenated vegetable o i l . . , Choline chloride (25$) Ground cellulose < Potassium phosphate, dibasic, Iodinated s a l t , Limestone , Manganese sulphate , Inositol Biotin Menadione Pyridoxine HC1 , Folacin . Riboflavin Calcium pantothenate........ Thiamin HC1 Niacin p_-aminobenzoic acid s<-tocopheryl acetate Ascorbic a c i d . . . . . . Vitamin D3 Vitamin A  27.33 64.02 4.5 0.5 2.0 0.4 0.5 0.75 6.0 45.4 0.0091 0.0454 0.182 0.182 0.361 0.908 0.454 4.54 9.08 0.68 10.0 120.0 2000.0  Natural Diet  20$ p r o t e i n /100 l b .  Herring meal Ground yellow corn Bonemeal Limestone Iodized s a l t Mangagese sulphate Menadione Niacin. Calcium pantothenate Riboflavin Vitamin D 3 Vitamin A  lb. 16.0 ti 82.0 tt 0.5 11 1.0 it 0.5 tt 0.02 0.022 gm. tt 0.6 tt 0.21 it 0.065 I.C.U./lb. 120.0 I.U./lb 2000.0*  lb.  gm.  I.C.U./lb. I.U./lb. 14$ protein /100 l b . lb. 5.0 tt 92.5 tt 1.0 tt 1.0 it 0.5 0.02 tt 0.022 gm. 0.6 11 tt 0.21 it 0.065 120.0 I.C U . 2000.0 I.U. +  The following vitamins were added i n t e s t s of the meals as protein supplements: r i b o f l a v i n 1.95, calcium pantothenate 6.3, n i a c i n 18.0, pyridoxine HC1 1.95, b i o t i n 0.06, f o l a c i n 0.375, vitamin B 0.006, choline chloride 900, «<-tocopheryl acetate 6, and thiamin HC1 1.2 mg. l b . 1 2  -80-  TABLE F-2 - Chemical analysis of stored herring meals. Date of sampling  % ether-extractable material (on dry weight basis)  Dec.10 Dec.19 Jan.l6 Mar.l6 Sept.24  % acetone-extract- Dec.10 Dec.19 able material not Jan.l6 soluble i n ethyl Mar.l6 ether (on dry Sept.24 weight basis) % acetone-extractable material afte r acid treatment of ether and acetone extracted meal (on dry weight basis)  Dec.10 Dec.19 Jan.16 Mar.16 Sep.24  Iodine no. of ether extract  Dec.10 Dec.19 Jan.l6 Mar.l6 Sept.24  B A normal normal stored stored 25.5°C -20°C.  10.2 10.0 9.9 10.1 9.4 1.49 1.16 0.96  0.65 0.77  C chilled stored 25.5°C  10.1 9.4 8.7  116 117  % indigestible protein (on dry weight basis) determined by pepsin digestion of whole meal  Dec.10 Dec.19 Jan.16 Mar.l6 Sept.24  4.3 5.1 5.4 6.0 5.8  % indigestible protein (on dry weight basis) determined on ether extracted meal  Dec.10 Dec.19 Jan.16 Mar.16 Sept.24  3.9 4.6 4.4 4.7 5.2  BHT after 1 week stored 25.5°C.  F BHT to hot meal stored 25.5°C.  9.0 9.0 8.7  10.0 9.9 10.3 10.0  10.1 10.0 10.4 10.3  2.30 0.97 0.65 0.83  0.89 0.93 0.66 0.69  2.21  2.25 2.24  1.23  0.87  1.03  0.72 0.72  0.68 0.89  1.07 1.17 0.69 0.95  2.22 1.86 1.78 1.75  2.16 2.02 1.87 1.53  130 130 126 128  143 138 132 126  2.27  1.95  1.92  134 135  127  E  10.2 10.0 9.6 9.5 8.9  2.24 2.02 2.46 1.80 1.66  D chilled stored -20°C.  1.89 2.01  124 131 81 66  122 112 108  2.77 1.97 1.77  135 79  60 62  4.7 6.2 6.1 6.2  5.3 5.8 5.9 6.3  4.6 6.0 5.5  4.7 4.7 4.9 5.5  6.4 6.4 6.4  5.1 4.9 5.1 5.0  4.5 4.6 4.6 4.7  4.7 4.1 4.6 4.7  4.3 3.9 4.4 4.5  3.7 5.0 6.3 5.3  -81TABLE F-3 - Ranking o f ideals a c c o r d i n g t o growth r a t e o f c h i c k s i n the various b i o l o g i c a l t e s t s .  T e s t s o f p r o t e i n q u a l i t y u s i n g meals as supplements t o a n a t u r a l d i e t  20% protein diet  Dec.  19  Jan.  16  Mar.  16  D  104  A A  1 0 0  100  Sept.  24  104  C c  9 9  100  plOl  Sept. 24 14$ protein diet  C  plOl  pl03  E  F  98 100  B  102  F  98  E  E D  96  D  97  B  98  9 9  E  100  A  100  B  ioo  A  100  1 0 1  D  A  1 0 0  B  95  D  97  100 C  97  T e s t s o f p r o t e i n q u a l i t y u s i n g meals as source o f s o l e p r o t e i n s y n t h e t i c diet'"  20$ protein diets  Dec.  19  Jan.  16  Mar. 16 Sept.  24  c  103  plOl  A B  A  I00  F  106  Dl05  B  102  A  iop  E  F  95  Tests o f vitamin Dec. 1 9 Jan. 16 Mar. 16 Sept  . 24  103  A  ioo  plOl  A  ioo  D  c  101  F  107  pLOl c  106  6  F  93  B  100  9 8  9  1 0 0  C99 E  101  D  92  E97 A  100  C  9 2  D  9 0  D92  cioo B  90  F  96  supplementary valuer E  ioo  c  98  A E  1 0 0  106  c  100  £97 E  99  A  1 0 0  B  98  D D  9 6  98  D  9 9  B3 9  B ? 9  B  99  * H e r r i n g meals not underscored by the same l i n e a r e s i g n i f i c a n t l y different (p<0.05).  - 82 -  FIGURE I.  Extractability of fat from herring meal  - 83 -  FIGURE IT,  Iodine values of ether-extractable fat i n herring meal  

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