The Use of Chicks for the Biological Assay of Members of the Vitamin B Complex

I. The construction is described of a simple constant-temperature room for rearing 2. The internal dimensions were 7 ft. o in. x 5 ft. o in. x 9 ft. 6 in., and this size #### 3. The temperature variation was within i- 1.j' F. throughout the room. #### 4. Young chicks were successfully reared in the room from hatching to 4 weeks of age. young chicks used in small-scale laboratory experiments. proved adequate to contain a rack of twenty-four small laboratory cages. REFERENCE Barott, H. G. & Pringle, E. M. (1949). J. Nutrit. 37, 153. ## The Use of Chicks for the Biological Assay of Members ## of the Vitamin B Complex #### 1. Tests with Pure Substances BY M. E. COATES, S. K. KON AND E. E. SHEPHEARD Cod-Liver Oil (Poultry) Standardization Laboratory, National Institute for Research in Dairying, University of Reading and the Agricultural Research Council (Received 10 May 19jo) During recent years rapid developments in the isolation and identification of the various R-complex vitamins have called for reliable methods of determining these factors and for some means of estimating their physiological availability. Chemical determinations have proved possible for aneurin, riboflavin and nicotinic acid, but the methods, although they may be perfectly reliable for the pure vitamins, are of questionable specificity with naturally occurring materials, as the chemical reactions may be masked, inhibited or enhanced by interfering substances or may fail to detect the vitamin if it is present in a bound form. The advancing knowledge of the nutrient requirements of micro-organisms has presented a means of measuring certain food factors essential to them; the foundations of the present-day technique of microbiological assay were laid by Snell & Strong (1939), who used the production of lactic acid by Lactobacillus casei as a measure of response to riboflavin. Many procedures involving different organisms and conditions are now in current use. The apparent simplicity of these methods has resulted in their widespread application to determinations of vitamins in a great variety of natural materials, and sometimes more confidence has been placed in the results than seemed justified. Although the methods have to some extent been checked by chemical or biological means, no consistent examination of their validity has so far been made, and it was largely to provide 204 I950 a biological check for results by microbiological assay that the work reported here was begun. Microbiological techniques, in common with chemical methods, can only rarely be directly applied to the assay of natural materials. Some preliminary treatment of the sample is usually necessary, involving an extraction process; this may fail to remove completely the vitamin to be measured or may be so drastic as to alter it profoundly or free it from a combined state in which it is not available to animals. It is now generally recognized that the total amount of a vitamin present in a food may differ markedly from the amount available to an animal and that it is only by biological assay that such a difference may be detected. Harris (1948-9) has recently ably reviewed some underlying causes of such discrepancies. The rat has usually been the animal of choice for nutritional biological tests, but its ability to dispense with an extraneous source of some members of the vitamin B, complex has limited its usefulness. It is normally able to do without exogenous nicotinic acid either because of the activity of its intestinal flora or because it can use tryptophan instead. Hence the suggested tests for this vitamin have depended on increasing the nicotinic-acid requirements of the animal by including a high proportion of maize in the diet. Successful assays with rats have been carried out for riboflavin and pyridoxin, but they are time-consuming, 6-8 weeks being needed for deficiency signs to appear. No reliable test with rats has yet been devised for the ‘newer’ members of the vitamin B, complex, some of which are synthesized to an appreciable extent by bacterial activity in the rat’s intestine. It seemed probable that the chick would be more suitable than the rat for biological standardization of the water-soluble vitamins. From the first day of its life, the chick is nutritionally independent of the mother except for certain reserves in the yolk-sac, and it can be given an experimental diet immediately after hatching. The rapid growth rate of the chick indicates that its requirements for the water-soluble vitamins may be high and our experience agrees with this view. According to Jukes & Stokstad (1948) ‘chicks appear to derive very little of their vitamin B-complex requirement from One of the factors contributing most to the error of a biological assay is the variation between animals; it can be considerably reduced if large numbers from a uniform strain can be used. Chicks are readily available in large numbers, and their cost is low, especially if use is made of surplus cockerels; when purchased from a reputable hatchery, they are generally from a fairly homogeneous stock. Chicks have been used in the past for the biological assay of some members of the vitamin B complex: vitamin B, (Arnold & Elvehjem, 1938; Jukes & Heitman, 1940), riboflavin (Jukes, 1937), pantothenic acid (Jukes, 1941), vitamin B, (Sarma, Snell & Elvehjem, 1946) and folic acid (Lillie & Briggs, 1947). The diets used in the earlier work were far from pure. Although with the improved knowledge of the chick’s nutrient requirements later workers designed more adequate ‘synthetic diets’, as far as we know none of these methods has been subjected to thorough statistical analysis, and no attempt has been made to assess their accuracy. In the present work the aim has been to devise a general method that could be M. E. COATES, S. K. KON AND E. E. SHEPHEARD intestinal synthesis ’’ ’ ; unpublished work in this laboratory supports their view. Vol. 4 B-vitamin assays with chicks. I adapted to the determination of as many different vitamins as possible, and to choose a diet with components of known chemical composition whose quality could be standardized. The results were submitted to statistical analysis, and the experiments were designed to yield the maximal amount of data for such an analysis, although to do this it was necessary to use rather larger numbers of birds than might be required for a routine biological assay. EXPERIMENTAL AXD RESULTS Part I. General methods Management of the birds. Sex-linked Rhode Island Red x Light Sussex cockerels, bred by the Agricultural Research Council's Field Station, Compton, Berkshire, were used throughout the investigations. They were housed in Hearson three-tier electrically heated brooders (Spratts Patent Ltd.) of a pattern commonly used in commercial poultry farms. The temperature of the hover was maintained at 95' F. for the first 2-3 days and was then gradually reduced until the supplementary heating was turned off completely during the 3rd week. The room temperature was maintained at about 65' F. by thermostatically controlled steam radiators. Lighting was provided partly by daylight and partly by tungsten lamps controlled by a time-switch to give 12 hr. of light every day. The birds were kept on wire-mesh floors and the droppings, falling through on to paper-lined metal sheets, were removed two or three times weekly. Food was provided dry and unrestricted in hoppers covered with a light wire mesh to reduce waste by scattering. Water was supplied at the end of each brooder in troughs, which were removed and scrubbed daily. The brooders were originally designed to house fifty to a hundred chicks on each floor; for experiments where only fifteen to twenty chicks were used in a group, the floors were divided longitudinally by wire-gauze partitions. Until they were 7 days old, the chicks in these subdivided brooders were only allowed half the usual length of the run, to prevent risk of chill. A modified brooder (Pl. I, I) was designed to contain even smaller groups of birds; it had the heated compartment in the middle and half the standard run at each end. When divided throughout its length and width, each floor provided four compartments suitable for groups of ten to fifteen chicks. For pilot experiments where only two or three chicks could be used at a time, a small constant-temperature room was designed in which birds could be kept in ordinary small-animal cages and the temperature of the room varied according to the age of the birds (Coates, Hall & Thiel, 1950). The chicks were usually received on the day of hatching, and all the experiments described here were begun within the first 2 days of life. Deaths occurring during the 1st week on test were not ascribed to the experimental treatment. The birds were weighed and examined for signs of vitamin deficiency twice a week. Preparation and storage of the diets. It was necessary to disperse very small amounts of potent materials, such as vitamins, evenly throughout all the diets used. A pre liminary mixture was made by hand in a mortar by triturating the substance to be dispersed with a small quantity of some finely divided constituent of the diet, such as 206 ‘950 dextrin. ‘I’he contents of the mortar were then added to the rest of the diet and mixed mechanically. For quantities up to 10 kg. a bench model mechanical dough mixer (EIobart Manufacturing Co. 1,td.) was used. Larger amounts were mixed in a 200 lb. horizontal-paddle mixer (Wm. Gardner and Sons (Gloucester) Ltd.). Oily substances were best incorporated by pouring them on to a rather granular material like gelatin or casein for mixing with the bulk of the diet. Before mechanical mixing, aqueous solutions were poured on to sufficient dry constituents in a mortar to make a paste suitable for blending. Refrigerator space was not available for storing large quan