9.4 ,I THE UTILIZATION OF VITAMIN A AND CAROTENIE BY .RUMINANTS VITAMIN A POTENCY 0F CAROTENES IN CORN S‘ILAGE FED T0 LAMBS. THE EFFECT OF TIME AND RDUTE 0F ADMINISTRATION OF VITAMIN A UN LIVER VI'IAMIN A STORAGE BY STEERS. THE EFFECT OF ROUTE 0F ADMINISTRATION OF VITAMIN A 0N BLOOD SERUM ANB LIVER VITAMIN A CONCENTRATIGNS IN STEERS AT SUBSEQUENT TIME INTERVALS Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY F. HEINRICH MARTIN 1967 ‘Hasm L [8 RA R Y Michigan State U IliVCfSiW This is to certify that the , thesis entitled ) THE UTILIZATION OF VITAMIN A I AND CAROTENE BY RUMINANTS presented by F. HEINRICH MARTIN has been accepted towards fulfillment of the requirements for Ph. 0. degree in Animal Husbandry and» I . Ell .. Major professor Date November I6, I967 0-169 elude-3W " r“ get/:3 ‘3. l ABSTRACT 'In.the first experiment 56 Western lambs with an average initial weight of 22.6 kg were fed a pelleted vitamin A depletion ration _§._c_l_ libitum for 110 days. This ration was composed of 40% soft winter wheat, 41% corn cobs, 14% soybean meal, 4% cane molasses and 1% minerals. The initial serum.vitamin A levels were 28.3 meg/100 ml and dropped to 20.0 meg/100 m1 at the end of the depletion period. The lambs were allotted at random to one of four repletion diets. Lot 1 received corn silage ad libitum plus 0.16 kg/lamb/day of a protein-mineral supplement. The total carotene content of the corn silage (AOAC procedure) was 8.12 mg/kg fresh silage. Lots 2, 3 and 4 received an average of 2.15 kg of pellets/lamb/day of the modified depletion ration supplemented with 937, 2815 and 8448 IU of vitamin A palmitate/kg, respectively, and were restricted attempting to provide the same dry matter intake of lot 1. This was not possible because the pellet-fed lambs began to eat their straw bedding. The average total consumption of carotenes from silage per lamb in lot 1 was 2.10 gm. The average number of IU of vitamin A consumed per lamb in lots 2, 3 and 4 was 138,876, 417,280 and 1,252,114, respectively. After 70 days final average serum vitamin A values were 41.6, 20.8, 40.2 and 48.3 meg/100 ml and the final vita- min A contents per liver were 12.32, 1.86, 8.25 and 15.41 mg for lots 1, 2, 3 and 4, respectively. In the second experiment 108 Hereford steer-calves averaging 244.5 kg initial weight were allotted to a 3 x 3 factorial design with 12 replicates and a covariate of initial weight which was used for treat- ments while time was assigned at random. This experiment was superim- POBP—d on a study of the effect of stage of maturity of corn silage and finaness of chop on beef cattle performance in feedlots. The particle sizes were 1 to 2 cm for the coarse silage and 1 cm for the fine silage. The steers were fed the appropriate silage, I% of body weight rolled shelled corn plus 0.45 kg/head/day of a protein-mineral supplement. Initial serum vitamin A level was 24.3 meg/100 ml. The average dry matter content of the ingoing and outgoing silages for the September, October and November harvest were 28.21 and 27.54%, 48.14 and 46.25%, and 59.55 and 54.18%, respectively. The total carotene content for the three harvest periods of the fine cut silage (AOAC procedure) was 11.99 and 7.70, 4.38 and 2.61, and 4.33 and 3.49 mg/kg of fresh silage, while the coarse cut silage contained 15.03 and 11.24, 4.63 and 3.12, and 4.42 and 3.32 mg/kg, respectively. Three steers out of every lot served as controls while the remaining steers were injected with 7,000,000 IU of a water miscible vitamin A preparation either intraruminally (IR) or intramuscularly (IM) at either 147, 63 or 35 days antemortem. Final serum vitamin A concentrations were 32.8, 37.4 and 40.1 meg/100 ml while final liver vitamin A stores were 7.56, 36.2 and 80.3 meg/gm liver (fresh basis) for control, IR and IM treatments, respectively. The effect of time of administration resulted in liver vitamin A stores of 25.8, 47.8 and 50.3 meg/gm (fresh basis) for 147, 63 and 35 days antemortem in- jection periods, respectively. In the third experiment 10 Hereford steers averaging 365 kg initial weight were used to determine the absorption and decay curve of serum vitamin A when vitamin A was administered either IM or IR. In trial I, 5 steers selected at random were injected IM with 1,500,000 10 of a water miscible vitamin A preparation and the remaining steers were in- Seeted In, Liver biopsies were performed to assess initial and final liVer vitamin A stores in all steers. The changes in serum vitamin A concentration were determined at 0, 1.5, 3, 6, 12, and 24 hr post-in- jection (PI) and at 2, 4, 8, l6 and 32 days PI. Average initial and final liver vitamin A stores were 7.37 and 23.3 mcg/gm and 7.63 and 12.9 meg/gm liver (fresh basis) for IM and IR treatments, respectively. Serum vitamin A concentration was significantly greater at 3 hr P1 with In than IR (44.9 vs. 36.8 meg/100 m1). Both treatments peaked at 12 hr PI (58.8 vs. 69.6 mcg/100 m1), while IR was significantly greater than IM at 24 hr (44.0 vs. 54.7 meg/100 ml). In trial II only 9 steers were used since one died accidentally during liver biopsy. 3,000,000 IU of vitamin A were injected. Treat- ment and sampling procedures were similar to those of trial I. Final average liver vitamin A concentrations were 34.5 and 24.9 meg/gm. IM serum vitamin A concentration was significantly greater at 3 hr PI than in IR; they peaked at 12 hr PI and IR was significantly greater than IM (58.6 and 83.1 meg/100 m1). In trial III the same steers received 4,500,000 IU of vitamin A, IM and IR. Final average liver vitamin A stores were 119.5 and 46.4 mcg/gm for IM and IR, respectively. Serum vitamin A concentrations peaked again at 12 hr PI (71.1 vs. 92.9 mcg/lOO ml) and at 24 hr PI IM was significantly lower than IR (51.0 and 67.6 meg/100 ml). mad ~.J I' “'23. - _. THE UTILIZATION OF VITAMIN A AND CAROTENE BY RUMINANTS l. VITAMIN A POTENCY OF CAROTENES IN CORN SILAGE FED TO LAMBS. THE EFFECT OF TIME AND ROUTE 0F ADMINISTRATION OF VITAMIN A ON LIVER VITAMIN A STORAGE BY STEERS. THE EFFECT OF ROUTE OF ADMINISTRATION OF VITAMIN A ON BLOOD SERUM AND LIVER VITAMIN A CONCENTRATIONS IN STEERS AT SUBSEQUENT TIME INTERVALS. It” By \ F.‘ Heinrich Mart in A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry 1967 7, -.—-...—_-‘.r ACKNOWLEDGEMENT The author expresses his sincere appreciation to Dr. D. E. Ullrey and Dr. W. H. Newland for their guidance and assistance throughout this work. The writer wishes to express his thanks to the members of his guidance committee, Drs. D. E. Ullrey, E. R. Miller, R. W. Luecke and E. P. Reineke for their suggestions and critical reading of this manu- script. The author is deeply grateful to the Animal Husbandry Department of Michigan State University for the use of facilities and animals and financial support through an assistantship. Sincere appreciation is also expressed to the fellow graduate students, members of the Beef Cattle Research Center, laboratory assistants and department secretaries, who offered a great deal of assistance and encouragement during the course of this study. A special note of appreciation is extended to Dr. Stanley R. Ames of D.P.I., a Division of Eastman Kodak, for supplying the vitamin A preparations used in this study. Special thanks are due to Mrs. Kathryn Ide, who very skillfully and efficiently typed this manuscript. Above all, the author is indebted to his wife, Nonnie, whose sacri- fices, patience and encouragement made the completion of this study possible. F. Heinrich Martin candidate for the degree of Doctor of Philosophy DISSERTATION! The Utilization of Vitamin A and Carotene by Ruminants. OUTLINE OF STUDIES: Major area of study: Animal Husbandry (Animal Nutrition). Supporting areas of study: Physiology, Biochemistry BIOGRAPHICAL ITEMS: Born: April 12, 1938, Bremen, Germany Undergraduate studies: National University, Bogoté, Colombia 1955-1959. Graduate studies: Michigan State University, 1962-1967. EXPERIENCE! DVM, Ministry of Agriculture, Bogota, Colombia, 1959-1962. Graduate Assistant, Michigan State University, 1962-1967. MEMBER: American Society of Animal Science. iii TABLE OF CONTENTS INTRODUCTION . . . . . . LITERATURE REVIEW . . . . . . . . . . EXPERIMENT I . . . . . . 'Materials and'Methods. . . . . . . . Experiment . . . . . . . Analytical Procedures . . . . . . . Results and Discussion . . . . . . . EXPERIMENT II . . . . . . . . . . Materials and Methods. . . . . . Experiment . . . . . Criteria 0 o o o o o o o o o 0 Results and Discussion . . . . . . . EXPERIMENT III . . Materials and Methods. . . . . . . Experiment . . . . . . . . . Results and Discussion . CONCLUSIONS . . . . . . Experiment I . . . . . . . . . . Experiment II . . . . . . . . . Experiment III . . . . . . . . . . SUMMARY . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . MNDH O O 0 O O O O O O I O 0 iv 31 31 32 33 34 40 40 41 45 46 56 56 56 58 68 68 69 70 71 74 81 Table la. lb. 2a. 2b. 3a. 3b. 10. ll. 12. 13. 14. free diet for 110 days . Weight gain and feed consumption of lambs fed rations con- LIST OF TABLES O taining carotenes or retinyl palmitate for 70 days . Serum vitamin A concentration of lambs fed a vitamin A free diet for 110 days . Serum vitamin A concentration of lambs fed rations con- 0 taining carotene or retinyl palmitate for 70 days. Liver vitamin A stores of lambs fed a vitamin A free diet for 110 days . . . Liver vitamin A stores of lambs fed rations containing carotene or retinyl palmitate for 70 days. Weight outcome groups . . . . The effect of vitamin A injection 147 days antemortem on final liver vitamin A concentration . The effect of vitamin A injection 147 days antemortem on final liver carotene stores . . . . . The effect of time of antemortem injections on final liver vitamin A stores in steers . The effect of treatment x time interaction on final liver vitamin A concentration Summary of simple correlation coefficients between variables . . . . Total carotene content of silages fed and average daily carotene intake by experimental steers . The effect of harvest time on total carotene content of ingoing corn silage . The effect of storage and degree of chop on the total carotene content of outcoming silage (fresh basis) Changes in average serum vitamin A concentration after an injection of 1,500,000, 3,000,000 and 4,500,000 IU of vitamin A . . . . Initial and final liver vitamin A concentration of liver biopsies (Trial I) V 0 .Weight gain and feed consumption of lambs fed a vitamin A Page 34 34 35 35 37 37 42 46 47 48 49 51 52 53 53 58 60 NI.- Table Page 15. Initial and final liver vitamin A concentration of liver biopsies (Trial II) . . . . . . . . . . . . . . . . . . 63 16. Initial and final liver vitamin A concentration of liver biopsies (Trial III). . . . . . . . . . . . . . . . . . 66 vi Figure 1. 4a. 69 LIST OF FIGURES —— Lul- F:.u—-W.- Regression of liver vitamin.A storage on dietary retinyl palmitate . . . . . . . . 39 Steer 4103, lot 21 (vitamin A deficient). . . . . . . 44 Steer 1122, lot 21 (normal) . . . . . . . . . . . . . 44 Serum vitamin.A.concentration changes following an IM and IR injection of 1,500,000 IU of vitamin A (linear) o o e o o o o o o o o o o 59 Serum vitamin A concentration changes following an IM and IR injection of 1,500,000 IU of vitamin A (low I O O O O O O O O O O I O O I O O O O .....59 Serum vitamin A concentration changes following an IM and IR injection of 3,000,000 IU of vitamin A (linear). . . . . . . . . . . . . . . . O O O O O 62 Serum.vitamin.A concentration changes following an IM and IR injection of 3,000,000 IU of vitamin A (108) O O O O O O O O O O I I O O O O O O O O O I O o 62 Serum vitamin A concentration changes following an IM.and IR injection of 4,500,000 IU of vitamin.A ......64 Serum vitamin A.concentration changes following an IM.and IR injection of 4,500,000 IU of vitamin A (log) . . . . . . . . . . . . 64 LIST OF APPENDIX TABLES Serum vitamin A levels of lambs during depletion (Exp. I) Serum vitamin A levels of lambs during repletion (Exp. I) Final liver vitamin A of lambs (Exp. I) . . . Initial serum vitamin A and carotene of steers (Exp. II), meg/100 ml . . . . . . . . . . . . Final serum vitamin A and carotene of steers (Exp. II), meg/100 ml. . . . . . . . . . . . . . . . . Final liver vitamin.A.and carotene of steers (Exp. II). . Serum vitamin A concentration changes, mcg/lOO ml, of steers (Exp. III) following an injection of 1,500,000 1U Of Vitamin A (Trial I) o o o o o o e c c c o o 0 Serum vitamin.A concentration changes, meg/100 ml, of steers (Exp. 111) following an injection of 3,000,000 IU of vitamin.A (Trial II) . . . . . . . . . . . . . Serum vitamin A concentration changes, mcg/100 ml, of steers following and injection of 4,500,000 IU of vitamin.A (Trial III) . . . . . . . . . . . . . . . viii 84 85 86 87 91 91 92 INTRODUCTION The following discussion of the discovery of vitamin A and its precursors, and the determination of their structures, has been excerpted largely from textbooks on vitamins (see Vogel and Knobloch, 1950; ‘McCollum, 1957; Wagner and Folkers, 1964; Moore, 1957; Zechmeister, 1962). The biological activity of vitamin.A was discovered during investi- gations of accessory factors in foods and not as a result of studies of a particular disease state. Stepp in 1909 observed that mice became ill when fed a diet which had been extracted with ether and ethanol, and the addition of lecithin or other fats did not prevent the onset of deficiency symptoms. Similar observations were recorded by Hopkins in 1912 and McCollum and Davis in 1913 who, working with rats, showed the existence of a growth promoting factor present in cod liver oil and butterfat but absent in lard. Young animals ceased to grow and a characteristic inflammation of the conjunctiva appeared (xerophthalmia). The disease symptoms were alleviated if lard were replaced by butter or whole milk. Because vitamin A was found in cod liver oil, which was known to have antirachitic properties, it was initially thought that the vitamin A activity was due to the antirachitic factor, but McCollum in 1922 and Steenbock in 1923 showed that the passage of air through cod liver oil did not affect the antirachitic properties but destroyed the growth promoting factor. In 1919, Steenbock found a relationship between the vitamin A activity of plants and their carotene fraction. He theorized that caro- tene in a leuco (colorless) form was the vitamin A carrier, and he was 83312 to alleviate vitamin A deficiency symptoms by administering caro- tene. His trials could not be duplicated at the time by other research- ers, and his work was criticized by Palmer who with Kempster in 1919 had raised two generations of chickens on a diet, virtually devoid of yellow pigments, composed of white corn, white squash, white onions and small amounts of pork liver. Since there were no carotenoids present which could be converted to the hypothetical leuco form and the true signifi- cance of the supplement of pork liver was not known, it appeared that Steenbock's theory was incorrect. It took another 10 years of intensive research before the true relationship between carotene and vitamin A was established. In 1928 Von Euler and Karrer demonstrated in rats that purified carotene in daily doses of 5 to 10 mcg possessed vitamin A activity. At short intervals thereafter, the chemical structures of carotene and vitamin A were determined and the relationship between the two compounds was explained. Eventually it was found that vitamin A activity was shared by a large group of structurally related compounds (polyenes). The different polyenes were either geometrical isomers of retinol, the first A-vitamin isolated, or functional group variants of this compound, each of which might also exist in several stereoisomeric forms (Zechmeister, 1962; Wagner and Folkers, 1964). It was also shown that there were several forms of provitamin A, all structurally related to either alpha, beta or gamma carotene. The provitamin was converted in vivo to the corres- ponding A-vitamin in the intestinal tract and in vitro by careful oxidation. While provitamins were generously distributed throughout the plant kingdom, the A-vitamins were found only in animals, especially in r. J-w the liver and viscera of fish and mammals. In addition to the research on the chemical properties and struc- tures of vitamin A, a large amount of work has been done pertaining to the physiological aspects of the vitamin in many animal species. It has been found that vitamin A is required by all animals for maintenance of epithelial tissues, for vision and normal bone development. The research done with sheep has been mainly concerned with factors affecting the utilization of vitamin A rather than establishing the efficiency of lambs in converting carotenes to vitamin A. These same problems have been studied even more extensively in beef cattle. Some research has been done with beef cattle to determine the rela— tive efficacy of two routes of vitamin A administration. The question of how long a single massive dose of fitamin A in a water miscible base with vitamins D and E will support normal serum vitamin A levels, under normal feeding conditions has not been answered. Neither has the form of the absorption and decay curve of serum vitamin A after a single in- jection of vitamin A in a water miscible base with vitamins D and E been determined. With the above points in mind, the following experiments were designed: 1. To determine the vitamin A potency of carotene in corn silage when fed to lambs. 2. To determine whether a single massive dose of vitamin A in a water miscible base with vitamins D and E will support normal serum vitamin A levels and adequate liver vitamin A stores for an extended period of time. 3. To determine the relative efficacy of intraruminal and intra- muscular administration of vitamin A. 4 4. To determine the absorption and decay curve of serum vitamin A when vitamin A is administered using either of these two routes. LITERATURE REVIEW Reguirements . Guilbert gt; a}. (1935, 1937, 1940) did much of the early work that established the minimum vitamin A and carotene requirements of farm animals. Their work was based upon night blindness as a criterion, supplemented by checks on storage. Evidence was presented which indi- cated that the amounts of vitamin A or carotene that just prevented night blindness represented a physiological minimm. Based on this criterion, they indicated requirements for sheep, cattle and swine which fell within the same relative ranges. The minimum requirement of caro- tene was found to be 25 to 35 mcg per kg of body weight daily, and the minimum vitamin A requirement was found to be 5 to 8 mcg per kg of body weight daily. Normal growth and deficiency symptoms disappeared at these levels, but storage after prolonged periods was low. When storage was studied at different levels of intake, it was found that at least 3 to 5 times the minimum level would be a desirable minimum for practi- cal purposes of storage and reproduction. Although the requirements per unit of body weight were about the same for young and mature animals, the young were more susceptible to pathological changes during low intake periods. From their data it can be seen that there is a tendency for the rate of depletion of vitamin A stores to decrease as depletion advances and reserves become smaller. Madsen 31:, _a_l. (1949) reported on the carotene requirement of beef cattle for reproduction. They found that cows that failed to have normal calves when given 30, 45 or 60 mcg of carotene reproduced nor- mally when they were given 90 mcg or more per kg of body weight. When the plasma vitamin A content of the dam was below 18 mcg per 100 ml at 5 or near the end of the gestation period, the chances of producing a normal living calf were poor. Parrish 35.31. (1950) studied the comparative value of vitamin A and carotene for supplying the vitamin A requirements of swine during gestation and beginning lactation. In each of two trials, 15 Duroc gilts were divided into three comparable lots. They found, as judged by con- centrations of vitamin A in serum and in colostrum of the gilts at farrowing time, and in livers of newborn and 5-day-old pigs, that daily supplements of 6500 to 7100 IU of vitamin A (2.2 to 2.4 mg vitamin acetate) were almost as effective as the yellow corn, tankage and alfalfa hay diet for supplying vitamin A during gestation and beginning lactation. Carotene appeared to be approximately one-half as effective as vitamin A, except for liver storage. Livers of pigs whose dams received caro- tene contained only one-third to one-fifth as much vitamin A as did those from pigs whose dams received vitamin A. Size, health and vigor of the pigs from sows of the three lots used in each trial did not differ markedly. Frape £5 21- (1959) studied the vitamin A requirement of the young pig up to 8 weeks of age, since early weaning partially excluded the sow as a source of vitamin A to the young pig. In each of the four experiments, involving 224 pigs, mature crossbred sows were fed a vita- min A-deficient ration from shortly after breeding until the weaning of their pigs at one week of age. Several criteria of adequacy were in- vestigated and their sensitivity, precision and validity discussed. These criteria were weight gain, feed efficiency, blood plasma and liver vitamin A, and cerebrospinal fluid pressure. Of the criteria studied, the plasma response was the most and weight gain the least sensitive, but the minimum point of normality that was most readily detectable, was cerebrospinal fluid pressure, and this point occurred at a similar dietary intake of vitamin A in all experiments (600 to 800 IU or 0.21 to 0.28 mg vitamin A acetate per lb of feed). The liver vitamin A storage, which was estimated in fewer animals, was found to be extremely sensitive to different intake levels and very consistent between experi- ments. The minimum requirement of the young pig for a stabilized source of vitamin A palmitate on a dry carrier was judged to be, based on cerebrospinal fluid pressure, 800 IU/lb of feed under the experimental conditions used. Normal weight gain was found at as low as 100 IU/lb of feed. The effects of marginal vitamin A intake during gestation in swine were investigated by Heaney (1960) who found that a dietary intake of 3 to 3.5 mcg of vitamin A per kg of body weight was apparently adequate to meet the daily requirements of gilts. This level prevented deficiency symptoms, restored plasma vitamin A values but was, according to the author, not high enough for optimum reproduction or liver storage. His data indicated that 5 to 6 mcg of dietary vitamin A per day per kg of body weight were needed for reproduction. Selke gt £1. (1967) studied the vitamin A requirements of the ges- tating and lactating sow. Vitamin A was added to the basal diet at levels of 0, 1/2, 2, 4, and 8 times the requirement listed by the N.R.C. (1959) which was 2939 IU per kg of feed (1.01 mg of vitamin A acetate per kg of feed). The breeding performance of the sows was con- sidered to be subnormal from the standpoint of the number of matings per conception, but apparently treatment differences had no effect upon this phenomenon. No trends in number of pigs farrowed (live, stillborn, or total) were established for the different treatments. No definite trends in cerebrospinal fluid pressures were established among the treatments when recorded for l4-day-old pigs. Contrary to the conclu- sions of Frape g; 5;. (1960), the levels of plasma vitamin A from either gilts or pigs were not considered a good measure of the vitamin A status of the animals. Carotene and‘Titamin A Levels in Plasma and Liver. Davis anthadsen (1941) reported an extensive study of vitamin A and carotene in cattle blood plasma. They found that long continued inadequate carotene intake and vitamin.A deficiency can be determined by blood analysis. The critical level of carotene in the plasma was found to be 20 to 25 mcg per 100 ml plasma and for vitamin.A in the same sample about 16 mcg per 100 m1 plasma. Cattle having vitamin A and carotene values at these levels, or above, usually did not show symptoms of vitamin A deficiency. The relative importance of dietary carotene and liver stores of carotene and vitamin.A for reproduction and lactation of beef cows was reported by Baker 25 5;. (1953). After a depletion period, one group of cows received 60 mg of carotene per head daily during gestation, another group received 300 mg of carotene per head daily during lactation, a third group received both carotene treatments, while a fourth group served as a negative control. There was a rapid decrease in liver vita- min.A stores during gestation which was not retarded by supplementation; whereas, carotene supplementation during lactation spared liver stores of vitamin A. Carotene supplementation during lactation increased the vitamin A content of the milk and of the calves' plasma and livers; the liver vitamin A reserves of the cows were less effective in this re- spect. Several cases of vitamin.A deficiency were observed in the 9 calves from cows receiving low or no carotene supplementation. Embry gt _a_l. (1962) after depleting steers to an average plasma value of 4.8 to 7.5 mcg of vitamin A per 100 ml, fed daily levels ranging from 1000 IU to 5000 IU of vitamin A per 100 lb of body weight, in increments of 1000 IU, to study the requirements of fattening cattle. Carotene as present in dehydrated alfalfa meal was also fed at levels of 2.5 mg, alone or with 1000 or 2000 IU of vitamin A, per 100 lb of body weight. The final plasma vitamin A values were 13.4, 20.5, 29.0, 33.5, and 32.5 mcg per 100 ml of plasma while liver vitamin A concen- trations were 0.92, 1.17, 2.67, 6.07 and 4.62 mcg per gram fresh liver tissue for the 1000 to 5000 IU levels of supplementation, respectively. No plasma carotene data were presented. Based on gains and liver storage, 2.5 mg of carotene appeared to be equal to 1000 IU of vitamin A but was more effective on the basis of plasma vitamin A values. The low plasma vitamin A concentration as well as the low liver storage make the adequacy of 1000 IU of vitamin A per 100 1b of body weight seem questionable. Roberts 35 a], (1963), working with beef cattle and using dietary vitamin A levels which ranged from 0 to 72,700 IU and beta-carotene from 15 to 63.3 mg per head daily, found no significant differences in aver- age daily gains. Liver vitamin A stores decreased in all groups but the decrease was greatest relative to the initial value in the group receiving no vitamin A. No vitamin deficiency symptoms were noticed. Williams et §__l.. (1963) when feeding steers 1, 2 or 4 times the levels recomended by N.R.C. for beta-carotene, with and without antioxidants, and l and 4 times the recomended vitamin A levels, found that there was increased plasma carotene, plasma vitamin A and liver vitamin A 10 storage and daily gains with increased carotene intake. Only their highest levels of dietary carotene or vitamin A supported maximum growth. Braun (1945a), studying the seasonal changes of plasma carotenoids and vitamin A levels in cattle, found that seasonal changes of the carotenoid level were mainly dependent upon the diet while the seasonal changes of the vitamin A level were dependent on the carotenoid and vitamin A intake and were modified during parturition, abortion and acute infections, at which time vitamin A levels showed a sharp, tempor- ary decrease. The relation of carotenoids and vitamin A in the blood was analyzed. A linear increase of vitamin A with increasing carotenoid level was found. The ratio of vitamin A to carotenoids at various caro- tenoid levels was established and was found to decrease with increasing carotenoid levels reaching a constant value at high carotenoid levels. The minimum normal A/carotene ratios went from 500 to 89 and were ob- tained by dividing IU of vitamin A per 100 ml plasma by mg of carotene per 100 m1 plasma. Animals which were supplemented with vitamin A showed, on the average, lower carotenoid levels than unsupplemented animals. Similarly to the A/carotene ratios in the blood, Braun (1945b) found that there existed a typical relationship between carotenoid and vitamin A levels in the liver. This relationship between vitamin A stores and vitamin A level in blood was direct only when liver stores fell below normal levels. Ralston and Dyer (1959) showed that there was no apparent relationship between plasma and liver vitamin A regard- less of season. The plasma carotenoid relationship to liver carotenoids and plasma vitamin A was most significant during the winter and unre- lated throughout the fall. The liver carotenoids were not related to Iv WW- ':V~.. .. . ‘_—¢ ..... I 11 liver vitamin A but showed a relationship to plasma vitamin A and temperature. The correlation between plasma vitamin A and liver caro- tenoids varied throughout the year from highly significant during the winter, with little or no relationship during the spring and fall, to a highly significant negative correlation through the summer. Diven gt __1_. (1960) who also investigated these relationships, found significant correlations between hepatic carotenoid and plasma carotenoid, hepatic carotenoid and hepatic vitamin A, plasma carotenoid and plasma vitamin A and hepatic vitamin A and plasma carotenoid, but due to the limited magnitude of the correlation coefficients, suggested that the formula- tion of prediction equations would be impractical. According to these authors, the results obtained from such equations would depend upon which variable was designated as dependent and which was designated as independent . Routes of Administration and Nature of Solvents. Hentges gt a}. (1952) studied the effects of carotene administered orally, IM and IV on vitamin A deficiency in pigs. It was found that, with aqueous preparations of carotene, all three methods afforded com- plete recovery from vitamin A deficiency symptoms and normal plasma vitamin A values were obtained. Orally administered carotene was con- verted most rapidly while IV injections were utilized before IM injec- tions. Carotene in cottonseed oil administered IM remained at the site of injection and was ineffective in relieving avitaminosis A. Bieri and Pollard (1953) demonstrated rapid conversion of carotene to vitamin A in rats using IV injections of carotene dispersed in water. Church 31; 311. (1954) found that normally circulating carotene was not 12 utilized by Hereford calves. The IV injection of carotene dispersed in Ween 40, caused no significant differences in plasma vitamin A values. Plasma carotene remained quite constant near 85 mcg per 100 ml of plasma for 2 to 24 hr following injection, then slowly decreased. No liver storage was evident from the comparison of liver samples taken by biopsy 1 month before and 3 days after injection. Advanced deficiency symptoms present in some calves were not alleviated and appeared to progress during the 10 day experiment. Using sheep, the results were quite different. Nine hr after the IV injection of beta-carotene, the plasma vitamin A concentration was more than twice the initial value. After a period of 10 days it had almost returned to the initial mean plasma value. The liver vitamin A values were highly variable. The liver carotene values were not significantly different from those of untreated wethers which served as controls. Quite different were the results ob- tained by Klosterman gt _e_l. (1949) who injected carotene, suspended in water or in cottonseed oil, IV in sheep. The injected carotene was very rapidly removed from the blood stream. However, this carotene was not converted to vitamin A as no increase of the vitamin in the blood or liver could be detected. The discrepancy of results obtained by the above authors was probably due to the different nature of the solvents employed to dis- perse the carotene preparation. Eaton _e_t_:_ _§_]_.. (1951) reported that IV carotene dispersed in coconut oil gave no beneficial effects when administered to vitamin A deficient dairy calves. However, in a second trial, with aqueous colloidal carotene, also administered IV, signifi- cant increases in plasma vitamin A were obtained. The efficiency of vitamin A or carotene utilization when administered “<---v-,._--_3.w- a... .. 13 through different routes varies considerably. Bentges _e_; _e_l. (1952), using pigs, determined that when carotene dispersed in water was admin- istered orally, IV and IM, all three routes of administration permitted complete recovery from vitamin A deficiency symptoms, and plasma values returned to normal. Orally supplied carotene was utilized most rapidly, while IV injections were converted before IM injections. When carotene was administered IM using cottonseed oil as the carrier, it was in- effective in relieving avitaminosis A symptoms because it remained at the site of injection. Studying the effect of method of administration on the absorption and storage of vitamin A by dairy calves, Blake _e_ §_l_. (1950) found that when vitamin A was fed dispersed in water and mixed with milk, it had not only a more rapid uptake, but the absorption was also more efficient than when administered in capsules. Jacobean _e_; _l. (1954) reported that vitamin A administered to calves in the presence of an emulsifying agent was absorbed and stored to a greater extent than when similar amounts of the vitamin were fed in oily solution. This occurred both when vitamin A was administered in milk by nipple feeder and when it was administered by capsule. Wise __t_: _e_l. (1949) had observed previously that a more rapid absorption of aqueous dispersions of vitamin A as compared to oily solutions, occurred in calves, which was also apparently indicative of a more complete absorption and stor- age. According to Jacobsen _t _l_.. (1950) , the rate of absorption was much slower when vitamin A was homogenized in milk and fed by stomach tube into the rumino-reticulum cavity than when this dispersion was fed by nipple feeder into the abomasum, and that both of these methods of administration promoted a more rapid absorption than when the vitamin was fed by capsule. Thus, the rate of absorption does not seem to be a l4 reliable criterion of efficiency of vitamin A utilization in cattle, because of the compound nature of the ruminant stomach. Varnell and Erwin (1960) used the IR, intraperitoneal, IM.and subcutaneous routes to study the influence of injection site on blood vitamin A and carotene levels in sheep. With vitamin A there was an increase of plasma vita- min A concentration in all treatments at 3 and 24 hr. When carotene was injected by the same routes in another group of sheep, plasma vita- min A levels increased only in those animals receiving either the IP or IM injections and did not increase in animals treated with IR and subcutaneous carotene injections. Roberts and Stringam (1962) studied the vitamin A stores and weight gains of cattle following the IR injection of 1,000,000 IU of vitamin A palmitate (550 mg) in maize oil as compared with 10,000 IU fed orally per head per day. The liver vitamin A concentration had trebled within 7 to 21 days following the IR injection. The values then declined gradually to the initial level as determined by liver biopsies performed periodically. A similar pattern was followed by those re- ceiving the vitamin A orally, but the rise was much smaller. No sig- nificant differences were observed for weight gains. Work by Record 25 21. (1963) showed that cattle receiving between 1,000 and 3,000 IU of vitamin A palmitate per 100 lb body weight per day could maintain normal serum vitamin A levels, but that 1000 IU per 100 1b of body weight per day were not sufficient to maintain adequate liver storage. Using beta-carotene in feed at levels ranging from 2.5 to 10 mg per 100 1b of body weight, normal serum vitamin A concentrations were main- tained, but liver concentrations were not maintained by the lowest level of carotene supplementation. Using a single massive dose of 15 6,000,000 IU of vitamin A injected IN, the vitamin A concentration in the liver at the end of 56 days was 6.5 times the initial level. At the end of 175 days the liver vitamin A concentration had dropped to 8 mch gm which was below the initial level. Serum vitamin A levels were al- most twice the initial levels and had returned to almost the original level at the end of 175 days. In another trial, these authors compared the efficacy of the IM and IR routes of administration. Using a single injection of 2,000,000 IU of vitamin A they found that liver vitamin A concentration was increased by the IM injection as compared to the initial level; this was not the case with the IR injection which did not maintain the original liver vitamin A concentration for 56 days. Roberts gt a}. (1965) established that the administration of 1,000,000 IU of vitamin A in an emulsified preparation maintained liver vitamin A stores above preinjection levels for only 30 to 35 days post- injection (PI). This was considerably less than that reported previously by Roberts and Stringam (1962) where vitamin A stores were maintained above preinjection levels for approximately 60 days. This disparity could be explained by relative differences in preinjection and maximum PI liver vitamin A levels of cattle used in those studies. In this present study the initial stores were 52 meg/gm and increased after injection to 88 mcg/gm fresh liver tissue. On the other hand, in the earlier experiment Roberts and Stringam (1962) observed levels of 9 mcg/gm and these increased to a maximum level of 29 meg/gm fresh tissue. As Guilbert 55 El. (1935) pointed out in their data, there was a tendency for the rate of depletion of vitamin A stores to decrease as depletion advanced and reserves became smaller. Ames t al. (1965) tested injectable preparations of vitamins A, D and E which contained 16 either vegetable oils or various polyoxyethylene derivatives as primary diluents. The vitamin A sources were the alcohol, palmitate ester and acetate ester. They found, as did Hentges gggl. (1951), that injectable preparations containing substantial amounts of vegetable oils had poor biological utilization. The maximum biological utilization of IM in- jected vitamin A was obtained with vitamin A acetate ester along with polysorbate 80 or certain other polyoxyethylene derivatives as absorp- tion promoters. The inclusion of d-alphaetocopheryl acetate in the in- jectable formulation increased the biological utilization of vitamin A. Absorption of VigaLmin A and Carotene. Reifman gt; _e_l. (1943) in working with rats found that the rate of absorption of vitamin A was proportional to the concentration of the administered material. No relationship was found between the rate of absorption of neutral fat and vitamin A. Barrick gt; _a_l. (1948) studied the absorption of carotene and vitamin A from the gastrointestinal tract of sheep. They found that relatively large amounts of vitamin A were required to cause a noticeable rise of vitamin A in the blood plasma. The change in the vitamin A content of the blood was much slower and less pronounced following the administration of carotene than in the case of vitamin A. No absorption was observed as a result of administering either vitamin A or carotene into the caecum or colon. Esh gg g1. (1948) reported that, in dairy animals, there was an indica- tion that lecithin enhanced absorption of vitamin A. Lecithin added to vitamin A increased vitamin A levels in colostrum and these levels re- flected those found in the plasma. Recent research, as sumarized by Lawrence g3 g1. (1966) and also found in Nutrition Reviews (1967), - —'~ fis‘ ‘ 17 indicates that prior to absorption the vitamin A esters are hydrolyzed in the gut by a digestive esterase, enter the mucosal cells as retinol which is primarily esterified with a saturated acid (mainly palmitic) in the microsomal fraction of the cell (Lawrence gg.gl., 1966). It is then transported via the lymphatics in the chylomicron fraction. Murray and Erody (1966) found that retrocvitamin.A acetate undergoes relatively little hydrolysis. It is absorbed, transported, stored in the liver and converted to vitamin A acetate. That this sequence is not very efficient is suggested by the low biological potency of retro-vitamin A. Huang and Goodman (1965) cannulated the thoracic ducts of rats and collected the chyle after administration of labeled vitamin A alcohol and beta-carotene. The lipid fraction‘was extracted and chromatographed on alumina columns. The washed chylomicrons contained 82% of the recovered radioactivity after administration of either retinol or beta- carotene. Retinyl esters were the prominent labeled compounds in all samples of lymph and contained approximately 90% of the radio-activity recovered after administration of the labeled retinol or highly purified beta-carotene. Small ammunts of labeled retinol were also recovered in all samples of lymph. The composition of the retinyl esters was ‘very constant regardless of the fatty acid composition of the test meal and regardless whether the retinyl esters were derived from retinol or from beta-carotene. Retinyl palmitate predominated in all samples and the saturated retinyl esters (palmitate and stearate in a 2:1 ratio) consistently comprised 2/3 to 3/4 of the labeled esters. Koizumi et al. (1963) using the intestine of rats, under anaerobic conditions, studied the metabolic pathways of beta-carotene, vitamin A aldehyde and vitamin A.alcohol, and demonstrated that terminal fission 18 0f beta-carotene can take place, yielding protein bound vitamin A aldehyde and beta-ionone. Under aerobic conditions the results obtained indicated that at the final step of vitamin A metabolism in the intes- tine, protein—bound vitamin A aldehyde might be converted to vitamin A alcohol and vitamin A acid. Based on electrophoretic and chromatographic separations of serum proteins, Glover and Walker (1964) showed that vitamin A was trans- ported after absorption by a specific protein carrier with properties, such as ionic mobility and isoelectric point, very similar to those of the copper carrying protein, ceruloplasmin, and according to these authors it behaved like an alpha-Z-globulin rather than an alpha-1- globulin as suggested by Veen and Beaton (1966). As a further check, Glover and Walker (1964) tested the livers of two sheep suffering from swayback, a disease associated with copper deficiency. One animal had moderate vitamin A reserves (354 IU/ng while the other one was low (26 IU/gm), but was not considered to be deficient. For this reason, the authors concluded that the copper deficiency and vitamin A status were unrelated. Preintestinal Destruction of Vitamin A and Carotene. In ruminants vitamin A is exposed to extensive microbial fermenta- tion in the rumen and reticulum and a changing chemical and enzymatic environment in the abomasum before reaching the primary site of absorp- tion in the small intestine. King g5 gl. (1962) conducted an in vitro and in vivo trial to determine the extent of carotene and vitamin A losses which occur in the rumen and reticulum. When vitamin A and caro- tene were incubated in vitro with rumen fluid, destruction was observed. When carotene was added to inoculated tubes, but not incubated, 97.5% 19 0f the carotene was recovered, whereas, carotene recovery after incuba- tion of the inoculated tubes was only 65.5%. Similar recoveries were obtained using vitamin A. When antioxidants such as ethoxyquin (l, 2- dihydro-6-ethoxy-2,2,4-trimethyl quinoline) or tocopherol were added to a comercially stabilized vitamin A preparation and incubated for ten hours losses were significantly reduced. In in vivo experiments using wethers which had been fed known amounts of carotene, vitamin A and Cr203, these authors found that after slaughter at 12, 24 or 48 hr following the meal, approximately 40% of the initial activity present had been lost at 12 hr, in addition to those losses resulting from passage into the lower tract. Klatte gg g1. (1963) injected 3 normal vitamin A and 3 depleted wethers IR with 250,000 IU of vitamin A acetate with Ween 80. Tim of the normal and two of the depleted sheep were surgically ligated immediately anterior to the pyloric valve before vitamin A injection. Blood sanples were collected initially and at 2, 4, 8, l2 and 24 hr after treatment. No consistent serum vitamin A increases could be ob- served in the operated wethers, while the unoperated wethers showed a marked increase in serum vitamin A. After 24 hr the contents of the rumen, reticulum, omasum and abomasum were removed from the operated wethers and analyzed for vitamin A. Fifteen to 26% of the injected vitamin A was recovered suggesting extensive preintestinal destruction of vitamin A in sheep. Resting gg g_1_. (1964) reported that the addition of ethoxyquin in vitro to rumen liquor collected from steers fed an all roughage ration positively influenced the retention of vitamin A, while the addition of 1% KNO3 affected adversely the retention of vitamin A with both a high 20 and low roughage ration. The destruction of vitamin A by rumen liquor from.steers on a high-roughage ration was significantly greater than‘with rumen liquor obtained from steers receiving a high-grain ration. Klatte l. (1964a, b) used ruminal and abomasal fluids obtained from dual- SE fistulated cattle and sheep on low pigment diets without vitamin A. Using 10 ml of water, ruminal, abomasal or autoclaved rumen juice, test tubes were either incubated after the addition of 20 IU of vitamin A oil dispersed in 20% Tween 80 or immediately analyzed for vitamin A content. The average percentage of vitamin.A recovered was 84% for water, 66.5% for abomasal juice, 63.9% for ruminal fluid and 87.8% for autoclaved rumen fluid. The results obtained with the autoclaved rumen fluid suggest that the destruction by rumen fluid is dependent on the microbial activity. The vitamin A activities recovered from steers and 'wethers were similar. In 1967 Mitchelliggmgl. used the chromic oxide technique to study the apparent in vivo destruction of vitamin A acetate anterior to the small intestine of steers. Two dual-fistulated steers were used and the ration fed contained KN03. The preintestinal destruction of vitamin A‘was estimated by administering 1,000,000 IU of vitamin A acetate in 20 ml of 20% Tween 80 in distilled water and 20 gm of Cr203 through the rumen fistula. After 24 hr the abomasal contents were withdrawn through the permanent fistula. The change in the ratio of Cr203 to vitamin A.was used to estimate the percentage of administered vitamin A reaching the abomasumm The average recovery of vitamin A was 50.4% when nitrate was fed and 43.0% when no nitrate was fed. Carotene Utilization and Conversion. It was once believed, and stated as a fact bthorrison (1948), that w ‘.V_,-—._—.r -._,‘ mas-mi- .. - 21 the change of carotene into vitamin A occurs chiefly in the liver. In recent years, however, considerable data have accumulated indicating that the intestinal wall is the primary site of conversion. After extensive studies, Sexton ggigl. (1946) showed that no caro- tene could be demonstrated in the livers of rats after oral administra- tion of carotene suspended in plasma, in a lecithin-stabilized solu- tion or in cottonseed oil or after feeding a diet containing 25% of alfalfa for 14 days. Under these conditions, increased levels of vita- min A were observed in all livers. The intrasplenic injection of caro- tene caused the storage of large amounts of carotene which was not utilized by the rats, while vitamin A injected by the same pathway was utilized. These authors concluded that in the rat, carotene was trans- formed into vitamin A before reaching the blood stream and the site of transformation suggested was the intestinal wall. Ron and Thompson (1951), on the basis of evidence presented in an extensive literature review, concluded that carotene is transformed to vitamin A mainly in the intestinal wall and that it is carried from there by the lymphatics to the blood stream and finally to the liver. Klosterman gg g}. (1949) had shown that intravenously injected carotene was rapidly removed from the blood stream, but it was not converted to vitamin A since no increase of vitamin A could be found in the plasma or liver. Lambs when given carotene orally or allowed green grass for a short period showed increased serum vitamin A levels. These observa- tions, together with the fact that no measurable amount of carotene was found in the blood of normal sheep, suggested that carotene was con- verted to vitamin A by sheep during digestion or absorption rather than by the liver. _‘W‘kp Jo certain improve demonst intestiu rats win marked i could be of care: marked 1 the mese testinal into vit made ex: found th Pancrea: proximal Here was} exelusive Va11 rat} °f Cenvez Vhi] that the: Blaed 0n “itemin A need“ ‘ ( H°1lte1n 22 Johnson and Bauman (1947) in studying depletion and utilization of certain carotene isomers in rats found that the retention of vitamin A improved when food intake was restricted. Alexander and Goodwin (1950) demonstrated conversion of carotene to vitamin A in rats in either the intestine or the intestinal wall. Oral administration of carotene to rats with the intestinal lymphatic vessels cannulated resulted in a marked increase in vitamin A content of the lymph while no carotene could be observed. Swick ££.£l' (1952) reported that when large doses of carotene were fed to pigs 6 to 7 hr before slaughter there was a marked increase (up to 20 fold) in the concentration of vitamin A in the mesenteric lymph with a smaller rise in the blood plasma and in- testinal wall. According to the authors, carotene thus was converted into vitamin A in the intestinal wall of the pig. Thompson _3 g1. (1950) made extensive studies of intestinal conversion in rats and pigs. They found that very little conversion took place until after the bile and pancreatic juice entered the intestine, with the peak conversion just proximal to the middle of the intestine. When the intestinal contents were washed out from the living intestine, vitamin A appeared almost exclusively in the wall of the intestine, indicating conversion in the wall rather than in the contents of the digestive tract. The efficiency of conversion of carotenes increased with the state of dispersion. While studying the conversion of carotene to vitamin A, we find that there are not only differences in species, but also between breeds. Based on the carotene intakes required to maintain borderline plasma vitamin A concentrations, Elliot (1949) observed that Guernsey calves needed a daily intake of 100 mcg of carotene per lb of body weight and Holstein calves only 60 mcg, or a requirement for the Guernseys 1.7 23 times that for Holsteins. Eaton.g£‘gl. (1959), in a similar study, found that Guernseys had higher concentrations of carotenoids in the plasma and liver than Holsteins. Conversely, Holsteins had higher con- centrations of vitamin A. They calculated that Holsteins converted carotene to vitamin A 1.4 times more efficiently than did Guernseys, when using plasma vitamin A as the criterion. There is evidence indicating that cattle fed deficient carotene intakes for extended periods of time, or cattle fed carotene-free rations and exhibiting low plasma and liver vitamin A concentrations do not appear to utilize carotene as efficiently as cattle fed rations adequate with respect to carotene intake, probably because of irrevers- ible bone pathology. Diven and Erwin (1958) showed that wethers having an average initial plasma vitamin A value of approximately 25 meg/100 ml, and given a single IR dose of 6 mg of carotene dispersed in water, had considerably greater increases in liver concentrations of vitamin A than deficient sheep with average initial values of approximately 10 mcg/lOO m1 of plasma. An increase of 10 meg/100 ml of plasma vitamin A concentration was found in both the normal and the deficient sheep. Grifo g5 g}. (1960) using cerebrospinal fluid pressures greater than 120 mm of saline as indicative of vitamin A deficiency, found that the feeding of as little as 12 mcg of carotene per 1b of live weight per day for 4 weeks, to Holstein calves did not result in deficiency. How- ever, extending this period to 16 weeks resulted in deficiency of such severity that subsequent feeding of 60 mcg carotene intake for 12 weeks, approximately twice the necessary intake to prevent elevated cerebro- spinal fluid pressure, did not result in the pressures returning to the normal level of 120 mm or less. 24 Many authors have shown that feeder steers are depleted of liver Vitamin A stores while consuming corn silage rations supplying carotene considered to be in excess of requirements. Deficiency symptoms may appear because either the corn silage carotene is poorly utilized, the expenditure of vitamin A is increased with silage rations or the feed- ing of silage somehow affects adversely the steer's capacity to main- tain liver vitamin A reserves. Jordan gg g1. (1963) studied the vitamin A nutrition of beef cattle fed corn silage. They found that feeder steers were depleted of liver vitamin A stores while wintered on corn silages containing carotene in levels considerably in excess of supposed needs. Vitamin A deficiency appeared even though the steers were supplemented with 7000 to 8000 IU of vitamin A palmitate per head per day. When later these steers were fed 32,000 IU of vitamin A during the final supplementation period the liver vitamin A stores failed to im- prove significantly unless the steers were supplemented with triiodothy- ronine. The results obtained by Klosterman g5 g1. (1964) when they studied the utilization of carotenes by steers fed corn silage were considerably different. After a depletion period of 137 days, 56 steers were fed a corn silage ration for 126 days. Blood plasma carotenoid and vitamin A levels quickly returned to normal after the silage rations were fed and supplemental vitamin A had no measurable effect. In another trial, non-depleted steers were fed silage rations for 182 days followed by a full feed of ground ear corn for 56 days. Feeding of 20,000 IU of vitamin A per head daily brought about no improvement in performance of carcass value. However, plasma vitamin A values of non-supplemented steers declined when the ear corn ration fed contained suboptimum 25 amounts of carotene. Similar results were obtained by Miller _g‘_l. (1967) who studied the utilization of carotene by dairy calves which were fed corn silage. They obtained linear increases in plasma vitamin A with increasing levels of dietary carotene. This was accompanied by a linear decrease in cerebrospinal fluid pressure. Thus, their data suggested that corn silage is as effective as dehydrated alfalfa leaf meal as a carotene source for maintaining normal cerebrospinal fluid pressure in Holstein calves. Storage and Stability of Vitamin A and Carotene Stores. During periods when the carotene or vitamin A intake is high, animals have the ability to store large amounts of vitamin A and carotene to help them weather periods of low intake, such as the winter months or during prolonged periods of drought. Maynard (1951) stated that 67 to 93% of the storage occurs in the liver, with storage also occurring in other areas of the body such as the depot fat and kidneys. In studies with rats, Davies and Moore showed that the adult rat is able to store, with massive doses, enough vitamin A in its liver to supply its theoretical requirement for a century, but these superfluous stores are eliminated at a very rapid rate until a state of stable storage is reached. There is some evidence indicating that in storage, as in absorption, an aqueous media may be superior to an oily media. Sobel gg‘gl. (1948), in experiments with rats, demonstrated that oral vitamin A was more effective when dispersed in aqueous media, as measured by liver storage than oily solutions. Sobel and Rosenberg (1950) reported that in rats, orally ingested vitamin A in an aqueous dispersion was more effectively transferred to milk and stored in the suckling than vitamin A in oil 26 solution. Braun (1945a) presented data which suggested that during periods of low vitamin A intake, first the stores of carotene are con- verted to vitamin A, thus decreasing the liver carotenoid levels without decreasing the vitamin A levels, and consequently, the A/carotene ratio rises rapidly. There was no correlation between carotenoid and vitamin A values for the liver and those for the blood under normal conditions. Only upon rapid depletion and when liver vitamin A concentration was below normal were these levels reflected in a low plasma concentration. Johnson and Baumann (1947) while feeding rats the low carotene level of 35 IU per day, found that more vitamin A was stored in the kid- ney than in the liver; at higher levels of intake more vitamin A appeared in the liver than in the kidney. The quantity of dietary vitamin A required to maintain liver vita- min A levels in farm animals (sheep, cattle, swine) is of interest. Guilbert g5 a1. (1940) indicated that 5 to 8 mg of vitamin A per kg of body weight was sufficient to prevent vitamin A deficiency symptoms, and that 3 to 5 times that level was required for storage. Hale _£.él- (1962) reported that 40,000 IU of vitamin A daily would maintain initial vitamin A levels (98 mcg/gm of fresh liver) in fattening steers for 168 days. Roberts and Phillips (1962) noted that higher vitamin A consump- tion (72,000 IU of vitamin A daily) did not maintain lower vitamin A concentration in the liver (73.3 meg/gm fresh liver) during 112 days. Liver vitamin A stores are widely used to evaluate the vitamin A status of farm animals and their ability to withstand short term de- ficiency without deleterious effects. Several workers studied the stability of these stores when the total vitamin A intake was not changing. Mitchell g5 g1. (1967b) studied the stability and turnover 27 rates of vitamin.A stored in the liver in 6 crossbred lambs which, prior to an IV injection of 4.8 mg of labeled vitamin A acetate, had been fed green leafy alfalfa hay for 60 days. Despite the_gg 1322529 feeding of the alfalfa, a continuous decrease in vitamin A stores occurred during the 75 days following treatment. This depletion was stopped by vitamin A supplementation. The liver was sampled by aspira- tion biopsy after 5, 33 and 61 days and then at 14 day intervals until 187 days after treatment. Liver vitamin A reserves decreased from 260 meg/gm of liver at 5 days to 89 meg/gm at 89 days. The radioactivity decreased a proportionate amount during this period. A half-time of 75 days was calculated from the decrease in the specific activity of liver vitamin A between 89 and 187 days. The detection of radioactivity in all blood samples supported the conclusion that there was a continuous turnover of vitamin A stores. A similar study was carried out with 6 Hereford steers by Hayes 2£.El- (1967). The steers received 125 mg of labeled vitamin A acetate IV and blood samples were collected at 5 and 30 min and at 1 and 6 hr PI of the labeled vitamin A acetate as well as before every liver biopsy. The first liver biopsy was performed 9 days after injection and then at 3 week intervals. From their data it can be seen that the liver vitamin A concentration after the first liver biopsy 9 days PI (62.2 meg/gm) remained relatively unchanged during four subsequent biopsies at 3 week intervals. After 93 days the liver vitamin A concentration was 59.4 meg/gm. The specific acti- vity of vitamin A declined in a linear fashion from the ninth day to the 93rd day. Half-time was calculated to be 48 days. Radioactivity 'was detected in the blood throughout the 93 day experiment which indi- cated a continuous turnover of liver vitamin A. 28 Interrelationshi Between Vitamins A D E and K. Kohlmeier and Burroughs (1962) have studied the use of supplemental vitamins A, E and K for beef cattle and found, when supplemental vitamin A was injected or fed to cattle in recovery experiments with animals known to be essentially depleted of vitamin A body stores, there were improved liveweight gains and feed conversion per unit of gain. Despite this improvement, the performance of cattle during recovery was inferior to similar cattle not depleted of vitamin.A body stores. Similar effects, during recovery in depleted Holstein calves, were observed by Grifo g3 'g1. (1960) using carotene. Perry‘ggmgl. (1964) were unable to show sig- nificant differences in weight gains between steers receiving 20,000 IU of vitamin A orally per head daily and those which received 4,000,000 IU injected IM as compared to control animals which received none. The feeding of vitamin.E at a rate of 200 mg per day per animal did not affect gains; supplemental vitamin K at 25 mg per head per day alone or in combination with vitamin E significantly depressed gains and feed efficiency. Kohlmeier and Burroughs (1962), feeding supplemental vitamin A to beef cattle, found improved liveweight gains, but these were only half as large as when vitamins E and K were included. Perry g§.gl. (1965) again studied the effects of vitamins E and K, alone and in combination, and found no effects on gains and feed efficiency when compared with the gains obtained by feeding vitamin A alone. Supplemental E and K had no effect on plasma carotene and vita- min A levels, while supplemental vitamin A and dehydrated alfalfa meal increased carotene levels. A possible explanation for some of the con- tradictory results obtained with beef cattle when vitamin K was included 29 in the ration could be that at low vitamin A intakes no antagonism be— tween vitamins A and K occurs while at higher intakes this antagonism becomes apparent and causes a depression in weight gains. Wostman and Knight (1965) used the germ free rat to study this possible antagonism. When vitamin A intake exceeded the optimum level by a factor of 10 or more, the feeding of 2000 IU/day caused the appearance of antagonism. Rats receiving a daily dose of 0.5 mcg of vitamin K plus 2000 IU of vitamin A had a shorter lifespan than the controls and they died with all the symptoms of the hemorrhagic syndrome typical of vitamin K de- ficiency. The effect of supplemental vitamins A, E, carotene and dehydrated alfalfa meal was studied by Beeson gg g1. (1962) who found that the daily addition of 0.5 lb of dehydrated alfalfa meal pellets per steer resulted in highly significant increases in daily gains when compared to lots not receiving alfalfa meal. The control lots receiving either no vitamin A or only vitamin E developed vitamin A deficiency symptoms. Final serum vitamin A values were lower than initial ones, but only those lots which showed deficiency symptoms had critically low serum levels, based on the levels suggested by Davis and Madsen (1941) of 16 meg/100 m1 of plasma. E The effect of administration of vitamins A and E on cattle, in- jected or fed orally, singly or in combination, were evaluated by Chap- man‘ggigl. (1965) who found that both vitamins stimulated gains, with vitamin E being more consistent. Oral vitamin E stimulated gains more than did the injected vitamin, suggesting that there was a possible ruminal effect of this vitamin. Newland gglgl. (1966) also studied the effects of injectable vitamins A and E in beef cattle, alone and in 30 combination and compared them to the corn oil carrier which served as control. Combining all data, vitamin A supplementation significantly increased liver vitamin A values over those injected with corn oil alone, while the response to vitamin E was nonsignificant. No signifi- cant increases in daily gains were obtained with either treatment. O'Donovan g£.gl. (1966) not only studied the interrelationships between A and E but also investigated the efficacy of the IM and IR routes of administration in beef cattle. They found that serum and liver vitamin A values increased rapidly after treatment, peaked at 2 weeks and then gradually decreased. Vitamin E had no effect on serum or liver vitamin A levels. Liver vitamin A storage lasted twice as long when vitamin A was administered IM.as when given IR. Chapman gg g1. (1964) studied the effects of supplemental vitamin A and E when steers were fattened on pasture. The inclusion of 25,000 IU of vitamin A significantly increased the rate of gain of steers during two winter periods but not during a summer trial. Vitamin E was inconsistent in improving gains during winter, since in one trial sig- nificant increases were obtained, while in another no differences were observed. Neither vitamin.A nor E had a significant effect upon steer performance during a summer feeding trial. According to the authors, the response to the vitamins appeared to be related to the level of carotene and KNO3 in the forage. Supplemental vitamins A or E, in- creased vitamin A and copper in the liver tissue. The growth response obtained by combining vitamins A and E was not as good as when fed separately. EXPERIMENT I Materials and Methods The sources of vitamin A activity used in this study were as follows: Natural carotenes: These were supplied by corn silage and the total carotene content was 8.12 mg/kg of fresh silage. This level was deter- mined by adapting AOAC (1965) procedures. Five gm of fresh silage were homogenized in 100 m1 of acetone using a Virtis mixer. Suitable aliquots of the pigments were extracted in 5 m1 of hexane and placed on Hyflo Super Cel:Sea Sorb 43 (111) columns (10 x 1.8 cm) followed by elution ‘with 10% acetone in hexane. The eluant'was appropriately diluted and the optical density measured in a Bausch and Lomb Spectronic 20 spectro- photometer at 440 mu. Retinyl palmitate: This material was obtained commercially as a vitamin A feed supplement. It contained 358,500 IU all-trans retinyl palmitate/ gm plus edible tallow, gelatin, glucose, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and soybean feed. Particle surfaces ‘were treated with sodium silico aluminate. Feed and feed premixes: The corn silage was harvested, finely cut and ensiled at 29.1% dry matter in a concrete silo. The average dry matter content of the silage at feeding time was 28.1%. This corn silage was supplemented with 0.16 kg/lamb/day of a protein-mineral mix which con- sisted of 93.7% dehulled solvent soybean meal, 1.8% limestone, 4.5% trace mineral salt andl gm irradiated yeast (0.22 gm ergocalciferol/kg) and was calculated to be isonitrogenous and isocaloric on a dry basis when compared with the pelleted repletion ration. The depletion ration consisted of 40% ground soft winter wheat, 41% ground corn cobs, 14% de- hulled solvent soybean meal, 3.75% molasses, 0.5% trace mineral salt, 31 32 0.5% limestone, 0.25% yellow grease and 1 gm irradiated yeast. The vitamin A feed supplement was diluted to appropriate premix concentra- tions with ground soft winter wheat and these premixes were added to the modified vitamin A depletion ration to supply 0.52, 1.55 and 4.65 mg of retinyl palmitate/kg of diet. Both the depletion and the repletion diets were pelleted. Experiment: Sixty-one Western lambs, averaging 22.6 kg initial‘weight, ‘were fed the pelleted vitamin Ardepletion ration 59,1ibiggg for 110 days. The lambs were housed in four 4 x 7 m pens and had free access to water. Blood samples were drawn from the jugular vein at the beginning of the depletion period and thereafter at 35, 70, and 110 days. The serum was frozen and stored at -20°C until analyzed for carotene and vitamin A. No vitamin.A-deficiency symptoms were observed during the depletion period, but five lambs were killed due to poor performance. Upon post- mortem examination it was found that two had suffered from hemorragic enteritis, one exhibited white muscle disease and the other two exhibited no detectable pathology. The remaining 56 lambs were allotted at random to one of four repletion diets. Lot 1 received corn silage gd_libitum plus the protein-mineral supplement, while lots 2, 3, and 4 received the pelleted repletion diet which consisted of 45% ground corn cobs, 40% ground soft winter wheat, 12% dehulled solvent soybean meal, 1.72% cane molasses, 0.5% limestone, 0.5% trace mineral salt, 0.28% lard and 1 gm irradiated yeast, supplemented with 0.52, 1.55 and 4.65 mg of retinyl palmitate/kg, respectively. An attempt was made to provide similar dry matter intakes for the four lots by restricting the pellets offered to lots 2, 3 and 4 to the dry weight of silage consumed by lambs in lot 1. This was impossible, however, because the lambs fed the pelleted rations began.to eat their straw bedding when the amount of feed offered was restricted to less than 1.75 kg/lamb/day. The pellet-fed lambs thus consumed 56% more dry matter than the lambs fed silage. The average daily consumption of carotene from silage per lamb in lot 1 was 31.5 mg‘while the average number of mg of retinyl palmitate consumed per lamb/ day in lots 2, 3 and 4 were 1.1, 3.3 and 9.9. Blood samples were ob- tained at 35 and 70 days after the start of the repletion period, the serum was frozen and stored at -20°C until analyzed for vitamin A and carotene. On the 7lst day of repletion, 53 lambs were slaughtered (3 suffocated while in transit to the slaughterhouse), their livers ob- tained, weighed and samples taken from the ventral lobe starting at the umbilical fissure excising in a downward direction, for vitamin A assay. Analytical_procedures: Serum vitamin A concentrations were determined using the antimony trichloride method according to Embree (1957). Carotene was measured in the petroleum ether extract of the serum at 440 mu and compared against a petroleum ether blank. Liver vitamin A concentrations were determined according to the method of Gallup and Hoefer (1946) with the following modifications! liver slices were homogenized in 45 ml of freshly prepared 9% ethanolic potassium hydroxide for 3 min in a Virtis mixer, diluted quantitatively to 100 ml with dis- tilled water and after complete mixing, duplicate 10 ml aliquots were taken. These were extracted by shaking for l min in 10 ml of petroleum ether (30-60°C b.p.) and centrifuging for 1 min. Appropriate aliquots 'were taken from the petroleum ether layer and carotene was measured against a petroleum ether blank at 440 mu. These aliquots were then evaporated in a vaccuum.oven at 55°C, the dried samples were dissolved in 1 ml of chloroform, 2 drops of acetic anhydride were added and the WHW- . r..._—=-n ‘1 “mars fi-J mixture reading were re Statist: polynom: Results The used are Table 1a Inil Fin: Ave: 1" 2 Number: Feed e) Table 1b, \ Initial t WEIghtl “Ml bod ks Amiga d 1.32 1 2 Sign“ Feed e 34 mixture reacted with 2 m1 of antimony trichloride. The maximum stable readings at 620 mu in a Bausch and Lomb Spectronic 20 spectrophotometer were recorded. Statistical analysis: Mean differences were compared using nonorthogonal polynomial coefficients according to Li (1964). Results and Discussion The effects of depletion and repletion upon‘weight gains and feed used are shown in Tables la and 1b. The differences in weight at the Table la. Weight gain and feed consumption of lambs fed a vitamin A free diet for 110 days. Initial body weight, kg 22.6 (61)1 Final body weight, kg 41.4 (56) Average daily feed, kg2 1.42 Numbers in parenthesis indicate number of lambs. Feed expressed on 100% dry matter basis. Table 1b. Weight gain and feed consumption of lambs fed rations con- taining carotenes or retinyl palmitate for 70 days. Lot 1 Lot 2 Lot 3 Lot 4 C. Silage Basal + 1.1 Basal-+ 3.3 Basal + 9.9 & Protein mg Retinyl mg Retinyl mg Retinyl Suppl. Palm/day Palm/day Palm/day Initial body ‘weight, kg 39.8 40.0 43.3 42.1 Final bod weight 1 ks y ' 47.1 48.4 50.41 50.5 Avera e dail feed ks28 y ' 1.24 1.93 1.93 1.93 1 Significantly greater (P (.01) than lot 1. Feed expressed on 100% dry matter basis. F— end of repleti lots 1 but not initial pletion The fact differen appears and 0.52 normal 94 Weight. The tratmn a Table 2a_ \ Tm. 2b, 35 dais 70 day. 2 31mm 3 31mm ignisich 35 end Of the depletion period were not significant. At the end of the repletion period no significant differences in weight were found between lots 1 and 2, but lots 3 and 4 were significantly different from lot 1 but not from lot 2. These differences may have been due to the higher initial weight of lots 3 and 4 over 1 and 2 at the beginning of the re- pletion period. Lots 1 and 2 gained more efficiently than lots 3 and 4. The fact that lot 1 did not gain more than lot 2 is attributed to large differences in dry matter intake between these two lots. Thus, it appears that 8.12 mg total carotene as supplied by 1 kg of corn silage and 0.52 mg/kg retinyl palmitate in the feed are adequate to support normal weight gains of vitamin A depleted lambs from 40 to 50 kg body weight. The effect of depletion and repletion upon serum vitamin A concen- tration are shown in Table 2a and 2b. Serum vitamin A decreased from Table 2a. Serum vitamin A concentration of lambs fed a vitamin A free diet for 110 days. EEIUQéELlEL Initial 28.3 70 days 23.6 110 days 20.0 Table 2b. Serum vitamin A concentration of lambs fed rations containing carotene or retinyl palmitate for 70 days (mcg/lOO ml). Lot 1 Lot 2 Lot 3 Lot 4 C. Silage & Basal + 1.1 Basal + 3.3 Basal + 9.9 Protein mg Retinyl mg Retinyl mg Retinyl Suppl. Palm/day Palm/day Palm/day 35 days 41.1 20.8 32.8 49.7 70 days 41.61 20.7 42.21,2 48.33 1 Significantly greater (P <1.01) than lot 2. 2 Significantly greater (P <:.05) than lot 1. Significantly greater (P <:.01) than lot 1. 28.3 days vitam the r: ugd: reach the pa in lot lot 3 1 from 1: Sponse the rep showed 1 tion anc by linea Vitamin . and tend: Concentra replerion depletion from the 0f the de SEIeCted "3 818m kn 3‘Has 36 28.3 to 20.0 mcg/lOO ml during 110 days of depletion. At the end of 35 days of repletion lots 1, 3 and 4 showed significant increases in serum vitamin A concentration. These levels did not increase further during the remaining 35 days of repletion, except in lot 3. Lot 2 showed a non- significant increase of serum vitamin A concentration which did not reach predepletion levels. When the silage-fed lambs were compared with the pellet-fed lambs it was found that the serum vitamin A concentration in lots 1 and 4 were significantly (P4<:.005) greater than in lot 2. In lot 3 the serum vitamin concentration was not significantly different from lot 1, but significnatly (P<::.005) greater than lot 2. The re- sponse of serum vitamin A concentration to treatments was linear during the repletion period at 35 and 70 days for lots 1, 2 and 4 while lot 3 showed a significant (P<::.005) quadratic response at 70 days of reple- tion and the treatment by period within group interaction was quadratic by linear (P'<:.05). This is a reflection of the fact that the serum vitamin A concentration in lots 1, 2, and 4 reached a maximum at 35 days and tended to decline at 70 days, while lot 3 continued to increase in concentration up to the end of the repletion period. The effects of repletion on liver vitamin A are shown in Table 3b. The level of liver depletion can be judged only on the basis of the liver values obtained from the animals sacrificed for reasons of performance prior to the end of the depletion period (Table 3a). These animals were, of course, not selected at random. The liver vitamin A stored at the end of the reple- tion period reflects the treatment effects. The lowest average liver vitamin A concentration was found in lot 2 (3.1 meg/gm fresh liver) and ‘was significnatly (P‘<:.01) different from either lot 1 or lots 3 and 4. Lot 3 was also significantly (P‘<:.01) lower than lots 1 and 4 but higher Table 3 In 71 Table 3% Fresh, m. Dry, 1mg, TOtsl, m! ._.______ 1313mm 2 Signif than lot There Wag t“"3911 lot respectix Pattern a‘ min A sto ”03 and linear. lots 1’ 2 IOt 4 was that: 10t 37 Tdble 3a. Liver vitamin A stores of lambs fed a vitamin A free diet for 110 days. Fresh Dry Total mcg/gm meg/gm mg/liver Initial value (3) 5.45 27.5 2.53 71 days depletion (l) 1.16 4.70 0.66 Table 3b. Liver vitamin A stores of lambs fed rations containing caro- tenes or retinyl palmitate for 70 days. Lot 1 Lot 2 Lot 3 Lot 4 C. Silage Basal + 1.1 Basal + 3.3 Basal + 9.9 & Protein mg Retinyl mg Retinyl mg Retinyl Suppl. Palm/day Palm/day Palm/day Fresh, meg/gm 19.9 3.11 12.42 24.6 Dry, meg/gm 69.9 9.8 42.3 79.1 Total, mg/liver 12.3 1.86 8.40 15.4 1 Significantly lower (P <:.01) than lots 1, 3 and 4. Significantly lower (P <:.01) than lots 1 and 4. than lot 2, with an average concentration of 12.4 meg/gm fresh liver. There was no significant difference in concentration of vitamin A be- tween lots 1 and 4 which averaged 19.9 and 24.6 meg/gm of fresh liver, respectively. The total vitamin A stored (mg/liver) followed the same pattern and the correlation between concentration and total liver vita- min A storage was r = .96. The response of liver vitamin A concentra- tion and total vitamin A storage in the liver due to treatments was linear. No significant differences due to treatments were found between lots 1, 2 and 3 Which averaged, respectively, 29.9, 30.7 and 29.6%, but lot 4 was significantly (P <:.025) higher in dry matter, with 31.1%, than lot 1. 38 To estimate the conversion efficiency with which the carotenes present in corn silage were converted to vitamin A stored in the liver, the regression of total liver vitamin A stored on dietary retinyl palmi- tate intake was calculated. The regression equation fitted to the data by the method of least squares (Figure 1) is as follows: ‘9 = 2.2693 + 1.3120 X where Y = mg vitamin A/liver and X = mg of all trans retinyl palmitate/lamb/day. Using total vitamin A stored after 70 days of re- pletion as the criterion, the relative biopotency of carotenes present in corn silage was 26.2% when compared with the efficiency of conversion of beta-carotene to vitamin A by the rat. This was calculated as follows: 31.5 mg total carotenes consumed by lambs/day corresponded to a liver storage of 12.3 mg retinal. Using the regression equation, such a storage is obtained by feeding 7.6 mg of retinyl palmitate/lamb/ day. Thus, 4.15 mg of total carotenes are equal to 1 mg retinyl palmi- tate or 1 mg total carotene = 438 IU of retinyl palmitate. The data presented here indicate that 1 mg of total carotene from corn silage is equivalent to 438 IU of all trans retinyl palmitate for the support of a normal liver storage of vitamin A in the depleted lamb from 40 to 50 kg of body weight. This conforms to the relationship currently suggested by the N.R.C. that "this value seems to vary from between 400 and 550 IU of vitamin A activity per mg of carotene". 1.1. 1.41%. 8 l 6 l 4 l 2 l 0 8 an ¢m>.a\< 2.21h; ME 4 2 "I! H u—s 3"" a as on s—s N mg VITAMIN A/llVER ‘19 ah N 0 2 4 6 8 10 12 m2 RETINYL PALMITATE/lAMB/DAY Figure l Material The follows: M1. aration palmitat acetate ‘ oxidants total ca: E! A ratio“ c and supp ‘dJuated receind . tained 1 (0.32 kg gm calci vitamin 1 EXPERIMENT II Materials and Methods The sources of vitamin A activity used in this study were as follows: Vitamin A palmitate: This material was an.experimental injectable prep- aration in a water miscible base. It contained 571,000 IU of retinyl palmitate, 50,000 IU of ergocalciferol and 50 IU of alphatocopherol acetate per m1 plus 2% benzyl alcohol as well as BHT and BHA as anti- oxidants. Natural carotenes: These were supplied by corn silage and the average total carotene content (AOAC procedure) per silo was as follows: Silo mg carotene/kg corn No. silagefresh basis 1 4.38 2 4.63 3 11.99 4 15.03 10 4.42 11 4.33 E333: All lots were fed a completely mixed ration twice daily. The ration consisted of the apprOpriate corn silage, rolled shelled corn and supplement fed at the rate of 1% of body weight. The amount was adjusted every 4 weeks according to the average lot weight. Each steer received 0.45 kg/day of M80 supplement 64. Each kg of supplement con- tained levels of additives adequate to supply 0.64 kg of crude protein, (0.32 kg from soy and 0.32 kg crude protein equivalent from urea), 44 gm calcium, 34.1 gm phosphorus, 165 mg chlortetracycline, 19,800 IU vitamin D2, 110 gm trace mineral salt and 22 mg stilbestrol. 40 .4211}. ment I Elm weight to det the qu weight at ram to; The “any" . t0 the h( Analytical procedures: ment I for corn silage. Experiment: ‘weight, were fed the appropriate to determine the effect of stage 108 choice Hereford 41 These were similar to those described in Experi- steer calves, averaging 244.5 kg initial silage plus the supplement for 180 days of maturity and fineness of chop on the quality of corn silage. The cattle had been randomly assigned by ‘weight to 12 lots of 9 head each at random as follows: Lot No. l3 14 15 16 17 18 19 20 21 22 23 24 Harvest Date Nov 14 Sept 15-16 Oct 17-18 Nov 15 Oct 17-18 Nov 14 Oct 19-20 Sept 13-14 Sept 15-16 Sept 13-14 Oct 19-20 Nov 15 'Qggree of Chop Coarse Fine Fine Fine Fine Coarse Coarse Coarse Fine Coarse Coarse Fine and treatment combinations were assigned Preparation at Feeding_ as ensiled as ensiled as ensiled as ensiled reground reground reground as ensiled as ensiled as ensiled as ensiled reground The vitamin A stude was superimposed on the above experiment by arranging the allotted steers by weight outcome groups from the lightest to the heaviest as can be seen in the following table: Table 4. Weight outcome groups. A 3 x 3 factorial design with 12 replicates and a covariate of Lightest Heaviest Lot 1%, 2 3 4 5 6 7 8 9 15 1139 1104 1168 1127 1329 1346 1215 1208 1194 23 1288 1305 1117 1321 4071 1284 1187 1120 1114 147D 35D 63D 16 1155 4110 1158 4195 1131 1207 1197 849 1167 35D 632_ 1479. 20 1191 1221 824 1270 1105 831 1126 1134 1153 63D 35D 147D 17 1181 1212 1302 1141 1217 847 699(0) 1237 1304 147D 63D 35D 24 1320 1118 1202 1124 1144 4223 1160 1218 4231 35D 63D 147D 19 1184 839 1291 657 825 685 1190 4156 1229 63D 147D 35D 21 1328 1222 1334 1163 1322 1135 1300 1192 4103 147D 63D 35D 18 1183 1228 4242 1166 1162 4217 733 1156 1177 35D 63D 147D 14 814 787 1227 852 1204 697 811 1106 722 147D 35D 63D 22 1230 1178 1220 4209 1169 4240 1256 815 1224 630 147,, 351, 13 808 1317 1138 1152 1186 1129 1150 703 1272 63D 35D 147D l 2 3 4 5 6 7 8 9 Treat . -C IN IR IR C 1M 1M IR C initial weight was used for treatments while time was assigned at random. The treatments were imposed uniformly within each of the 12 lots and an example (lot 17) follows: 147 days antemortem No. l - saline placebo injected intramuscularly (IM), and intra- ruminally (IR). N. T! 11.4 cm below th tion. 1‘ time by BL drawn fn meat. tene and 'ymPtOms only Via: Cau be 86 tion; We: lacrificE fr” the Ital-ting 43 No. 2 - 7,000,000 IU vit. A IM and saline placebo IR No. 3 - 7,000,000 IU vit. A IR and saline placebo IM 63 days antemortem No. 4 - 7,000,000 IU vit. A IR and saline placebo IM No. 5 - saline placebo injected IM and IR No. 6 - 7,000,000 IU vit. A IM and saline placebo IR 35 days antemortem No. 7 — 7,000,000 IU vit. A IM and saline placebo IR No. 8 - 7,000,000 IU vit. A IR and saline placebo IM No. 9 - saline placebo injected IM and IR The IR injection was made on the left side using a 16 gauge needle, 11.4 cm long, approximately 8 cm behind the last rib and about 10 cm below the edge of the loin, introducing the needle in a downward direc- tion. The precision of the needle location could be determined each time by the escape of ruminal gas. Blood samples for serum carotene and vitamin A analysis were drawn from the jugular vein at the beginning and the end of the experi- ment. The serum was frozen and stored at —20°C until analyzed for caro- tene and vitamin A. Only one steer showed mild vitamin A deficiency symptoms after 100 days on the experiment (steer 4103 in lot 21). The only visible symptom was a pair of very protruding eyes. One of these can be seen in the following picture. No further diagnostic determina- tions were done. On the 147th day of the experiment the steers were sacrificed and their livers were obtained, weighed and samples taken from the edge of the ventral lobe, excising in a downward direction starting at the esophageal notch for vitamin A and carotene analysis. a .1 ma.4 .1 . q... .. . — a. an; . div . .1. IL ...~....._EIL-l. . alert. a... _ .3. . _ a, 1. a .. . .._ . 2.1.5:??? . wt. Ill 45 The analytical procedures followed for liver vitamin A and caro- tene analysis were the same as those described for the sheep experiment. Criteria: It was recognized that the injections of vitamin A necessarily included vitamins D and E as well. The formulation of the preparation was designed to improve upon injectable products currently used at more frequent intervals because of poor absorbability. Vitamin E may affect the utilization of vitamin A or protect it from destruction. The presence of vitamin D probably has no bearing on vitamin A utilization. Only criteria related to vitamin A effects were measured. The primary criteria were liver vitamin A concentration and total liver vitamin A. Initial serum vitamin A levels were determined to provide some indica- tion of beginning vitamin A status. Final serum.vitamin A levels were analyzed for treatment effects. The silage used in this study was harvested on September 13 to 16, October 17 to 20 and November 14 to 15, 1966. On each harvest date 2 silos were filled, one with fine chop silage (1 cm) and one with medium chop silage (1 to 2 cm). The corn field was divided into eight row plots with two rows harvested in September, the adjacent two rows in October and the follow- ing two in November. To establish grain yield per acre the remaining two rows were harvested immediately following the November harvest as ear corn. Each load of silage was weighed and sampled. From these data, yield per acre, silo storage capacity and dry matter values were determined. Adverse weather conditions encountered while harvesting silages late in the fall may cause field losses as well as difficulty in harvesting. There was no frost prior to the September 13 harvest date. Between September 16 and October 20 (the end of the October har- Lawn—was}? ga' -'*r*_t-Ji T:- , _ 46 vest) freezing occurred on 5 different nights. There was no snowfall and the wind velocity reached 20.3 mph on one day. Between October 20 and November 15 freezing occurred on 14 of 26 nights. It snowed a total of 4 days with a maximum.accumu1ation of 23 cm. All snow had melted prior to the November harvest. Maximnm‘wind velocity reached 19.7 mph on one day during this time. Results and Discussion Although no significant differences in weight gains were obtained 'with the vitamin A treatments (2.79 and 2.81 1b/head/day) for the con- trol and injected groups, treatment resulted in highly significant differences in vitamin A.stores at the end of the experimental period (Table 5). Table 5. The effect of vitamin A injections 147 days antemortem on final liver vitamin A concentration. Control _l§_ _lfla Vitamin A meg/gm, fresh 7.561 36.22 80.3 Vitamin A meg/gm, dry 27.71 130.42 292.2 Vitamin A mg/liver 35.31 165.32 367 .7 ; Significantly lower (P <:.0005) than IR and IM. Significantly lower (P <:.0005) than IM.and higher than control. The control group which received no supplemental vitamin A had the lowest vitamin A stores with 7.56 meg/gm of fresh liver as compared to those which were injected with vitamin A either IR or IM (36.2 and 80.3 meg/gm). This relationship was true also when the storage was ex- pressed either on a dry basis or as total storage. The low vitamin A concentration found in the control group is considerably higher than the 47 stores found by Jordan _£ _1. (1963) after wintering steers on corn si- lage for 134 days, and those reported by Miller 25 51. (1967) in Holstein calves. The differences found between routes of administration (IR or IMD were highly significant (36.2 vs. 80.3 meg/gm, fresh liver) which indicated that the IM route was twice as effective in providing adequate liver storage as the IR route. This was probably due to the destruction of the vitamin A preparation by rumen microbial population as shown by Klatte st 31. (1964a, b). King 25 51. (1962) have found that the inclu- sion of antioxidants such as tocopherol significantly reduced preintes- tinal losses, while in their absence 40% of the initial activity was lost at 12 hr after treatment. The analysis of liver carotene storage showed that steers which had received supplemental vitamin A by either route, had significantly higher (P <:.0005) carotene concentrations on a fresh basis that did those which only received carotene from corn silage (1.1 vs. 3.4 and 3.7 mcg/gm fresh liver, for control, IR and IM, respectively) as can be seen in the following table: Table 6. The effect of vitamin A injections 147 days antemortem on final liver carotene stores. Control __I_R; __Igi_ Carotene meg/gm, fresh 1.11 3.4 3.7 Carotene meg/gm, dry 3 .91 12.3 13 .6 Carotene mg/liver 5.21 15.4 17.3 1 Significantly lower (P < .0005) than IM or IR. No significant differences were found in liver carotene concentration between those steers which received either the IM or IR injection of vitamin A. 48 As can be observed from the above table, the liver carotene of un- treated steers was only 1/3 that of the treated steers, which suggests that in order to meet the normal vitamin A requirements of the animal,a large proportion of the ingested carotene was converted to vitamin A. Since no classical vitamin A deficiency symptoms were observed (with possibly one exception) it can be assumed that the carotene supplied by all silages was sufficient to meet the dietary requirements for carotene. This is further substantiated by the average vitamin A serum con- centrations found which were within normal ranges, although the un- treated steers had significantly lower (P‘<:}002) serum vitamin A con- centrations than did the treated steers (32.8‘vs. 37.9 and 40.1 for IR and IM, respectively). The results of the effect of time of antemortem injections are shown in Table 7. Table 7. The effect of time and antemortem injections on final liver vitamin A stores in steers. 147 days 63 days 35 days antemortem antemortem antemortem Vitamin A mcg/gm, fresh 25.81 47.8 50.3 Vitamin A meg/gm, dry 93.91 170.3 183.2 Vitamin A mg/liver 121.41 218.9 228.6 1 Significantly lower (P <.0005) than 63 and 35 days antemortem. The final liver vitamin A concentration for the 147 day antemortem (AM) period was significantly lower than the 63 and the 35 day period, but based on the results obtained, the absence of vitamin A deficiency symptoms and the normal gains of these cattle, the injection of 7,000,000 IU of a water miscible preparation of vitamin A, plus vitamin D and E was adequate to maintain normal liver stores for at least 147 .‘ mg! 3. 1". ~Ild: a’ ”I :1 i3. -M'T~ "A 31". 49 days, if not for an even longer period of time. Roberts 25 21. (1965) have shown that the administration of 1,000,000 IU of vitamin A.in an emulsifiable preparation maintained liver stores above preinjection levels for only 30 to 35 days. How long preinjection levels of vitamin A.can be maintained depends not only on the magnitude of the injection, but also on the carotene intake of the animals (if any is present). Roberts 2; a1. (1962) observed preinjection liver levels of 9 meg/gm of vitamin.A which increased to a maximum.level of 29 meg/gm of fresh liver and were maintained for approximately 60 days. Since the preinjection liver vitamin A stores'were not known, the changes produced in liver stroage cannot be determined. Based on the initial serum vitamin A concentration, these stores did not appear to be deficient. The correlation between initial serum vitamin A and final liver vitamin A concentration (fresh basis) was 0.283 which was highly significant (P1<:.001). No significant differences were found in liver vitamin A stores between the 63 and the 35 day injection periods (47.8 vs. 50.3 meg/gm, fresh basis). The treatment x time interaction effects can be seen in Table 8. Table 8. The effect of treatment x time interaction on final liver vitamin A concentration. * Trix Trlx Trlx Trzx Trzx Trzx Tr3x Tr3x Tr3x T1 T2 T3 T1 T2 T3 T1 T2 T3 Vilfée::§lgm 7.61 7.71 7.31 16.61 37.72 54.22 53.22 98.13 95.93 V1t<firglglgm 28.01 28.31 72.81 69.01 142.02 197.32 194.72 346.23 325.63 Vitiivifi’ 35.21 37.61 34.61 77.61 176.92 241.32 231.92 442.13 409.93 1,2,3 'Means with different superscripts are significantly different (P <.0005) . A: . . x 1* a, .‘I I - x .' ‘a, . 1“. . _'-- -‘I_JT_ -—“ ' 50 There was no significant difference in liver vitamin A stores be- tween the control steers for all 3 treatment periods and the steers in- jected IR at 147 days AM, even though liver vitamin A concentration was higher for the latter. A highly significant difference was found for liver vitamin A stores between the first and second IR injection periods (16.6 vs. 37.7 meg/gm fresh liver), which indicates that an IR injection of 7,000,000 IU of a'water miscible preparation of vitamin A 63 days before slaughter is capable of producing liver stores in beef cattle ‘which are adequate to meet the requirements for vitamin A for at least 63 days. No significant differences were found between the 2nd and 3rd IR injection periods, although the stores for the last injection (35 days AM) were higher (37.7 vs. 54.2 meg/gm, fresh basis). The differences between the last IR injection period and the 1st IM injection period ‘were also nonsignificant (54.3 vs. 53.2 meg/gm, fresh basis), indicating that an IM injection of vitamin A in a water miscible base 147 days before slaughter produced liver vitamin A stores not only comparable to those produced by an IR injection 35 days before slaughter, but that the IM route of administration was 4.2 times more efficient than the IR route. No significant differences were found between the 2nd and 3rd IM injection periods although the 2nd period showed liver vitamin.A stores ‘which were slightly higher than those of the third period (98.1 vs. 95.9 mcg/gm, fresh basis). A possible explanation for this difference could be the greater fatness of the steers at the site of injection during the last injection period as compared to those injected 63 days AM. If the vitamin A “afi3! ____.P_‘ *flo-Wn..'. .l . ~ .~ L 1-: fl 4‘- 51 ‘Preparation'were partly lodged in a fat deposit, this would decrease the rate and efficiency of absorption of the preparation, since adipose tissue has fewer blood vessels than muscle tissue. In studying the correlations between the different variables analyzed, we find that some of these are highly significant as can be seen in Table 9. Table 9. Summary of simple correlation coefficients between variables. Carotene ISC ISVA FSC FSVA FLCF FLVAF Intake Initial serum caro- tene (ISC) 1.000 0.5381 0.8371 0.176 0.150 0.261 0.8061 Initial serum 1 1 1 1 vitamin A (ISVA) 1.000 0.519 0.404 0.191 0.283 0.542 Final serum caro- tene (FSC) 1.000 0.181 0.063 0.090 0.7901 Final serum vita- 1 min.A (FSVA) 1.000 0.275 0.187 0.134 Final liver caro- tene, fresh (FLCF) 1.000 0.4131 0.022 Final liver vita- min.A, fresh (FLVAF) 1.000 0.220 Ice <:.001) Contrary to the observations by Ralston and Dyer (1959), liver carotenoids were significantly related to liver vitamin A levels while there was no relationship between serum carotenoids and liver carotenoids as found by Diven.g£'gl. (1960). While the initial serum carotenoids ‘were significantly correlated with initial serum vitamin A, which con- firms the observations by Diven 25.21. (1960), there was no relationship between initial serum.carotene and final serum vitamin A, nor were hepatic vitamin A and plasma carotenoids significantly related. The initial high correlation between ISVA and ISCA changed to a .._.-—... - 'a.‘_"' ‘1. “'f " 52 nonsignificant relationship between these two variables at the end of the experimental period, not only due to some low daily intakes, but probably also due to the injection of vitamin A which possibly suppressed the absorption or the utilization of the available carotene. The following table reflects the average daily carotene intake by the different lots as well as the carotene content of the different silages. Table 10. Total carotene content of silages fed and average daily caro- tene intake by experimental steers. L25; Silo No. ggpgarotene/head/day 15 a 17 1 50.13 23 & 19 2 60.18 14 & 21 3 254.17 20 & 22 4 367.59 16 & 24 10 56.61 13 & 18 11 61.04 The calculated daily intakes of total carotene meet the minimum requirements set by the N.R.C. (1964). Based on the liver carotene con- centrations in the control animals and the fact that these animals did not develop vitamin A deficiency symptoms, the suggested carotene intakes seem to meet daily carotene requirements for fattening yearling cattle between 260 and 500 kg of body weight. The effect of harvest time on carotene content in corn silage can be seen in the following table. ~“A 4 53 Table.ll. Effect of harvest time on total carotene content of ingoing corn silage. mg carotene/kg corn M_c_3_r_i___tl'i Silo No. Chop silage (fresh basis) September 3 Fine 11.998‘ 4 Coarse 15.03b October 1 Fine 4.38 2 Coarse 4.63 November 10 Coarse 4.42 11 Fine 4.33 ‘ Significantly greater (P 2:.005) than silos 1 a 11. Significantly greater (P‘<:.005) than silos 2 & 10. From the preceding table we can observe that there was a signifi- cant decrease in carotene content from the September to the October har- vest, while the decrease between October and November was not signifi- cant. The decrease seemed to be directly related to the browning of the leaves of the corn plant. The percent dry matter increased from 28.21 in September to 48.15 in October to 59.55% at the end of the November harvest. The traces of carotene still found are those which are present in the lower portions of the stalk which may still be green and in the corn kernel. In the following table the effect of storage and degree of chop on the total carotene content of corn silage is summarized. Table 12. Effect of storage and degree of chop on the total carotene content of outcoming corn silage (fresh basis). mg carotene/kg corn si- Percent of ingoing Silo No. Chop lage (fresh basis) total carotene 3 Fine 7.701 60.05 4 Coarse 11.242 74.78 1 Fine 2.61 59.70 2 Coarse 3.12 67.38 10 Coarse 3.32 75.03 11 Fine 3.50 80.66 ESignificantly greater (P <:.005) than silos 1 and 11. Significantly greater (P.<:.005) than silos 2 and 10. .-——-, a View“ £4,511; .. .1 54 Trhe effect of storage on the total carotene content was very marked. ‘With exception of silo 4, the decrease in carotene content in all silos 'was significant. While there was no difference within each harvest period, a trend was established that suggested that degree of chap affected the carotene content of the outcoming silage. The carotene content for the l to 2 cm cut silage was higher than that of the fine cut silage (1 cm). This was true for silos 3 and 4 ensiled in Septemr ber. This may be explained by the type of fermentation occurring in each silo. In the coarsely cut silage there is less packing with more air trapped, but the particle surface area eXposed to oxidation is much smaller than that of the fine cut silage. Other factors not considered are the pH, the type and proportions of volatile fatty acids produced and the temperature changes which occur during fermentation, all of which may affect the total carotene content of corn silage. In silos 10 and 11 this relationship was inverted, that is, the fine cut silage had a higher, though nonsignificant, carotene content than did the coarse silage. Although the reason for this inverted rela- tionship was not apparent, a possible explanation is the fact that ‘water had to be added to the silage from the November harvest, which ‘was too dry to permit adequate packing and fermentation. The samples of ingoing silage collected did not contain the additional water, while the outcoming silage did. The loss of total carotene due to storage ‘was less in these silos than in the silos filled in September and October. ' Due to the hardness of the corn kernel at this advanced stage of maturity (November harvest), the carotene extraction by the modified AOAC procedure was quite difficult. Some kernels remained almost intact “‘17‘ 3 fl 55 in the homogenized residue. For this reason it is recomended that in future analyses Gillingham's (1967) freeze drying procedure be used. :r?’ *“9 EXPERIMENT III Materials and Methods The sources of vitamin A activity used in this study were as follows: Retinyl palmitate: The injectable preparation used in this experiment was the same as that described for experiment II. 'Ngtural carotenes: These were supplied by coarsely ground shelled corn and the carotene content was 2.95 mg/kg on an air dry basis (Quacken- bush, 1961). Eggg: .All steers were fed.§d_libitum a mixed ration consisting of 10 parts of coarsely ground shelled corn and 1 part of wheat straw, plus trace mineral salt free choice. This ration was supplemented with MSU supplement 64 (as described in experiment II) at the rate of 0.45 kg/ head/day. The protein content of the corn-straw ration as determined by the semi-micro Kjeldhal nitrogen method (Kirk, 1950) was 10.8%. Experiment: Ten Hereford steers, averaging 365 kg initial weight were divided into two groups at random and fed the corn-straw ration during the entire period of experimentation. Blood samples were drawn from the jugular vein at the beginning of every experimental period (0 hour sample) and thereafter at 1.5, 3, 6, 12, 24 and 48 hr, and 4, 8, l6 and 32 days post injection. In the first trial 1,500,000 IU of the vitamin A preparation were injected using the IM and IR routes. In the second trial, 3,000,000 IU were used and 4,500,000 10 were injected in the third trial. Liver biopsies were performed according to the method described by Whitehair‘gg'gl. (1952) to determine the initial carotene and vitamin A stores and at the end of every experimental period to assess carotene 56 57 and vitamin A storage during the experimental period. The only pre-operative care consisted in clipping a 60 cm2 area on the right side of each steer. After careful washing of this area with a germicidal solution, local anesthesia was obtained by using a 2% novocaine solution injected into the intercostal space between the 11th and the 12th ribs. After the onset of anesthesia a 3 cm incision was made in the skin of the anesthesized area, between the ribs starting 30 cm from the midline of the back. After the incision, the rib cage was perforated using a cannula and a trocar. In most cases the liver was located very easily, with exception of steer 1154 which had a thick layer of adipose tissue covering the liver. No liver samples were obtained from this animal throughout the experimental periods. After locating the liver, a sample was obtained through the cannula using the biopsy instrument designed by Whitehair g; 51. (1952). Four gm of liver tissue were usually obtained. The sample was blotted dry and placed in a plastic bag on ice to be frozen later to -20°C until analyzed for carotene and vitamin A. The incision was sutured with a nonabsorbable thread. Every steer received 3,000,000 units of procaine penicillin IM at 3 day intervals for 3 times. No infections were ob- served nor could any adhesions be detected during subsequent biopsies. Other drugs were used in some cases in order to accelerate clotting of blood at the site of biopsy, such as Azium (dexamethasone, Schering, 10 cc IM) and Klot stainless (40 cc IV). This biopsy procedure was followed each time with little difficulty except for steer 1154 as mentioned earlier, and steer 1151 which was killed accidentally during the second series of biopsies. At the pre- cise instant that the biopsy instrument touched the liver, the steer J 58 moved abruptly and the instrument went through the liver and struck a portal vein causing an internal hemorrhage which could not be contained, and the animal bled to death. Results and Discussion The basic pattern followed by each absorption and decay curve of serum vitamin A was similar as can be seen in Figures 2, 3 and 4, al- though the response in time was slightly different. Table 13. Changes in average serum vitamin A concentration after an injection of 1,500,000, 3,000,000 and 4,500,000 IU of vitamin A. Hour 1,500,000 101 3,000,000 102 4,500,000 102 PI IM IR IM IR IM IR mcg vit A/lOO m1 mcg vit A/lOO ml mcg vit A/lOO ml 0 41.5 37.7 30.9 29.6 39.9 38.9 1.5 43.3 37.7 58.93 30.9 53.9 39.2 3 44.93 36.8 50.4 52.7 54.4 61.9 6 48.3 49.9 56.9 70.6 52.9 71.1 12 58.8 69.6 58.6 83.14 71.1 92.9 24 44.0 54.74 52.4 67.9 51.0 67.64 48 25.7 34.2 47.9 48.5 43.0 46.7 96 25.1 23.9 47.4 43.5 38.8 35.8 192 32.5 30.9 36.5 38.9 44.2 40.3 384 30.4 30.2 41.9 44.7 44.4 44.2 768 47.8 43.8 37.8 41.7 28.8 30.4 1 5 steers per treatment. 4 steers per treatment. 3 Significantly greater (P <:.05) than IR. 4 Significantly greater (P (.05) than IM. In the first trail a significant difference in serum vitamin A concentration between IM and IR injections was obtained at 3 hr PI (44.9 vs. 36.8 meg/100 ml serum) (see Figure 2). The 2 curves continued increasing in vitamin A levels and peaked at 12 hr with 58.8 and 69.6 ‘4‘“. . .1! 1.3““. 'an ' tw-Hmmu 5's];- u-ow -. - “F... um; nursing-x1 Lesun- vitamin! meg l00 II V *‘O. 59 mt . . 1,500,000 Ill Vila-Ill 80- 0 3 6 12 2 2 4 I 10 32 IOIIS lays Figure 3a. Saul Vitasis A, It: [100 II Serum vitamin A concentration changes following an IM and IR injection of 1,500,000 IU of vitamin A 1.500.000 II Vitalil I Figu 3 3b. 6 I! IIIIS day: Is: .1 tin Serum vitamin A concentration changes following an IM and IR injection of 1,500,000 IU of vitamin A ~ ~ in, ’ 4 - IL _‘kv‘-o- *‘4 60 meg vitamin A/lOO m1 serum for IM and IR respectively, and there was no significant difference due to the overlap of the individual values. At 24 hr PI the serum vitamin.A concentration for IR treatment was signi- ficantly greater than the IM level (54.7 vs. 44.0 mcg vitamin A/lOO ml serum). These serum vitamin A concentrations continued to decrease and attained their lowest level at 96 hr PI, after which they increased somewhat again and maintained a stable level for 8 days between the 192 and 384 hr PI period. The final serum.vitamin A level was higher than that found at 384 hr P1 with 47.8 and 43.8 mcg vitamin A/lOO m1 serum for the IM and IR injections, respectively. There is no explanation for this final rise in serum vitamin.A concentration. Table 14. Initial and final liver vitamin A concentration of liver biopsies (Trial 1). Initial liver vitamin A Final liver vitamin A Steer No. meg/gm. fresh basis ggglgm,fresh basis 1122 3.23 i 41.9 IM1176 7.08 24.6 1195 14.2 18.7 1285 5.03 8.1 Avg 7.37 23.31 1330 2.93 9.9 726 20.8 23.1 IR1136 3.41 8.3 1351 3.36 10.5 Avg 7.63 12.9 10 Significantly greater (P <:.OS) than initial. No significant differences in liver vitamin A concentration were found in the first trial in.initia1 or final levels between.IM.and IR treatments. The initial levels were low on the average, bordering on a deficiency level, but no deficiency symptoms were found. No measurable 61 liver carotene storage was present initially and, although some was found later, no levels are reported here since they did not differ significantly. A significant increase in vitamin A concentration was found between the IM injected steers (7.37 vs. 23.3 mcg vitamin A/gm liver, fresh basis) 768 hr PI, while the IR injection showed no signi- ficant increase in vitamin A stores after an equal treatment period (7.63 vs. 12.9 mcg vitamin A/gm liver, fresh basis). These results [ suggest that when liver vitamin A stores are low and bordering on de- I ficiency, the IR route of administration is less effective in restoring vitamin A stores than is the 111 route. i i g. The absorption and decay curve of serum vitamin A concentration for the second trial showed an earlier response than that obtained in the first trial (see Figure 3). At 1.5 hr PI the IM injection showed a significant (P (.05) increase in serum vitamin A concentration over the IR treated steers (58.9 vs 30.9 meg/100 ml) . After this initial peak, the IM curve declined somewhat, but no sharp changes were observed throughout the experimental period. As in the first trial, the IR route peaked at 12 hr PI, rising from 30.9 to 83.1 meg/100 ml, and this level was also higher than that of the IM treatment. The observed difference between the IM and IR routes of administration at this time was significant (P (.05) (58.6 vs 83.1 meg/100 ml). The IR serum vitamin A level declined rapidly and reached a more stable concentration at 192 hr PI. The later changes in serum vitamin A concentration were minor and the levels were maintained at about 40 meg/100 m1 serum. The effect of the injection of 3,000,000 11'! of vitamin A, either IM or IR, on liver vitamin A storage can be seen in Table 15. There were significant differences (P <.05) between the initial and final 62 E 3,000,000 II Vitalil A g 00 . "ll'llllllll m 5..» ""ln. 1.‘ 3 so 5 «x h :5 f’fl.’. ”a.“ 2". E 9’ -' axis“ E ‘0 l 5': h‘.} .fl'“I""'“gz'lunl"""flnunuusss—ssuun E :3 Ngrxl’lb’I” '/.,'~, "A, Nun-us: E W: = z 20 - .. II m nuns- ' a 1 u. I s l 1 1 r 0 3 0 ll 24 Z 4 0 l0 32 halts Serum Vitamiancg/lflo sl Fi days Figure 4a. Serum vitamin A concentration changes following an 1M and IR injection of 3,000,000 IU of vitamin A 3.000.000 Ill Vita-ill 1.5 3 0 II II 32 hm: m: In at tin gure 4b. Serum vitamin A concentration changes following an IM and IR injection of 3,000,000 IU of vitamin A 63 liver vitamin A stores after 32 days of treatment. A significant difference (P<.05) was also found between the IM and IR route of ad- ministration in the final liver vitamin A storage (34.5 vs 24.9 meg/gm liver, fresh basis). Table 15. Initial and final liver vitamin A concentration of liver biopsies (Trial II). Initial liver vitamin A Final liver vitamin A Steer No. 913—8188! liverkfresh basis mcglg liver, fresh basis 1122 41.9 61.6 1176 24.6 24.3 m 1195 18.7 31.5 1285 8.1 20.7 Avg 23.3 34.51 1330 9.9 29.8 726 23.1 25.6 IR 1136 8.3 26.3 1351 10.5 18.1 Avg 12.9 24.92 T IM significantly greater (P <.05) than initial and IR. 2 IR significantly greater (P< .05) than initial. The results obtained in the third trial were quite similar to those obtained in the second trial (see Figure 4). There was a large nonsignificant difference at 1.5 and 12 hr, similar to the changes ob- served in the second trial (which were significant at 1.5 hr PI but not at 12 hr PI). Again, both curves peaked at 12 hr PI and then declined more rapidly than in the second trial, leveling at about 40 mcg/ 100 ml serum between 192 and 384 hr PI. The only significant difference (P (.05) found between the m and IR routes of administration occurred 24 hr PI (51.0 vs 67.6 meg/100 m1 serum). Of interest was the fact that the response of the serum vitamin A SNIIIII vitamin, meg/100 ll I00 4.5!..." II Vita-ill 00 .0" sf 1' 00 4o /"'"" zo- -" _ll . 4 . . , i 0 3 0 12 m4 I II 32 hours lays Serum vitamin A concentraLion changes following an Figur: Sa. IM and IR injection of 4,500,000 IU of vitamin A III 4.50M" ll Imsil A ‘5. ‘ .,\ Sens Vitamin A, mg [100 ll 40 w / 20 -- I. 0 - 1.5 3 0 12 2 0 0 I0 32 hours days In H tin Figure 5b. Serum v? amin A concentration changes following an IM aufl IR gnj‘ction of 4,500.000 IU of vitamin A concentration to the IM route of administration was consistently lower than the IR treatment and this occurred in all three trials. In the first two trials the highest average serum vitamin A concentration ob- tained with the IM treatment reached 60.0 mcg/lOO m1 serum only after injecting 4,500,000 IU vitamin A did the highest average serum vitamin A level surpass that level and reach 71.1 meg/100 ml serum. The response of serum vitamin A concentration to the injection level was more direct in the IR than in the IM treatment. In the first trial, the highest average serum vitamin A concentration reached 69.6 mcg/lOO m1 serum, in the second trial it was 83.1 meg/100 ml and in the third trial it reached 92.9 mcg/lOO ml serum. These three maximum levels were obtained at 12 hr P1 in each trial, after which they de- clined rather rapidly to the 96 hr PI period and leveled off for about 8 days, maintaining a close relationship with the values obtained for the IM treatment. The reason for the large differences between the mo routes of administration is the site of absorption. The IM injection has, under normal conditions, one of the fastest absorption rates, while the oral route, that is, the IR route, is one of the slowest (Jones, 1959). For this reason, the IM route of administration was expected to show both faster and greater changes in serum vitamin A levels, since the muscle tissue has an ample blood supply. As expected, the serum vitamin A concentration changed rapidly following the IM injection. Not expected, though, was the magnitude of the observed changes, which, when compared to the response obtained with the IR injection, was smaller. The following reasons may explain these results. First, the IM site of in- jection may be infiltrated with adipose tissue which has a minimal blood supply (as compared to the muscle), and secondly, the area exposed to the blood is very small as compared to the large absorption surface present at the intestinal wall. Consequently, the IR route showed a greater magnitude in the response obtained, since a larger quantity of the vitamin A preparation can be absorbed at one time. Table 16 shows the final liver vitamin A concentration in fresh tissue which was 2.6 times and significantly (P<.05) greater for the Pl] IM than the IR injection. Using the initial and final liver values for I I Table 16. Initial and final liver vitamin A concentration of liver ; tissue (Trial III). 2 f E Initial liver vitamin A Final liver vitamin A g“; Steer No. mcgzg liver, fresh basis m liver, fresh basis 1122 61.6 111.4 IM 1176 24.3 117.7 1195 31.5 153.3 1285 20.7 95.4 Avg 35.1.1 119.51 1330 29.8 42.5 IR 726 25.6 79.4 1136 26.3 35.7 1351 18.1 27.8 Avg 24.9 46.4 1 IM.significantly CPs<:.05) greater than IR. *n this experimental period, a net storage can be calculated if it is E? assumed that the liver did not change in weight the last 32 days of the experimental period and that the liver is the only storage organ. The net storage was 38.9 vs 101 mg of vitamin A per liver (IR and IM routes, respectively). The dose of 4,500,000 IU of retinyl palmitate equals 2,475.25 mg of retinyl palmitate (1 mg - 1818 ID). If the net storage is divided by 67 ‘mg of retinyl palmitate injected, the efficiency of utilization of the final injection is obtained. The result indicates that the IM route of administration had a storage efficiency of 15.7% as compared to only 4.0% for the IR route. This indicates that the IM route is 3.9 times more efficient than the IR route under these conditions of vitamin A status in the steer. CONCLUSIONS From the results of the three experiments several conclusions can be drawn. Certain of these conclusions can be arrived at with confi- dence while others must remain tentative pending further research. Experiment I A vitamin.A depletion period of 110 days in lambs reduced serum vitamin A concentration by only 8.3 meg/100 m1 from 28.3 to 20.0 mcg/ 100 m1. At this level no vitamin A deficiency symptoms were found. The liver vitamin A concentration of one lamb sacrificed after 71 days of depletion was 1.16 mcg/gm (fresh basis). Although this level ‘was considered to be deficient, no symptoms of vitamin A deficiency were apparent. A longer depletion period might be advantageous in or- der to find at what serum vitamin A level deficiency symptoms appear, since vitamin A concentration can be a useful criterion in assessing vitamin A status. Once the lambs were placed on their respective repletion diets, the serum vitamin A concentrations of the lambs in lot 2 (receiving 1.1 mg retinyl palmitate/head/day) did not reach predepletion levels. Based on the current standards suggested by the N.R.C. (1964), this level is adequate for the normal growth of fattening lambs. However, although no deficiency symptoms were present, the liver vitamin A stor- age of 3.1 meg/gm liver (fresh basis) attained after 71 days of reple- tion indicates a marginal deficiency status. With the exception of lot 3, the serum vitamin A concentration in lambs increased linearly with increasing dietary vitamin A levels and reached a maximum concentration after 35 days, while lot 3 continued to increase until the final day of repletion. The response of liver 68 69 vitamin.A concentration as well as total liver vitamin A storage due to treatments was linear. Based on the regression of total liver vitamin A stored on dietary retinyl palmitate, it was found that the feeding of 1 mg total carotene from corn silage was equivalent to 438 IU of retinyl palmitate. The biopotency of the carotenes present in corn silage was 26.2% when compared with the efficiency of conversion of beta-carotene to vitamin A by the rat. Experiment II In experiment II, where a water miscible vitamin A preparation was used in steers, it was found that an IM injection of 7,000,000 IU ‘was more efficient in producing adequate liver vitamin A storage than an equal amount injected IR. Similarly, the IR injection produced a significantly higher liver vitamin A storage than did the feeding of carotenes as present in corn silage. This indicates that, in order to meet daily vitamin A.requirement of the steer, a large proportion of the ingested carotene was converted to vitamin A. Although the feeding of corn silage for 147 days produced lower vitamin A storage levels than did the IR injection of 7,000,000 IU of vitamin.A, the difference was not significant. A similar IM injection at 147 days antemortem was as efficient in producing adequate liver vitamin A storage as an IR injection 35 days antemortem. This indicates that there is considerable preintestinal vitamin A disappearance even though vitamin E, BRA and BHT have been added as antioxidants to the vitamin A preparation in order to increase its stability and prevent vitamin A losses. The effect of harvest time on the total carotene content in corn silage was significant since late harvested corn silage had lower total carotene content. The effect of storage was noticeable too, since all the outgoing silage samples had markedly lower total carotene concentra- tions. Interesting was the fact that corn silage of finer particle size produced a feed which was lower in total carotene content than did the silage which had been cut coarsely. Before definite conclusions are drawn here, it is necessary to establish first the effects of cer- tain other factors which were not analyzed, such as the pH of the si- lage and its possible role in carotene destruction, as well as the effects of the temperatures attained during the fermentation process in the 8110. Experiment III In each trial of this study it was found that an IR injection al- ways caused higher serum vitamin A concentrations than did a similar IM injection after a 6 to 12 hr period. Despite the higher serum vita- min.A concentration, the IM.treatment was more efficient in increasing liver storage. The PI serum.vitamin A changes using the IM route were consistently faster (1.5 to 3 vs 6 to 12 hr) than those obtained with the IR administration. At 6 and 12 hr PI the response to the IR in- jection.was consistently greater than that of the IM route of adminis- tration. Both treatments peaked at 12 hr PI and started to decline rather rapidly, tending to reach a stable level 2 to 4 days PI. They remained fairly stable at this level for about 8 days. At the end of each trial the serum concentrations tended to reach levels comparable to those found at the beginning of the trial, suggesting that the nor- mal serum vitamin A concentration, under these vitamin A intakes, is found at about 40 meg/100 m1 of serum. SUMMARY Three experiments were conducted to study the utilization of vita- min A and carotene by the ruminants. In the first experiment, 56 west- ern lambs were fed a vitamin A depletion ration for 110 days. Although the serum vitamin A concentration decreased markedly, no deficiency symptoms were observed at the end of the depletion period. The lambs 'were allotted at random to 4 repletion diets. One lot received corn silage gg libitum, supplemented with a protein-mineral mixture, while the three remaining lots received the modified depletion ration con- taining three levels of vitamin A (1.1, 3.3 and 9.9 mg/head/day). These rations were fed for 70 days after which the lambs were slaugh- tered and the livers assayed for vitamin A stores. The serum and liver vitamin A concentrations reflected the dietary treatments. The lowest vitamin A level fed was adequate to prevent onset of deficiency symp- toms. The corn silage carotene was utilized by the lambs with an efficiency of 26.2% as compared to the utilization of beta-carotene by the rat. In the second experiment, 108 Hereford steer calves were used to determine whether a single massive dose of vitamin A in a water misci- ble base would support normal serum.vitamin A levels and adequate li- ver vitamin A.stores for an extended period of time. This tudy was superimposed on a corn silage experiment designed to determine the effects of stage of maturity and fineness of chop on the quality of corn silage and the performance of beef cattle in the feed lot. The steers received 7,000,000 IU of vitamin A at 147, 63 and 35 days ante- mortem, either IR or IN. The initial and final serum carotene and 71 72 ‘vitamin A levels, as well as liver carotene and vitamin A stores, ‘were determined. The lowest liver carotene and vitamin A concentra- tions were found in the control steers which had received only corn silage as a vitamin A source. The IR injection at 147 days ante- mortem and the control treatments showed no significant differences, while the IM injection at 147 days antemortem was comparable to the IR injection 35 days antemortem. The corn silages harvested in October and November had a signifi- cantly lower total carotene content than those of the September harvest. In studying the effect of storage on corn silage, it was found that the carotene content of the outcoming silages was significantly lower than that of the ingoing silage. The degree of chop did not affect signi- ficantly the carotene content of the outcoming silages, although a trend appeared which indicated that the coarser silages had a higher carotene content than did the corresponding fine cut silages. Ten Hereford steers were used in the third experiment to deter- mine the absorption and decay curve of serum vitamin A when vitamin A is administered either IM or IR. Three 32-day trials were conducted in which 1,500,000, 3,000,000 and 4,500,000 IU of vitamin A were in- jected in trials 1, 2 and 3, respectively. At the beginning and the end of every trial liver biopsies were performed to assess liver vita- min.A stores. Blood samples were collected prior to the injection and at 1.5, 3, 6, 12, 24 and 48 hr PI and at 4, 8, l6 and 32 days PI. In each trial the serum vitamin A concentration peaked at 12 hr PI for both routes of administration. Although the IM treatment consistently lproduced a higher serum vitamin A concentration in the first 3 hr PI, at 12 hr the response of the serum vitamin A levels following the IR A e35: $733131 I s‘ .U. 'eflb J "1 fl 1 l . h 73 injection was greater in each trial. The liver stores at the end of the final experimental period indicated that the IM injection of vita- min A was utilized 3.9 times more efficiently than the IR injection. BIBLIOGRAPHY Alexander, J. and T. W. Goodwin. 1950. A demonstration of the con- version of carotene into vitamin A in conscious rats. Brit. J. Nutr. 4:421. Ames, Stanley R. and Willard F. O'Rourke. 1965. Biological utiliza- tion of vitamin ADE injectables. J. Animal Sci. 24:871. (Abstr.). AOAC. 1965. Officical Methods of Analysis of the Association of Offi- cial Agricultural Chemists. 10th ed. Association of Official Agri— cultural Chemists, Washington, D. C. Baker, F. H., L. S. Pope and R. MacVicar. 1953. Relative importance of dietary carotene and liver stores of carotene and vitamin A for reproduction and lactation of beef cows. J. Animal Sci. 12:906. Barrick, E. R., F. N. Andrews and J. F. Bullard. 1948. The absorption of carotene and vitamin A from various levels of the gastrointesti- nal tract of sheep. J. Animal Sci. 7:539. Beeson, W. M., T. W. Perry, W. H. Smith and M. T. Mohler. 1962. Effect of vitamins A and E, carotene and dehydrated alfalfa on the perform- ance of steers. J. Animal Sci. 21:988. (Abstr.). Bieri, J. G. and C. J. Pollard. 1953. Efficient utilization of intra- venous carotene by the rat. Federation Proc. 12:409. (Abstr.). Blake, J. T., N. L. Jacobson and R. S. Allen. 1950. The effect of method of administration on the absorption and storage of vitamin A by dairy calves. J. Animal Sci. 9:648. Braun, W. 1945a. Studies of the carotenoid and vitamin A levels in cattle. I. Seasonal changes of the carotenoid and vitamin A levels a and the normal carotenoid and vitamin A ratio of the blood. J. Nutr. 29:61. Braun, W. 1945b. Studies of the carotenoid and vitamin A levels in cattle. II. Carotenoids and vitamin A in the liver, their ratio and their relationship to blood levels. J. Nutr. 29:73. Chapman, H. L., Jr., R. L. Shirley, A. Z. Palmer, C. E. Haynes, J. W. Carpenter and R. J. Cunha. 1964. Vitamins A and E in steer fatten- ing rations on pasture. J. Animal Sci. 23:669. Chapman, H. L., Jr., R. L. Shirley, G. H. Taki, A. Z. Palmer and J. W. Carpenter. 1965. Value of injected and orally administered vita- mins A and E for steers. J. Animal Sci. 24:878. (Abstr.). 74 we“. $2., . 75 Church, D. C., R. MacVicar, J. G. Bieri, F. H. Baker and L. S. Pope. 1954. 'Utilization of intravenously administered carotene by sheep and cattle. J. Animal Sci. 13:677. Davis, R. E. and L. L. Madsen. 1941. Carotene and vitamin A in cattle blood plasma with observations on reproduction at restricted levels of carotene intake. J. Nutr. 21:135. Diven, R. H. and E. S. Erwin. 1958. Utilization of vitamin A and caro— tene by normal and deficient sheep. Proc. Soc. Exp. Biol. Med. 97:601. Diven, R. H., O. F. Pahnish, C. B. Roubicek, E. S. Erwin and H. M. Page. E- 1960. Vitamin.A and carotenoid interrelationship in bovine plasma ~ and liver. J. Dairy Sci. 43:1632. Eaton, H. D., L. D. Matterson, L. Decker, C. F. Helmboldt and E. L. 5 Jungherr. 1951. Intravenous and oral administration of an aqueous A suspension of carotene to calves depleted of their vitamin A stores. J. Dairy Sci. 34:1073. Eaton, H. D., G. S. Meyers, Jr., MI W. Dicks, B. A. Dehority, A. P. Grifo, Jr., R. Teichman, C. F. Helmholdt, E. L. Jungherr and D. G. Gosslee. 1959. Conversion of carotene from alfalfa to vitamin A by Guernsey and Holstein calves. J. Dairy Sci. 42:642. Elliot, R. F. 1949. Carotene requirements for young dairy calves. J. Dairy Sci. 32:710. Embree, N. D., S. R. Ames, R. W. Lehman and P. L. Harris. 1957. De- termination of vitamin A. Methods of Biochemical Analysis, ed., David Glick, Interscience Publishers, Inc., New York. Vol. 4, p43. Embry, L. B., R. J. Emerick, R. A. Weichenthal and F. W. Whetzal. 1962 Vitamin A requirements of fattening cattle. J. Animal Sci. 21:994. (Abstr.). Esh, G. C., T. 8. Sutton, J. W. Hibbs and W. E. Krauss. 1948. The effect of soya-phosphatides on the absorption and utilization of vitamin A in dairy animals. J. Dairy Sci. 31:461. Frape, D. L., V.C. Speer, V. W. Hayes and D. V. Catron. 1959. The vitamin A requirement of the young pig. J. Nutr. 68:173. Gallup, W. D. and J. A. Hoefer. 1946. Determination of vitandn A in liver. Ind. Eng., Chem., Anal. Ed. 18:288. Gillingham, J. T. 1967. Determination of carotene in fresh forages and silages following freeze-drying and grinding. J.A.O.A.C. 50:827. Glover, J. and R. J. Walker. 1964. Absorption and transport of vita- min A. Exp. Eye Res. 3:327. w ‘ (30rdon, H. A., G. S. Smith, A. L. Neumann, J. E. Zimmerman and G. W. Breniman. 1963. Vitamin A nutrition of beef cattle fed corn silages. J. Animal Sci. 22:738. Grifo, A. P., Jr., J. E. Rousseau, Jr., H. D. Eaton, B. A. Dehority, D. G. Hazzard, C. F. Helmboldt and D. G. Gosslee. 1960. Effect of duration of deficient carotene intake upon subsequent utilization of carotene from alfalfa by Holstein calves. J. Dairy Sci. 43:1809. Guilbert, H. R. and G. H. Hart. 1935. Minimum vitamin A requirements with particular reference to cattle. J. Nutr.. 10:409. Guilbert, H. R., R. F. Miller and E. H. Hughes. 1937. The minimum vitamin A and carotene requirement of cattle, sheep and swine. J. Nutr. 13:543. Guilbert, H. R., C. E. Howell and G. H. Hart. 1940. Minimum vitamin A and carotene requirements of mammalian species. J. Nutr. 19:91. Hale, W. H., F. Hubbert, Jr., R. E. Taylor, R. A. Anderson and B. - Taylor. 1962. Performance and tissue vitamin A levels in steers 4* fed high levels of vitamin A. Am. J. Vet. Res. 23:992. Hayes, B. W., G. E. Mitchell and H. B. Sewell. 1967. Turnover of li- ver vitamin A in steers. J. Animal Sci. 26:855. Heaney, D. P. 1960. Effects of marginal vitamin A intake during gestation in swine. Ph.D. Thesis, Michigan State University. Hentges, J. F., Jr., R. H. Grummer and D. K. Sorenson. 1952. Effects of carotene administered orally, intramuscularly and intravenously on avitaminosis A in pigs- J. Animal Sci. 11:794. Huang, H. S. and DeWitt Goodman. 1965. Vitamin A and carotenoids. I. Intestinal absorption and metabolism of 14C—labeled vitamin A alcohol and beta-carotene in the rat. J. Bio. Chem. 240:2839. Jacobson, N. L., G. H. Wise, R. S. Allen and 0. Kempthorne. 1950. Rate of absorption of carotene and of vitamin A from the alimentary tract of dairy calves. I. Effect of method of administration. J. Dairy Sci. 33:645. Jacobson, N. L., R. S. Allen, J. T. Blake and P. G. Homeyer. 1954. The effect of method of administration on the absorption and stor- age of vitamin A by dairy calves. J. Nutr. 54:143. Johnson, R. M. and C. A Baumann. 1947. Storage and distribution of vitamin A in rats fed certain isomers of carotene. Arch. Biochem. 14:361. Jones, L. M. 1959. Farmacologia y Terapéutica Veterinarias. Uteha, Mexico, D. F. 77 Jordan, H. A., G. S. Smith, A. L. Neumann, J. E. Zimmerman and G. W. Breniman. 1963. Vitamin A nutrition of beef cattle fed corn si- lages. J. Animal Sci. 22:738. Rearing, E. A., W. H. Hale and Farris Hubbert, Jr. 1964. In vitro de- gradation of vitamin A and carotene by rumen liquor. J. Animal Sci. 23:111. King, T. B., T. G. Lohman and G. S. Smith. 1962. Evidence of rumeno- reticular losses of vitamin A and carotene. J. Animal Sci. 21:1002. (Abstr.). Kirk, P. L. 1950. Kjeldahl method for total nitrogen. Anal. Chem. 22:354. Klatte, Fred J., T. L. Huber, C. 0. Little and G. E. Mitchell, Jr. 1963. Preintestinal disappearance of vitamin A in sheep. J. Animal Sci. 22:239. (Abstr.). Klatte, F. J., G. E. Mitchell, Jr. and C. 0. Little. 1964a. In vitro destruction of vitamin A by abomasal and ruminal contents. J. Agri. Food Chem. 12:420. Klatte, F. J., G. E. Mitchell, Jr. and C. 0. Little. 1964b. Stability of vitamin A acetate in ruminal and abomasal fluids. J. Animal Sci. 23:296. Klosterman, E. W., D. W. Bolin and M. R. Light. 1949. Carotene and vitamin A studies with sheep. J. Animal Sci. 8:624. Klosterman, E. W., L. J. Johnson, A. L. Moxon and A. P. Grifo, Jr. 1964. Utilization of carotene from corn silage by steers. J. Animal Sci. 23:723. Kohlmeier, R. H. and W. Burroughs. 1962. Supplemental vitamins A, E and K for beef cattle. J. Animal Sci. 21:1003. (Abstr.). Koizumi, 1., T. Suzuki and Y.Sahasi. 1963. Metabolic pathways of carotene to vitamin A. III. Degradation of carotene and vitamin A in rat intestine. J. Vitaminol. 9:154. Kon, S. K. and S. Y. Thompson. 1951. Site of conversion of carotene to vitamin A. Brit. J. Nutr. 5:114. Lawrence, C. W., F. D. Carin, F. J. Lotspeich and R. F. Krause. 1966. Absorption, transport and storage of retinyl-15-14C palmitate-9, 103s in the rat. J. Lipid. Res. 7:226. Li, C. C. 1964. Introduction to Experimental Statistics. McGraw-Hill Book Co., New York. Madsen, L. L. and R. E. Davis. 1949. Carotene requirements of beef cattle for reproduction. J. Animal Sci. 8:625. 78 liaynard, L. A. 1951. Animal Nutrtion, 3rd. ed. McGraw-Hill Book Co., New York. McCollum, E. V. 1957. A History of Nutrition. The Riverside Press, Cambridge, Massachusetts. Miller, R. W., L. A. Moore, D. R. Waldo and T. R. Wrenn. 1967. Utili- zation of corn silage carotene by dairy calves. J. Animal Sci. 26:624. Mitchell, G. E., Jr., C. 0. Little and B. W. Hayes. 1967a. Pre-intes- tinal destruction of vitamin A by ruminants fed nitrate. J. Animal Sci. 26:827. Mitchell, G. E., Jr., C. 0. Little, H. B. Sewell and B. W. Hayes. 1967b. Mobilization of liver vitamin A in sheep. J. Nutr. 91:371. Moore, T. 1957. Vitamin A. Elsevier Publishing Co., New York. Morrison, F. B. 1948. Feeds and Feeding, 21st. ed. The Morrison Pub- lishing Co., Ithaca, New York. Murray, T. K. and P. Erody. 1966. Absorption and storage of retro- vitamin A acetate (rat). Biochem. and Biophys. Acta. 124:190. N. R. C. 1959. Nutrient Requirements of Domestic Animals, No. 4. Nutrient Requirements of Beef Cattle. National Research Council, mmmgm,mc. N. R. C. 1964. Nutrient Requirements of Domestic Animals, No. 5. Nutrient Requirements of Sheep. National Research Council, mmmym,mc. Newland, H. W., H. E. Henderson and D. E. Ullrey. 1966. Injectable vitamins A and E for finishing cattle. J. Animal Sci. 25:907. (Abstr.). Nutrition Reviews. 1967. Vitamin A transport in man. 25:199. O'Donovan, J. P., W. H. Smith and W. M. Beeson. 1966. Interrelation- ship between vitamin A and E in steers. J. Animal Sci. 25:907. (Abstr.). Parrish, D. B., C. E. Aubel, I. D. Wheat and J. S. Hughes. 1950. Comparative value of vitamin A and carotene for supplying the vita- min A requirements of swine during gestation and beginning lactation. J. Animal Sci. 9:664. Perry, T. W., W. M. Beeson, W. H. Smith and M. T. Mohler. 1964. Oral vitamin A, vitamin E or vitamin K and injectable vitamins for grow- ing yearling steers. J. Animal Sci. 23:888. (Abstr.). 79 Perry, '1‘, W, , W, M. Beeson, W. H. Smith and M. T. Hohler. 1965. Vita- mins A. E, and K and dehydrated alfalfa meal for fattening beef cattle. J. Animal Sci. 24:899. (Abstr.). Ralston, A. T. and I. A. Dyer. 1959. Relationship of liver and plas- ma carotenoid and vitamin.A content in cattle as affected by loca- tion and season. J. Animal Sci. 18:874. Record, R. E., W. M. Beeson and W. H. Smith. 1963. Utilization of carotene, oral and injectable vitamin A in beef. J. Animal Sci. 22:845. (Abstr.). Reifman,.A. G., L. F. Hallman and H. J. Duel, Jr. 1943. The effect of concentration on the absorption of vitamin A. J. Nutr. 26:33. Roberts, W. K. and E. W. Stringam. 1962. Note on liver vitamin A stores and weight gains in beef cattle following intraruminal in- jection or oral administration of vitamin A. Can. J. Animal Sci. 42:110. Roberts, W. K. and G. D. Phillips. 1963. Vitamin.A and carotene studies with fattening beef cattle. Can J..Animal Sci. 43:31. Roberts, W. K., G. M. Findlay and E. W. Stringam. 1965. Liver vitamin A storage in fattening cattle following intraruminal or intramuscu- lar injection of vitamin A. Can. J. Comp. Med. Vet. Sci. 29:43. Selke, M. R., C. E. Barnhart and C. H. Chaney. 1967. Vitamin A re- quirement of the gestating and lactating sow. J. Animal Sci. 26: 759. Sexton, E. L., J. W. Mehl and H. J. Deuel, Jr. 1946. Studies on caro- tenoid metabolism. J. Nutr. 31:299. Sobel, A. E., M; Sherman, J. Lichtblau, S. Snow and B. Kramer. 1948. Comparison of vitamin A liver storage following administration of vitamin A in oily and aqueous media. J. Nutr. 35:225. Sobel, A. E. and A. A. Rosenberg. 1950. Vitamin A stores of sucklings following administration to the dams of vitamin.A in oily and aque- ous media. J. Nutr. 42:557. Swick, R. W., R. H. Grummer and C. A. Baumann. 1952. The effect of thyroid and carotenoid metabolism in swine. J..Animal Sci. 11:273. Thompson, S. Y., R. Brande, M. E. Coats, A. T. Cowie, J. Ganguly and S. K. Kon. 1950. Further studies of the conversion of carotene to vitamin A in the intestine. Brit. J. Nutr. 4:398. Varnell, T. R. and E. S. Erwin. 1960. Influence of injection site on blood vitamin A and carotene levels in sheep. J. Animal Sci. 19:960. (Abstr.). 80 'Veen, M. J. and G. H. Beaton. 1966. Vitamin A transport in the rat. Can. J. Physiol. Pharmacol. 44:521. Vogel, H. and H. Knobloch. 1950. Chemie und Technik der Vitamine. 3rd. ed. Ferdinand Enke Verlag, Stuttgart. Wagner, A. F. and K. Folkers. 1964. Vitamins and Coenzymes. John Wiley & Sons, New York. Whitehair, C. K., D. R. Peterson, W. J. Van Arsdall and O. 0. Thomas. 1952. A biopsy technique for cattle. J. Am. Vet. Med. Assn. 121: 285. Wise, H. C., N. L. Hacobson, R. S. Allen and S. P. Yang. 1949. Effect of type of dispersion on rate of absorption of carotene and vitamin A by dairy calves. J. Dairy Sci. 32:711. Williams, C. M., J. M. Bell and L. J. Fisher. 1963. Levels of caro- tene and vitamin A for steers. J. Animals Sci. 22:849. (Abstr.). Wostmann, B. S. and P. L. Knight. 1965. Antagonism between vitamins A and K in the germfree rat. J. Nutr. 87:155. Zechmeister, L. 1962. Cis-Trans Isomeric Carotenoids Vitamins A and Arylpolyenes. Academic Press, New York. --' I"3Q~l‘v. ‘- 2 .. ._ w “Wye 3"“. ‘u'. . ~ -. A . Appendix Table 1. 9/21/66 Lot 1 Lamb # mcg/ 100 ml 57 25.8 47 29.9 17 40.6 34 20.0 7 33.6 27 30.1 50 29.1 36 25.9 61 34.6 33 34.6 11 27.9 31 37.4 1 31.3 8 21.9 Avg 30.2 10/26/66 Lot 1 Lamb # mcg/ 100 ml 57 35.6 47 23.6 17 34.6 34 38.3 7 15.5 27 38.3 50 41.9 36 60.0 61 41.0 33 39.3 11 24.5 31 33.9 1 19.1 8 22.8 Avg 33.5 81 Serum vitamin A levels of lambs during depletion (Exp I). Lot 2 Lamb # mcg/ 100 m1 5 31.0 45 12.8 18 21.0 35 21.9 30 37.4 26 36.5 39 34.6 23 24.6 40 32.8 32 24.6 2 24.6 16 32.4 56 20.8 48 24.0 27.1 Lot 2 Lamb # mcg/ 100 ml 4 46.6 45 28.3 18 22.8 35 21.4 30 15.5 26 39.3 39 36.5 23 41.9 40 37.4 32 37.4 2 24.6 16 48.4 56 29.1 48 27.9 32.7 26.6 23.6 29.9 24.6 20.3 23.3 23.4 19.6 27.7 Lot 3 # mcg/ 100 ml 40.1 51.8 36.9 26.4 45.5 37.4 46.6 25.5 62.4 23.4 23.6 34.6 44.6 38.3 38.4 mcg/ 100 m1 19.6 28.3 36.9 40.1 32.8 24.6 27.0 24.6 30.1 24.6 31.6 20.8 35.3 16.6 28.1 ‘wvwm J \n ,v-;;§> Aggendix Table la. 11/30/66 Lot 1 Lamb # mcg/ 100 ml 57 14.3 47 26.8 17 27.3 34 16.3 7 15.3 27 26.8 50 36.9 36 26.6 61 18.3 33 19.4 11 14.3 31 21.9 1 21.4 8 21.4 Avg 21.9 1/10/67 Lo 1 Lamb # mcg/ 100 ml 57 18.9 47 12.0 17 31.6 34 19.6 7 18.3 27 16.0 50 16.0 36 38.6 61 19.6 33 21.0 11 14.9 31 18.6 1 15.6 8 11.6 Avg 19.5 Serum vitamin A levels of lambs during depletion (Exp I). Lot 2 Lamb # mcg/ 100 ml 5 24.7 45 20.0 18 16.3 35 21.9 30 10.9 26 18.3 39 18.9 23 13.1 40 16.3 32 20.7 2 10.7 16 17.5 56 10.5 48 19.7 19.4 Lot 2 Lamb # mcg/ 100 m1 5 17.1 45 19.6 18 12.4 35 25.5 30 17.9 26 19.6 39 18.6 23 28.0 40 14.9 32 18.3 2 7.3 16 9.5 56 6.9 48 13.5 16.4 82 Lot 3 Lamb # mcg/ 100 ml 38 28.6 52 30.0 21 38.3 54 28.8 55 24.8 28 26.5 46 21.5 9 31.0 49 32.8 12 15.4 53 14.5 3 31.0 42 36.5 60 20.1 27.1 Lot 3 Lamb # mcg/ 100 ml 38 22.6 52 17.1 21 37.3 54 14.9 55 23.0 28 21.5 46 29.1 9 14.6 49 22.6 12 10.9 53 12.4 3 19.3 42 33.1 60 31.0 22.1 Lot 4 Lamb # mcg/ 100 ml 43 23.3 12 40.8 51 18.1 59 24.8 29 39.1 10 31.8 41 24.8 25 17.5 4 16.1 22 31.5 15 28.5 44 25.9 20 28.1 37 10.9 25.8 Lot 4 Lamb # mcg/ 100 ml 43 19.0 13 25.1 52 18.9 59 12.4 29 23.6 10 25.1 41 32.7 25 34.5 4 17.5 22 37.5 15 18.1 44 12.4 20 16.0 37 14.6 21.9 Aggendix Table 2. (Exp I). 2/14/67 Lot 1 Lot 2 Lamb # mcg/ Lamb # mcg/ 100 m1 100 ml 57 44.6 5 14.6 47 41.0 45 28.3 17 39.8 18 17.4 34 40.1 35 19.3 7 40.1 30 18.3 27 53.8 26 21.9 50 51.1 39 16.4 36 47.4 23 31.0 61 45.6 40 20.0 33 41.9 32 , 33.8 11 34.6 2 17.4 31 40.1 16 19.9 1 38.3 56 17.4 8 20.1 48 15.5 Avg 41.1 20.8 3/20/67 Lot 1 Lot 2 Lamb # mcg/ Lamb # mcg/ 100 m1 100 ml 57 36.4 5 21.0 47 40.1 45 21.0 17 37.4 18 20.0 34 47.4 35 21.0 7 39.3 30 21.0 27 47.4 26 20.0 50 52.0 39 21.0 36 46.5 23 27.4 61 41.1 40 19.1 33 42.0 32 32.9 11 36.5 2 20.0 31 36.5 16 15.5 1 48.4 56 12.8 8 32.0 48 12.3 Avg 41.6 20.7 83 Lamb # mcg/ 100 ml 38 34.6 52 37.4 21 29.3 54 22.8 55 29.1 28 35.8 46 28.3 9 46.5 49 30.1 12 39.3 53 25.5 3 32.8 42 37.4 60 30.1 32.8 Lot 3 Lamb # mcg/ 100 ml 38 40.3 52 33.6 21 33.6 54 32.9 55 32.9 28 52.0 46 46.1 9 48.4 49 37.1 12 37.4 53 47.4 3 46.5 42 40.1 60 34.8 40.2 Lo 4 Lamb # mcg/ 100 ml 43 47.5 13 56.5 51 39.3 59 65.6 29 64.8 10 44.6 41 55.6 25 36.5 4 67.4 22 41.9 15 42.9 44 53.8 20 52.9 37 26.5 49.7 Lot 4 Lamb # mcg/ 100 ml 43 44.8 13 51.1 51 41.1 59 49.3 29 41.1 10 48.4 41 47.4 25 55.8 4 49.3 22 39.3 15 61.1 44 60.3 20 50.3 37 37.4 48 3 Serum vitamin A levels of lambs during repletion Agnendix Table 3. Lot 1 Lamb # DM% 57 30.7 47 29.6 17 31.1 34 31.4 7 29.6 27 30.5 50 29.4 36 29.8 61 29.3 33 29.7 11 29.3 31 29.8 8 29.0 Avg 29.9 Lot 3 Lamb # DMZ 38 28.8 52 28.9 21 31.1 54 30.8 28 29.8 46 29.8 9 29.3 49 31.7 12 28.3 53 29.6 3 27.9 42 30.1 60 28.9 Avg 29.6 Vitamin A mes/8m Fresh h‘hlhl Vitamin A meg/gm Fresh H IA . . H omboxoo‘oooouo . . s o . . HNWHO‘UIUINUIWN k-h-a\ha J.‘ \l on 43.7 F CHOWGOQOUQWWU H n. I O. “\DUO\€D\INUIU\IUIO\O\ Hid a)\1\nuah>a>\lua«>a\uuuaua CD b Lot 2 Lamb # Lot Lamb # 43 13 51 59 29 10 41 25 4 15 44 20 37 4 DMZ 31.3 33.1 31.0 32.4 30.1 30.5 29.2 30.3 29.8 30.8 31.6 31.0 27.8 30.7 DM% 32.4 29.4 32.1 31.6 30.2 29.7 31.8 31.9 32.9 29.9 30.3 31.9 29.8 31.1 Final liver vitamin A of lambs (Exp I). Vitamin A mcg/gm Fresh H CDF‘CDCDUJh3£~h‘h>h‘hl\3h‘ U) D" Vitamin A meg/gm Fresh 16.4 37.3 37.1 19.3 15.9 17.2 31.3 41.9 17.7 15.4 26.2 23.1 20.7 24.6 a 0(DCDCDBDFINDOrHLIrJUIO .b a>uaLb\o Ula>O\a\\Ic~h-a> c H o \D @umbmuo‘v-belwwm 3. Angendix Table 4. Lot 13 Steer # 808 1317 1138 1152 1186 1129 1150 703 1272 Lot 16 1155 4110 1158 4195 1131 1207 1197 849 1167 Lot 19 1184 836 1291 657 825 685 1190 4156 1229 Lot'22 1230 1178 1220 4209 1169 4240 1256 815 1224 Caro- tene 18.9 17.1 16.0 18.3 14.5 27.0 15.8 22.1 17.9 17.8 20.4 17.1 24.0 16.8 19.9 11.7 20.3 26.3 18.8 21.0 11.4 14.1 12.9 19.1 18.4 24.6 18.4 46.6 33.8 23.1 52.0 41.5 57.8 41.6 26.3 55.1 Vit A 23.6 23.7 30.4 26.4 22.3 21.1 23.1 26.4 23.6 25.3 33.8 28.3 30.0 25.5 37.4 25.5 23.6 23.6 25.3 26.4 31.1 26.5 21.4 34.8 28.4 29.8 23.6 32.9 74.5 34.0 40.3 47.1 42.9 29.3 26.5 42.9 Lot 14 Steer # 814 787 1228 852 1204 697 811 1106 722 Lot 17 1181 1212 1302 1141 1217 847 699 1237 1304 Lot 20 1191 1221 824 1270 1105 831 1126 1134 1153 Lot—23 1288 1305 1117 1321 4071 1284 1187 1120 1114 85 Caro- tene 24.9 40.7 38.5 25.6 40.1 32.0 34.6 25.9 29.6 19.7 21.4 16.2 17.1 18.1 16.3 31.4 19.2 24.2 41.5 57.1 26.5 34.1 34.6 45.6 30.3 32.7 38.9 15.1 18.6 20.1 23.4 24.1 12.5 21.1 13.4 14.4 Vit A 17.4 31.9 20.1 25.3 27.4 32.8 29.3 32.9 25.5 21.4 18.3 20.1 22.8 21.9 28.4 18.3 24.6 30.1 38.4 49.3 38.4 40.3 36.6 33.9 32.6 41.5 38.4 30.0 25.5 24.6 41.9 26.4 23.2 36.4 37.4 25.5 Lot 15 Steer 1139 1104 1168 1127 1287 1346 1215 1208 1194 Lot 18 1183 1228 4242 1166 1162 4217 733 1156 1177 LEE—1’1 1328 1222 1334 1163 1322 1135 1300 1192 4103 Lot 24 1320 1118 1202 1124 1144 4223 1160 1218 4231 Caro- tene 23.6 16.8 20.6 14.6 21.3 18.8 17.5 16.8 23.4 10.9 17.7 13.0 24.7 12.3 20.4 27.6 24.7 14.5 Initial serum vitamin A and carotene of steers (Exp II), meg/100 ml. Vit A 29.5 22. 32. 29 22. 27 27 31 31 oo~1>ooo~oxo N o WO‘HJ-‘HHUIWN 30.8 34.6 25.6 20.1 25.5 27.3 21.9 27.3 24.6 42.8 35.5 33.7 28.0 86 Appendix Table 5. Final serum vitamin A and carotene of steers (Exp II), meg/100 ml. Lot 13 Lot 14 Lot 15 Steer Caro- Vit A Steer Caro- Vit A Steer Caro- Vit A # tene # tene # tene 808 40.7 44.7 814 27.6 26.6 1139 42.7 32.8 1317 10.6 34.7 787 66.3 36.1 . 1104 23.6 37.4 1138 15.8 36.5 1227 42.7 18.2 1168 28.9 34.7 1152 15.8 44.7 852 35.1 27.7 1127 20.6 27.3 1186 14.4 27.3 1204 38.1 38.3 1329 27.3 18.2 1129 26.5 29.7 697 31.5 34.6 1346 28.3 40.8 1150 1.4 51.9 811 27.6 28.3 1215 22.9 39.2 703 8.6 52.9 1106 42.7 48.3 1208 31.5 40.1 1272 32.8 27.3 722 51.8 34.6 1194 28.3 31.0 Lot 16 Lot 17 Lot 18 1155 29.6 24.6 1181 15.4 30.9 1183 17.0 30.9 4110 26.3 45.6 1212 18.4 36.4 1228 26.3 42.8 1158 32.6 40.1 1302 15.4 33.7 4242 8.8 22.8 4195 32.8 37.4 1141 12.5 34.7 1166 15.1 37.4 1131 26.3 36.5 1217 24.7 39.2 1162 8.9 18.2 1207 26.9 47.4 847 9.2 47.4 4217 6.6 58.3 1197 22.1 55.6 699 21.3 37.4 733 22.1 36.5 849 26.9 45.6 1237 21.0 36.6 1156 13.1 60.4 1167 53.8 32.8 1304 25.0 30.1 1177 27.3 40.9 Lot 19 Lot 20 Lot 21 1184 13.7 30.9 1191 95.8 43.2 1328 29.6 33.7 836 23.6 27.4 1221 95.9 50.5 1222 51.3 24.6 1291 13.4 20.9 824 31.9 43.2 1334 24.9 51.1 657 23.3 35.6 1270 41.4 39.2 1163 41.4 34.1 825 3.1 24.6 1105 56.9 44.3 1322 37.4 28.2 685 3.3 37.3 831 46.6 24.1 1135 73.5 38.3 1190 16.5 41.0 1126 47.3 39.7 1300 37.2 29.7 4156 12.9 31.5 1134 45.9 44.6 1192 32.2 31.9 1229 19.7 19.1 1153 79.4 43.7 4103 52.3 31.9 Lot 22 Lot 23 L3: 24 1230 88.0 43.7 1288 22.4 29.2 1320 17.2 33.2 1178 74.9 51.0 1305 20.3 43.7 1118 14.6 39.2 1220 40.7 43.7 1117 21.1 48.3 1202 4.6 29.2 4209 64.3 38.3 1321 17.1 41.9 1124 20.4 43.7 1169 83.4 45.5 4071 26.3 35.6 1144 12.8 27.3 4240 78.1 44.7 1284 7.9 36.5 4223 5.7 40.1 1256 49.9 40.1 1187 17.9 52.9 1160 20.1 44.6 815 51.9 37.4 1120 5.3 26.4 1218 17.1 45.5 1224 84.0 42.8 1114 5.9 39.2 4231 13.6 23.7 87 Appendix Table 6. Final liver vitamin A and carotene of steers (Exp II)- Lot 13 Vitamin A Carotene Steer # DMZ meg/gm mg/liver meg/gm mg/liver Fresh Fresh Fresh Fresh 808 29.9 3.6 17.9 0.4 2.1 1317 28.4 73.4 307.6 1.8 7.4 1138 27.3 26.0 118.1 6.8 31.0 1152 27.1 41.6 185.9 1.5 6.7 1186 28.0 1.4 6.3 1.9 8.7 1129 27.4 45.6 220.4 6.5 31.3 g 1150 25.4 33.2 186.1 3.2 17.7 E 703 25.6 4.3 21.6 1.8 8.8 L 1272 28.1 1.5 7.2 3.6 16.9 I Lot 14 H 814 27.0 21.1 99.2 1.6 7.4 L 787 29.5 35.0 158.4 5.2 23.3 g 1227 28.6 69.8 333.5 1.1 5.5 g 852 27.1 57.3 250.1 0.7 2.9 1204 27.1 16.8 83.1 1.5 7.4 697 27.7 92.1 472.9 1.5 7.4 811 27.3 178.3 802.4 1.3 5.7 1106 27.9 82.4 441.6 5.1 27.3 722 28.0 20.2 97.5 1.4 6.9 Lot 15 1139 28.9 3.2 16.3 1.5 7 5 1104 27.5 55.8 279.9 4.1 20.8 1168 27.4 18.1 79.3 1.9 8.5 1127 29.3 9.8 49.3 3.9 19.6 1329 26.4 0.6 2.4 0.3 1.2 1346 27.4 50.2 228.0 4.0 18.3 1215 26.4 93.5 403.2 2.6 10.9 1208 27.8 59.4 271.7 1.7 8.0 1194 27.7 3.8 20.1 1.2 6.7 Lot 16 1155 27.9 6.3 30.1 0.6 2.7 4110 28.9 99.9 480.3 1.8 8.4 1158 29.3 48.9 210.5 7.7 32.8 4195 28.5 28.3 119.5 4.2 17.5 1131 27.0 5.5 23.6 0.9 3.8 1207 26.3 73.6 340.0 2.6 12.1 1197 28.7 53.3 254.9 3.9 18.5 849 28.9 10.3 43.1 3.1 12.8 1167 27.2 4.3 19.1 0.8 3.5 Appendix Table 6a. Lot 17 Steer # 1181 1212 1302 1141 1217 847 699 1237 1304 Lot 18 1183 1228 4242 1166 1162 4217 733 1156 1177 Lot 19 1184 839 1291 657 825 685 1190 4156 1229 Lot 20 1191 1221 824 1270 1105 831 1126 1134 1153 28.5 26.9 27.7 27.3 27.7 26.6 28.5 26.8 28.7 28.1 28.6 29.6 26.3 28.4 26.5 30.2 28.7 28.6 28.3 27.3 28.0 26.2 26.1 26.4 28.7 28.5 29.2 25.6 27.5 Final liver vitamin A and carotene of steers xp II). Vitamin A meg/gm mg/liver Fresh Fresh 2.2 9.9 13.8 66.1 9.1 38.9 38.3 181.4 4.4 24.8 63.5 327.6 73.9 372.1 77.5 378.9 3.8 19.2 3.1 12.8 69.8 304.9 63.1 224.9 49.1 240.9 3.5 14.3 102.8 461.4 66.3 291.4 13.5 65.7 3.9 20.8 3.9 9.8 103.4 384.7 17.5 76.2 6.6 30.5 3.4 14.9 60.0 244.6 86.8 407.9 28.3 119.5 1.5 5.9 13.3 67.8 77.4 327.2 32.9 141.9 86.4 407.9 19.9 102.9 135.2 539.4 66.9 309.9 7.9 37.7 23.6 109.9 Carotene meg/gm mg/liver Fresh Fresh 0.9 3.8 4.9 23.3 3.0 13.1 5.5 25.8 0.7 3.8 1.7 8.8 7.2 36.3 5.4 23.2 0.4 1.9 1.5 6.1 5.7 24.8 3.8 13.0 4.2 20.5 1.3 5.3 2.8 12.4 2.3 10.1 2.5 12.2 1.2 9.0 0.6 2.1 1.6 6.0 1.3 5.6 4.6 20.9 0.5 2.3 3.2 13.1 5.2 24.2 5.2 24.9 0.5 2.0 1.1 5.6 8.1 34.3 2.1 8.9 5.5 26.0 1.0 5.4 3.8 14.9 2.1 9.8 1.8 8.8 1.6 7.2 Appendix Table 6b. Lot 21 Steer # 1328 1222 1334 1163 1322 1135 1300 1192 4103 Lot 22 1230 1178 1220 4209 1169 4240 1256 815 1224 Lot 23 1288 1305 1117 1321 4071 1284 1187 1120 1114 Lot 24 1320 1118 1202 1124 1144 4223 1160 1218 4231 DMZ 26.4 25.7 27.9 28.8 27.6 27.9 26.3 28.2 25.6 26.8 27.3 29.6 27.5 26.4 27.4 24.7 25.1 25.8 26.2 27.4 26.8 28.8 28.0 27.3 28.3 27.2 28.5 27.0 28.3 26.6 29.2 28.8 28.6 26.4 28.2 27.8 Final liver vitamin.A and carotene of steers (Exp II). Vitamin.A meg/gm mg/liver Fresh Fresh 9.9 42.9 62.1 325.9 16.2 81.7 48.9 214.6 7.3 33.9 93.1 432.1 91.7 467.9 33.2 153.2 4.3 23.7 17.1 83.5 120.2 611.8 47.6 215.8 25.9 109.0 6.9 33.5 126.5 591.3 98.9 480.9 49.8 233.9 21.9 92.2 9.4 36.1 26.3 122.1 17.4 81.9 64.0 265.6 8.4 39.5 110.2 454.9 136.2 596.4 27.5 116.8 1.1 5.3 5.3 27.0 73.1 325.9 45.9 219.3 28.6 139.9 2.3 10.8 94.0 406.1 45.2 235.5 10.2 50.5 9.2 50.9 HwNJ-‘I-ANNUIH HMUIL‘HJ-‘pr-I O O O C O O 0 O O. O. O UIuam>oss~c>m>m>ha mo‘xlI-IUINxot-le I e O 0.0 NmmwaNGO‘ Ok-l-‘nPl-‘UNUO UIN§U|O-FU|NN oweh-Obr-woo Carotene mg/liver Fresh rarer: HwN \JOOHO\\IUUM \ONNMb-L‘HHN FIB) mmnwxnowuoo I I O I O O O O. umubJ-‘wwmn F‘h‘h‘ h‘h‘h‘ HNH OVOONUbOO‘N wmwkaJ-‘xoto H I N UMNO‘NHVUH p:n1 c>c>¢>a-a1¥-haLaL: .zH can“ Amo.uv mv umuuuum afluamuwwaawam «H N .mH cusp Amo.uv mv noumouw mauchHwacme 2H H w.m¢ ~.on m.om ¢.m~ N.¢m Nu.¢m o.mo m.mq m.om n.5m n.nm w>< o.n¢ m.mn 0.0m m.am w.~¢ w.mo m.nw o.Hq o.¢m ¢.om «.om HmMH o.~m «.0N m.n~ o.¢a m.mN N.mq «.wo w.~m N.mm m.Hm m.~m owed n.m¢ H.0m o.Hm m.mN ¢.Hm H.0m m.mo o.Hm m.qm m.wm o.H< «mHH MH 00. 0.?» «.mm w.~m m.o~ cow m.$ H60 now 14.2” mdm «on can m.wm m.m~ w.Nn ¢.o~ o.¢¢ n.m¢ m.~o m.wq m.wm n.m¢ m.H¢ omma w.n¢ ¢.om m.~m H.m~ n.m~ o.¢¢ w.wm n.w¢ Hm.q¢ m.mq m.H¢ w>< m.Hq m.mN m.n~ w.- N.w~ o.H¢ H.om m.w¢ H.o¢ m.mm o.¢¢ mwua m.~¢ m.om m.om w.NN «.oN o.mm o.Hm n.m¢ m.H¢ o.H¢ ¢.em mmHH ¢.nq N.mN m.m~ N.mH o.o~ H.0q «.no ¢.on m.wn o.¢n n.nn mNHH =9 H.om ~.m~ 0.8m o. mo DH 090.com.H «0 acauoufinw no wnwaoHHow aHHH .umme unwoum mo .HE oo~\maa .mowausu coaumuunouaou < :Hamua> Enuom .m manna Nquaomn< .zn saga Amo.uv mvuouwouw mHuamOHwHeme MH 6 .MH awsu Amo.uv.mv nouoouw zHuewUHmHamHm 2H H 6.66 6.66 6.66 6.66 6.66 6.66 NH66 6.66 6.66 6.66 6.66 6>6 6.66 6.66 H.66 6.66 6.66 6.66 6.66 6.66 6.66 6.H6 6.66 H66H 6.66 H.66 6.66 6.66 6.H6 6.66 6.66 H.H6 6.66 «.66 6.66 66~H 6H 6.66 6.66 6.66 2.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 666 1. 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6666 9 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 H6.66 6.66 6>6 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6666 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 66HH an 6.66 6.66 5.66 6.66 6.66 6.H6 H.66 5.66 6.H6 H.66 6.66 66HH 6.66 6.66 6.66 6.66 6.66 6.66 6.66 5.66 6.66 6.66 6.66 NNHH . oz Hmwu m 666 666 66H 66 66 66 NH 6 6 6.5 6 66:66 66\6H\6 .666 666666 6.66a666> 66 pH 666.666.6 66 666666666 cm waHsoHHow AHHH .umme mumoum mo .HE ooH\wuB .moweoso :oHumuucmoaoo < cHEwuH> asuom .w mHndH vaaommd .ZH amsu Amo.UV my uwumouw hHuamo6MchHm MH 6 6.66 6.66 6.66 6.66 6.66 66.66 6.66 6.66 6.66 6.66 6.66 6>< 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6666 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6666 66 6.66 6.66 6.66 6.66 6.66 6.66 6.666 6.66 6.66 6.66 6.66 666 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6666 m" 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6>¢ 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6666 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6666 :6 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6666 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6.66 6666 .oz Mouum 666 666 666 66 66 66 66 6 6 6.6 6 66:66 6666666 .6666 666666 6 a6aau6> 66 66 666.666.6 66 666666666 a waHsoHHom aHHH .umxmv muooum mo .HE 00H\wua .momawno aowuwuuamocou < cHEmHH> Enuom .m oHan xwvaommd (U F 1.. I ”u” ”Jul. .1! I .. “r 1"“ “—5: ',..~ ,."-~ .~ ICHIGAN STRTE UNIV. LIBRARIES LLILLLLLLILILILLLLLILLLILLLLLIILLLI 31293104425909