WWWWW1WWHUHflHIHWHIHHill \l L0 —iO 2‘ UWBY Whig“ State i» University, l. o ,. ‘p-__.'-r‘- . f This is to certify that the thesis entitled VITAMIN D METABOLITES IN THE UTERUS (SHELL GLAND) OF THE LAYING HEN presented by SANDRA I. AMBRUS has been accepted towards fulfillment of the requirements for M.S. degree inAm‘maI Science 1 Major profeskir Date 5-4—83 0.7639 MS U is an Affirmative Action/Equal Opportunity Institution .,-_.__... _.... _... . MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. VITAMIN D METABOLITES IN THE UTERUS. (SHELL GLAND) OF THE LAYING HEN By Sandra 1. Ambrus A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science I983 /3&-9Q?J:3 ABSTRACT VITAMIN D METABOLITES IN THE UTERUS (SHELL GLAND) OF THE LAYING HEN By Sandra 1. Ambrus Single Comb White Leghorn hens, approximately one year of age were divided into two groups. One group received a control diet, the other received the same ration, except that the vitamin mix contained no vitamin D3. All birds were fed their respective rations for three to ten weeks. Egg production and shell quality measurements were taken. Para- meters-for shell quality included whole egg weight, shell weight, shell thickness,and egg length and breadth. After hens had been on the deficient diet for at least three weeks, they were injected intravenously with two microcuries of 3H-D3. Liver, kidney and uterine tissues were collected 15 to l8 hours later and sub- jected to total lipid extraction. Lipid samples were chromatographed using Sephadex LH-20 column chromatography. Egg production and shell quality were not markedly affected during the experimental period. 3H-D3 and 25-0H-D3 were recovered from most of the lipid samples. l,25-(OH)2-D3 was not recovered from any sample. Several liver, kidney and uterine samples had unidentified peaks appearing between 100 and 260 ml. ACKNOWLEDGEMENTS The author wishes to express her sincere appreciation to Dr. Donald Polin for his help and encouragement during this period of study and research. Sincere thanks are also extended to Drs. R. K. Ringer and T. H. Coleman for their valuable criticisms and help in the preparation of this manuscript. The author is also grateful to her parents for their moral support. . Finally, the author is indebted to her friend, Kathy, for her support and understanding during this period of research. ii TABLE OF CONTENTS Page LIST OF TABLES, FIGURES AND GRAPHS ................ iv INTRODUCTION ........................... l OBJECTIVES ............................ 2 REVIEW OF LITERATURE ....................... 3 PROCEDURES ............................ l9 Column Preparation and Standardization ........... 23 Injection of Hens ...................... 27 Collection of Tissue Samples ................ 27 Sample Processing and Lipid Extraction ........... 27 Sample Analysis by Column Chromatography .......... 28 RESULTS AND DISCUSSION ...................... 35 Egg Quality ......................... 36 Recovery of Radioactivity .................. 36 Chromatography of Tissues .................. 40 SUMMARY ............................. 50 BIBLIOGRAPHY ........................... 5l Table 10. lla. llb. Figure LIST OF TABLES, FIGURES AND GRAPHS Layer Ration for Vitamin D3 Experiment ......................... Calculated Analysis of Layer Ration for Vitamin D3 Experiment ..................................................... Feed Intake (grams per bird per day) Control Diet vs. D3 Deficient Diet .......................................... Total Weekly Egg Production of Hens in Preliminary . Feeding Trial .................................................. Relationship of Diet to Egg Production Collected on a Once Weekly Basis ............................................ Calcium (mg) per Shell Surface Area (mm2) Calculated from Egg Quality Measurementsa ............................ ..... Shell Quality Change as Related to Egg Shell Thickness ......... Changes in Percent Shell Weight as Compared with Whole Egg Weight Based on Dietary Regime ............................. Tissue Weights and Presence of Egg in Uterus, Collected at Time of Sacrificing ......................................... Recovery of Radioactivity in Tissues Collected from Injected Hens .................................................. Uterine dpm as Percent of Total dpm Recovered, as Related to Dietary Treatment ................................... Uterine dpm as Percent of Total dpm Recovered as Related to Presence of Egg in Uterus ........................... Metabolism of Vitamin D3 ........................................ Mechanism of Vitamin D3 Action ................................. The Action of l,25-(OH)2-D3 on Calcium Transport in the Intestinal Cell ......................................... Effects of Low Serum Calcium ................................... Effects of Low Serum Phosphorus ................................ iv Page 20 2T 29 3O 3T 32 34 37 38 4T 42 6 8 l6 18 List of Tables, Figures and Graphs (continued) Graphs Page l. Column standardization-vitamin D3 and 25—OH-D3 ....... 24 2. Column standardization-l,25-(OH)2-D3 ............ 25 3. Column standardization-vitamin D3. 25-0H-D3, l,25-(OH)2-D3 26 4a. Chromatography pattern-liver ................ 43 4b. Chromatography pattern-liver ....... _ ......... 44 5. Chromatography pattern-kidney ............... 45 6. Chromatography pattern-uterus . . . ............. 46 INTRODUCTION Thin-shelled eggs and egg breakage are of concern to the poultry industry. The incidence of poor quality egg shells tends to increase as the hen ages, with estimates up to l6%. It is currently known that the liver and kidney are involved in vitamin D3 metabolism, and ultimately calcium absorption from the birds' intestine. Research has also been done to determine what effects the addition of different calcium sources or vitamin D3 metabolites to the hen's diet have on maintaining or improving egg shell quality. However, none of these studies has yet been able to elucidate the exact cause of declining shell quality in the aging hen. This study will attempt to determine if the uterus of the laying hen contains metabolites of vitamin D3 which vary in concentration during the egg-forming cycle and to determine if vitamin D3 deficiency influences these metabolites. OBJECTIVES To feed hens a vitamin D3 deficient diet until they begin laying eggs with poor quality shells. To compare the chromatography patterns of vitamin D3 metabolites in the liver, kidney and uterine tissue of vitamin 03 deficient hens with hens that have been fed diets with adequate levels of vitamin D3: REVIEW OF LITERATURE ,A hen's body store of calcium is approximately 20 grams. The major nutritional component involved in egg shell deposition is calcium, which comprises approximately 40% of the egg shell. If the hen lays an egg daily, which contains 2 grams of calcium, she will turn over approximately lO% of her body store of calcium each day (Hudson §t_al,, 197l). Egg breakage, shell—less egg, and thin-shell eggs are a persistent and continuing problem for the poultry industry.' As the hen ages, her ability to maintain adequate shell quality decreases. In a study by Roland (l977), the percent of uncollectible eggs (shell-less and thin- shell) was found to increase as the hen aged. During the winter months, birds 8 months of age had 2.39% uncollectible eggs while birds l7 months of age had 14.74% uncollectible eggs. The percentages increased during the summer months with 2.86% for birds 8 months old and l6.ll% for birds 17 months old. In addition, Roland determined that as the hen aged, the percent of eggs laid with cracks in the shell tended to increase. His study also indicated that out of every lOO eggs, 7.77 were uncollectible. In additiOn to poor shell quality, egg handling practices can also result in fewer marketable eggs. Orr et_al,, (l977), reported that "the incidence of cracks which occurred during the laying, gathering and handling prior to shipment ranged from l.l to l.7 percent of all eggs examined". There was also an increase in shell damage while the eggs were being graded, washed and packed. In addition, this study showed that during laying, gathering and packing the highest percent of shell cracks were in the small end (27.l%), while during shipping, washing, grading and packing, most cracks occurred from the large end of the egg to the center (34.0%). The metabolism of vitamin D to its active form, l,25-dihydroxy- vitamin D3, which promotes calcium absorption from the intestine and mobilization of calcium from bone, occurs in several sequential steps. This metabolic pathway is regulated by several factors, including diet, enzymes, and hormones. Vitamin D3 is produced in the epidermal layer of the skin. Pro- vitamin D3, 7-dehydrocholesterol undergoes cleavage of the B-ring-carbon- carbon bond between C 9 and C l0 to form provitamin D3. Ultraviolet light, at wavelengths of 280-305 nm, is required for the cleavage to occur (Editorial, Lancet, l974). Provitamin D3 is then slowly converted to vitamin D3, without any further action by ultraviolet light. Vitamin D3 is then carried to the liver by means of an alphaz-globulin. Vitamin D3 may also be ingested in the diet. It is absorbed primarily from the duodenum and jejunum through the aid of bile salts and intraluminal lipids. Bile salts play an essential role in the intestinal absorption of vitamin D3 and calcium. If a bile-salt deficiency occurs, the result could be a decreased absorption of dietary calcium. The absorbed vitamin D3 is transported in lymph by means of chylomicrons (Avioli and Haddad, l973; Holick and Clark, I978). At the liver, vitamin D3 is first hydroxylated at the C-25 position on the side chain to produce 25-hydroxyvitamin D3 [25-OH-Dg], which is the major circulating metabolite of vitamin D3. Haussler and Rassmussen (1972) stated that the kidney is also capable of converting vitamin D3 to 25-OH-D3. 25-OH-D3 is then transported to the mitochondria of the renal tubule cells, where it is hydroxylated at either the C-1 or C-24 position to form 1,25-dihydroxyvitamin D3 [1,25-(OH)2-D3] (Figure l) or 24,25-dihydroxyvitamin D3 [24,25-(OH)2-D3] (Ghazarian and DeLuca, 1974; Henry and Norman, 1978; Swaminathan et_§l,, 1977; Holick and Clark, 1978). The enzymatic reaction involved in the hydroxylation of 25-OH-D3 to 1,25-(OH)2-D3 has been studied extensively. 25—0H-D3-lm-hydroxy1ase and 25-OH-D3-24R hydroxylase are stereospecific mixed function oxidases that are located in the renal mitochondria. Reduced nucleotide (NADPH) and molecular oxygen are required for enzymatic activity. Cytochrome P450 is required by 25-OH-Dg-lc-hydroxylase for its activity. In vitamin D3 metabolism, the rate limiting step is the renal 1a-hydroxylation of 25-0H-D3 (Ghazarian and DeLuca, 1974; Holick and Clark, 1978; Avioli and Haddad, 1973; DeLuca, 1974). The mode of action of 1,25-(0H)2-D3 is best summed up by Kumar (1982). He states that, "1,25-(OH)2-D3 probably enters the intestinal cell by diffusion across the cell membrane. In the cytosol it combines with a receptor ...... that is specific for l,25-(OH)2-D3. After it binds with the receptor, 1,25-(DH)2-D3 is translocated into the nucleus. Once in the nucleus, the 1,25-(OH)2-D3 receptor complex acts to increase the activity of the chromatin template and of a RNA II polymerase, an enzyme that makes messenger RNA that is specific for intestinal calcium binding protein (CaBP)". l,25-(OH)2-D3 also has other effects on the intestinal cell. These 6 Figure 1. Metabolism of vitamin D (DeLuca, l974). 3 VITAMIN 03 AS A PROHORMONE .. Skin —___’ .0 uv Light HO 7- DEHYDROCHOLESTEROL *— Duel Kidney ‘____ I) Normal P1 2) Normal Co” la,25*(OH)203 l 1,24,25-(OHl303 Intestine Bone 7 include an increase in the activity of alkaline phosphatase and calcium- dependent ATPase, and an increase in the uptake of calcium by the vesicles of the brush border membrane, the Golgi apparatus, and the basolateral membrane." The CaBP formed in response to vitamin D metabolism which is respon- sib1e for the transfer of calcium across the intestinal wall, and into the bloodstream, is present in the intestinal mucosa of chicks. (Figures 2, 3) A similar CaBP is also present in the uterus and intestine of the laying hen. Bar and Hurwitz (1975) found that ”calcium absorption and CaBP levels increase under conditions of long-term calcium depletion and at the onset of egg production. On the other hand, the increase in intestinal absorption and uterine secretion of calcium during periods of egg shell formation are not associated with any detectable changes of CaBP level in the two organs." In other words, the uterine and intestinal calcium-binding activity showed no significant change during the egg formation cycle. They also concluded that "the CaBP level of the differ- ent intestinal segments... also did not change significantly (P>0.05) between periods of egg shell calcification and periods of uterine inacti- vity.” Finally, they determined that the reason for the increased absorption of calcium during shell formation was due primarily to an increase in permeability for calcium and not any change in CaBP. Wasserman and Coombs (1978) studied calcium absorption in Japanese quail, and reported results similar to those by Hurwitz and Bar (1975). Wasserman and Coombs determined that there was no change in the efficiency of calcium absorption between the calcifying and non-calcifying stage in egg formation. They stated that "this finding is consistent with the observation that the kidney 25-hydroxycholecalciferol-l~hydroxy1ase activity does not differ during shell formation and in the noncalcifying Mechanism of vitamin D3 action (Kodicek, 1972). Figure 2. on: ¢+a0 &.IU|¢OQU meowas 4w203. on: Tau coautht .38 93:55 v 87.3.? a w 2.3.0.... .38 r. <2: Al. 2.30.... \ «:0 30:93.... . 330:: (302050»... 30:92. 3. w 1.: F 9 3.535: amazon .3305» .Smu 442:.mmbz. 22:... 3:98.. Al 00:.3 Al 4 >wzo3. cm): 1.. l .o 2.2:; 1.1 2.3.8:... rL 4.50 wzom _ .uo:n-3.r. 9 on calcium transport mar, l982). )2'0 The action of 1,25-(OH in the intestinal cell Figure 3. (K8 mDmJUDZ 5205 9.5596238 o 5* 50on (ZN. 89.039: *0 P552395 3.0.365 meEOU .0389 50.5033 2. ++OU 832.2 .38 g\. ‘X «2.... v ‘/ cotton :35 . :0 Soto tote .0389 ozomoio of 2 8:5 momfoIm. . =8 _oczmflfi opt :_ toamcoh 5:53 5 «enigma; .0 .528 a: 10 period, i.e., up to 4 hours after ovulation.” Bar et_al.(l978) stated that "intestinal CaBP synthesis is con— trolled by the concentration of l,25-dihydroxycholecalciferol in the cells of this organ.” They found that uterine and intestinal CaBP increased at the onset of egg production in birds, but that uterine CaBP concentration did not change, while intestinal CaBP concentration increased When laying hens were subjected to calcium restriction. Also, kidney-l-hydroxylase activity, as well as, intestinal CaBP levels were higher in the calcium restricted hens, while no change in uterine or renal CaBP was noted. However, the increased production of l,25-(OH)2-D3 did not result in a corresponding increase in calcium deposition into shells. They therefore concluded that, ”calcium deposition into egg shell is not dependent upon cholecalciferol metabolism.” In addition to calcium ingested from the diet, Hurwitz and Bar (l966) found that the ends of the femurs of the hen were the principal site of storage of available calcium that could be utilized during early egg production, even when the hen's diet contained a sufficient level of calcium. When the hens were fed diets that were depleted in calcium (l.7%),eaprogressive decrease in blood and egg shell calcium resulted. Both ends and medullary segments of the femur showed a marked decrease in calcium content during the period of calcium restriction, however, the calcium level in the cortical segment of the bone was not affected to a great extent. When the birds were placed on a repletion diet that con- tained 3.7% calcium, the researchers found that the egg shell calcium returned to normal levels after 6-8 days on the repletion-level diet. They also found that the bones returned to normal in 3 weeks on the repletion diet, despite the rapid increase in egg shell calcium levels. ll The recovery of the bones led them to suggest that the hen's ability to retain calcium following a period of calcium depletion was greater than normal. This assumption was based on a trial in which hens were fed either a high-calcium (3.7%) or low-calcium (l.7%) diet for one week. Then, all birds were fed calcium “free" diets for two days, and then returned to their initial diets for one week. The excreta were collected and analyzed for calcium content. ‘In both the low-calcium and high-calcium groups, the calcium excretion was markedly reduced while the repletion diet was being fed, resulting in a higher calcium retention. Hurwitz and Bar (l966a) further found that, "a greater absorption of calcium seems to be responsible for the increased retention, since the magnitude of this increase exceeded the total amount of calcium of 140-200 mg per day nor- mally found in the urine of laying hens". In a previous study, Hurwitz and Bar (l965) determined that the majority of calcium and phosphorus absorption occurred in the anterior portion of the intestinal tract, and that the absorption and phosphurus appeared to be related to, as well as influenced by, the absorption of calcium. They also reported that, "the absence of a calcifying shell was associated with a reduced rate of calcium absorption”. More recently, Hurwitz et 31. (l973) reported that a duirnal fluctuation in calcium absorp- tion occurred, which is generally associated with the laying cycle; also that the absorption of calcium tended to increase during shell calcification in all intestinal segments, except the lower ileum. Older hens tend to have a greater percent of cracked or broken eggs. This problem is more apparent in warm weather. ~McLaughlin and Soares (1976) showed that "hen-sized calcium carbonate, regardless of the source had a definite influence on improving shell quality during warm weather". These l2 researchers also experimented with feeding different calcium sources (lime- stone or oyster shell) at 3.5 or 4.0% of diet, in combination with either 600 or 3000 I.U. of vitamin D or 25-0H-D3 per kg. of diet. This study 3 showed that ”...600 I.U. of 25-0H-D3 per kg. when fed in combination with either limeStone or oyster shell as the calcium source to laying hens in an advanced state of production results in an improvement (P<.05) in shell quality as measured by specific gravity and shell thickness.” In addition, they determined that 25-0H-D3 was more effective than vitamin D3 in promo- ting the mobilization of calcium in older hens and also "in the formation of CaBP in the uterus during egg shell formation.” Their study led them to postulate that the reason for the more efficient effect of 25-0H-D3 on the mobilization of calcium and subsequent egg shell calcification might be due to a decreased ability of the older hen to hydroxylate vitamin D to 25-0H-D . 3 3 In another study involving the effect of calcium source on shell quality, Scott et_ a1, (l97l) demonstrated that when hen-sized oyster shell was substituted for a portion of pulverized limestone in the diet of laying hens, an improvement in shell strength resulted. The improvement appeared to be due to the fact that the oyster shell allowed for a constant metering of calcium from the gizzard, therefore allowing the hen to absorb calcium during the entire day. This was compared to the fact that hens with the pulverized limestone in their diet must absorb all their calcium during the l4-l6 hours of light. The metering of calcium from the crop and gizzard was studied by Roland et_ 91. (l972a, l972b). Most of the calcium source was metered from the gizzard during the early morning hours, with little at night. In addi- tion, it was observed that when granular limestone was the calcium source, l3 it was metered fairly uniformly, but at a decreasing rate during the night. They concluded that "less total calcium is metered into the lower digestive system from the cr0p and gizzard during the night when oyster shell is fed than when fine granular limestone is fed”. Many factors are responsible for the regulation of calcium absorption and vitamin D3 metabolism. Hurwitz §t_al, (l973) believe that there exist two types of regulation for intestinal calcium absorption. These differ in stimulus, site and time of response. The first type is based on the increase of calcium absorption during egg shell formation. The time of response to this stimulus is very short, about 2 hours or less. This first type of regulation is "characterized by a general increase in calcium absorption at all levels of the intestine, except for the ileum. Within the small intestine, the response is greatest in the upper jejunum. This mechanism seems to be available during the first few days of egg produc- tion, and is found at similar intensity in both young and old hens". The second type of regulation described occurs only in the duodenum and is stimulated by the onset-of egg production from a nonproducing state, and also by the amount of body calcium stores. The response time for duo- denal regulation is l.5 days. Also, the duodenum had a higher leVel of calcium absorption in the older lyaing hen than in the younger laying hen (Hurwitz gt_al,, 1973). Polin and Ringer (l977) conducted a study to determine the effects of 25-0H-D3, vitamin D3 and varying levels of phosphorus on egg shell quality. While D3 is involved in the transport of phosphorus across the intestinal wall, the absorption of phosphorus is not dependent upon calcium absorption. This study found that when no phosphorus was added, or when the level of added available phosphorus was 0.28%, adding 25-0H-D3 to the diet appeared T4 to yield better quality shells than when vitamin D3 was added. The D3 forms resulted in an increased availability of phosphorusfrom diets that consisted primarily of plant-type ingredients. Both D3 forms resulted in comparable quality egg shells when targeted levels of 0.42 or 0.5 % phos— phorus was included in the diet. In addition, ”25-0H-D3 did not protect shell quality any better than D3 during hot weather.“ Both 03 forms were unable to prevent the decrease in shell quality, brought on by aging and hot weather, that occurred when the hens were 32 to 52 weeks of age. And, when both D forms were added at 25ug/kg of diet, 25-0H-D3 tended to result 3 in higher egg production and better shell quality at submarginal levels of phosphorus. Polin and Sturkie (l957) studied the effect of the parathyroid glands on blood calcium levels and shell deposition. They found that a decrease in the level of diffusible calcium occurred at the time of shell deposition, but that total calcium level showed no change. When the parathyroid glands were removed from laying hens, a marked decrease in plasma calcium was noted, and subsequently, eggs that were present in the uterus were expelled pre- maturely and showed little or no evidence of shell calcification. They further found that ”shell deposition is dependent upon the maintenance of the diffusible calcium level in the blood and that the parathyroid glands are involved in the maintenance of this level.“ When the rate of calcium mobilization was less than the rate of shell deposition, the lowered con- centration of diffusible calcium acts as a stimulus for an increase in cal- cium mobilization. Another study by Polin and Sturkie (l958) showed that a rise in the non-diffusible calcium level in blood occurred in response to estrogen- like substances. The level of non-diffusible calcium was found to be 15 higher in laying birds than in non-laying birds. Parathyroid hormone was involved primarily with the regulation of the level of diffusible calcium. When the parathyroids were removed, the diffusible level decreased, with or without the administration of estrogen. Their study concluded that “para- thyroid hormone and estrogen are indirectly related in their action on blood calcium. Parathyroid hormone maintains diffusible calcium levels so that estrogens can increase non-diffusible calcium levels.“ Many physiological mechanisms are involved in the control of vitamin D3 metabolism. Colston et_ a1, (l973) proposed two possible mechanisms. First, that hydroxylase activity is regulated by the concentration of intra- cellular calcium. The hydroxylase activity responds rapidly and inversely to changes in calcium concentration. The intracellular calcium concentra- tion is influenced by parathyroid hormone and calcitonin. The second regu- latory mechanism is based on the concentration of 25-hydroxycholecalciferol- binding protein in the cytoplasm. This leads to the accumulation of 25-0H-D3, thereby raising its concentration in the region of the enzyme, 25-hydroxycholecalciferol-l-hydroxylase, resulting in an increased rate of formation of l,25-(0H) D 2' 3‘ The plasma calcium levels control the parathyroid activity. When the bird is in a state of hypocalcemia, the production and release of para- thyroid hormone is stimulated (Figure 4). When parathyroid hormone is secreted, the increased amount stimulates the activity of 25-hydroxychole- calciferol-l-hydroxylase, thereby resulting in an increased production of l,25-(OH)2-D3 by the kidney. It has been shoWn that l,25-(0H) is l3-15 2‘03 times as effective as vitamin D3 in stimulating intestinal absorption of calcium, and 5.5 times as effective as vitamin D3 in stimulating the eleva- tion of serum calcium. When the calcium level returns to normal, the para- 16 Figure 4. Effects of low serum calcium (DeLuca, l974).- LOW SERUM CO” l Serum Ff —-» PTH W |,25-(OH)203 CALCIUM CALCIUM R FROM FROM FROM INTESTINE, BONE INTESTINE BONEFTC. l“ P in urine L ' 4 V Serum CO”? l7 thyroid hormone secretion is suppressed, the activity of 25-hydroxycholecal- ciferol-l-hydroxylase decreases while the activity of the enzyme 25-hydroxy- cholecalciferol~24-hydroxylase increases. Once formed, the 24-hydroxylated metabolites are rapidly metabolized and excreted (Swaminathan et_ al,, l977, Holick and Clark, l978, Suda e: _a__l_., 1973, Henry and Norman, 1978, Norman and Henry, l974). Holick and Clark (1978) also demonstrated that in rats constantly infused with calcitonin, no direct role of calcitonin in vitamin D metabolism could be elucidated. The role of phosphate depletion in vitamin D3 metabolism was studied by Haussler et_ a1, (l977) (Figure 5). They observed that l,25—(0H)2-D3 is able to mobilize phosphate in gut, bone, and possibly kidney, but whether phosphate could control the formation of l,25-(0H)2-D3 was undeter- mined. Also, several controls on the stimulation activity of 25-hydroxy- cholecalciferol-l-hydroxylase were noted, including, low dietary phosphate, vitamin D3 deficient diet, and hypophosphatemia and hypocalcemia in rela- tion to improper bone calcification. 0n the other hand, birds with a nor- mal level of vitamin D3 and a low phosphorus level in the diet, had low enzyme activity. They also found that a high level of enzyme activity was present in birds on a vitamin D3 deficient diet, regardless of the calcium level in the diet (0.2, 0.7, or 3.0%). 18 Figure 5. Effects of low serum phosphorus (DeLuca, l974). LOW SERUMR i Lea-(onto, nO PTH ‘ ‘ CALClUM P FROM * FROM, iNTESTINE 8 Ca” FROM iNTESTiNE ELSEWHERE BONE nO PTH I nO PTH CALCIUM i L J V Calcium in serum increases slightly. P greatly increases in blood. PROCEDURES This experiment was conducted in two trials. Both studies were to determine which vitamin D3 metabolites, if any, were present in the uterus of the laying hen. The first trial was conducted primarily to determine how the experimental diet affected shell quality and the uterine metabo— lites of vitamin D3. The second trial utilized the information gathered in the first and, additionally, investigated the fate of vitamin D3 in the liver, kidney, as well as in the uterus of the laying hen. Single Comb White Leghorn mature laying hens, approximately one year of age, were subjected to one of two dietary regimes. The rations were either a control diet, which contained an adequate level of vitamin D3 (l500 ICU/kg of diet) or a diet that was nutritionally inadequate in vitamin D3 (no vitamin D3 added to diet). Feed and water were supplied ad libitum. The diets were formulated to equal or exceed the nutritional require- ments of laying hens (Tables l and 2) according to values given in Nutri- tion of the Chicken (Scott _t__l,, l976). The values used to formulate the vitamin mixture were based on recommendations from the Department of of Animal Science, Michigan State University. In the deficient diet, a separate vitamin premix was used, which had no vitamin 03 added. The hens were fed diets with or without adequate 03 for a minimum of three weeks to a maximum of ten weeks. 20 Table l. Layer Ration for Vitamin D3 Experiment Ingredients Parts per l000 Corn #2 yellow, grnd. 573.5 Soybean meal, 40% 240.0 Alfalfa 42.0 Corn 011, stab1.a 42.0‘ Limestone 74.0 Defluorinated phosphate l8.0 Salt, iodized 3.0 Methionine hydroxy analogue l.0 Choline chloride, 50% l.5 Vitamin mixb 3.0 Mineral mec 0.5 Selenium mixd 0.5 Ethoxyquin added to supply l25 mg/kg diet. Supplies per kg diet: vitamin A-l2,000 I.U.: vitamin D -1500 I.C.U.: vitamin E-l5 I.U.: Menadione sodium bisulfite complex-l.5 mg: Thiamine—2.4 mg: Riboflavin-6.6 mg: Pantothenic acid 6.6 mg: Niacin 30 mg: Pyridoxine-9.0 mg: Biotin-0.3 mg: Folic acid~l.25 mg: B -.009 mg: carrier of corn gluten meal with 4% corn oil to 3 grams. Vitamin D omitted from vitamin premix in ration fed to hens on D3 deficient diet. Trace mineral premix obtained from Calcium Carbonate Company (CCC), Quincy, Illinois, 6230l. From CCC. 21 Table 2. Calculated Analysis of Layer Ration for Vitamin D3 Experiment Nutrient ‘ Value Metabolizable energy - Kcal/g 2.9l ‘ Protein - % l6.40 Calcium - % 3.55 Phosphorus, available - % 0.46 Fat - % 6.78 Fiber - % 3.59 Lysine - % 0.84 Methionine — % 0.36 Methionine - % of protein 2.l9 Methionine plus cystine — % of protein 3.89 22 Feed intake was monitored weekly for each group of birds during the experimental period. Feed intake (grams/bird/day) was calculated from the weekly consumption measurements. During the summer months, the hens were housed in individual cages in house number 5 at the Michigan State University Poultry Research and Teaching Center. When the weather turned cooler along with significant day-night temperature fluctuations, the birds were moved into cages in the cage room at Anthony Hall and housed one per cage. There, the temperature was maintained at 22:2OC. In both housing situations, the hens were sub- jected to a photoperiod of l5 hours light, 9 hours dark. Eggs were collected at day 0 and once weekly during the experimental period. These eggs were then subjected to shell quality measurements. Production records were kept for each hen according to dietary regime. Shell quality was measured based on the following parameters: whole egg weight (grams), shell weight (grams), and egg length and breadth (cm). These values were used in a formula (Carter, l975) to calculate mg of cal- cium per square millimeter of shell surface area. To determine shell weight, eggs were broken at the equator, rinsed and allowed to air dry. Shell membranes were left intact, and the entire shell was weighed. Shell thickness was also determined, and in the following manner. The shell, with the membranes attached, was measured using a micrometer (Federal, Providence, R.I.) at two places along the equator. The average of these two measurements was used as the value for the thickness of the shell. Egg length and breadth, in cm, were measured using vernier calipers. The length was based on the distance between the large and small end of the egg, and the breadth was taken at the widest point along the egg's equator. 23 Column Preparation and_$tandardization Chromatography columns were prepared according to the procedure of Holick and DeLuca (l97l). Twenty grams of Sephadex LH-20 (Pharmacia Fine Chemicals, Inc., Piscataway, W.J.) was slurried in 70 mls of chloroform- hexane, 65:35 (v/v). The slurry was allowed to equilibrate for 24 hours. It was then poured into a glass column measuring 60 x 1.5 cm, that con— tained 20 ml of the solvent. The Sephadex was allowed to settle by gravity, with solvent flowing until it was completely settled. Each column was washed with 50-l00 ml of solvent prior to the application of any sample. To determine the elution pattern of vitamin D3 (D3) and its metabo- lites, 25-0H-D3 and l,25-(0H)2—D3, known radioactive, tritiated standards (Amersham, Arlington Heights, Illinois.) were applied to the chromatography columns and eluted with CHCl3-hexane. Up to 75 elution fractions, 5 ml each, were collected after the standard(s) were applied to the column. The fractions were then transferred to scintillation vials, and the solvent was allowed to evaporate by air drying in a hood. Ten mls of Scinti-verse® Liquid scintillation counting solution (Fisher Scientific Company, Pitts- burgh, PA) was added to each vial. Radioactivity of each sample was deter- mined by liquid scintillation counting in a Searle Isocap 300 counter, for one minute per vial. Using radioactive standards, vitamin D3 was found to elute off the column at 40-45 ml, 25-0H-D at 65-70 ml, and l,25-(OH) at 295-300 ml 3 2‘03 of solvent flowing through the chromatography column (Graphs l, 2, and 3). An additional peak was present at 140-150 ml. It was initially apparent in the chromatography pattern of the l,25-(OH)2—D3 standard, and appeared again when 03 and both metabolites were concurrently chromatographed. The identity and significance of this peak is unknown at this time. 24 -OH-03 .) Column standardization-vitamin D. and 25 Graph 1. 23000 i 20000 I _- I .1 15000‘ lGOOO 5000 :o_uuegu _a m Lwa Env um: ..II!\ Il‘l’ll Iii“ l000 Mia 66 80 ml eluted from column 40 20 Graph 2. Column standardization-l,25—(CH)2-D3 4000“ C: .2 3000 {J 3 L 14.. LO 5 200a; i 5‘- l 6/ 1 a. ’ I l l I '0 I _I' i g l I i 1000: i I ' I’ i f i E A ’4‘\\ | ‘\ I_..../\vvn-v—~~*vn...../ ),_j IL a»..- L, J I I I I ' 0 so 100 150 290 zéo 300 350 ml eluted from cclumn 25-0H—03, l,25-(0H)2-D3 Column standardization-vitamin D3. Graph 3. 3 D _ H O . r3 2 0......) HH.....I..MI....IH. . .111 Vitamin 03 i 2 D3 _ \d 2 . . \ )/ mw I I\II\\ Iii JI SWIIII I} 5 1.1.1.. 7» 1.1.1.4174. . 1‘ n" I.) / , ~I\. nWI/r S \\ n.\\\\ \I\ 1., / II liililil‘. if. it'll ill. 1. l.l! .IlII )1111‘1.‘ IIIII II. II I I I i Ilitlii’ll I \\ ll}1ll.ll¢\ IIIIJIIII' ‘lnuliillillllllI 350 :4 2 nm W U l. O C 0m 30C 2r. :1 d e t U .II e l. m”. 15b 10b 5'0 I i _- .— 6000 3000 1000 :owuumgw F: m can 51c pm: 27 Injection pf ngs Preliminary feeding trials using D3-deficient diets showed that hens which had been fed the deficient diet for a minimum of three weeks had lowered egg production as compared with the control birds (Table 4). A similar trend was not seen for egg production and shell quality in the second trial (Tables 5, 6, 7, and 8). When hens being fed the D3-deficient diet during the second trial period began showing lowered egg production and an apparent decrease in shell quality, 6 control and 6 deficient hens were injected intravenously (brachial vein) with two microcuries of 3H-D3. A non—injected hen fed a diet adequate in 03 was sacrificed for use as a control. Collection pf_Tissue Samples Hens were sacrificed by cervical dislocation l5 to l8 hours after injection with 3H-D3. This was to allow time for egg shell calcification to be occurring in each hen. The following data were collected for each hen at the time of sacrificing - presence of an egg in the uterus, and weight of liver, kidney and uterine tissues (Table 9). After weighing, the tissues were washed in physiological saline and then frozen individually until time of analysis. Sample Processing gpd Lipid Extraction The frozen tissue samples were processed as follows. The entire tissue sample was placed in a Waring Blendor‘3 that contained 200 ml of methanol and l00 ml of chloroform. The tissue was homogenized for two to three minutes. The homogenate was transferred to a beaker, and l00 ml of distilled water and 50 ml of chloroform were added to the homogenate 2 28 hours later. This procedure allowed for the separation of the lipid phase from the water soluble phase. The beaker contents were transferred to a separatory funnel and the lipid (chloroform) phase was collected in an Erlenmyer flask. An equal volume of water was added to the flask, and it was stored overnight at 4°C. The next day, the flask contents were trans- ferred to a separatory funnel, the chloroform phase was collected and allowed to air-dry under a hood at room temperature. Sample Analysis py Column Chromatography Each dried lipid sample was redissolved in 0.3 to 0.5 ml of CHCl3— hexane solvent. The entire sample was then applied to a Sephadex LH-20 chromatography column. Four hundred ml of CHCl3-hexane was run through the column to elute D3 and/or its metabolites. The 400 ml was collected in 5 ml fractions. The elution fractions were handled in the manner previously stated. 29 Table 3. Feed Intake (grams per bird per day) Control Diet vs. D3 Deficient Diet Intake—g/b/d Deficient as ngk_gf_lpigl Control Deficient % of Control l 99 103 104 2 127 155 122 3 117 118 101 4 108 95 88 5 107 84 79 6 111 85 76 7 111 94 85 8 116 86 74 9 130 85 65 10 123 97 79 30 Total Weekly Egg Production of Hens in Trial 1. Table 4. Week 1 Week 2 Week 3 Week 4 Week 5 Hen No. Treatment 556456565656 355555565634 445332445444 555456465554 455655555645 +D3 5:2 Mean + S.D. 30044035114110 030341321510 111452450510 435455551143] 655465576065 123456789012 .I..1I.l -D3 5:2 Mean + S.D. 3l Table 5. Relationship of Diet to Eggs Collected on a Once-per-Week Basis in Table 2. Hen No. Diet Day 0 Day 7 Day l4 Day 2l 57 Control +3 + + 8 Control + + - 59 Control + - - + 60 Control - + - - 93 Control + + + + 94 Control + + + + 95 Control + + + - 96 Control + + + + Total # ‘ 7 7 6 5 % of Production 87.5 87.5 75.0 62 5 65 Deficient + + + - 66 Deficient - + + — 67 Deficient + + + + 68 Deficient + + - - 69 Deficient + + - - 70 Deficient - + + + 7l Deficient -— + - - 72 Deficient + + + + 89 Deficient + + + + 90 Deficient + - - + 9l Deficient + + + + 92 Deficient + — - - Total # 9 l0 7 6 % of Production 75.0 83.3 58.3 50 a = laid egg; - = no egg laid on day of collection. 32 Tab1e 6. Ca1cium (mg) per She11 Surface Area (mmz) Ca1cu1ated from Egg Qua1ity Measurementsa (Tria1 2). Ca1cium (mg)/S (mm2) Treatment Hen No. Day 0 Day 7 Day 14 Day 21 +03 57 .30 .29 .32 .32 58 .32 .32 .34 no egg 59 .32 no egg no egg .33 60 no egg .30 no egg no egg 93 .28 .27 .31 .32 94 .32 .30 .32 .30 95 .32 .34 .38 no egg 96 .34 33 34 .37 Mean : S.D. .31 i .02 .31 :_O.3 .34 :_.02 .33 :_.02 % Production 87.5 _ 87.5 75.0 62.5 -03 65 .30 .29 .32 no egg 66 no egg .30 .34 no egg 67 .32 .30 .32 .30 68 .27 .26 no egg no egg 69 .34 .37 no egg no egg 70 no egg .30 .34 .34 71 no egg .31 no egg no egg 72 .30 .30 .3 3 89 .30 .30 .34 31 9O .31 no egg no egg 34 91 .32 . 34 92 .32 no egg no egg no egg Mean :_S.D. .31 :_.02 .30 :_.03 .33 :_.01 .32 :_.02 % Production 76.0 83.3 58.3 50.0 a = Carter, British Pou1try Science, 1975. 33 no egg 1aid or co11ected on that day. no data on day 21 to a11ow ca1cu1ation. Tab1e 7. She11 Qua1ity Change as Re1ated to Egg She11 Thickness (Tria1 2). Egg She11 Thickness (mm) Treatment Hen N0. Day 0 Day 7 Day 14 Day 21 Diy 21-Day 0 +03 57 37 36 38 36 -1 58 39 38 42 -- *b 59 40 -- —- 39 —1 60 -a 35 __ __ * 93 36 33 36 36 0 94 39 36 36 36 -3 95 42 44 47 -— * 96 43 40 39 40 -3 Mean 39.4 37 6 39.7 37.2 —2.0 —D3 65 48 37 38 -- * 66 -- 38 39 37 * 67 4O 36 36 37 -3 68 34 32 —- —- * 69 45 45 -— -- * 70 -- 38 42 39 * 71 —- 35 -- -— * 72 39 38 36 38 -1 89 40 39 40 35 -5 9O 40 37 40 37 -3 91 40 37 40 37 -3 92 42 -- —- —- * Mean 41 37 5 38.8 37.0 -3.0 34 Tab1e 8. Changes in Percent She11 Weight as Compared with Who1e Egg Weight Based on Dietary Regime. (She11 Weight/Who1e Egg Weight) x 100 Treatment Hen N0. Day 0 Day 7 Day 14 Day 21 Day 21-Day 0 +03 57 10.2 9.7 11.8 11.9 1.7 58 10.4 10.3 12.1 -- *b 59 11.1 -— -- 12.2 1.1 60 -a 10.1 -— -- * 93 9.7 9.2 11.8 12.1 2.4 94 11.4 10.2 12.1 11.1 -0.3 95 10.8 11.3 13.7 -- * 96 11.3 10.9 12.9 13.9 2.6 Mean 10.7 10.2 12.4 12.2 +1.5 -03 65 9.9 9.6 11.8 -- * 66 -- 9.4 12.0 12.4 * 67 10.8 10.2 12.0 10.6 -0.2 68 8.9 8.5 -- -- * 69 11.2 11.9 -- -- * 70 * -- 10.3 12.3 13.2 * 71 -- 10.0 -- -- * 72 10.1 9.8 12.2 11.5 1.4 89 9.7 9.5 12.6 10.9 1.2 90 10.6 -- -- 12.6 2.0 91 10.1 9.3 12.7 12.3 2.2 92 10.2 -- -- -- * Mean 10.2 9.8 12.2 11.9 +1.3 n0 egg 1aid or c011ected on that day. no data on day 21 to a11ow ca1cu1ation. RESULTS AND DISCUSSION Feed Consumption Tab1e 3 shows that from the fourth week of the tria1, through the termination, hens being fed a diet that contained no vitamin D3 ate 1ess feed than did the contro1 birds. The deficient birds ate 65% to 88% of the quantity consumed by the contro1 group. The decrease in feed consump- tion was brought about by the vitamin D3 deficiency which 1ed to a decreased ca1cium absorption. The decrease in ca1cium absorption, in turn, resu1ted in 1ess feed consumption. Egg Production In the pre1iminary feeding tria1 (Tab1e 4) the data show that after hens had been fed a vitamin D3 deficient diet for 3 weeks, their week1y egg production was 1ess than that of hens fed the diet which had an adequate 1eve1 of vitamin D3. In the second tria1, in which eggs were c011ected once week1y, (Tab1e 5) the vitamin D3 deficient hens again showed a 10wer percent production on the days that eggs were c011ected. Egg_Qua1ity The data presented in Tab1es 6, 7, and 8 show that 0n1y s1ight changes in she11 qua1ity occurred during the course of the second tria1. The quan- tity of ca1cium per mm of she11 surface area was .01 mg 1ess in the vitamin D3 deficient group in days 7, 14, and 21, than for the contro1 group. Egg she11 thickness decreased for both the contro1 and vitamin D3 deficient hens. The thickness decreased by an average of 2.0 mm for the contro1 group; for the deficient hens an average decrease of 3.0 mm resu1ted. 35 36 The data on the change in % she11 of tota1 weight (Tab1e 8), again show the minima1 changes that took p1ace during the second experimenta1 period. The average tota1 change from day 0 to day 21 for the contro1 group was an increase in she11 weight of 1.5%; the change for the deficient birds was an increase of 1.3%. These s1ight differences suggest that the hens were not subjected to the vitamin D3 deficient diet for an adequate 1ength of time to bring about the c1assica1 changes of vitamin D3 defi- ciency: thin-she11ed and soft-she11ed eggs and a decrease in egg produc- tion. These symptoms have been reported to occur about 1—2 months after the birds are on a deficient diet (Scott §t_§l., 1976). The resu1ts of the second tria1 wou1d be improved had egg qua1ity data been c011ected for a 10nger period of time, i.e. 28 or 35 days. Another reason for the minima1 changes might be that a1though the hens were being fed a vitamin D3 deficient diet, they attempted to maintain she11 qua1ity during the experimenta1 period by producing fewer eggs, that were sma11er in size, and who1e egg weight. In this manner, they were ab1e to conserve avai1ab1e ca1cium for she11 deposition onto the eggs that were produced. Recovery of Radioactivity The data in Tab1e 10 show that, a1though a11 birds were injected intra— venous1y with equa1 amounts (2 microcuries) of 3H—vitamin D3, the recovery of radioactive metabo1ites was 10w and varied with each hen. One contro1 hen and one deficient hen, both sacrificed with an egg present in the uterus, had the best recovery of 3H-vitamin 03, with 3.02% and 3.87%, respective1y. The data from the remaining birds fo11owed no predictab1e patter, in that 37 Tab1e 9. Tissue Weights and Presence of Egg in Uterus, Co11ected at Time of Sacrificing Egg in Hen No. Diet Uterus Liver Kidney Uterus 57 Contro1 + 29.0 11.0 12.5 58 Contro1 + 21.8 10.9 13.8 59 Contro1 - 40.8 12.9 11.3 *60 Contro1 - 19.8 11.2 12.0 93 Contro1 - 27.0 11.2 10.5 94 Contro1 - 20.0 11 2 13.0 Mean :_S.D. 26.4 :_8.0 11.4 :_0.7 12.2 i 66 Deficient - 24.0 10.5 15.0 *67 Deficient - 24.8 9.3 10.0 68 Deficient + 24.5 11.5 17.5 69 Deficient + 22.8 9.5 10.0 70 Deficient + 25.5 9.1 12.0 91 Deficient + 34.5 14.5 15.5 Mean :_S.D. 26.0 :_4.2 10.7 i 2.0 13.3 : Hon-injected hen ----- + 44.0 14.5 18.0 *Hens 60 and 67 1aid eggs 5-10 minutes prior to sacrificing. 38 33¢ mmae o.o_ 3:268: mm.m mom omem m.¢ amcuwx mm_ oemm m.¢m cm>wS - peacowcmo No mmp omem o.m_ 6:268: mm.~ emw meow m.o_ ameuwx «em mewm o.¢m em>wu - pcawuacmo we 03 wow o.m_ 6:268: 5m. 88 we“ N.__ ameuwx mm wom_ o.o~ 26>.4 - Foepcou em oFm mmmm m.o_ 3326»: Rm.m vow emme N.__ smeewx em memm o.NN em>wu - Peepeou mm N om o.N. 3:268: mo. m_ Ne_ N.FP xmcewx o o m.m_ 26>.u - _oepcou so u om m._P macaw: mo. 8, ma_ m.NF ameuwx 3 oo_ m.o¢ Lm>23 - _ocpcou mm 3_ Nm_ w.m_ magma: N_. mm com m.o_ xmcewx m «N m.PN em>wu + _oepcou mm . oNN _mwm m.mp macaw: No.m mew Doom o.__ zmcuwg c_m Nm_m o.m~ em>wu + _oepcou Km empowncw Emu quoe mammwp .m vmcm>ouwm .m pgmwmz mammwe magma: pm?o .oz co: co, xAumgw>oomL saw _mpopv cma Emu Emu Peach mammwh :H mom mam: cmpumwcH soc; empumppoo mmzmmwh cw xuw>wpomowumm 4o >Lm>oomm .o_ m_nmp 39 NR mNFF Fm.p wow mmom mm mmFm mam FmF¢ mw.m _wm «mam vmq owe,— o o _o. m we 0 o o o _o. m ca 0 o \ umpomncw Eav _wpop 63mm?“ .m umgm>oomm 00— x,uwcm>ouwc Emu FaHOHV 2mg Eqn. Emu Pouch LDLOLO o (fir-r— LDv—O ,._ mscwp: sacuex Lw>w4 magma: >6:u_¥ Lm>w4 magma: smeewx Lw>w4 mscwp: socewx Lm>w4 ozmmwh + peacowcwo + “cawowcmg + pcmwuwcmo + #:8282469 ngwga wove :H mom Aumzcwucouv Om mm mm .62 cm: .op wFth 40 percent recovery appeared to be independent of diet and presence of egg in the uterus at time of sacrificing. These resu1ts may be due to indivi- dua1 bird variation, inabi1ity of these hens to metabo1ize the 3H-vitamin D3 or improper 1ength of time between injection of 3H-vitamin D3 and sacrificing of birds to a11ow for accumu1ation radioactive vitamin D3 or its metabo1ites in the 1iver, kidney, or uterus. The data presented in Tab1e 11a indicates that a greater percent recovery of radioactivity occurred in uterine tissue from the hens on the. contro1 diet than from the uterine tissue of deficient hens. The data in Tab1e 11b indicate that a greater percent recovery of radioactivity occurred in hens sacrificed without an egg than those birds which had an egg. This tab1e a1so revea1s that 0% recovery of radioactivity was associated with 2 deficient hens sacrificed when an egg was present in the uterus. This resu1t was not expected as it seems more probab1e that hens with ca1cifying she11s present at time of sacrificing wou1d a1so have 3H-D3 or its meta— bo1ites present, as opposed to those birds sacrificed without an egg. Chromatography 9f_Tissues The resu1ts of the co1umn chromatography of the 1ipid extract from the 1iver, kidney and uterine tissue c011ected from the hens are depicted in Graphs 4a, 4b, 5 and 6. Except for the 1iver of one bird, a11 other samp1es showed recovery of some of the injected 3H-vitamin D3. This was usua11y the prominent peak in the chromatographic pattern. The 1iver patterns a11 had evidence of the 25-OH-D3 metabo1ite. These peaks were usua11y 1ess than 500 dpm and were e1uted between 60 and 80 m1. The two contro1 hens showed 25-0H-D3 peaks of 1000 dpm and 2500 dpm, respective1y. One of these hens a1so had a peak of 700 dpm appear at 100 41 Tab1e 11a. Uterine dpm as Percent of Tota1 dpm Recovered, as Re1ated to Dietary Treatment Egg In Treatment Hen No. Ute_rus_ % Uterine dpm +03 57 + 19 58 + 34 59 — 19 60 - 38 93 - ‘ 43 94 - 23 Mean : S.D. 29 i 10 -D3 66 - 25 67 - 46 68 + 0 69 + 0 70 + 22 91 + 15 Mean : S.D. 18 i 17 42 Tab1e 11b. Uterine dpm as Percent of Tota1 dpm Recovered as Re1ated to Presence of Egg in Uterus Egg In Treatment Hen No. Uterus % Uterine dpm +03 59 - 19 +03 60 - 38 +03 93 - 43 +03 94 - 23 -D3 66 - 25 -D3 67 - 46 Mean :_S.D. 32 :_11 +03 57 + 19 +03 58 + 34 -D3 68 + 0 -D3 69 + 0 —D3 70 + 22 -D3 91 + 15 Mean :_S.D. 15 i 13 net dpm per 5 m1 fraction Graph 43. vitamin D3 43 Chromatography pattern-1iver 3 2000 1 -0 ’\ -Egg ‘1000 / 1. 25-0H-D, ‘ u 1‘ 1. "N E-WMJ' W W”""M“M-- “"'""“A.mmuwmwm 4000 " 3000 '03 -599 _ vitamin D7 2000 A J .. {2’1“ 1000 /’ 1 2508-03 ”4000 $1 3000 g ) v1tam1n 03 +03 1 ) *E99 ”2000 1 , 1 1‘ 25-011-03 7000 ,1! ‘ ‘1 ? )’ k‘xpl"u; \\__ "3065‘" _4 2000 I, ‘ , \25-014-03 +07 '1000 I \ +E99 j . a T . 1 r 1 1 0 20 60 100 140 180 220 260 e1uted from c010mn Net dpm per 5 m1 fraction Graph 40. Chromatography pattern-1iver $5000 1‘5 32000 g \ vitamin D3 1 i 1 - 1 1 1 D3 {1000 1 a +Egg I " Z 5 -0H - D . 3 :t‘r‘m K‘d‘n. .12000 1 . ,_ F 11000 H ’ 1 ,10000 )1 vitamin D '2" y ‘ 3 {4000 1 1 1 1 1 . a 1 1 3000 g 1 1 1 1 "2000 I Z ‘03 ) +E99 ‘fiooo ! 1 25-011-03 ? 4,! l 1“:wa ix . . F i H I ' J 20 60 100 140 18 220 260 m1 e1uted from cqumn Net dpm per 5 m1 fraction 45 Graph 5. Chromatography pattern-kidney 1.. EZOUO vitamin D3 'D3 1 'Egg ?‘ x 1‘000 “fl; \ O 2x _...] \v‘M 22000 >~ _. , . 1 /) vitamin D3 ‘ ,1 " 3'1000 . \ 25014-03 '03 1 / 1 -Egg 0 ”_d' ‘x‘m‘yN 13000 2 ,3 vitamin 03 _U3 (2000 ‘1 +599 1'1000 I! ‘ O 2 \‘J Ext-_NAK 14000 § 4 3000 it Vitamin D3 -D3 1' 1 +E99 "2000 I ) 1 1 1 1000 I \ 25-0H-D3 c .,_ _/ «c ‘2000 j/\ +03 1000 A vitamin D3 +Egg 0 ~,// {g {4000 ‘ 6 +0 ‘ 1 ? 3 3000 11k +599 '2000 _ . D 11 " Vitamin . ‘1 3 1 ‘1 11000 ‘1 j ‘1. ? _.-.- / \— \‘1 r1 ‘ . 26.-...- 0 20 60 100 140_ 180 220 260 m1 e1uted from co1umn net dpm per 5 m1 fraction 46 Graph 6. Chromatography pattern-uterus ‘1 1 1.. $2000 vitamin 03 -D 3 .5000 f\ 25-0H—03 -Egg 5 firm \‘ ”JMNWN :nE"“"“‘j V I». 13000 E, \ vitamin D -D g2000 /?E 3 E3 1 I "1, - 99 $1000 . '1: 25-011-03 :em.r-,«m)ji Ray/Jr’sfith £2000 % 25-0H-D3 +Egg .3.me cm M 2000 7000 v1tam1n D3 -03 I 25-0H-03 ? +Egg EM‘M/mj’ . /6\\ IN 2000 . . +03 1000 [/S‘A\K\v1tamin D3 +Egg '3~—._“__ Fr_n_ 4000 i 3000 a 1 I) vitamin D3 :5 +Egg 2000 I 1000 I 0 213 60 100 140 180 220 250 m1 e1uted from co1umn 47 m1. One deficient bird had a sma11 peak of 300 dpm at 260 m1. The identity of this peak and the peak at 100 m1 are unknown at this time. These peaks may be significant due to their re1ative prominence in the e1ution patterns of 2 hens, both sacrificed with an egg present in the uterus. The e1ution pattern for the kidneys shows 25-0H—D3 peaks in 0n1y two of the birds. As in the 1iver there peaks are 1ess than 500 dpm. The pattern from one contro1 bird has two additiona1 peaks, one of 3400 dpm at 100 m1 and one of 350 dpm at 260 m1. The uterine chromatography pattern appears to fo11ow the trend of the previous two tissues, in that 3H-vitamin 03 was recovered from a11 samp1es and that 4 0f 6 showed sma11 25-0H-D3 peaks (Graph 6). One contro1 bird had a prominent peak (3500 dpm) at 150 m1. This was the same bird which had a significant peak at 100 m1 in the kidney. These resu1ts show that the injected vitamin 03 was transported to a11 tissues examined. In addition, some metabo1ism occurred, which resu1ted in the production of the 25—0H-D3 metabo1ite. However, because of the re1ative sma11 recovery of dpm in the 25-0H-D3 peaks, the time interva1 a11owed between injection and sacrificing may have been incorrect. The hens were either sacrificed too soon to a11ow for comp1ete metabo1ism of the injected samp1e, or too 1ate to a11ow for metabo1ite recovery. This is probab1y a1so the reason why none of the 1,25-(0H)2-D3 metabo1ite was recovered. A study by Ho1ick et a1. (1976) did show that after a singTe intravenous injection of 0.125 mg of either 25—0H-26, 27-3H-D3, or 24,25- (OH)2[26,27—3H]D3, the tissue concentrations of 25-0H-D3 and 1,25-(0H)2-D3 were greater at 24 hours than at 48 hours. The 1iver concentration of 48 25-0H-Dé was 77 pg-gram of tissue at 24 hours, and 28 pg/g at 48 hours. Simi1ar resu1ts were obtained for the 1,25-(0H)2-D3 metabo1ite in the Tiver and intestine. The peaks at 100 m1, 150 m1, and 260 m1 were observed in hens that were sacrificed with an egg in the uterus. It is possib1e that these peaks represent the 24-hydroxy metabo1ites which are produced when no additiona1 1,25-(OH)2-D3 is required for ca1cium absorption and subse- quent egg she11 deposition. Work done by Ho1ick gt_al, (1976) shows that 24,25-(0H)2-D3 is e1uted from a Sephadex LH-20 coTumn at 72 m1, and the 1,24,25-(0H)2-D3 metabo1ite is e1uted between 180 and 250 m1, with the peak occurring at 216 m1. Many factors inf1uence ca1cium absorption, transport, and deposition. The parathyroid g1ands are invo1ved in the maintenance of the diffusib1e ca1cium 1eve1 in the b1ood. They therefore have an indirect inf1uence on egg she11 deposition (Po1in and Sturkie, 1957). The metabo1ism of vitamin D3 resu1ts in the production of a ca1cium- binding protein, which mediates the active transport of ca1cium in the intestine. A simi1ar ca1cium-binding protein is a1so present in the uterus of hens (Corradino §t_al,, 1968). The activity of both intestina1 and uterine ca1cium-binding protein was found not to change during the egg formation cyc1e or during periods of uterine inactivity (Bar and Hurwitz, 1975). This study shows that the uterus of the 1aying hen is ab1e to take up vitamin D3. The presence of 25-0H-D3 in the uterine 1ipid extract indicates that the uterus is either ab1e to take up the metaboTite or metabo1ize vitamin 03. l‘" 49 The presence of a peak at 150 m1 in the chromatography pattern of the uterine tissue of one hen suggests that the uterus may be invo1ved in the metabo1ism of vitamin D3. The identity of this peak is undetermined at this time, however, it is possib1e that it is the 1,24,25-(0H)2-D3 meta- bo1ite, since it occurred in a position on the e1ution pattern simi1ar to that described by Ho1ick et_ al,, (1976). In addition, the peak occurred in a hen that was sacrificed with an egg in her uterus. This study therefore suggests that the uterus of the hen may be ab1e to reguTate the production of its own ca1cium-binding protein. Further, the uterus wou1d then be ab1e to regu1ate the transport of ca1cium it requires ‘ for egg she11 ca1cification. SUMMARY The purpose of this experiment was to determine if the uterus of the 1aying hen is ab1e to metabo1ize the vitamin D3 to its active form, 1,25-(OH)2-D3, and therefore regu1ate its own ca1cium transport. The effects of vitamin D3 deficiency on egg production and she11 qua1ity were a1so noted. Production records showed that vitamin D3 deficiency wi11 cause a decreased egg production. She11 qua1ity ana1ysis was inconc1usive, but eggs from hens on deficient diets tended to have thinner she11s, with 1ess ca1cium per mm of surface area, and the she11 weight comprised 1ess of the who1e egg weight. It was observed on co1umn chromatography of 1ipid extracts from 1iver, kidney and uterus, that these tissues were a11 ab1e to take up vitamin 03. Most of these organs were a150 either ab1e to participate in the meta— bo1ism of vitamin D3 of take up the metabo1ites from the b1ood as evidenced by the presence of 25-0H-D3 and other undetermined metabo1ites in their chromatographic patterns. 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