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Electronic Theses and Dissertations
1983
Vitamin A and Carotene Supplementation on Feedlot Lambs
Donald F. Samuel
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Recommended Citation Samuel, Donald F., "Vitamin A and Carotene Supplementation on Feedlot Lambs" (1983). Electronic Theses and Dissertations. 4392. https://openprairie.sdstate.edu/etd/4392
This Thesis - Open Access is brought to you for free and open access by Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contact [email protected]. VITAMIN A AND CAROTENE SUPPLEMENTATION
OF FEEDLOT LAMBS
BY
DONALD F. SAMUEL
A thesis submitted in partial fulfillment of the requirements for the degree Master of Science Major in Animal Science South Dakota St�te University 1983
SOUTH DA: OF FEEDLOT LAMBS This thesis is approved as a creditable and independent investigation by a candidate for the degree, Master of Science, and is acceptable for meeting the thesis requirements for this degree. Acceptance of this thesis does not imply that the conclusions reached by the candidate are necessarily the conclusions of the major department. L-awrence IB/ Emb"ry uaL.e Thesis Adviser Richard M. Luther Date hesis Adviser T --- John R. Romans 'uate Head, Department of· Animal and Range Sciences ACKNOWLEDGMENTS Appreciation is extended by the author to Dr . Lawrence B. Embry and to Dr . Richard M. Luther , Professors of Animal Science , for their guidance , encouragement , criticisms , patience and friend ship throughout the long period of time involved wi th the research and write-up of this thes is . Dr . Luther is also recognized for assistance in the collection and laboratory analys es of tissue samples. Special thanks to Dr . John R. Romans , Head of the Department of Animal and Range Sciences, and others of the staff at South Dakota State University for their encouragement and interest in this work . Special thanks also to Dr . William L. Tucker , Station Stat istician , for his assist�nce in the statistical analysis of the da ta . Thanks to the many animal science graduate students with whom - I have been acquainted for their advice and friendship . Ap precia tion is also extended to Marjorie Thorn for her expertise in the final typ ing of this thes is . DFS TABLE OF CONTENTS Page INTRODUCTION . . . . . 1 REVIEW OF LITERATURE • 3 The Ro le of Vitamin � and Caro tene in Body Processes . 3 Ef fects of Vitamin A Deficiency (Hypovitamino sis A) ...... 4 Ef fects of Vitamin � Toxicity (Hypervi tamino sis A) .. 9 Me thods of Determining Vitamin· A Status and Re quirements . . . • . . 10 Growth and Deficiency S ymptoms . 11 Blood and Liver Vitamin A Levels . 12 Cerebrospinal Fluid Pressure 17 Factors Affecting the Utilization of Vi tamin A . 18 Vitamin A Precursors ...... 18 Ab so rption, Transport and Storage 18 Preintestinal Losses . 22 Protein and Urea . . 23 Thyroxine, Thiourea and Thiouracil . 25 Ni trate and Ni trite 26 Other Factors 29 Supplemental Vi tamin A for Sheep . 31 Me thods of Supplementing Vitamin A and Carotene . . 31 Reproduction . 35 Page Maintenance of Blood and Liver Vitamin A Levels . . . . . 38 Lamb Performance . 41 ME THODS OF PROCEDURE . . • • • . 44 Trial 1: Vi tamin A Depletion Followed EY Supplementation • . • . 44 Depletion Period . 44 Supplementation Period . 47 Trial 2: Vitamin A S upplementation Without Previous Depletion . so RE SULTS AND DISCUSSION . • . 54 Trial 1: Vitamin A Depletion Followed £y S upplementation . . . • 54 Depletion Period . 54 Su pplementation Period . 59 Blood Vitamin A 64 Blood Carotenoids 66 Liver Vitamin A 67 Trial 2: Vitamin� Supplementation Wi thout Previous Depletion . . . . . 69 S UMMARY 74 LITERATURE CITED • 78 APPENDIX . 89 LIST OF TABLES Table Page 1. COMPOSITION OF THE DEHYDRATED ALFALFA MEAL SUBSTITUTE 48 2. INGREDIENT COMPOSITION OF DIETS , SUPPLEMENTATION PERIOD FOLLOWING DEPLETION , TRIAL 1 ...... 49 3. INGREDIENT COMPOSI TION OF DIETS , SUPPLEMENTATION PERIOD WITHOUT PREVIOUS DEPLETION , TRIAL 2 ... 52 4. FEEDLOT PERFORMANCE, BLOOD AND LIVER DATA DURING VITAMIN A DEPLETION OF LAMBS (TRIAL 1: JANUARY IS- DECEMBER 13, 1976; 333 DAYS) ...... 55 5. AV ERAGE WEIGHTS , FEED CONSUMPTION AND DAILY GAINS DURING SUPPLEMENTATION PERIOD FOLLOWING DEPLETION (TRIAL 1: DECEMBER 14, 1976 , TO MARCH 14, 1977 ; 91 DAYS) ...... 60 6. LEAST-SQUARES MEANS OF SUPPLEMENT SOURCE , LEVEL OF SUPPLEMENTATION AND DAYS OF SUPPLEMENTATION FOR BLOOD VITAMIN A, BLOOD CAROTENOIDS AND LIVER VITAMIN A FOR SHEEP FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A FOLLOWING DEPLETION , TRIAL 1 ...... 61 7. LEAST-SQUARES MEANS OF SOURCE X LEVEL INTERACTION FOR BLOOD VITAMIN A, BLOOD CAROTENOIDS AND LIVER VITAMIN A FOR SHEEP FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A FOLLOWING DEPLETION , TRIAL 1 . . . . • . . . . 62 8. LEAST-SQUARES MEANS OF DAY X LEVEL INTERACTION FOR BLOOD VITAMIN A AND BLOOD CAROTENOIDS FOR SHEEP FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A FOLLOWING DEPLETION , TRIAL 1 ...... • ...... 63 9. AVERAGE WEIGHTS , FEED CONSUMPTION AND DAILY GAINS OF LAMBS FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A DURING SUPPLEMENTATION PERIOD WI THOUT PREVIOUS DEPLETION (TRIAL 2: FEBRUARY 10-MAY 19, 1976; 99 DAYS) . . .- ...... 70 10. LEAST-SQUARES MEANS OF SUPPLEMENT SOURCE , LEVEL OF SUPPLEMENTATION AND SOURCE X LEVEL INTERACTION FOR BLOOD VITAMIN A, BLOOD CAROTENOIDS AND LIVER VITAMIN A FOR LAMB S FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A WITHOUT PREVIOUS DEPLETION , TRIAL 2 ...... 71 LIST OF FIGURES Figure Page 1. Blood vitamin A concentration of 10 lamb s slaughtered at approxima tely 2-ino intervals during depletion pe riod of trial 1 . . • . . .. • . . . . • ...... 56 2. Liver vitamin A concentration of 10 lambs slaughtered at approximately 2-mo interval s during depletion period of trial 1 • • • . . • ...... • . . • • . . 57 LIST OF APPENDIX TABLES Table Page 1. BLOOD AND LIVER DATA OF LAMBS FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A, SUPPLEMENTATION PERIOD FOLLOWING DEPLETION (TRIAL 1: DECEMBER 14, 1976 , TO MARCH 14 , 1977; 91 DAYS) ...... o o o • o 89 2. ANALYSIS OF VARIANCE FOR BLOOD VITAMIN A AND BLOOD CAROTENOIDS DURING SUPPLEMENTATION PERIOD FOLLOWING DEPLETION, TRIAL 1 . o • o o • o o ••• o • o o 90 3o ANALYSIS OF VARIANCE FOR LIVER VITAMIN A DURING SUPPLEMENTATION PERIOD FOLLOWING DEPLETION , TRIAL 1 90 4. BLOOD AND LIVER DATA OF LAMBS FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A DURING SUPPLEMENTATION PERIOD WITHOUT PREVIOUS DEPLETION (TRIAL 2: FEBRUARY 10- MAY 19, 1976 ; 99 DAYS) . . o • o ••• o • • • • 91 5. ANALYSIS OF VARIANCE F�R BLOOD VITAMIN A, BLOOD CAROTENOIDS AND LIVER VITAMIN A DURING SUPPLEMENTATION PERIOD WITHOUT PREVIOUS DEPLETION, TRIAL 2 . o o o • o • 92 1 INTRODUCTION Af ter the discovery and identification of vi tamin A as an essential nutrient , the role of the vitamin and its precursor carotene in the nutrition of animals came under widespread study . Many experi ments have been designed in the past to examine the interrelationships concerning vitamin A and carotene in animal feeding . In view of the similarity of digestive systems between cattle and sheep , it wa s reasonable to assume that vi tamin A and carotene might func tion much the same in the two species . As numerous invest igators have demonstrated, there are many similarities in vitamin A me tabolism between cattle and sheep . However , there are also important differences and some conflicting reports . Generally , among the like ch aracteristics of vitamin A and carotene nutrition are source and me thod of administration, de ficiency and toxicity symptoms , metabolic functions of the vitamin and site of maj or storage of vitamin A in the body . Differences occur in the conversion of carotene to vi tamin A, storage of carotene in the body and length of time for deficiency symp toms to occur wh en consuming diets lo w in vitamin A or carot ene . An apparent greater efficiency in ut ilization of vitamin A and carotene by sheep as compared to cattle has no t be en fully exp lained. This di fference coupled wi th limited evidence as to the full ro le of vitamin A an d carotene in me tabolism leaves the story of vitamin A nutrition far from comp lete. 2 The experiments conducted and reported in this thesis were designed to study the. va lue of supplemental vi tamin A and carotene for feedlo t lambs. The diets used were balanced for known required nutrients except vi tamin A. Various levels of vi tamin A palmitate or carotene from dehydrated alfalfa meal formed the treatments . The level of vi tamin A or carotene intake was based on amount of feed consumed . The objectives of these experiments were to study the length of time involved in depleting vi tamin A reser ves of sheep consuming diets low in carotene and to pro vide information as to the effects of feeding supplemental vi tamin A or carotene to -either previously depleted or apparently normal sheep . Evalua tion criteria were blood and liver vi tamin A and caro tenoid leve�s cons idering sex differences and length of time involved in depleting and repleting liver vi tamin A stores . Blood and liver samples were collected periodically throughout the course of the experiment for evaluation of the vi tamin A status . 3 REVIEW OF LITERATURE ThP. Role � Vitamin A and Carotene in Body Processes Considerab le research has been conducted to study the role of vitamin A and carotene in animal feeding and biochemical systems . Several review articles have been published wh ich summarize much of this research (Hart, 1940 ; Mc Gillivray , 1960 ; Owen , 1965 ; Roels , 1967; Ullrey , 1972; Thompson, 1975) . Various forms of vitamin A exist in animal tissues , wh ile none have been found in plant life (Ganguly and Mu rthy , 1967) . Herbivorous animals such as ruminants by na ture satisfy mo st of their nutrient requirements through consump tion of plants and plant products . Therefore , it was concluded t hat one or more fac tors pr esent in vegetation must be converted to vitamin A in the animal body (Moore , 1930) . These factors were identified as carotene and other caro tenoid pigments and collectively are known as vitamin A precursors . The primary pigment involved is carotene , and this precurs or is often termed provitamin A (Thompson , 1975 ). Laboratory synthesis has made available preformed sources of vitamin A which can be used to supplement diets low in carotene . Any evaluation of vitamin A requirements should take into consideration both carotene and preformed vitamin A. Vitamin A is required for a numb er of body processes . A reaction that takes place in the retina of the eye invo lves vitamin A comb ining with a protein to form visual purp le , an important intermediary product 4 in vi sion. Lack of vi tamin A interrup ts the reaction sequence , resulting in a deficiency symp tom termed night blindness (Maynar d and Loosli , 1969) . Func tions of vitamin A in body processes other than vision are much less understood , although many researchers ha ve examined the vi tamin interrelationships in many species of animals . Thompson (1975) reviewed the role of carot ene and vitamin A in animal feeding. A maj or function of vi tamin A involves body growth and ma intenance of epithelial tissues and thus it plays important roles in many bo dy processes � The vi tamin 's mechanism of action in these processes has no t yet been fully explaine d. Effects of Vitamin� Deficiency (Hypovitaminosis �· Various . symp toms occurring in vi tamin A-deficient animals have been reported in the literature . Wo lbach and Ho we (1925) de scribed in detail the physical characteristics and the location of keratinized epithelium in wh ite ra ts suffering from advanced avitamino sis-A . Symp toms co mmonly found we re humped posture , rough coat , emaciation and encrusted eyelids . Internally , extensively keratinized epithelium had formed . Affected tissues we re located in the respiratory tract , alimentary tract , genito-urinary system, the eyes and their accessory glands and in the thymus gland . Anzano et al . (1979) studied the sequence of appearance of individual signs of deficiency in rats follo wing the induction of synchronous vi tamin A de ficiency . Their da ta we re highly reproducible and the order of appearance of de ficiency signs wa s dep ressed gro wth an d appetite , de creased intestinal goblet cell numbers , de creased pilocarpine induced salivation, tracheal me taplasia , tr ansient periocular porphyria , 5 altered salivary gland morpho logy, decreased stomach emp tying in force fed animals and twisting and crippling of legs . In cattle studies, Guilbert and Hart (1934) recorded clinical symptoms of deficiency after 225 to 240 d of feeding a diet formulated to be deficient in vitamin A. After 282 d, analysis of liver samples indicated the reserves to be prac tically exhausted. In ano ther study by the same wo rkers (1935) , it wa s determined tha t the first deficiency symp tom to occur and the last to disappear following vitamin A supple mentation wa s night blindness . Other wo rkers have also found night blindness to be one of the first symptoms to develop from vitamin A -deficiency (Schmidt , 1941; Jones et al ., 1943) . Embry and Emerick (1964) noted deficiency symptoms in feedlot cattle after 60 d of vitamin A deple tion in one experiment and 75 d in another . Deficiency symp toms included incoordination, ulcerated eyes and excessive salivation. This wa s in agreement wi th earlier wo rk by Halverson and Sherwood (1930) wh ich resulted in various eye lesions and blindness after 88 d of feeding steers co ttonseed hulls and meal . The difference in length of time before deficiency symptoms we re noted by various wo rkers wa s probably due to the na ture of previous feeding of the experimental animals, the ini tial amount of vitamin A in body stores and the carotene level of the de p letion di et. As wi th cattle, Guilbert et al . (1937) established that night blindness wa s usually the first sign of vi tamin A de ficiency to appear in sheep . Several animals we re killed or di ed after 27 to 30 mo of dep letion at wh ich time no liver vita m1n A wa s de tected . 6 In addition to night blindness , Eveleth et al . (1949) reported other symp toms of vitamin A deficiency in ewes wh ich started the experiment as yearlings . Symp toms noted after 175 d we re progres sive excitability , pica , incoordination and corneal opacity . One ewe was slaughtered and examined internally . Findings included enlarged la ryngeal lymph nodes , thyroid gland , left ureter and kidneys ; hemorrhages in the subcutaneous tissues of the legs , lymph nodes , ep iglottis , ep icardium, endocardium, lungs, omasum , liver and duodenum ; inflamed nasal passages and pyloric valve area ; slight cons triction of the left optic nerve in the region of the optic foramen and cqngestion of the lungs . Other wo rkers have no ted similar symptoms in vitamin A deficient sheep ( Schmidt, 1941 ; Klosterman et al ., 1949 ; Dutt and Sawhney , 1969 ; Bayfield et al ., 1972) . It appears that vitamin A may be actively involved in ma intaining ep ithelial tissues , although no mechanism of act ion has been determined . As reported by We bb et al . (1968) , polyurea can occur in vitamin A deficiency . In two trials, deficient we thers produced twice as much urine as control we thers . According to the authors , the results suggest vitamin A deficiency might alter renal function in sheep . Webb et al . (1970) studied renal function in ewes during mild and severe vitamin A depletion. As long as plasma vitamin A levels we re not different , no differences we re found in glomerular fil tration rate (GFR) , renal plasma flow (RPF) , urea clearance or phosphate clearance . Af ter severe depletion of vitamin A stores , GFR , RPF and urea clearance were found signif icantly 7 lo wer, wh ile phosphate clearance tended to be higher , although not significantly different . Ur inary calculi are often found in vitamin A deficient animals. Dutt and Sawhney (1969) we aned lambs at 60 d of age and then maintained them on a lo w carotene diet for 8 to 9 months . Histopathological changes we re examined in the kidneys and related tissues . The epithelial cells in the medullary zone sho wed extensive keratinization impregnated wi th some calcium salts . They conc luded that thi s condition may predispose formation of urinary calculi in conj unction wi th other factors such as . hard wa ter , mineral imbalance, urinary . infection , hyperparathyroidism and alkaline urine . According to Schmidt (1941 ), urinary calculi are frequently found in vitamin A deficiencies of ruminants. Lindley et al . (1949) found urinary calculi in several ram lambs consuming either a deficient or a vitamin A supplemented diet . Ho we ver , Eveleth et al . (1949) found no cases of calculi in ewes or lamb s dying from chronic vitamin A deficiency . Reproduction of ruminants may be adversely affected by vitamin A deficiency . Foss and Eveleth (1944) reported observations of pregnancy disease in several ewe flocks occurring concurrently wi th vitamin A de ficiency symp toms . Many of the ewes we re suffering from ketosis, co rneal opacity and , in extreme cases , comp lete blindness. Abortion wa s co mmon . Pos tmortem examination of several ewes sho we d characteristic signs of vi tamin A defic iency . Many ewes recovered satisfactorily following vi tamin A supplementation . · These wo rkers conc luded that 8 vitamin A deficiency may be a predisposing factor but not the primary cause in the production of ketosis in pregnant ewes . Schmidt .(1941) found that pregnant animals suffering from vitamin A deficiency may abort or give birth to dead or weak young that live only a short time . If the yo ung survive for several days, severe scours often develop before death . Similar results we re reported by Guilbert et al . (1937) and Eveleth et al . (1949). Sooud et al . (1973)· compared the reproductive performance of vitamin A deficient and supplemented ewes . Heat symptoms were normal for all ewes. Ho wever , significant differences we re fo und in conception ra te , numb er of lambs dropped at full term , numb er of lambs born alive and in the number of viable lambs . Hart (1940) and Peirce (1954 ) foun d that cattle and sheep co uld be dep leted to the point of night blindness yet show normal heat , ov ulation and conception . Degeneration of the germinal ep ithe lium of the testes has been linked wi th vitamin A deficiency in bulls (Madsen et al ., 1948) and in rams (Lindley et al ., 1949). Dutt and Sawhney (1969) observed that the testes of vitamin A deficient rams we re much smaller in size and we ight than those of properly supplemented rams . It has been shown that a severe deficiency of vitamin A may be accompanied by widespread nerve degeneration . This is often associated wi th abnormal bone growth near the affected area (Mellanby , 1947). Constriction of the op tic foramen in deficient animals ha s been fo und in conj unc tion wi th cons triction and degeneration of the op tic nerve and 9 night blindness in cattle (Moore , 1939; Moore and Sykes, 1940) and in sheep (Eveleth et al ., 1949). Abnormal development of bones in the skull and spinal column could account for an increase in cerebrospinal fluid pressure found by some workers in vitamin A deficient animals. As reported by Rousseau et al . (1 954), an increase in the cerebrospinal fluid pressure was one of the early changes occurring in calves as they became deficient in vitamin A. Eveleth et al . (1 949) found that the cerebrospinal fluid pressure in sheep disp laying vitamin A deficiency symp toms was much higher than in normal sheep . Other symp toms that may be seen in animals suf fering from vitamin A deficiency are reduc�d feed intake ; pronounced edema of the coronary band , corpus , hock and brisket; lacrimation; xeropthalmia ; convuls ive seizures ; abnorma l semen and marked suscep tibility to respiratory and other infections (Mitchell , 1967). It is unlikely that all the symp toms related to vitamin A deficiency as noted in the literature could ever be found in any single animal . Whenever several of these signs show up in a flock or herd, vi tamin A deficiency should be suspected . Various methods of clinical verification have been developed . Effects of Vitamin A Toxicity (Hypervitaminosis A) . Vitamin A toxicity may occur following ma ssive or prolonged excessive intakes of vitamin A concentrates . This disorder may be acute, but more co mmonly the chronic form is found . 10 Nieman and Obbink (1954) reviewed the toxicity symptoms found in chronic hypervitamino sis A in rats. Generally , there is decreased feed consump tion accompanied by decreased growth, thickening of the skin , loss of hair and skin peeling , skeletal lesions with spontaneous fractures, hypertrophy of many endocrine glands, degeneration of various organs , anemia and other blood changes and damage to the central nervous system. These descriptions of vitamin A toxicity were supported in studies by Hazzard et al . (1964) and Gorgacz et al . (1971) with calves , Fr ier et al. (1974) with goats , Ma llia et al . (1975) with.rats and Donoghue et al . (1979) with ewes. Vitamin A toxicity is no t a common problem because of the wide range in tolerance of the vitamin by animals . However , due to error in formulation and mixing of feeds and mechanized feeding of livestock, vitamin A toxicity could occur . Me thods of Determining Vitamin � Status and Requirements According to Thompson (1975) , the preformed vitamins A, retinyl esters , retinol, retinaldehyde and the provitamins containing a B-ionone ring such as B-carotene can all be converted to retinol , and retinol will satisfy all the known funct ions of vitamin A in animals . As a basis for comparison, the following international standards have been set up for vitamin A activity as related to vitamin A and B-carotene (NRC , 1975) . One Internat ional Unit (IU) or one United States Pharmacopoeia Unit (USP) of vitamin A equals the ac tivity of .300 meg of vitamin A alcohol , .344 meg of vitamin A acetate or .5 50 meg of vitamin A palmitate. Th e standard for provitamin A is B-carotene . One IU of vitamin A act ivity 11 is considered equal to .600 meg of B-carotene , and 1 mg of S-carotene equals 1,667 IU of vitamin A. This is based on conversion of S-carotene to vitamin A by the rat . A further considerati- on is that the biological value of dietary vitamin A or carotene is often influenced by several factors . Different species of animals ut ilize vitamin A and its precursors wi th varying degrees of efficiency , and values determined for rats should not be applied directly to mo st other animals (Ullrey , 1972) . The recommendation for sheep (NRC, 1975) is that 1 mg of feed carotene equals 400 to 700 IU of vitamin A activity. Adequacy of dietary source in fulfilling vitamin A requirements has been evaluated by several.criteria inc luding gro wth rate and deficiency symptoms , blood and liver vitamin A assays and variations in cerebrospinal fluid pressure . Gro wth and Deficiency S ymptoms . Animal requirements for particular nutrients are co mmonly determined after deficiency symp toms occur due to lack of a nutrient and favorable response is sho wn following supplementation of the nutrient . Growth rates are frequently used as a measure of treatment effectiveness in many experimental studies . Numerous researchers have reported that the gro wth rate of animals consuming vitamin A deficient diets often remains satisfactory until decreased feed consumption occurs . Several symptoms develop follo wing reduced feed consump tion (Guilbert and Hart , 1934) . Critical studies wi th cat tle , sheep and swine by Guilbert et al . (1937) indicated that the minimum amount of vitamin A or carotene consumed that wo uld prevent the onset of night blindness appeared to represent a true physiological 12 minimum . Within the different species tested , animals cont inued in a state of thrift for several months and made excellent gains . These results applied equally well with both young and mature animals . Peirce (1945 ) observed that the body weight of sheep consuming vitamin A deficient diets increased steadily and satisfactorily for about 20 months . An imals in an advanced state of deficiency lost weight gradually and , in some cases , suddenly during the last few weeks of their lives . Lindley et al . (1949) no ted partial loss of appetite , general weakness and unthriftiness in 3- to 5-mo �o ld ram lambs that developed deficiency symptoms after 7 to 21 wk on experime nt . However , differences in rate of gain compared to vitamin A supplemented rams did no t become apparent for several months . Late in the exp eriment , all deficient rams had slower rates of gain. These works indicate that growth rates may no t be satisfactory early measures of vitamin A status in deficiency studies . However , growth rates could be useful as an indication of severe dep letion of vitamin A body stores . Blood and Liver Vitamin A Levels . Vitamin A is stored primarily in the liver of animals . Extremely high levels can be built up during ex tended periods of liberal intakes of carotene or vitamin A. This is particularly so in ruminants grazing good pasture or consuming high quality hay . The amount of liver storage plays a maj or role in the length of time before deficiency symp toms appear after animals start to consume feeds low in carotene or vitamin A. 13 Normally , the blood also contains measurable amounts of vitamin A. However,.it is believed that the levels found in the blood are normal physiological amounts and are not appreciably affected by body stores except under condit ions of extremes in storage levels. Carotene and other carotenoid pigments are stored to a lesser degree than vitamin A, and there is greater variability among animal species in the ability or need to store these pigments as compared to vitamin A. Da ta reported by Moore and Payne (1942) indicated substantial differences in liver vitamin A cont ent between cattle and sheep grazing grass pas ture over summer and wint er periods . Liver vitamin A values for the two periods were 468 and 450 IU per g, respectively, for sheep , wh ile values for cattle were 198 and 89 IU per g, respectively . No carotene was found in the livers of sheep , but selected cattle livers were estimated to contain very small amounts. Guilber t and Hart (1934) found the liver vitamin A content of beef steers consuming diets of grain, green fodder and hay or green pasture to be ab out 500 blue units per g of liver . Ac cording to Guilbert et al . (1937), 1 blue unit was equal to approximately .25 IU . Therefore , 500 blue units eq �al approximately 125 IU . Da ta reported by Byers et al . (1955) indicated that cows grazing green pasture may have liver vitamin A values of 75 to 200 meg per gram . We swig et al . (1958) also reported values wi thin this range . Cons iderab ly higher liver va lues have been reported for sheep . Guilbert et al . (1937) found that 7-yr-old ewes coming off green pasture stored 2,500 to 4,000 blue uni ts of vitamin A per g of liver . Based on the SOUTH DAKO A STA E UNIVERSITY LIBRAR'l 3 1...)0 ((\) 0 L_'"' L') 14 given conversion factor (1 blue unit equals .25 IU) , this would be 625 to 1,000 IU of vitamin A per g of liver. Af ter 10 to 16 mo of depletion, the ewes had liver stores of 200 to 600 blue units (50 to 150 IU) per g of liver. Peirce (1945) reported an av erage value of 200 meg of vitamin A per g of liver for sheep coming off summer grazing at a time when the pasture was dry . Corresponding blood values averaged 25 meg per 100 ml of plasma . In fur ther studies , Peirce (1946) duplicated these values for blood plasma but found liver values ranging from 230 to 810 meg per gram . These liver data were supported in ·work by Eveleth et al . (1949) , but the lamb s used in this study had blood vitamin A levels of about 100 IU per 100 ml of serum . The lambs had been feeding on good pasture . Sheep ma intained in arid zones where pastures are dry and poor for mo st of the year have been found to have blood vitamin A levels of about 41 meg per 100 ml of plasma (Dwa raknath and Pareek, 1971) . In a drought feeding experiment , Bayfield et al . (1972) fed vitamin A deficient sheep having blood and liver values ranging from 7.7 to 8.3 meg per 100 ml and 9.0 to 39 .0 me g per g, respectively , for 5 wk on green pasture . At that time , blood and liver values were 22.9 to 28.1 meg per 100 ml and 149 to 210 meg per g, respec tively . Sooud et al . (1973) studied the vitamin A status of ewes raised under desert conditions . Blood values obtained in this work averaged about 34 me g per 100 ml of plasma . The relationship between blood and liver values has been studied extensively in attempts to determine the vitamin A status of animals . There is general agreement that the no rmal range of blood vitamin A for 15 mammals is 20 to 45 me g per 100 ml of plasma as long as there is adequate storage in the liver (Peirce , 1945 ; Ho efer and Gallup , 1947 ; Pope et al ., 1949 ; McGillivray , 1960; Martin et al . , 1968 ; Dwaraknath and Pareek , 1971; Bayfield et al . , 1972) . Wright and Hall (1979) found a linear relationship between plasma and liver vitamin A concentrations . Their data were expressed as logarithms and simple correlation coeffic ients we re .92, .60, .93, .92 and .75 for calves , weanling pigs, rabbits, rats and adult goats , respectively . Du ring periods of low carotene intake , liver stores of vitamin A are utilized to maintain blood levels within the no rmal range . Liver stores of vitamin A must be depleted to a cons iderable extent before blood values fall below about 20 meg per 100 ml (Peirce, 1945 ; Hoefer and Gallup , 1947) . Peirce (1945) reported on a deple tion study with 4- to 8-mo-old lamb s having initial blood and liver vitamin A values of 35 meg per 100 ml and 200 meg per g, respectively . The liver concentration fell to 100 meg per g at 2 mo , 35 meg per g at 4 mo and 4 meg pe r g at 9 mo of depletion. The blood levels remained at the initial level for 3 mo , then fell slowly to 23 meg per 100 ml at 6 mo , 13 meg per 100 ml at 9 mo and remained constant at about 6 meg per 100 ml after the 13th month. Similar results we re reported by Weir et al . (194 9) . The results of another study by Peirce (1954) showed that liver vi tamin A reserves in mature sheep consuming low carotene diets were depleted very slowly . Bl ood vitamin A levels did no t fall below 15 meg 16 per 100 ml of plasma until 16 mo into the depletion period . These data support earlier work by Guilbert et al. (193 7) . The normal vitamin A status of matur e animals is quite different from that of the newborn . Numerous workers have shown that newborn animals have low plasma levels and little or no liver vitamin A stores (Moore and Payne , 1942; Pope et al. , 1944 , 1949; Eveleth et al ., 1949 ; Weir et al ., 1949 ; Sooud et al ., 1973) . However , following the ingestion of colostrum , which is normally high in vitamin A, plasma vitamin A concentration in the newborn reaches a level comparable to. that of adult animals as demonstrated in calves (Guilbert and Hart, 1934) and in lambs (Pope et al. , 1949 ; Weir et al ., 1949; Sooud et al ., 1973) . Similarly; once the young animal starts to consume green feeds high in caro tene content, the liver stores appreciable amounts of vitamin A. Eve leth et al . (1949) demonstrated tha t young lamb s on good pasture had liver vitamin A values in excess of 500 IU per gram . Significant sex differences have sometimes been found in blood and liver concentrations of vitamin A. Brenner et al . (1942) reported that , on equal intakes , ma le rats stored only two-thirds as much vitamin A as females . Also , the ma le rats were depleted of their liver stores more rapidly than the females . "After 6 wk of depletion , the males had lost 91% of their original stores as compared to 79% for the females . At the end of the 13th wk , losses for male rats were 98% vs 91% for females . According to the authors , these da ta suggested a more conservative utilization of vitamin A by the fema le rats. Further studies by Popper and Brenner (1942) verif ied these results . 17 Dw araknath and Pareek (1971) found significantly higher blood values in ewes as compared to rams . Plasma vitamin A values were 44 and 37 meg per 100 ml for ewes and rams , respec tively. Research has shown that there is little direct relat ionship between blood and liver concentrations of vitamin A as long as blood plasma values are in excess of about 20 meg per 100 milliliters . A state of deficiency is indicated when blood plasma values are found below about 20 meg per 100 ml as demonstrated in sheep (Peirce, 1945 ; Hoefer and Gallup , 1947 ; Ev eleth et al ., 1949 ) and in cattle (Madsen and Davis , 1949 ; Pope et al., 1959) . The carotene content of blood plasma and liver tissue in ruminant animals has been studied by numerous experimenters . Heasurable amounts of carotene have been found in cattle (Madsen and Davis , 1949; Pope et al ., 1959) but little or none in sheep (Peirce, 1946; Eveleth et al ., 1949 ; Pope et al ., 1949 ; Har tin et al ., 1968 ; Dw araknath and Pareek, 1971) . According to Haynard and Lo osli (1969) , only traces of carotene are ab sorbed into the blood stream of ra t s, sheep , goats and pigs , while cattle and horses may absorb substantial amounts . Cerebrospinal Fluid Pressure . Several studies have shown that , as an animal becomes depleted of vitamin A, one of the first measurable changes to occur is an increase in cerebrospinal fluid pressure . This has been demonstrated in cattle (Moore and Sykes , 1940; Mo ore et al ., 1943 , 1948 ; Rousseau et al., 1954; Woelfel et al ., 1965) and in sheep (Eveleth et al ., 1949) . 18 Wo elfel et al. (1965) found the av erage cerebrospinal fluid pressure in a calf given an adequate carotene intake to be 76 mm of _ saline , while the value for a deficient animal averaged 250 mm of saline . Similar changes were found in older animals . Eveleth et al . (1949) reported that the normal cerebrospinal fluid pressure in sheep was about 65 mm of fluid , while in deficient animals the value was as high as 152 mm of fluid . Factors Affecting the Utilization of Vitamin A Vitamin A Precursors . Beta-carotene has been found to be the primary precursor of vitamin A and the mo st abundant in natural feedstuffs . However , there are other carotenoid pigments having some degree of vitamin A activity . According to Bauernfeind (1972) , there are over 100 naturally occurring carotenoids, many of which are contained in foods and feeds consumed by man and animals . Only a few of these are known to have vitamin A activity , and some are of minor significance due to rarity in dis tribution or low concentration. Those carotenoid pigments having significant vitamin A activity are alpha-carotene , beta-caro tene , gamma-carotene , the epoxy-8-carotenes , the monohydroxy carotenes (cryptoxanthin) , the mono keto-B-carotenes (echinenone) and the apocarotenals . Ab sorption, Transport and Storage. The conversion of dietary vitamin A precursors to vitamin A takes place primarily in the mucosa of the small intestine as demons trated ·in the ra t (Huang and Goodman , 19 1965 ; Ganguly, 1969), in cattle (Stallcup and Herman, 1950), in the goat (Niedermeier et al ., 1949) and in sheep (Barrick et al ., 1948b ; Klosterman et al ., 1949). According to Goodman (1969b ), the conversion of dietary S-carotene to vitamin A involves a two-step reaction sequence . First, S-caro tene is cleaved to form two mo lecules of retinal . Second , the retinal is reduced to retinol. The retinol then becomes esterified with long chain fatty acids , incorporated into lymph chylomicrons and is transported from the intest ine in the lymph . Huang and Goodman (1965) examined the composition of lymph 14 vitamin A es ters derived from dietary sources using 15- c- retino l and 14 C-S-carotene . Eighty-two percent of the recovered radioactivity was contained in the lymph . Retinyl esters constituted the maj ority of labeled comp ounds and contained approximately 90% of the recovered radioactivity. The predominant ester was retinyl palmitate . All samples of lymph also contained small amounts of labeled retinol. From the lymph , vitamin A es ters pass into the blood stream and on into the liver where retinyl palmitate is removed and stored (Thompson, 1975). Retinol is the circulating form of vitamin A derived by hydrolysis of liver retinyl palmitate and comb ined with a spec ific retinol binding protein respons ible for its transport (Goodman, 1969a) . Other workers have shown that the primary storage form is retinol . Aguilar and Parrish (1971) analyzed liver samp les from cattle , sheep , swine and chickens . They found an average of 96% all-trans retinol wi th a range of 91 to 100% . 20 Mitchell et al . (1967b) demons trated a continual turnover of liver vitamin A stores . Six crossbred ram lambs were given 4.8-mg intrajugular injection� of vitamin A acetate labeled with 384 microcuries of tritium . They found a continuous decrease in tritium activity in the liver and detec ted tritium activity in the blood throughout the trial . The fate of other forms of vitamin A in the diet has also been studied . Retinaldehyde is mostly reduced in the intestinal mucosa to retino l and then transported in the same way as dietary retinol (Thompson, 1975) . Retinoic acid has been found to be absorbed primarily through the portal circulation rather than the lymphat ics , converted to B-glucuronide in the liver and excreted into the bile (Ganguly , 1969) . This study also demonstrated that significant portions of dietary retinol can be absorbed via the portal blood and excreted in the bile . Barrick et al . (1948b) reported on the absorp t ion rate of vitamin A and carotene from various levels of the gastrointestinal tract of sheep . Treatments were administered orally or directly through fis tulas in the rumen, small intest ine , caecum and colon . No effect on blood vitamin A or carotene was observed following direct administration into the caecum or colon. Absorption of orally administered vitamin A was comparab le to that given via the rumen fis tula . The vitamin A level of the blood increased sooner following direc t do sage into the small intest ine as compared to either oral or rumen administration. Changes in blood vitamin A were much slower and less pronounced in cases of carotene administration as compared to vitamin A administration. 21 Klosterman et al . (1949) no ted that blood vitamin A levels were increased in sheep given oral doses of carotene or allowed to consume green grass for short periods of time . In cases where no carotene was found in the intestinal contents, they found no vitamin A in the intestinal wall. However , when carotene was present in the intestinal wa ll , vitamin A was also present . The enterophepatic circulation may play an important role in the vitamin A metabolism of sheep as demonstrated by Bo ling et al. (1969) . · · · · 14 • • T r1t1um 1 a b e 1 e d v1tam1n A an d c- �o -carotene were ln Jected 1n· travenous 1 y to sheep with bile duct cannulas . Du ring the first 12 hr of collection, 87% of the labeled vitamin A and 83% of the labeled carotene were recovered in the bile . When this radioactive bile was inj ected into the duodenum of an anesthetized lamb , blood samples indicated appreciable ab sorption of vitamin A metabolites . Similar results were reported by Hume et al . (1971) . Work by �1cGillivray (1951) indicated the possibility of intest inal synthesis of carotene by sheep . De termination of carotene to lignin ratios during a digestibility trial showed that the ratios decreased through the upper portions of the small intestine , increased through the ileum, reached a maximum in the caecum and decreased slightly through the colon and rectum. It was suggested by the au thors that microorganisms of the ileum and caecum were capab le of carotene synthesis . Kirton et al . (1975) stated that lamb carcasses may some times have yellow fat rather than the mo re common wh ite fat . This may be caused by the presence of bi le pigmenti from damaged livers or the 22 deposition of natural plant pigments . A survey indicated that approxi mately one in 1,000 New Zealand lambs may be rej ected for export due to excessive yellow fat . All analyses of yellow fat samp les indicated the presence of xanthophyll pigments . Crane and Clare (1 975) found lutein and flavoxanthin as the chief caroteno ids in yellow fat from sheep . Small amounts of auroxanthin, S-carotene and flavochrome were also present . Preintest inal Losses . Several workers have shown tha t pre intest inal losses of vitamin A and caro�ene can occur in ruminant animals as a resul t of ruminal degradation. In vitro studies with cattle and sheep by King et al . (1 962) showed significant differences between incubated and nonincubated tubes of rumen fluid containing carotene or vitamin A. Carotene recovery from inoculated but nonincubated tubes averaged 97.5%, while tubes incubated for 9 hr averaged 65 .6% recovery . Vitamin A recoveries for nonincubated and incubated tubes were 81 .1% and 60.4%, respectively . Measured amounts of carotene , vitamin A and chromic oxide were then fed to wethers and steers . At 12, 24 or 48 hr following consump tion, the animals were slaughtered and ingesta were collected from various parts of the gas trointestina l tract . Caro tene-to-chromic oxide and vitamin-to-chromic oxide ratios indicated approximately 40% loss from the rumen-reticulum after 12 hours . Klatte et al . (1964) reported losses of similar magnitude with little differenc e between cattle and sheep . Klatte et al . (1962 ) reported extensive preintestinal destruction of vitamin A in sheep . Mature we thers were either depleted of vitamin A 23 stores and surgically ligated anterio r to the pyloric valve or left normal . Four to 8 hr following intraruminal inj ection of 250, 000 IU of vitamin A acetate, marked increases in serum vitamin A were observed in the nonligated sheep . Only 15 to 26% of the inj ected dose could be recovered from mixed preintest inal contents after 24 hours . Long et al . (1971) studied the effect of added fat in the form of co ttons eed oil on preintest inal disappearance of vitamin A. Steers and wethers consuming a high starch diet averaged 60.4 and 62.1% for vitamin A disappearance, respectively . With the cottonseed oil diet , disappearance values were 70 .2 and 69 .3%, respec tively , for steers and we thers . Potkanski et al . (1974a) reported preintestinal losses of one- fourth to one-third of the dietary carotene intake in wethers consuming isocaloric and isonitrogenous diets containing either alfalfa hay or isolated carotene beadlets . Further studies by Potkanski et al . (1974b ) were carried out with wethers consuming high starch or high cellulose die ts . No differences were found between treatments. Preintestirral losses averaged 23 .1% on the high cellulose diet and 23 .3% on the high starch diet. Protein and Urea . Vitamin A is transported from the liver in the bloodstream bound to a specific retinol-b inding protein (Goodman , 1969a). The protein content of the diet , therefore, may have an effect on vitamin A metabolism . Dye et al . (1945) reported no signif icant effect of protein level on vitamin A utilization in rats fed 9, 18 or 36% protein in the diet . 24 Rechcigal et al. (1962) found that rats receiving no protein in their diet utilized less vitamin A than pro tein-supplemented rats . As the level of protein in the diet was increased, total vitamin A content of the liver decreased. In addition, these wo rkers found that pro tein quality had an effect on vitamin A utilization. Ra ts on an 18% casein diet lost mo re liver vitamin A and had higher concentrations of vitamin A in the kidneys than did slower gaining rats fed diets with 18% of either gluten or zein. Following supplementation of the gluten and zein diets with amino acids , they observed improved growth rates , increased depletion of hepatic vitamin A and greater total utilization of the vitamin. Berger et al . (1962) reported similar results in respec t to caro tene utilization in rats . In general, the proteins of higher quality and qu antity resul ted in greater conversion of carotene to vitamin A than did inferior proteins or protein fed in insufficient amounts . Further support of these results was reported by Faruque and Walker (1970a) on stud ies of vitamin A and pro tein interrela tionships in milk-fed lambs . Anderson et al . (1962) fed vitamin A-depleted sheep either a protein deficient (5 .9%) or protein adequate (10 .4%) diet for 8 wk followed by daily intraruminal inj ections of vitamin A or carotene for 4 weeks . Af ter both periods , the protein-deficient sheep had significant decreases in serum albumin, albumin to globulin ratio , plasma vitamin A and body weight . The protein adequate group stored more than twice as much vitamin A in the liver than did the low protein group . However , the level of pro tein had little effect on carotene conver sion to vitamin A. 25 Gallup et al . (1951) compared the effect on vitamin A metabolism in sheep with pro tein supplemented as soybean oil meal , cottonseed meal or urea . No statistical differences were found ; however , vitamin A storage tended to be greater with cottonseed meal diets than with soybean me al diets . Urea had no effect on carotene or vitamin A me tabolism . Similar results were reported by Love et al . (1961) and Du rdle et al . (1962). Smith et al . (1964) studied the vitamin A status of sheep receiving their maj or source of dietary nitrogen from urea . Compared to sheep consuming a similar diet supplemented with soybean protein in stead of urea, the urea-fed sheep had lower body weight gains and lower liver vitamin A stores . After 3 mo feeding , the urea-fed sheep had average liver stores of 68 meg of vitamin A per g versus 150 meg per g for the soyb ean-fed sheep . In further stud ies reported at the same time , these workers found no difference in we ight gains between lambs fed urea or soybean protein. Al so , no differences were found in plasma vitamin A concentrations or total liver vitamin A content . Thyroxine , Th iourea and Thiouracil . Several researchers have in dicated that a state of hyper- or hypothyro id ism may adversely affect vitamin A and carotene metabolism . Johnson and Baumann (1947a) in duced a hyperthyro id condition in weanling rats by feeding des iccated thyroid tissue . Other rats were fed thiourea or thiouracil to stimulate a hypothyro id cond it ion . Treatments consis ted of either 40 meg of B carotene or 40 IU of vitamin A daily fo� 15 days . There were no signif i cant differences in the ab il ity to store preformed vitamin A among the 26 hyperthyroid, hypothyroid or normal rats . However , when carot ene was fed , hyperthyroid rats stored more vitamin A than did normal rats , while hypothyroid rats stored very little. Thyroxine administration to the hypothyroid rats restored the ability to convert carotene to vitamin A. Similar results were reported by Gambhir et al . (1975). In contrast, Arnrich and Morgan (1954) observed equal efficiency of carotene utilization in supplying vitamin A for bo th maintenance and storage be tween no rmal and hypothyroid rats . Barrick et al . (1948a) no ted that thiourea and high levels of thiouracil administered to feeder lambs seemed to interfere with carotene conversion to vitamin A. Cline et al . (1963) reported that thyroid treatments in the form of 1.05 g of tapazole or 700 meg of triiodo-L-thyronine given as weekly inj ections over a 60-d period had no significant effects on vitamin A storage in feeder lambs . Nitrate and Nitrite . Soil nitrogen , an important nu trient for plant growth , is found primarily in the form of nitrates . Usually , soil nitrates are taken up by plant tissues and converted into amino acids , proteins and other ni trogenous compounds with no excess ive accumulation of nitrates . However, excessive amounts of nitrates may occur in forages during growth under stres s or heavy nitrogen fertili zation . Many studies have been conducted to check the effect of nitrates on the utilization of carotene and vitamin A in animals . O'Dell et al . (1960) reported that weanling rats were more rapidly depleted of vitamin A reserves when fed a vitamin A deficient basal diet plus .3% potassium nitrate than those fed the ba sal die� alone . 27 Emerick and Olson (1962) used vitamin A depleted rats to study the effect of nitrate and ni trite on liver storage during supplementation of the vitamin . Oral administration of vitamin A resulted in average liver levels of 26 .2 and 26 .6% of the administered dose in rats receiving the control and nitrate diets, respectively . However, only 15 .7% of the administered dose wa s stored in rats receiving the nitrite treatment . Inj ection of vitamin A resul ted in very little liver vitamin A storage in rats fed the control and nitrate diets , while tho se receiving nitrite stored significantly higher levels . When carotene was administered, both nitrate and nitrite signif icantly reduced vitamin A storage as compared to the controls . Mitchell et al . (1965) reported increased disappearance of caro tene from the small intestine of rats receiving either .4% potass ium nitrate or .5 to 1% potassium nitrite as compared to controls . Smith et al . (1962) fed 60 sheep for 30 d on basic diets of hay or silage wi th or without vitamin A or carotene supplementation. Four percent of potassium nitrate had no effect on blood plasma or liver contents of vi tamin A. In vitro studies by Pugh et al . (1962) demons trated S-carotene destruction in all cases when incubated with molar ratios of potass ium nitrite ranging from 1:. 5 to 1:100 . Destruction of S-carotene was greatest at a pH of 1 to 3 and least at a pH of 5 to 7. Similarly , Roberts and Sell (1963) showed that potassium nitrite added to sheep abomasum fluid in which the pH wa s less than 4 resulted in destruct ion of most of the added vitamin A during a · 60-min incubation period . 28 However, in rumen fluid having a pH above 6, nitrite destroyed very little of the added vitamin A. Control samples without nitrite also had little destruction of vitamin A. Olson et al. (1963) reported on a series of experiments concerning the effect of nitrate and some of its reduction products on carotene stability . Nitrate did not interfere with carotene metabolism . Ni trite itself caused little destruction of carotene in neutral or alkaline medium . However, nitrite , nitric oxide and nitrogen tetroxide did de stroy carotene under acid conditions . When the pH was decreased below 6, destruction of carotene occurred rap idly wi th ni trite , apparently due to the decomposition of nitrous acid to yield gaseous oxides of nitrogen which caused rapid destruction of carotene . Embry and Emerick (1963) reported similar results on studies wi th rats , cattle and sheep . Their work indicated that nitrate may cause mo re significant carotene losses in feeds such as silages prior to their consumption than after ingestion . Goodrich et al . (1964) found that 2.5 and 3.0% sodium nitrate had no effect on plasma vitamin A levels in sheep . However , the sheep fed 3.0% sodium nitrate had 46 .0% less liver vitamin A stores than did the controls . Likewise, sheep receiving 3.0% ·sodium nitrate plus 4,100 IU of vitamin A daily had 45 .1% less liver vitamin A than did sheep receiving 4,100 IU of vitamin A without nitrate . Mitchell et al . (1967a) reported that potassium ni trate did no t significantly increase apparent preintestinal destruction of vitamin A in steers and we thers . Vi tamin A recovery from abomasal samples with 29 and without added potassium nitrate wa s 50 .4 and 53 .0%� .respectively , for steers and 50 .7 and 44 .2%, respectively, for we thers. Ho ar et al . (1968) found that 2.5% sodium ni trate significantly reduced plasma and liver vitamin A concentrations in lambs supplemented with either carotene or vitamin A following vitamin A depletion . Hydroxylamine is an intermediate compound formed during the reduction of nitrate to ammonia. Wald et al . (1955 ) demonstrated that hydroxy lamine is a retinene trapping agent , and Jamieson (1958) detected this compound in the rumen of sheep . Tillman et al . (1965) studied the effect on the vitamin A status in sheep of inj ected hydroxylamine . Daily inj ections of 8.0, 1.9 or 2.9 mg of hydroxylamine per kg of body weight in lambs resulted in significant reductions in plasma vitamin A concentrations , but no signifi cant differences were noted in liver vitamin A levels . Davison et al . (1965) reported on two experiments using pregnant ewes fed forages plus various levels of nitrate ranging from .4 to 2.6% . While plasma and liver vitamin A level s were related to the carotene content of the forage , there was no indication of nitrate interfering wi th either carotene utilization or vitamin A levels in the blood and liver tissue . Other Factors . Several other factors have been reported in the literature as having some effect on vitamin A and carotene me tabolism . 30 Klosterman et al. (1952) reported that, when rats we re fed equal amounts of carotene or vitamin A and either a low or high pho sphorus diet , ra ts receiving the low phosphorus diet stored significantly more liver vitamin A. With sheep , a highly significant negative correlation ( r = -.39) was found to exist between blood serum ino rganic phosphorus and vitamin A. Support for these results was found in work with lambs by Gallup et al. (1953). In vitro studies by Erwin and Page (1958) demonstrated that vitamin A, carotene and alfalfa pigments were ab sorbed by soft phosphate particles . In further studies , a comparison wa s made of soft phosphate , bone meal and dicalcium phosphate as sources of supplemental phosphorus for sheep . Soft pho sphate significantly reduced liver vitamin A stores mo re than did either bone meal or dicalcium phosphate. Ricke tts et al . (1965) reported on the effect of iron and vitamin A supplementation for newborn lambs. In one trial, lambs treated with 25 ,000 IU of vitamin A orally in capsule form or the same vitamin A treatment plus 225 mg of inj ectable iron dextran at birth had signifi cantly higher plasma vitamin A levels at 10 d of age than controls or lambs receiving iron dextran alone . Moo re et al . (1972) reported a direct relationship between copper and retinol concentration in the blood plasma of mature ewes at pasture . A correlation coeff icient of .511 wa s determined . However , in three lamb s fed to induce copper pois oning , an inverse relationship was found between blood copper and re tinol. Two lambs dosed for long periods with copper acetate but no t displaying copper toxicity symptoms had higher liver copper levels but no rmal retino l reserves . 31 Arora et al . (1973) reported results concerning the influence of dietary zinc on blood vitamin A. The concentration of blood serum vitamin A was less throughout the experiment in lambs fed a low zinc diet . Mean blood serum vitamin A levels were 37 .1 meg per 100 ml while on the low zinc diet . After supplementing with 50 ppm zinc in the basal diet , mean blood serum levels were 48 .5 meg vitamin A per 100 milliliters. Varnell and Erwin (1959) reported that adrenaline admini stered intramuscularly to cattle and sheep had no effect on the serum pro tein fractions or the carotene and vitamin A content of blood and liver . Eaton et al . (1949) observed the effects of added crude soybean lecithin on the absorp tion and utilization of vitamin A in pregnant ewes . Ewe supplementation with 200 , 000 IU of vitamin A daily or the same vitamin A treatment plus 4 g of crude soybean lecithin daily did no t significantly raise maternal or newborn lamb blood plasma vitamin A levels as compared to controls consuming a low vitamin A basal diet . However , the liver vitamin A levels of lambs at birth and at 30 d of age from the vitamin A and lecithin supplemented ewes were significantly higher than from lambs of the control ewes . There were no differences between the vitamin A and vitamin A plus lecithin treatments . Supplemental Vitamin A for Sheep Methods of Supplementing Vitamin ! and Carotene . Vitamin A and carotene have been used to supplement a wide variety of sheep diets . Methods of supplementation that have be�n used include intrasmuscular , intravenous and subcutaneous inj ections ; oral do sing and administration 32 in feed mineral mixes. A variety of different forms of vitamin A and carotene have also been used. Foss and Eveleth (1944) reported that 30,000 units of vitamin A daily administered by capsule to a pregnant ewe near parturition and displaying vitamin A defi ciency symptoms resulted in some improvement in locomotion and eyesight . However , the ewe was in a severe state of deficiency in conj unction with pregnancy disease symptoms and died after 5 d of vitamin A supplementation. Hoefer and Gallup (1947) compared the vitamin A value of a caro tene concentrate from alfalfa meal to a Research Carrot Oil in gelatin capsules and a fish liver oil. Administered at equivalent carotene levels , the alfalfa meal cons istently resulted in higher blood and liver values than did the Research Carrot Oil. In a second trial , the fish liver oil was superior to alfalfa meal , especially in liver storage of vitamin A. Klosterman et al . (1949) found that crystalline carotene suspended in water with Tween 20 or caro tene in cot tons eed oil inj ected into the bloodst ream of lambs was rapidly removed but did no t increase the vitamin A levels in the blood or liver . Also , Church et al . (1954) demons trated that aqueous carotene preparations dispersed in Tween 40 inj ected intravenously at a level of ap_proximately seven times the minimum daily requirement did result in significantly higher blood vitamin A levels than the controls . Af ter carotene inj ections , the mean blood values at 3, 6, 9 and 12 hr were approximately twice the initial 33 value. Differences in liver vitamin A values were highly variable but no t statistically significant . Peirce (1954) supplemented ewes during gestation with carotene via lucerne meal or in the form of a concentrate in oil . Blood vitamin A levels increased in amounts propor tional to carotene intake , but the supplements in the form of concentrate in oil resulted in smaller increases than equivalent amounts of carotene in the form of lucerne meal . Franklin et al . (1955) observed a high degree of protection against vitamin A deficiency in 18-kg weaned lamb s when individually drenched with a single dose of 500, 000 IU of vitamin A in the form of a shark liver oil emulsion . Beneficial effects were evident in blood plasma vitamin A levels and survival rates nearly 6 mo after vitamin A administration. Varnell and Erwin (1960) reported on a study involving intra rumina! , intraperitoneal , intramuscular and subcutaneous inj ections of aq ueous emulsions of carotene and vitamin A at levels of 2.2 mg per kg of body we ight . Av erage initial plasma vitamin A levels in lamb s receiving vitamin A inj ections by intraruminal , intraperitoneal, intra mu scular and subcutaneous routes were 35 .6, 35 .2, 34 .0 and 41.9 me g per 100 ml, respectively . At 24 hr , plasma levels were 48 .6, 46 .4, 46 .8 and 44 .7 meg per 100 ml , respectively . Intraruminal and subcutaneous inj ections of carotene did no t affect plasma vitamin A levels . However , intraperitoneal and intramuscular inj ections of carotene increased from initial levels of 28 .7 and 26 .1 meg per 100 ml to 29 .2 and 39 .7 meg per 100 ml at 24 hours . 34 Hayes et al. (1962) reported that gelatinized vitamin A mixed with salt lost 20 to 25% of its potency in a 2-wk period and almost all of its potency after 5 weeks . Calcium stearate added at a rate of .25% to similar mixtures resulted in a 20% loss of vitamin A activity the first week but no further losses during the 6-wk trial. The calcium stearate prevented caking and resulted in less mo isture being absorbed by the mixture. Anderson et al . (1962) dep leted sheep of liver vitamin A reserves while feeding protein deficient (5.9% crude protein) or protein adequate (10.4% crude protein) diets. Intraruminal inj ections of .1 mg of vitamin A acetate per kg of body weight were nine times more effective in the protein deficient group and more than 18 times more effective in the protein adequate group than intraruminal inj ec t ions of .2 mg of s carotene per kg of body weight . In a 21-d trial , Faruque and Walker (1970b) found that newborn lambs stored significantly more retinol in the liver when supplemented daily with 550 meg of retinyl palmitate per kg of body weight than similar lambs supplemented with 82,500 meg of retinyl palmitate per lamb on the first day of life. Bayfield et al . (1972) found that ewes in a state of vitamin A deficiency recovered and stored substantial amounts of vitamin A in the liver after being turned out to good quality green pasture for 5 weeks . Other deficient ewes did not respond to an inj ect ion of 150,000 IU of vitamin A. Three d after the inj ection, these ewes were drenched with . 1 million IU of vitamin A and ma intained on vitamin A def icient die.ts 35 for the same 5-wk period as the pastured ewes . At that time, liver vitamin A stores were similar in all ewes . Based on the reviewed literature, it. appears that oral adm�nis tration of either vitamin A or carotene tends to result in greater liver storage as compared to other methods of supplementation. Although data are limited, it also appears that vitamin A supplementation resul ts in higher blood and liver levels of the vitamin than does carotene supple mentation for a given set of test conditions . Either vitamin A or carotene sources can be used satisfac torily in supplying the vitamin A needs of sheep . Which source and method of administration to use depends on the quality of feedstuffs, condition of the animals and stage of produc tion. Reproduction . Supplemental vitamin A given to ewes before breeding or during gestation has been shown to have varying effects . The va lue of supplementing ewes with vitamin A depends on the time of supplementation in relation to breeding and the amount of body reserves of the vitamin at the time of supplementation. Hart and Mil ler (1937) reported no differenc e in the number of lamb s born between adult ewes fed a low vitamin A diet over a period of 5 mo prior to and includ ing the breeding season and similar ewes supplemented with vitamin A at levels above the minimum requirements during the breeding season. We ir et al . (1946) observed the effect of breed , season and age on the variation of blood vitamin A levels in sheep . Four Hampshire , four Shropshire and four Southdown ewes were selected and blood samples 36 were obtained every month for 2 years . Blood from the Hampshire ewes had higher vitamin A levels than the other breeds in all but one month . - All of the ewes tended to have higher blood vitamin A levels during the winter period while in drylot and lactation than any other time of the year or pasture periods . Weir et al . (1949) found that, if not supple- mented with vitamin A, good quality roughage was required in the winter diets of ewes in order to maintain optimum vitamin A levels of blood plasma and liver reserves of the ewes , their lambs and the ewes ' milk . Satterfield and Clegg (1944) determined the vitamin A content of colostrum from ewes that had been grazing on excellent pastures consisting of lezp edeza and native grasses . The vitamin A content was highest on the first day following parturition and diminished through day 7. The level of vitamin A in the milk during the second and third weeks wa s lower than during the first 7 days . Ricketts et al . (1965) studied the effect of supplementing ewes 40 d prepartum with 1,393 IU of vitamin A palmitate per kg of body weight . As compared to controls , the supplemented ewes had significantly higher plasma vitamin A levels both at 10 d prepartum and postpartum. The vitamin A level of colostrum was 129% higher for the treated ewes , and the vitamin A level of the 10-d pos tpartum milk samples was 96% higher in the treated ewes as compared to controls . Colby et al . (1950) compared the results of supplemental vitamin A administered to ewes after grazing green or dry summer ranges . No consistently significant differences were no ted in ewe blood plasma vitamin A levels , percent lamb crop , lamb birth weights and vigor or growth rates of the lambs . 37 Pope et al. (1949) observed that ewes wh ich were no t supple mented with vitamin A or carotene aside from that which was obtained from good quality feeds had consistently higher plasma vitamin A levels during lactation than at any other time . No significant drop in plasma vitamin A level was no ted immediately before or after parturition . The colostrum contained six to seven times as much vitamin A as did later milk samples . Also , the first day colostrum samp les averaged 224 and 255 meg per 100 ml for the ewes nursing singles and twins , respectively . The second day milk samples averaged 189 and 131 meg per 100 ml , respec tively . The authors indicated that the number of young may have had an effect on the rate of transfer of vitamin A from colosteral to normal milk , although no signif icant differences were noted in the plasma vitamin A levels of the lambs . Peirce (1954) conc luded that the reproductive performance of mature ewes was not likely to be adversely affected by low caro tene intake until after 18 mo of feeding under these conditions . Results showed that satisfactory rep roductive performance could be obtained when carotene intake was 50 meg or mo re per kg of body weight , provided at least 80% of the carotene was supplied as lucerne meal . Sooud et al . (1973) studied the effect of vitamin A supplementa tion on the productivity of ewes managed under desert conditions . Three months before the start of breeding , all ewes were started on a vitamin A deficient diet . Treatment groups consisted of a control, medium and high level of vitamin A supplementation containing 0, 50,000 IU and 100,000 IU , respectively , of vitamin A per head per month in one dose . The numb er of 38 lambs born alive per 100 ewes bred was 0, 50 and 70% for the control, medium and high treatment groups, respectively . The medium and high treatment groups showed no significant differences ; however , both were highly significantly different from the control groups . Reproductive performance of ewes consuming adequate diets and having normal vitamin A reserves apparently is no t affec ted by additional vitamin A supplementation. However , ewes managed under drought conditions or fed low carotene diets for prolonged periods have shown improved rep roductive performance following vitamin A or carot ene supplementation. Maintenance of Blood ---and Liver Vitamin -A Levels . The minimum requirements for vitamin A and carotene under a variety of conditions have been reasonably well established. Many workers have also studied the effect of supplementing vitamin A and caro tene in order to maintain adequate blood and liver levels for normal physiological functions . Generally , results of such studies indicate that preformed vitamin A is mo re effective than vitamin A precursors , and there appears to be a lesser effic iency of utilization in deficient animals as compared to normal animals . Guilbert et al . (1937) conc luded that the minimum requirements for caro tene and vitamin A of cattle , sheep and swine were 25 to 30 meg and 6 to 8 meg daily per kg of body weight, respectively. Further studies by Guilbert et al . (1940) support the previous work . In addition, these workers found that about three times the minimum vitamin A level and five times the minimum carotene level were required 39 before significant liver storage and no rmal reproduction could be demonstrated . In terms of the ratio of efficiency of carotene to vitamin A by weight , the level corresponding to minimum requirements wa s about 6:1. The ratio resulting in signif icant storage and successful reproduction was about 10:1. Peirce (1945) fed young sheep previously depleted of vitamin A reserves either a low , intermediate or high level of carotene for a period of 14 months . All sheep grew satisfactorily . Sheep consuming 10 meg caro tene per kg body weight developed several vitamin A deficiency signs . Sheep receiving 25 to 30 meg carotene per kg body weight also displayed night blindness. Sheep receiving 50 to 55 me g carot ene per kg body we ight showed no signs of vitamin A deficiency . Final blood vitamin A levels were 6, 11 and 24 meg per 100 ml plasma for the low , intermediate and high carotene treatments, respectively . None of the treatments had an appreciable effect on liver storage of vitamin A. Similar results were reported by Hoefer and Gallup (194 7) . In addition , these wo rkers found that lambs consuming 500 IU of vitamin A per kg body we ight daily from alfalfa meal or fish liver oil sources stored a considerable amount of vitamin A in the liver . Liver vitamin A levels were approximately 19 ,742 ± 1686 meg and 3,322 ± 136 meg per 100 g for lamb s receiving fish liver oil and alfalfa meal , respectively . Plasma vitamin A levels were 34 .3 and 33 .8 me g per 100 ml , respectively , for the alfalfa meal-fed and fish liver oil-fed lambs . Approximately ten times the minimum requirement for caro tene in the form of alfalfa meal was needed to maintain the initial level of liver vitamin A. 40 Gallup et al . (1951) reported that a level of intake approxi ma tely eight times minimum carotene requirements was necessary to result in appreciable liver storage of vitamin A in lambs . Diven and Erwin (1958) found that the ratio of biological value of vitamin A alcohol to S-carotene was 3.3 in sheep consuming a normal , vitamin A balanc ed diet and 4.6 in sheep consuming a vitamin A deficient diet . All animals received intraruminal inj ections containing 1.3 mg per kg body weight of either S-carotene , vitamin A alcohol or vitamin A acetate. Similar absorption rates were found for vitamin A alcohol and vitamin A ac etate. However, the sheep treated with 1.3 mg per kg body weight of the alcohol form stored 2.3 times mo re hepatic vitamin A than sheep treated with the same level of vitamin A acetate. Myers et al . (1959) fed lambs previously dep leted of vitamin A reserves the following treatments for six consecutive 7-d periods : 88 , 176, 352 or 704 meg carotene per kg live weight per day from dehydrated alfalfa leaf meal or 17.6, 35 .2, 52.8 or 70.4 meg vitamin A alcohol per kg live weight per day from a dry carrier . The effect on liver storage was greater with vitamin A supplementation than with carotene supplementation. Analyses of plasma vitamin A showed carotene to vitamin A ratios on a weight basis to be 8:1, 9:1, 11:1 and 13 :1 for carotene treatments 88 , 176, 352 and 704 meg per kg live weight per day , respectively . Liver vitamin A assays showed ratios of 6:1, 8:1, 10:1 and 13 :1 for the same carotene treatments . There is general agreement that the minimum requi rement to maintain adequate blood levels of vitamin A for normal physiological 41 func tions is 25 to 30 meg carotene daily per kg body weight or 6 to 8 me g daily vitamin A per kg body weight . Also , it appears that in order to obtain significant liver storage of vitamin A in deficient animals , mo re than three times the minimum vitamin A level and more than five times the minimum carotene level are required . Preformed vitamin A has been shown to be more effective than carot ene sources in terms of ma intaining blood levels and liver vitamin A storage . However , if administered in adequate amounts, both preformed vitamin A and carotene can be used to maintain and store normal vitamin A levels in the body . Lamb Performance . Numerous researchers have reported on the effects of vitamin A supplementation to young lambs during growth and fattening trials . Benefits of such supplementation seem to dep end on the level of body vi tamin A stores at the start of such trials . Hoefer and Gallup (1947) concluded that supplemental vitamin A has little effect on the gaining ability of sheep if they have appreciable body reserves of the vitamin . Eveleth et al . (1949) found that lamb s on good pasture maintained liver vitamin A levels of over 500 IU per g, but that weaned lamb s consuming a low carotene diet became depleted very rap idly and many died of intercurrent infections . The addition of carotene to the diets had an ins ignificant effect on weight gains . Lindley et al . (1949) reported highly significant differences in average daily gains between ram lambs treated daily with 20 ,000 IU of vitamin A in the form of percomorph liver oil and ram lambs consuming a basal diet with or without treatments other than vitamin A. Av erage 42 daily gains over the entire feeding period were higher for the vitamin A treated rams . However , these differences were no t evident until after several months of feeding . Myers et al . (1959) reported that daily gains of lambs fed differertt levels of vitamin A or carotene were no t affected by treatment . Similar results were obtained by Smith et al . (1962) . Bell et al . (1963) repor ted that lambs from ewes treated daily wi th vitamin A start ing approximately 40 d prior to and ending at lambing gained over 2.3 kg more from birth to 90 d of age than similar lambs from control ewes . The plasma vitamin A level at birth was 14 .7 and 6.3 me g per 100 ml , respectively , for lamb s from treated ewes and from contro l ewes . Bell and Johnson (1964) concluded that supplemental vitamin A given to ewes during late pregnancy had no effect on lamb weight gains at 10, 30 or 90 d of age as compared to lambs from control ewes consuming a good quality diet . Ri cketts et al . (1965) found that supplementation of newborn lamb s wi th 25 ,000 IU of vitamin A orally in capsule form compared to simi lar lamb s without vitamin A supplementation had no significant effect on we ight gains through 90 d of age . Bell et al . (1965) reported inconsistent results of supplementing 3- to 4-wk-old creep-fed suckling lambs wi th single injections of 250 ,000 IU preformed vitamin A. In this 2-yr study , only one group of treated lamb s during the second year had significantly higher daily gains than similar lamb s consuming a contro l diet . 43 Faruque and Walker (1970b ) found no significant differences in live weight gain among several groups of newborn lamb s fed for 21 d with or without vitamin A or carotene supplementation. Treatments consisted of a control diet, vitamin A given as retinyl palmitate at rates of 13 .75, 55 .0, 220 or 440 meg per kg live weight per 24 hr or caro tene given as 8-carotene at rates of 68 .5, 275 , 1,100 or 2,200 meg per kg live we ight per 24 hours . Martin et al . (1968) fed a vitamin A depletion diet to 61 crossbred lamb s for a period of 110 days . Following the depletion phase, the surviving lambs were fed for 71 d ei ther a bas al diet containing corn silage carotenes or the basal diet plus 1.1, 3.3 or 9.9 mg of retiny l palmitate per day . No significant differences were found in av erage daily gains during either the depletion or repletion periods . As discussed earlier in this report , growth rates of lambs consuming low carotene or vi tamin A deficient diets for pro longed periods have been satisfactory . It would also appear that supplemental vi tamin A or carotene has little or no effect on the growth rates of lamb s consuming diets considered adequate in vitamin A content . 44 METHODS OF PROCEDURE The obj ectives of these experiments were threefold . In trial 1, a depletion study was conducted to determine the rate and time involved in depleting feedlot lambs of their vitamin A body stores . The dep letion period was followed by a supplementation period to examine blood and liver response to various levels of carotene or vitamin A. In trial 2, the objec tive was to examine the effects of vitamin A or carotene supplementation to typical feeder lambs wh ich had no t been previously depleted of body vitamin A stores . The experimental animals were selected from a group of 423 native South Dakota lambs purchased in early December , 1975 . Shortly after arrival , all lambs were weighed , ear tagged , sex identified , vaccinated for prevention of enterotoxemia and drenched for control of in ternal paras ites . A few lambs had no t previously been castrated or docked , and these operations were performed during processing . The lambs were fed alfal fa-brome hay ad libitum unt il the start of each trial . Prio r to and throughout the experimental periods , they 2 were maintained in outside, unpaved pens measuring 4.88 m and bedded with straw as needed . Feed was offered in open bunks , and wa ter was provided by an automatic fountain equipped wi th electrical heat ing elements . Trial 1: Vitamin � Depletion Followed £y Supplementation Depletion Period . The depletion period of trial 1 began on January 15 , 1976 , and ended on December 13, 1976 , after 333 d on test . 45 The experimental animals consisted of 160 lambs with an equal numb er of ewes and wethers . The lambs were weighed on January 13, 1976 , and averaged 27 .3 kilograms . Five ewes and five wethers were slaughtered to obtain blood and liver samples in order to determine initial vitamin A levels . On January 15, 1976 , the remaining 150 lamb s were allotted to 10 pens of 15 head each on the basis of we ight wi th ewes and we thers penned separately . The depletion diet consisted ini tially of rolled oats plus free access to a mineral mix wi th equal parts of trace mineral salt and dicalcium phosphate . This diet was considered to be sufficiently low in carotene so that essentially no source of vitamin A was available to the lambs . At the beginning of the depletion phase , the hay offered was adj usted to .91 kg per head daily . The hay was gradually reduced with an increase in the vitamin A depletion diet until hay was eliminated from the diet in 10 d and the lambs we re essentially on a full feed in 15 days . Once on full feed , feeding wa s once daily to appetite for each pen of lambs . The depletion diet was later altered by providing a supplement containing ground limestone , dicalcium pho sphate , trace mineral salt and chlortetracycline wi th ground oats as a carrier . The supplement was top dressed at a rate of .09 kg per head daily at each feeding . This was thought necessary because there was very little consump tion of the free choice salt-dicalcium phosphorus mixture . Feedlot performance during the depletion period was no t considered to be a ma in measure of treatment effects due to the extended length of 46 time anticipated to deplete the lambs of liver vitamin A stores . Schmidt (1941) and Peirce (1945) reported normal growth of lambs during deficiency trial s last ing approximately 1 year . Hoefer and Gallup (1947) reported no deficiency symptoms and normal growth in lambs fed vitamin A deficient diets for 194 days . Similar resul ts we re reported by Klosterman et al . (1949) . Al l pens of the test animals were weighed at 2-mo intervals. After each weighing , the five heaviest ewe s and five heaviest wethers were selected for slaughter . This procedure was used in an attempt to keep equal numbers of each sex and for un iformity of weight wi thin the groups . This me thod of selection was considered to have advantages over a random selection, which wo uld greatly accentuate weight variation , in view of the extended depletion period expected . Periodic slaughter of animals from experimental groups to obtain blood and liver samples for vitamin A analyses during depletion studies has been reported by several other wo rkers (Peirce, 1945 ; Hoefer and Gallup , 1947; Gallup et al ., 1951; Martin et al ., 1968; Bayfield et al ., 1972) . Blood and liver samp les were obtained on each slaughter date in order to determine the lambs ' vitamin A nutritive state and liver stores . The blood samp les were allowed to clot so that serum could be obtained . The serum was drawn off and frozen for storage until analyses for vitamin A and carotene could be carried out . Liver samp les we re obtained by removing the caudate lobe of each liver so that in every case the same area of liver wa s sampled . The liver tissues were frozen and stored until analyses for vitamin A and carotene could be performed . 47 The analysis procedure followed was that based on the Carr-Price reaction as described for blood by Ko ehn and Sherman (1940) and for liver by Johnson and Baumann (1947b) . Ninety-one animals weighing an average of 67 .6 kg completed the 333-d depletion phase. Blood and liver vitamin A levels of the 10 slaughtered animals from the group at this time indicated a severely depleted state . Eighty animals were used for the supplementation phase of the experiment . Blood and liver data from the depletion phase were analyzed using linear regression as outlined by Steel and Torrie (1980) . Supplementat-ion Period . The supplementation period of trial 1 began on December 14, 1976, and ended on -March 14, 1977, after 91 don test . Forty ewes and 40 wethers weighing an average of 61 .7 kg were allotted to 16 pens of five animals each according to sex and weight . Blood samp les were taken from the jugular vein of all sheep at this time . Similarly , the lambs were weighed and blood samples taken every 28 d for three periods . Final blood and liver samples were obtained when the lambs were slaughtered upon completion of the trial . Al l samples were handled as in the depletion phase of the experiment . According to the des ign of the experiment , eight diets were calculated to be approximately equal in nutrients except for the level of vitamin A or caro tene . In order to do this , a feed mixture was formulated to be similar to dehydrated alfalfa meal in pro tein, energy and calcium but wi th essentially no carotene . The feed mixture was used to rep lace variou s portions of dehydrated alfalfa meal to provide carotene trea tment levels and as an ingredient rep lacement in diets wi th the vitamin A 48 treatments . Composition of this mixture is shown in table 1 and as percentages of the diets in table 2. E_ach of the eight diets supplied a different treatment level of either carotene or vitamin A. The source for carotene was from a supply of dehydrated alfalfa meal which , upon analysis , had a carotene concen- tration of 143 mg per kilogram. The vitamin A was from a product wh ich , upon analysis , had a concentration of 350 , 000 IU vitamin A palmitate per gram. The ingredient composition and treatment levels of the diets are shown in table 2. TABLE 1. COMPOSITION OF � THE DEHYDRATED ALFALFA MEAL SUBSTITUTE Ingredient % Dried beet pulp 40 Oat hulls 25 Soybean oil meal (44%) 32 Ground limestone 3 Carotene treatment levels 2.2, 4.4, 8.8 and 17 .6 mg per kg of diet correspond to vitamin A levels 880 , 1,760, 3,520 and 7,040 IU per kg of diet , respectively , based on the conversion factor of 1 mg feed carotene equals 400 IU vitamin A activity (NRC , 1975) . Because each diet contained some of the ingredients in small amounts , a premix was first prepared and then added to the other maj or ingredients at the feed mill . For the diets supplying carotene , a 32-kg TABLE 2. INGREDIENT COMPOSITION OF DIETS , SUPPLEMENTATION PERIOD FOLLOWING DEPLETION , TRIAL 1 a b Carotene treatment , Vitamin A treatment , mg/kg diet IU/kg diet Ingredients 2.2 4.4 8.8 17.6 880 1760 3520 7040 % Coarse ground oats 96 .20 94 .20 90 .40 82 .60 96 .20 94 .20 90.45 82 .70 Dehydrated alfal fa meal , 17% 1.54 3.08 6.16 12.32 Dehydrated alfal fa meal substitute .46 .92 1.84 3.68 2.00 4.00 8.00 16.00 Trace mineral salt .50 .50 .50 .50 .50 .50 .50 .50 Trace mineral premix .20 . 20 .20 .20 .20 .20 .20 .20 c Chlortetracycline .10 .10 .10 .10 .10 .10 .10 .10 Ground limestone 1.00 1.00 .80 .60 1.00 1.00 .75 .50 Vitamin A concentrate, mg/kg diet ------2.52 5.03 10.06 20 .12 a Calculated caro tene treatment levels based on 143 mg carotene per kg of dehydrated alfalfa meal . b Calculated vitamin A treatment levels based on 350,000 IU vitamin A palmitate per g of concentrate . c Twenty-two mg auromycin per kilogram. ,J::- \.0 50 premix was prepared . containing the commercial trace mineral premix and the chlortetraycline with ground oats as a carrier . For the diets supplying vitamin A, the vitamin A palmitate was first diluted in 6.8 kg of soybean oil meal and then added with the trace mineral premix and the chlortetraycline with ground oats as a carrier for a total of 32 kilograms . The animals were accustomed to an all-concentrate diet as fed during the dep letion period . Therefore, the feeding schedule began at a rate of .91 kg of diet per head daily . The diet was adjusted thereafter daily for each pen in amounts that would be nearly consumed by the next feeding . Upon termination of the trial, the animals weighed an average of 68.4 kilograms . All animals were slaughtered on March 15 , 1977 , at a commercial packing plant . Blood and liver data from this study were evaluated by least squares analysis of variance using a 2 x 4 factorial arrangement of treatments with average daily feed intake as a covariate . Comparisons between treatment means were made using Fisher 's test for least significant difference when a significant difference was indicated by a preliminary F-test (St eel and Torrie , 1980) . The level of signif icance was based on that found in the preliminary F-tests . Trial 2: Vi tamin � Supplementation Wi thout Previous Depletion Trial 2 began on February 10 , 1976 , and ended on May 19, 1976 , after a 99-d test period . A group of 168 lamb s weighing an average of 34 .9 kg and cons isting of unequal numb ers of ewe s and wethers were allotted to 24 pens of seven head each according to weight . 51 The experimental diets were prep ared as previously described for the supplementation phase of trial 1. According to the design of this experiment, there were three replications for each treatment . The ingredient composition and treatment levels of the diets are shown in tab le 3. The intention was to use the same treatment levels in trial 2 as in trial 1. However, an error in formulation occurred , and the actual treatment levels were about one-half of · the intended levels . The source of carotene was from a supply of dehydrated alfalfa meal which , upon analysis , had a carot ene concentration of 42.9 mg per kilogram . The vitamin A was from a product which , upon analysis , had a concentra tion of 15 ,000 IU o f vitamin A per gram . Prior to the start of the experiment , the lambs had been fed a diet of alfalfa-brome hay . In order to adj ust the lambs to the experi mental diets , the same feeding schedule was followed as described for the depletion phase of trial 1. At the end of the trial , the lambs weighed an average of 53 .6 kilograms . In order to have some idea of the vitamin A status of the lambs at the beginning of the trial , 10 lamb s that had been previous ly handled and fed in the same manner but not used in any previous trials were slaughtered so that blood and liver samples could be obtained . Blood and liver samples from all the test animals were obtained at the end of the trial when the animals were slaughtered at a commercial packing plant . Al l samp les were handled as previously described for the depletion phase of trial 1. TABLE 3. INGREDIENT COMPOSITION OF DIETS , SUPPLEMENTATION PERIOD WITHOUT PREVIOUS DEPLETION , TRIAL 2 a b Carotene treatment , v·1ta m1n· A treatment , mg/kg· diet IU/kg diet Ingredients .86 1.72 3.43 6.86 440 880 1760 3520 % Coarse ground oats 96.2 94 .2 90 .4 82.6 96 .2 94 .2 90 .4 82 .7 Dehydrate alfalfa meal , 17% 2.0 4.0 8.0 16 .0 Dehydrated alfalfa meal substitute ------2.0 4.0 8.0 16 .0 Trace mineral salt .5 .5 .5 .5 .5 .5 .5 .5 Trace mineral premix .2 .2 .2 .2 .2 .2 .2 .2 c Chlortetracycline .1 .1 .1 .1 .1 .1 .1 .1 Ground limestone 1.0 .10 .8 .6 . 1.0 1.0 .8 .5 Vitamin A concentrate, mg/kg diet ------29 .33 58 .66 117.33 234 .66 a Calcula ted carotene treatment levels based on 42 .9 mg carotene per kg of dehydrated alfalfa mea . S Calculated vitamin A treatment levels based on 15,000 IU vitamin A palmitate per g of concentrate . c Twenty-two mg auromycin per kilogram . V1 N 53 Blood and liver data from this study were evaluated by least squares analysis of variance using a 2 x 4 x 3 factorial arrangement of treatments with average daily feed intake as a covariate . Comparisons between treatment means were made using Fisher 's test for least signif icant difference when a significant difference was indicated by a preliminary F-test (Steel and Terrie , 1980) . Analysis of variance tables for all analyzed parameters of trials 1 and 2 appear in the appendix of this thesis. 54 RESULTS AND DISCUSSION Trial 1: Vitamin A Depletion Followed EY Supplementation Depletion Period . Feedlot performance and blood and liver vitamin A levels at periodic intervals during the depletion phase of trial 1 are shown in table 4. Results of regression analysis for blood and liver vitamin A values are shown in figures 1 and 2, respectively . Feed consump tion was at a satisfactory level for the oat diet throughout the 333 d of vitamin A depletion . ·There were no maj or variations during this time . Weight gain was satis factory through the first 180 d, after which there was . a marked reduction . This is in agreement with work by Peirce (1945) and Lindley et al . (1949) who reported satis factory gains by sheep consuming vitamin A deficient diets for several months . In their studies , rates of gain were not affected until an advanced state of deficiency was reached . The average weight of the lambs in this trial at 180 d was about 54 .5 kilograms . Body weight and the degree of finish were likely important factors affecting rate of gain after this time . While there were no physical symptoms of vitamin A deficiency at this point , the blood serum and liver vitamin A levels of the slaughtered lambs indicated that the entire group may have reached levels bordering on deficiency . Earlier research has shown that a state of deficiency is indicated when blood vitamin A values fall below about 20 meg per 100 ml as reported for cattle (Madsen and Davis , 1949 ; Pope et al ., 1959) and for sheep (Peirce , 1945 ; Hoefer and Gallup , 1947 ; Eveleth et al ., 194 9) . TABLE 4. FEEDLOT PERFORMANCE , BLOOD AND LIVER DATA DURING VITAMIN A DEPLETION OF LAMBS (TRIAL 1: JANUARY IS-DECEMBER 13, 1976; 333 DAY$) Weight and feed Blood and liver Avg Av g Av g serum Avg liver Time No . of daily daily No . of vitamin A, vitamin A, a b period animals gain, kg feed, kg animals mcg/ 100 ml ± SE mcg/g ± SE Initial 160 27 .3 (init . wt) -- 10 28 .10 ± 1.00 173.33 ± 17.78 0-61 d 14 7 .141 1.02 10 24 .41 ± 2.44 86 .94 ± 20 .81 62-117 135 .137 1.20 10 23 .29 ± 2.96 39.02 ± 8.90 118-180 124 .161 1.31 10 17 .44 ± 2.74 10.31 ± 2.7 9 181-228 114 .089 1.32 10 20 .91 ± 3.36 11.23 ± 3.42 229-284 103 .058 1.20 10 11.20 ± 3.16 2.91 ± .90 285-333 91 .096 1.27 10 13.86 ± 2.14 1.46 ± .29 0-333 -- .121 1.21 a Decreasing numb ers per period due to 10 animals slaughtered for blood and liver samples, two ewes lambed and were removed from the study and seven lambs died during the trial due to factors unrelated to gitamin A depletion . Standard error . V1 V1 56 - �0 y = 27 .803 .046X -- predicted va lues 35 means 30 -. e =' 1-1 Q) tJ) 25 � e 0 0 ...... 20 ...._ bO (.) ...... e � 15 � .,..... e m • .4-J ·r-1 10 :> "'C 0 0 � j:Q 5 - 1 0 �0 90 1�0 190 2�0 290 3�0 Days of depletion Figure 1. Blood vitamin A concentra�ion of 10 lambs slaughtered at approximately 2-mo intervals during deple tion period of trial 1. 57 2 Y = 165. 4053 + .0025X - 1.2924X --- predicted values means - bO ...... bO CJ '-""s < � •r-1 75 +J� •r-1 :> � Q) :> ·r-1 .....:1 25 0 - 2 5 -so -75 -100 -10 �0 90 1�0 190 240 290 3�0 Days of depletion · Figure 2. Liver vitamin A concentration of 10 lambs slaughtered at approximately 2-mo intervals during depletion period of trial 1. 58 Blood serum and liver vitamin A levels were regressed on days of deple tion. For blood data, there was a linear effect (P< .Ol ; 2 R = .28) and the regression equation was blood serum vitamin A = 27 .803 - .046X (figure 1) . For liver data , there was a quadratic effect 2 (P< .01; R = .74) and the regression equation was liver vitamin A 2 165 . 4053 + .0025X - 1.2924X (figure 2) . While decreasing wi th time , serum vitamin A values did not fall much below levels considered adequate for normal growth with absence of deficiency signs until 228 d (table 4) . Liver vitamin A levels were quite high initially and markedly lower at each sampling time through 180 days . The liver vitamin A values at 180 and 228 d, while representing marked depletion of initial stores , maintained plasma vitamin A at levels considered minimal for absence of deficiency signs . This is in agreement with studies by Peirce (1945) , Hoefer and Gallup (1947) and Weir et al . (1949) . Liver storage at 284 d wa s low and blood values were at a level associated with vitamin A deficiency . Signs of deficiency (tremb ling and unsteady gait) were observed in about one-third of the animals at this time . This is in agreement with work by Guilbert et al . (1937) , Peirce (1945 , 1954) , Hoefer and Gallup (1947) and Weir et al . (1949) . Blood vitamin A levels at 28 4 and 333 d were at levels repre senting depleted liver reserves . Corresponding liver vitamin A values indicated essentially depleted livers . The data show tha t a lengthy depletion period is required when initial liver vitamin A levels are high as wi th these lambs . It is 59 evident that time required for depletion of the vitamin would depend upon the initial liver level (figure 2) . A depletion period could be relatively short , since the lamb s in this experiment went from blood vitamin A levels (20.91 meg per 100 ml ) indicative of adequate vitamin A nutrition to levels (11.2 0 meg per 100 ml ) associated wi th deficiency signs during· a period of 56 d (228 to 284 d of · depletion) . The results of this study show blood vitamin A values are indicative of vitamin A nutrition at the time but no t of body stores until animals have reached a depleted state . Supplementation Period . Feed consumption and weight gain data for the supplementation period of this experiment are shown in table 5. Feed intake was at a higher level than during the depletion phase . The animals were larger and would be expected to have more feed capacity . Analysis of covariance showed no significant effect of feed consump tion on the various treatments , indicating a true effect of treatment on blood and liver response. Therefore , average daily feed intake was dropped from the model and statistical treatment of blood and liver data is summarized in tables 6, 7 and 8. Appendix table 1 shows the raw means for blood and liver data . Appendix tables 2 and 3 show least-squares analysis of variance for blood and liver values during supplementation following deple tion . Over the 91-d experiment , feed consump tion and rates of gain were similar for all group s (table 5) . The low average daily gains would no t be considered unusual for sheep of this weight and condition . Earlier research by Myers et al . (1959) � Smith et al . (1962) and Martin TABLE 5. AVERAGE WEIGHTS , FEED CONSUMPTION AND DAILY GAINS DURING SUPPLEMENTATION PERIOD FOLLOWING DEPLETION (TRIAL 1: DECEMBER 14 , 1976 , TO MARCH 14 , 1977; 91 DAYS) Carotene treatment , Vitamin A tre atment , mg/kg of feed IU/kg of feed Item 2.2 4.4 8.8 17 .6 880 1760 3520 7040 No . of lambs 10 10 10 10 10 10 10 10 Avg initial wt , kg 62 .3 62 .2 61 .3 62 .6 60 .5 60 .8 62 .3 61.7 Avg final wt , kg 68 .0 69 .9 69 .6 71.6 67 .2 66 .9 67 .0 66 .6 a Av g daily feed consumption , kg 0-29 d 1.36 1.30 1.41 ' 1.48 1.17 1.10 1.07 1.09 30-57 d 1.48 1.50 1.40 1.58 1.37 1.45 1.27 1.22 58-91 d 1.24 1.32 1.36 1�5o 1.21 1.36 1.34 1.33 0-9 1 d 1.35 1.36 1.39 1.52 1.25 1.31 1.2 2 1.22 a Avg daily gain , kg 0-29 d .107 .066 .117 .104 .065 .051 .010 .017 30-57 d -.075 -.026 .019 -.009 -.024 -.008 .036 .013 58-91 d .140 .190 .129 .184 .161 .141 .100 .119 0-9 1 d .063 .084 .091 .099 .073 .066 .051 .054 a Average daily feed consumption and average daily gain based on sheep days per period . 0'\ 0 TABLE 6. LEAST-SQUARES MEANS OF SUPPLEMENT SOURCE, LEVEL OF SUPPLEMENTATION AND DAYS OF SUPPLEMENTATION FOR BLOOD VITAMIN A, BLOOD CAROTENOIDS AND LIVER VITAMIN A FOR SHEEP FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A FOLLOWING DEPLETION , TRIAL 1 Blood Blood vitamin A, caro tenoids, Liver No . of mcg/100 ml mcg/100 ml No . of vitamin A, Item sampJ..�-- serum serum samples meg/g liver Supplement source Carotene from dehy d drated alfalfa meal 160 27 .12 9.31 40 9.02 e Vitamin A palmitate 160 27 .58 8.78 40 12.35 Standard error ±.55 ±.20 ±.51 Level of supplementation a a 1 80 21.79 8.66 20 4.60 . a a 2 80 21 .70 8.94 20 3.14 b b 3 80 31.07 ' 9.34 20 11.38 c c 4 80 34 .86 9 .23 20 23 .64 Standard error ±.78 ±.28 ±1 .44 Days of supplementation a a 0 80 12.00 6.40 b b 29 80 35 .95 10.04 b b 57 80 34.66 9.06 c b 91 80 26 .8l 10.67 80 10.69 Standard error ±.78 ±.28 ±.72 : ,b,c Means in the same column within item with different superscripts are different (P< .Ol) . ,e Means in the same column wi thin item wi th different superscripts are different (P< .05) . 0\ � TABLE 7. LEAST-SQUARES MEANS OF SOURCE X LEVEL INTERACTION FOR BLOOD VITAMIN A, BLOOD CAROTENOIDS AND LIVER VITAMIN A FOR SHEEP FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A FOLLOWING DEPLETION , TRIAL 1 Blood Blood vitamin A, carotenoids , Liver No . of mcg/100 ml mcg/100 ml No . of vitamin A, Source x level samples serum serum samples mcg/g liver a Carotene (1) 2.2 mg/kg diet 40 21.16 8.73 10 5.29 a (2) 4.4 40 20.26 9.04 10 2.63 b (3) 8.8 40 31 .80 . 9.64 10 12.28 b (4) 17.6 40 35 .28 9.83 10 15 .90 Standard error ±1.10 ±.40 ±2.04 a Vitamin A (1) 880 IU/kg diet 40 22.41 8.60 10 3.90 a (2) 1760 40 23 .14 8.85 10 3.64 a (3) 3520 40 30 .35 9.03 10 10.48 b (4) 7040 40 34 .44 8.64 10 31.38 Standard error ±1.10 ± .40 ±2.04 a,b Means in the same column within source x level with different superscripts are different (P< .01) . 0'. N 63 TABLE 8. LEAST-SQUARES MEANS OF DAY X LEVEL INTERACTION FOR BLOOD VITAMIN A AND BLOOD CAROTENOIDS FOR SHEEP FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A FO_LLOWING DEPLETION , TRIAL 1 Blood Blood vitamin A, carote noids , No . of mcg / 100 ml mcg / 100 ml Day x level samEl es serum serum 0 1 20 11 .46 6.79 2 20 10 .87 6.00 3 20 12.16 6.58 4 20 13 .54 6.24 a 29 1 20 28 .73 9.82 a 2 20 29 .92 10.69 b 3 20 42 .42 9.96 b 4 20 42 .72 9.70 a d 57 1 20 25 .40 7.81 a d 2 20 26 .94 8.10 b e 3 20 38 .80 10.52 c e 4 20 47 .48 9.82 a 91 1 20 21 .56 10.23 . a 2 20 19.06 10.98 b 3 20 30 .9l 10.30 b 4 20 35 .70 11 .17 Standard error ±1 .56 ±.57 a,b,c Means in the same column wi thin day x level wi th different sup r cripts are different (P< .01) . a ' � Means in the same column within day x level wi th different superscrip ts are different (P< .05) . 64 et al . (1968) showed no significant differences in average daily gain of lambs supplemented with vitamin A or carotene during repletion periods following depletion: The main measures of effects of carotene or vitamin A supplementation in this experiment were the concentrations of blood vitamin A, bloc� carotenoids and liver vitamin A. Liver carotenoid levels were detected- in very small amounts with l ittle variation between treatment levels of either vitamin A or carotene . Johnson and Baumann (1947b) determined that the small amount of to tal ether-soluble yellow pigments detec ted with the liver analysis procedure did no t contain either a- or 8-carotene . They concluded that these pigments were not involved in the formation or storage of vitamin A. Fo r these reasons , liver carotenoid data from this experiment will be omitted in the discussion. The raw means for liver carotenoid concentrations are shown in appendix table 1. Blood Vitamin A. There were no significant differences in blood vitamin A values between sexes , and the data presented in all tables are treatment averages for both ewes and wethers . Levels of supplementa tion 1, 2, 3 and 4 represent pooled data over levels between sources . Levels were 880 , 1,760 , 3,520 and 7,040 IU vitamin A per kg of diet, respectively , or the equivalent in carot ene using 400 IU of vitamin A ac tivity per mg of carot ene . Level 3 was greater than 1 and 2, and level 4 was greater than level 3 (P< .01) , showing a greater response in blood vitamin A values wi th increasing levels .of supplementation . This is in agreement with earlier work by Peirce (1945, 1954) and Hoefer and Th Gallup ( 194 7) . e same trend occurred· in the source x level in t�rac tion 65 (table 7) , although the differences were no t significant with the numb er of observations involved . Pooled blood vitamin A values over all treatments between days of supplementation (table 6) were also different (P< .01) . At days 29 and 57·, the blood vitamin A concentration was greater than the depleted value shown for day 0. The final blood vitamin A value shown at day 91 wa s also greater than at day 0 but was significantly less than at days 29 and 57. In the day x level interaction (table 8) , the blood vitamin A values for treatment levels 1, 2, 3 and 4 at day 0 were no t different . However , at day 29 of supplementation ,._ levels 3 and 4 were greater than levels 1 and 2 _ (P< .01) . At day 57 , level 3 wa s greater than levels 1 and 2, and level 4 was greater than level 3 (P< .01) . At day 91, levels 3 and 4 were greater than levels 1 and 2 (P< .01) . The trend of decreasing blood vi tamin A concentrations from day 29 through 91 could have been due to a decreas e in the vitamin A potency of the carotene in the dehydrated alfalfa meal or in the vitamin A palmitate over time . It has been reported that trace minerals can catalyze the oxidative destruction of carotene and vitamin A in mixed feeds (Halverson and Hart, 1950 ; Kamstra et al ., 1953 ; Luther , 1980) . Reduced potency of vitamin A supplements has been reported due to exposure to oxygen (Lowen et al ., 1937) , temperature fluc tuations (Reid et al ., 1955) and long periods of storage (Holder and Ford , 1939) . These effects could no t be positively determined in this study because the samples of aehydrated alfalfa meal and vitamin A palmi tate were pooled wi thin source and analyzed_ for carotene and vitamin A 66 concentrations . The supplies of dehydrated alfalfa meal and vitamin A palmitate were stored at cold temperatures to minimize storage losses , but such losses canno t be ruled out . It will be noted from tables 6, 7 and 8 that for all levels and days of supplementation there wa s a promp t rise in blood vitamin A from depleted concentrations to concentrations wi thin a range considered to represent adequate vitamin A nutrition (Peirce, 1945 ; Hoefer and Gallup , 1947 ; Pope et al ., 1949; McGillivray , 1960; Martin et al ., 1968 ; Dwaraknath and Pareek, 1971; Bayfield et al ., 1972) . This was - accomplished after only 1 mo of supple�entation with carotene and vitamin A treatments at level 1 which correspond to 2.2 mg of caro tene from dehydrated alfalfa meal or 880 IU of vitamin A palmitate per kg of diet (see appendix tab le 1) . Carotene treatment level 1 approximates minimum requirements and vitamin A treatment level 1 approximates one-half minimum requirements for sheep weighing between 60 and 70 kg (NRC , 1975) . Blood Carotenoids. Blood caro teno id concentrations were detected in a range of 5.69 to 12.82 meg per 100 ml of serum (appendix table 1) . This is in contrast to earlier wo rk wh i�h found little or no caro tene in the blood of sheep (Moore and Payne , 1942; Peirce , 1946 ; Eveleth et al ., 1949 ; Pope et al ., 1949 ; Martin et al ., 1968 ; Dwaraknath and Pareek , 1971) . Al though measurable , there were no significant differences in blood carotenoid concentrations between sources , levels or in the source x level interaction of carotene or vi tamin A supplementation (tab les 6 and 7) . Pooled blood caroteno id values . over 67 all treatments were greater at days 29, 57 and 91 of supplementation than the dep leted value at day 0 (P< .01, table 6) . In the day x level interaction, levels 3 and 4 were greater than levels 1 and 2 at day 57 (P< .05, table 8) . At this day of supplementation, the blood carotenoid concentrat ions between levels of supplementation were mo re variable and did no t fo llow the trend that was found at days 0, 29 or 91. In view of the similarity in blood serum carotenoid values between the various carotene and vitamin A treatments (appendix table 1) , blood carotenoids as a measure of treatment effects in this study wo uld be highly questionable . Liver Vitamin A. There were no differences in liver vitamin A concentrations between sexes . Statistically different liver vitamin A values were found between sources , levels and in the source x level interaction of carotene and vitamin A supplementation (tables 6 and 7) . Liver vitamin A concentrations were greater with vitamin A palmitate treatments than with carotene from dehydrated alfalfa meal (P< .05) . Supplementation level 3 was greater than 1 and 2, and level 4 was greater than level 3 (P<.Ol) . In the source x level interaction , carotene treatment levels 3 and 4 were similar but greater than 1 and 2 (P< .Ol) . The data from this experiment indicate that vitamin A palmitate was mo re effective than carotene from dehydrated alfal fa meal in terms of liver storage of vitamin A (table 6) . The da ta also show an increase in liver vitam�n A storage with increasing levels of either carotene or vi tamin A supplementation. This is in agreement wi th work by Hoefer and 68 Gallup (1947) , Gallup et al . (1951 ), Diven and Erwin (1958) and Myers et al . (1959) . Values for liver vitamin A (appendix table 1) indicate · that 2.2 and 4.4 mg of carotene per kg of diet or 880 and 1,7 60 IU of vitamin A were no t adequate levels to increase liver storage appreciably above the depleted levels (table 4) during the 91-d supplementation period . Caro tene at 8.8 mg or vitamin A at 3,520 IU per kg of diet resulted in elevated liver storage along with rather high plasma values . Up to this level of carotene supplementation, the vitamin A value of carotene would appear to be 400 IU per 1 mg of carotene as commonly used for cattle and as the low value in a range for sheep (NRC , 1975) . Carotene at 17.6 mg per kg of diet appeared to have somewhat less activity per unit of weight than the 8.8-mg level on the basis of liver vitamin A. Vi tamin A at 7,040 IU per kg of diet resulted in a substantial increase in liver storage over the 3,520 IU level . The differences in liver vi tamin A storage between 8.8 mg of carotene and 7,040 IU vitamin A per kg of diet would be of little practical importance because these levels of supplementation are much higher than levels that would normally be used in practice . It should be no ted that as little as 2.2 mg of carotene or 880 IU of vitamin A per kg of diet resulted in a prompt rise in blood vitamin A concentrations from dep leted levels to levels considered to represent adequa te vitamin A nu trition while having no apparent effect on liver storage . It would appear that liver vitamin A storage in sheep is of little importance if their daily requirement for vi tamin A is supplied in the diet . 69 Tr ial 2: Vi tamin � Supplementation Wi thout Previous Depletion Feed consumption and weight gain data from trial 2 are shown in tab le 9. As in trial 1, analysis of covariance showed no effect of feed consumption on the blood and liver data from each treatment group . Therefore , feed intake data were not considered important , and statistical treatment of blood and liver data is summarized in table 10 . Appendix table 4 shows the raw means of blood and liver data . Appendix table 5 shows least-squares analysis of variance for blood vitamin A, blood carotenoids and liver vitamin A concentrations . Feed consumption was accepted a.s normal for feedlot lambs of this weight with little variation among test groups . Average daily gains were simi lar and satisfactory for all groups with little variation throughout the 99-d trial . Le as t-squares analysis of variance showed no differences in blood caroteno id concentrations between sources of vitamin A supplementation as either carotene from dehydrated alfalfa meal or vitamin A palmitate (tab le 10) . Al so , there were no differences found between levels of supplementation or in the source x level interaction . Likewise, there were no differences in blood vitamin A values between sources of supplementation . Significant differences did occur between levels of supplementation. For final blood vitamin A values , levels 1, 2 and 3 were similar , levels 3 and 4 were similar , but level 4 was greater than 1 and 2 (P< .01) . In the source x level interaction , no significant differences were found between blood vitamin A values. However , the trend was the same within carotene and vitamin A treatment levels . The same results occurred for the final liver vitamin A values . TABLE 9. AVERAGE WEIGHTS , FEED CONSUMPTION AND DAILY GAINS OF LAMBS FED VARIOUS LEVELS OF CAROTENE OR VITAM_IN A DURING SUPPLEMENTATION PERIOD WITHOUT PREVIOUS DEPLETION (TRIAL 2: FEBRUARY 10-MAY 19, 1976; 99 DAYS ) Carotene treatment , Vitamin A treatment , mg/kg diet IU/kg diet Item . 86 1.72 3.43 6.86 440 880 1760 3520 a No . of lambs 16 21 18 20 17 18 19 19 Av g initial wt , kg 35 .6 34 .6 34 .1 34 .9 34 .4 34 .6 35 .6 35 .2 Av g final wt , kg 53.6 53.9 52 .8 57.9 50 .5 52.5 53.1 54 .8 Avg daily gain, kg .181 .194 .188 .232 .162 .181 .176 .197 b Av g 'daily feed , kg 1.38 1.56 1.57 1.75 1.34 1.40 1.51 1.60 Feed/gain ratio 7.64 8.04 8.38 7.56 8.28 7.7 6 8.55 8.13 a Variation in number of lambs per treatment group due to losses resulting from factors unrelated to reatments . All feed data adj usted for sheep days throughout the trial . E Alfal fa-brome hay fed the first 10 d until all lambs were on full feed of the all-concentrate diet . -....J 0 TABLE 10. LEAST-SQUARES MEANS OF SUPPLEMENT SOURCE, L�VEL OF SUPPLEMENTATION AND SOURCE X LEVEL INTERACTION FOR BLOOD VITAMIN A, BLOOD CAROTENOIDS AND LIVER VITAMIN A FOR LAMBS FED . VARIOUS LEVELS OF CAROTENE OR VITAMIN A WITHOUT PREVIOUS DEPLETION, TRIAL 2 Blood Blood Liver vitamin A, carotenoids , vitamin A, No . of mcg/ 100 ml mcg /100 ml mc g/g liver Item samples serum ± SE serum ± SE ± SE Supplement source Carotene from dehydrated alfalfa meal 75 39 .37 ± .34 4.91 ± .22 67.62 ± 3.38 Vitamin A palmitate 73 39 .67 . ± .34 4.76 ± .23 72.13 ± 3.43 Level of supplementation 1 ( .86 mg carotene + 440 IU a a vitamin A) 33 34 .60 ± 1.62 5.19 ± .27 60 .06 ± 4.29 2 (1.72 mg carotene + 880 a a IU vitamin A) 39 37 .57 ± 1.49 4.38 ± .25 60 .35 . ± 3.95 3 (3 .43 mg caro tene + 1760 ab ab IU vitamin A) 37 40 .42 ± 1.53 4.51 ± .26 73.74 ± 4.05 4 (6.86 mg caro tene + 3520 b b IU vitamin A) 39 45 .49 ± 1.49 5.2 7 ± • 25 85 .34 ± 3.95 Source x level Caro tene (1) .86 mg/kg diet 16 34 .38 � 2.03 5.25 ± .56 63 .71 ± 13.67 (2) 1.72 21 37 .38 ± 1.77 4.37 ± .48 62 .04 ;t 11.93 (3) 3.43 18 38 .80 ± 1.91 4.29 ± .52 71.18 ± 12.89 (4) 6.86 20 46 .94 ± 1.82 5.7 3 ± .50 73.53 ± 12.23 Vitamin A (1) 440 IU/kg diet 17 34 .81 ± 1.97 5.13 ± .54 56.42 ± 13.26 (2) 880 18 37 .77 ± 1.91 4.3 9 ± .52 58 .67 ± 12.89 (3) 1760 19 42 .05 ± 1.86 4.72 ± .51 7 6.30 ± 12.55 (4) 3520 19 44 .05 ± 1.86 4.81 ± .51 97 .16 ± 12 .55 --....J ...... a,b Means in the same column within item with different superscripts are different (P< .01) . 72 As suming the 10 lambs slaughtered initially were representative of the who le group , the vitamin A status of the lamb s beginning this trial was comparable to that for the lambs started on the depletion diet of trial 1. Average blood and liver vitamin A concentrations for those 10 lambs were 28 .79 �cg per 100 ml serum and 162.54 meg per g liver , respectively (appendix table 4) . Initial blood and liver samples were not taken from the. experimental animals in this trial . However , final blood serum vitamin A values were markedly higher , and final liver vitamin A values markedly lower for all treatment group s as compared to the initial slaughter group . Level 1 of both carotene and vitamin A supplementation approxi mates minimum requirements for lambs weighing 35 kg (NRC , 1975) as did the lamb s at the start of this trial . Accordingly , level 1 of carotene and level 2 of vitamin A supplementation approximate minimum require ments for lamb s weighing 55 kg as did the lambs at the end of the trial . On that basis, levels 2,. 3 and 4 of carotene supplementation correspond to two , four and eight times minimum requirements, respectively. Levels 3 and 4 of vitamin A supplementation correspond to two and four times minimum requirements , respectively . The data show (table 10) that final blood vitamin A values were well within a normal range representing adequate vitamin A nutrition as reported earlier by Peirce (194 5) , Pope et al . (1949) , McGillivray (1960) , Martin et al . (1968) , Dwaraknath and Pareek (19 71) and Bayfield et al . (1972) . It was also ap parent that the treatment levels were no t sufficient to prevent dep letion of liv�r vitamin A stores over the 73 99-d test period as compared to liver vitamin A concentrations for 10 lambs slaughtered at the start of the trial (appendix tab le 4) . The lambs in this study grew satisfactorily with no maj or variations between treatment groups . These results support the data reported for the previous study in the supplementation period following depletion of . vitamin A reserves in feedlot lambs . It is. concluded that the feeding of high levels of vitamin A or carotene to feedlo t lamb s wo uld not be a prac tical consideration if the lambs receive their minimum vitamin A requirement in their daily diet . 74 SUMMARY A depletion study wa s conducted in order to determine the length of time involved to deplete feedlot lambs of their vitamin A body stores . The experimental animals consisted of 160 lambs with equal numb ers of ewes and wethers . At 180 d of depletion, the lambs weighed approximately 54 .5 kg, and blood and liver vitamin A concen trations indicated the lambs were border ing on deficiency . Av erage blood vitamin A concentrations of 10 lambs slaughtered - at 0, 61, 117, 180, 2�8 , 284 and 333 d of depletion were 28 .10, 24 .41, 23 .29, 17.44, 20 .91, 11.20 and 13.8'6 meg per 100 ml serum, respectively . Corresponding liver vitamin A- concentrations were 173 .33 , 86 .94, 39 .02, 10 .31, 11.23, 2.91 and 1.46 meg per g of liver , respectively . Regression analysis of the data showed a linear effect (P< .Ol) for blood vitamin A values and a quadratic effect (P< .Ol) for liver vitamin A values . The deplet ion period was followed by a 91-d supplementation period in order to determine the lambs' blood and liver response to various levels of carotene or vitamin A. Forty ewes and 40 wethers were allo tted to 16 pens of five animals each according to sex and weight . Exp erimental diets contained either 2.2, 4.4, 8.8 or 17.6 mg of carotene per kg of diet from dehydrated alfalfa meal or 880 , 1,7 60 , 3,520 or 7,040 IU of vitamin A per kg of diet from vitamin A palmitate . Blood and liver data were evaluated by least-squares analysis of variance using a 2 x 4 factorial arrangement of treatments . For all levels and at all days of supplementation, there was a ma rked rise in blood vitamin A from 75 depleted concentrations to concentrations within a range cons idered to represent adequa te vitamin A nutrition . Final liver vitamin A concentrations were appreciably higher than the initial deple ted conc entrations in lambs consuming diets with either 8.8 mg of carotene or 3,520 IU of vitamin A per kg of diet , respectively . Up to this level of supplement�tion, it appeared tha t the appropriate value for the vitamin A activity of carotene from dehydrated alfalfa meal would be approximately 400 IU per 1 mg of carotene . The data from this experiment show that lamb s wi th high liver storage of vitamin A require a lengthy period for dep letion to levels indicative of a deficient state as measured by low blood levels of the vitamin and deficiency signs . However , liver storage at levels to give blood vitamin A considered adequate for growth and absence of deficiency signs may be depleted to deficient levels in as little as 2 months . The level of carotene or vitamin A supplementation necessary to result in appreciable liver storage of vitamin A under the conditions of this study was higher than tha t which wo uld be normally used in practice . The lamb s in this study grew satisfactorily with no maj or variations in feedlot performance between groups receiving treatment levels which represented near minimum requirements to several times minimum require ments . As little as 2.2 mg of carotene or 880 IU of vitamin A per kg of diet , whil� having no apparent effect on liver storage , raised blood vitamin A concentrations from depleted levels to levels considered to represent adequa te vitamin A nutrition . It wa s concluded that high 76 initial liver vitamin A stores or high levels of vitamin A supplementa tion were of little value or importance to the performance of feedlot lambs during growing and fini shing periods if the lambs received near minimum requirements for vitamin A in their daily diet . In ano ther study , a 99-d trial wa s conducted in order to examine the effect of vitamin A or carotene supplementation to typical feeder lamb s wh ich had no t been previously depleted of liver vitamin A stores . . A group of 168 lamb s consisting of unequal numbers of ewes and we thers were allotted to 24 pens of seven head each according to weight . Ac cording to experimental design, there were three replications per treatment . The experimental diets contained either .86 , 1.72, 3.43 or 6.86 mg of carot ene per kg of diet from dehydrated alfalfa meal or 440 , 880 , 1,7 60 or 3,520 IU of vitamin A per kg of diet from vitamin A palmitate . Blood and liver data were evaluated by least-squares analysis of variance using a 2 x 4 x 3 factorial arrangement of treatments . Results of this trial support the findings during the supple mentation period following vitamin A depletion as previously reported in this thesis . 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BLOOD AND LIVER DATA OF LAMBS FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A, SUPPLEMENTATION PERIOD FOLLOWING DEPLETION (TRIAL 1: DECEMBER 14 , 1976 , TO MARCH 14 , 1977; 91 DAYS) Caro tene treatment , Vitamin A treatment , mg/kg diet IU/kg diet Item 2.2 4.4 8�8 17.6 880 1760 3520 7040 No . of lambs 10 10 10 10 10 10 10 10 Blood serum vitamin A (mcg/100 ml ) Initial 12 .17 10.73 . 11.93 13.23 10. 74 11.01 12.40 13.84 29 d 25 .24 27 .97 45 .79 45 .75 32.19 31 .86 39 .05 39 .69 57 d 24 .46 24 .78 38 .66 48 .50 26 .35 29 .09 38 .94 46 .47 91 d 22 .79 17.54 30.82 33.64 20 .33 20.58 31 .00 37 .77 a Blood serum caro tenoids (mcg/ 100 ml ) Initial 6.51 5.69 6.72 6.11 7.07 6.31 6.44 6.37 29 d 9.40 9.78 10.15 10.53 10.25 11.60 9.77 8.87 57 d 8.52 8.83 11.13 9.87 7.10 7.38 9.90 9.78 91 d 10.48 11.85 10.58 12.82 9.98 10.11 10.02 9.52 Liver vitamin A (meg/g) 91 d 5.29 2.63 12.28 15.90 3.90 3.64 10.48 31.38 Liver "carotenoids" (meg/g) 91 d .39 .41 .58 .58 .44 .32 .33 .32 a Defined as the total ether-soluble yellow pigments present . 00 1..0 90 TAB LE 2.· ANALY SIS OF VA RIANCE FOR BLOOD VITAMIN A AND BLOOD CAROTENOID$ DURING SUPPLEMENTATION PERIOD FOLLOWING DEPLETION , TRIAL 1 Mean sguares Blood Blood Source df vitamin A carotenoids To tal 319 Sex 1 93 .96 .11 Sour ce 1 16 .84 22 .58 Sex x source 1 43 .66 13.20 Level 3 3552 .15** 7 . ·40 Sex x level 3 121 .29 4.38 Source x level 3 78 .76 4.88 Sex x source x level 3 221 .88 12.48 Days 3 9680 .01** 283 . 23** Sex x days 3 11.71 1 1.47 Source x days 3 14 .16 16 .02 Sex x sour ce x days 3 70 .80 .83 Days x level 9 350 . 86** 12.68* Sex x days x level 9 64 .60 2.31 Source x days x level 9 82 .41 6.0 2 Sex x source x days x level 9 28 .15 6.10 Error 256 48 .94 6.45 * P< .OS. ** P< .01. TABLE 3. ANALYSIS OF VARIANCE FOR LIVER VITAMIN A DURING SUPPLEMENTATION PERIOD FOLLOWING DEPLETION , TRIAL 1 Source df Mean squares To tal 79 Sex 1 11.40 Source 1 221 .11* Sex x source 1 .54 Level 3 1749 .37** Sex x level 3 24 .59 Source x level 3 336 .00** Sex x source x level 3 30 .54 Erro r 64 41 .77 * P< .05. ** P< .01 . TABLE 4. BLOOD AND LIVER DATA OF LAMBS FED VARIOUS LEVELS OF CAROTENE OR VITAMIN A DURING SUPPLEMENTATION PERIOD WITHOUT PREVIOUS DEPLETION a (TRIAL 2: FEBRUARY 10-}�Y 19, 1976; 99 DAYS) Carotene treatment , Vitamin A treatment , Initisl mg/ kg diet IU/kg diet Item group .86 1.72 3.43 6.86 440 880 1760 3520 No . of lambs 10 16 21 18 20 17 18 19 19 Blood serum vitamin A (mcg/ 100 ml ) 28.79 34 .33 37 .37 38 .93 46 .93 35 .38 37 .82 41 .99 44 .66 c Blood serum carotenoids (mcg/ 100 ml ) 6.73 5.25 4.37 4.28 5.70 5.2 4 5.96 4.63 4.71 Liver vitamin A (meg/ g) 162.54 63 .28 62 .04 70.69 74 .19 57 .64 59.28 77.00 93 .88 Liver carotenoids (meg/ g) .96 .51 .56 .67 . 76 .51 .41 .43 .43 -- a Average values obtained from blood and liver samples taken at time of slaughter upon completion of 5he trial . . Average values from five ewes and five wethers with average weight of 40.5 kg slaughtered February 3, 1976 , for initial comparison . c Defined as total ether-soluble yellow pigments present . 1..0 '"""" 92 TABLE 5. ANALYSIS OF VARIANCE FOR BLOOD VITAMIN A, BLOOD CAROTENOIDS AND LIVER VITAMIN A DURING SUPPLEMENTATION PERlOD WITHOUT PREVIOUS DEPLETION , TRIAL 2 Mean sguares Blood Blood Liver Source df vitamin A carotenoids vitamin A Total 147 Rep 2 5.45 2.19 1391 .19 Source 1 3.16 .78 734 .90 Rep x source 2 8.48 3.74 857 .40 a Level 3 770. 73** 7.67 5419.89** Rep x level 6 87 .01 2.39 607 .32 Source x level 3 58 .98 2.9 9 1736 .96 Rep x source x level 6 65 .90 4.94 2990 .85 Error 124 57 .54 3.49 1121 .45 a Test of hypothesis for level using mean square for rep x level as an error term . ** P< . 01.