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Ovulation and calcium metabolism in white leghorn hens (Gallus gallus) Ruschkowski, Sharon Rose 1990

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OVULATION AND CALCIUM METABOLISM IN WHITE LEGHORN HENS (GALLUS GALLUS) By SHARON ROSE RUSCHKOWSKI B.S.A., The University of Saskatchewan, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s thesis as conforming to the required standards THE UNIVERSITY OF BRITISH COLUMBIA August 1990 Sharon Rose Ruschkowski In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of fln-imal c ; r i - o n f . 0 The University of British Columbia Vancouver, Canada Date A u g u s t 31, 1990  DE-6 (2/88) ABSTRACT Calcium status i s a major factor i n the regulation of reproductive a c t i v i t y i n the hen. R e s t r i c t i o n of dietary calcium (Ca) or vitamin D (D) i s assumed to cause cessation of ovulation through decreased plasma calcium concentrations. Several studies suggest that there may be a threshold l e v e l of ionized calcium (CajJ below which ovulation w i l l not proceed. The objectives of t h i s thesis were to determine how Ca^ concentration i s involved i n the process of ovulation by comparing Ca and D-deficient hens, that had ceased laying, with control birds that were laying normally. A secondary objective was to determine the e f f e c t s of multiple blood sampling (MBS) on the hen's ovulatory cycle. SCWL hens were divided into three groups-control, Ca-d e f i c i e n t and D-deficient groups and fed respective d i e t s . Control birds were s e r i a l l y sampled every two hrs for 24-26 hrs immediately a f t e r o v i p o s i t i o n u n t i l the next o v i p o s i t i o n . Def i c i e n t birds, that had ceased laying for 10 to 14 days, were sampled at the same time. MBS was achieved with an indwelling vascular access port. Six birds/experimental group were used. Control birds were bled two weeks l a t e r from l a t e afternoon u n t i l the following day at the same time. Whole blood was analyzed for Cai. Separated plasma was analyzed f o r t o t a l calcium (Ca^), inorganic phosphorus (P^), estrogen (E2), progesterone (P4) and l,25(OH)2D3 concentrations. Tibiae were ashed for mineral content. In expt. 1, the e f f e c t of MBS on the ovulatory pattern of hormones and ions was observed by sampling control birds twice, using two d i f f e r e n t time courses. Patterns and concentrations of the hormones and ions, regardless of time course, were s i m i l a r to previous studies. Overall treatment e f f e c t s were only s i g n i f i c a n t between treatments with regards to t o t a l calcium and e s t r a d i o l concentrations. The large loss of plasma proteins during the bleeding regime resulted i n a steady decline i n Cat over the 2 6 hrs, however, i t was s t i l l within the physiological range of laying b i r d s . . E2 concentrations were also affected due to interruption of the laying sequence. However, t h i s can be avoided since some birds continued to lay. In expt. 2, the control group had s i g n i f i c a n t l y higher mean plasma Ca^ and P^ concentrations and bone ash than both the d e f i c i e n t groups. Control and D-deficient groups had s i m i l a r mean Ca^ concentrations, however, the ovulatory p r o f i l e of the control group had a s i g n i f i c a n t c y c l i c pattern over the 24-26 hrs, whereas, both d e f i c i e n t groups did not vary s i g n i f i c a n t l y over the 24 hrs. Plasma P^ concentration i n the control group, not previously described, was c y c l i c i n nature, related to the egg laying cycle. Plasma l,25(OH)2D3 concentrations were s i g n i f i c a n t l y higher i n the Ca-deficient group than the control group. D-deficient birds had detectable l e v e l s of plasma 1,25(OH)2D3, but i t was i i s i g n i f i c a n t l y lower than the control group. Plasma E2 and P4 concentrations were s i g n i f i c a n t l y higher i n the control group In conclusion, i t would appear that an i n t e r - r e l a t i o n s h i p e x i s t s among Cai, P^ and 1,25(0H)2D3 and the reproductive hormones. A threshold concentration of Ca^ may be the t r i g g e r for ovulation, perceived at the l e v e l of the p i t u i t a r y , hypothalamus or ovary. A threshold of P i and a window of l,25(OH)2D3 concentration may also have permissive roles i n ovulation. In addition, MBS, regardless of time course, can be used as a method for determining ovulatory p r o f i l e s i n i n d i v i d u a l birds without seriously a f f e c t i n g i o n i c and hormonal concentrations and patterns. i i i T A B L E OF CONTENTS Abstract . . . i i L i s t of Tables v i i i L i s t of Figures ix Acknowledgements x i i Literature Review 1 1. Introduction 2 1.1 Vitamin D 4 1.1.1 Action of Vitamin D 5 1.2 Parathyroid Hormone 6 1.3 Phosphorus Metabolism 7 1.4 The Ovulatory Cycle 8 2. Calcium Metabolism and Reproduction 10 2.1 l,25(OH) 2D 3 10 2.2 Parathyroid Hormone 14 2.3 Calcium Deficiency 15 2.4 Vitamin D Deficiency 17 2.5 Ionic Calcium Concentrations 18 2.5.1 Ionic Calcium Concentration and Brain Interaction 19 3. Objectives 23 iv Materials and Methods 24 1. Experimental Birds 25 1.1 Management 25 1.2 Diets 26 2. Experimental Protocol 26 3. Surgical Procedure 29 4. Blood Sampling 30 5. Hormone Assays 32 5.1 Determination of Plasma E s t r a d i o l Concentrations 32 5.1.1 Preparation of estradiol-17/3 standards i n hen plasma 33 5.1.2 Assay 33 5.1.3 Calculations 34 5.2 Determination of Plasma l,25(OH)2D3 Concentrations 34 5.2.1 Sep-Pak washing 34 5.2.2 Sample extraction 34 5.2.3 Assay 36 5.2.4 Calculations 36 5.3 Determination of Plasma Progesterone Concentrations 37 5.3.1 Assay 37 5.3.2 Calculations 37 6. Bone Analysis 38 v 7. Plasma Ion Analysis 38 The E f f e c t s of Multiple Blood Sampling on the Hen's Ovulatory Cycle 39 1. Introduction 40 2. Objectives 43 3. Methods 43 3.1 Sampling Time Course 43 4. S t a t i s t i c a l Analysis 44 5. Results 44 6. Discussion 57 Reproductive F a i l u r e i n Calcium-Deficient and Vitamin D-Deficient Hens 66 1. Introduction 67 2. Objectives 69 3. Methods 69 3.1 Calcium and Vitamin D-Deficient Diets 69 3.1.1 Methods 70 3.1.2 S t a t i s t i c a l analysis 70 3.1.3 Results and Discussion 71 4. S t a t i s t i c a l Analysis 76 5. Results 77 5.1 Plasma Ionized Calcium Concentrations 77 5.1.1 Correlations 77 5.2 Other Plasma Hormones and Ions 83 vi 5.2.1 T o t a l Calcium C o n c e n t r a t i o n s 83 5.2.2 I n o r g a n i c Phosphorus C o n c e n t r a t i o n s . . . 88 5.2.3 l,25(OH) 2D3 C o n c e n t r a t i o n s 88 5.2.4 E s t r a d i o l C o n c e n t r a t i o n s 97 5.2.5 Progesterone C o n c e n t r a t i o n s 97 5.3 Bone A n a l y s i s 106 6. D i s c u s s i o n I l l Concluding Remarks 125 References 128 vi i L I S T OF TABLES Table 1. Laying hen di e t compositions 27 Table 2. Correlations between plasma hormones and ions for control, Ca-deficient and D-deficient hens 82 v i i i L I S T O F F I G U R E S Figure 1. Mean hematocrit values of hens sampled over 24 hrs 31 Figure 2. Mean ionized plasma calcium concentration over two ovulatory cycles 46 Figure 3. Mean t o t a l plasma calcium concentration over two ovulatory cycles 47 Figure 4. Mean plasma phosphorus concentration over two ovulatory cycles 49 Figure 5. Mean plasma l,25(OH)2D3 concentration over two ovulatory cycles 50 Figure 6a. Mean plasma e s t r a d i o l concentration over two ovulatory cycles 52 Figure 6b. Mean plasma e s t r a d i o l concentration of layers vs. non-layers 53 Figure 7. Mean plasma progesterone concentration over two ovulatory cycles 55 Figure 8. Mean hematocrit values of control hens sampled over 24 hrs 56 Figure 9a. Average weight change over 8 weeks 72 Figure 9b. Average feed consumption over 8 weeks 73 Figure 9c. Average egg production over 8 weeks 74 Figure 10. Mean ionized plasma calcium concentration of control hens 78 ix Figure 11. Mean ionized plasma calcium concentration of calcium-deficient hens 79 Figure 12. Mean ionized plasma calcium concentration of D-deficient hens 80 Figure 13. Mean plasma ionized calcium concentrations . . . 81 Figure 14. Mean t o t a l plasma calcium concentration of control hens 84 Figure 15. Mean t o t a l plasma calcium concentration of Ca-deficient hens 85 Figure 16. Mean t o t a l plasma calcium concentration of D-deficient hens 86 Figure 17. Mean t o t a l plasma calcium concentrations . . . . 87 Figure 18. Mean plasma phosphorus concentration of control hens 89 Figure 19. Mean plasma phosphorus concentration of D-deficient hens 90 Figure 20. Mean plasma phosphorus concentration of calcium-deficient hens 91 Figure 21. Mean plasma phosphorus concentrations 92 Figure 22. Mean plasma l,25(OH)2D3 concentration of control hens 93 Figure 23. Mean plasma l,25(OH)2D3 concentration of Ca-deficient hens 94 Figure 24. Mean plasma l,25(OH)2D3 concentration of D-deficient hens 95 Figure 25. Mean plasma l,25(OH)2D3 concentrations 96 x Figure 26. Mean plasma e s t r a d i o l concentration of control hens 98 Figure 27. Mean plasma e s t r a d i o l concentration of Ca-deficient hens 99 Figure 28. Mean plasma e s t r a d i o l concentration of D-deficient hens 100 Figure 29. Mean plasma e s t r a d i o l concentrations 101 Figure 30. Mean plasma progesterone concentration of control hens 102 Figure 31. Mean plasma progesterone concentration of calcium-deficient hens 103 Figure 32. Mean plasma progesterone concentration of D-deficient hens 104 Figure 33. Mean plasma progesterone concentrations . . . . 105 Figure 34. Bone ash as a percentage of t o t a l bone weight 107 Figure 35. F i n a l mean body weights of a l l three groups 108 Figure 36. Mean ovary weight as a percentage of mean body weight 109 Figure 37. Mean oviduct weight as a percentage of mean body weight 110 xi ACKNOWLEDGEMENTS I would l i k e to thank Dr. L e s l i e Hart for making t h i s project one of great i n t e r e s t . Her i n s i g h t f u l advice and c r i t i c i s m contributed greatly to the f i n a l product and was much appreciated. I would also l i k e to thank my committee members, Drs. Baimbridge, B l a i r , Cheng and Rajamahendran for t h e i r comments and suggestions. My thanks to Darin Bennet, Dr. Chris Harvey-Clark, G i l l e s Galzy, S y l v i a Leung and Dr. Frank Robinson for a l l t h e i r h e l p f u l advice and assistance. In addition, thanks to Ron B l a i r , Lynn Weber and the Poultry Farm s t a f f f or t h e i r help. My sincere gratitude to my mother, father and family for giving me the encouragement to pursue my goals. F i n a l l y , I wish to thank Agriculture Canada and the B.C. Egg Marketing Board for funding and scholarships. xii Chapter 1. LITERATURE REVIEW 1 1. INTRODUCTION The calcium metabolism of the laying hen i s of p a r t i c u l a r i n t e r e s t because of the large demand for calcium that i s required during reproduction for egg s h e l l formation. The average egg s h e l l contains 2.0 g of calcium. This represents about 10 per cent of the t o t a l body calcium of the hen. Considering that the domestic hen lays an egg every 24-2 8 hrs, approximately 1000 mg of calcium/kg of body weight i s needed d a i l y j u s t for s h e l l formation (Soares, 1984). The immediate source of calcium for egg s h e l l and yolk formation i s the blood. Riddle and Reinhart (1926) f i r s t noticed that female pigeons prepare for the sudden calcium demand i n egg s h e l l formation by c y c l i c l y undergoing hypercalcemia. Male pigeons have a low basal plasma calcium concentration of approximately 8-10 mg/100 ml. However, the female pigeon exhibits a pre-ovulatory r i s e i n plasma calcium concentration from a mean of 9.3 mg/100 ml to a peak of 19.9 mg/100 ml. Likewise, plasma calcium concentrations of the laying female i n other avian species are at l e a s t twice as high as those of the non-laying female or male (Soares, 1984). This increase i n plasma calcium concentration i s stimulated by increased l e v e l s of s t e r o i d hormones secreted by the developing f o l l i c l e s about two weeks before the onset of egg production. Both male and immature female birds exhibit a hypercalcemic response to estrogen administration (Baksi and 2 Kenny, 1977). This extra calcium i n the plasma i s complexed to yolk precursor proteins released by the l i v e r i n response to estrogen (Riddle, 1942; Dacke et a l . , 1973). Increasing plasma calcium concentration i s i n part a r e s u l t of a concomitant increase i n i n t e s t i n a l calcium absorption with the onset of sexual maturation (Hurwitz et a l . , 1973). This increase i n calcium absorption i s associated with an increase i n the concentration of a vitamin D-dependent i n t e s t i n a l calcium binding protein (CaBP), which i s twice as high i n laying as non-laying hens (Bar and Hurwitz, 1972; Bar et a l . , 1976). Another mechanism responsible f o r the increase i n plasma calcium concentration i s the increase i n the reabsorption of calcium ions at the kidney tubules, which i s under the d i r e c t control of parathyroid hormone (Clark and Sasayama, 1981). I n t e s t i n a l absorption can adapt to d i f f e r e n t conditions of calcium a v a i l a b i l i t y , but i t alone cannot provide s u f f i c i e n t calcium ions to meet the d a i l y demands of egg s h e l l formation (Simkiss, 1975). This necessitates a reservoir from which to draw upon during the period of egg s h e l l formation. Besides normal s t r u c t u r a l bone, there i s a highly l a b i l e store of non-structural bone present i n the marrow c a v i t i e s of the skeleton. I t i s unique to birds. This medullary bone i s deposited i n proportion to the increase i n s i z e of the developing f o l l i c l e s (Thaeler, 1979). I t s formation i s dependent on both androgens and estrogens (Common et a l . , 3 1948). This reservoir provides an additional supply of calcium, which i s drawn upon during the period of egg s h e l l formation. Calcium metabolism i n egg-laying birds i s greatly affected by vitamin D status and parathyroid hormone (PTH). These two hormones function together to regulate the plasma calcium concentrations that are necessary for normal phys i o l o g i c a l function as well as egg s h e l l formation. l . l Vitamin D Vitamin D i s essential for calcium metabolism. Its precursor, 7-dehydrocholesterol, i s synthesized i n the l i v e r during normal steroi d synthesis. This compound i s then transported v i a the c i r c u l a t o r y system on a vitamin D-transport protein to the skin. Here, i r r a d i a t i o n with u l t r a v i o l e t l i g h t r e s u l t s i n the synthesis of vitamin D 3 . A binding protein then c a r r i e s vitamin D3 to the l i v e r for further metabolism. Vitamin D3 can also be obtained from dietary sources v i a gut absorption. Vitamin D3 w i l l ultimately reach the l i v e r with fats absorbed from the small in t e s t i n e (DeLuca, 1980). Vitamin D3 accumulates i n the l i v e r , where i t i s further metabolized to 25-hydroxyvitamin D3 (25-OH-D3)(Lund and Deluca, 1966). 25-hydroxylation occurs i n the microsomal f r a c t i o n of the l i v e r c e l l s catalyzed by the enzyme vitamin D3~25-hydroxylase. In the chicken, the i n t e s t i n e and kidney 4 are also capable of 25-hydroxylation of vitamin D3 to some extent (Tucker et a l . , 1973). The vitamin D-transport protein c a r r i e s 25-OH-D3 to the kidney for further metabolism. The kidney i s the major s i t e for metabolism of 25-OH-D3. The most important step i s the la-hydroxylation of 25-OH-D3 to produce la,25-dihydroxyvitamin D3 (1,25(OH)2^3)• l/25(OH)2 n3 i s considered the active form of vitamin D 3 (Fraser and Kodicek, 1970; Holick et a l . , 1971). The vitamin D3 endocrine system i s cont r o l l e d by plasma calcium and phosphorus concentrations (Tanaka and Deluca, 1973). Hypocalcemia i n d i r e c t l y stimulates the a c t i v i t y of the 25-OH-D3-la-hydroxylase system through PTH, thereby increasing the production of l,25(OH)2D3 (Garabedien et a l . , 1972). Upon normocalcemia or hypercalcemia, the renal 25-OH-D3~la-hydroxylase system declines i n a c t i v i t y and there i s a resultant decrease i n c i r c u l a t i n g l,25(OH) 2D3. 1 . 1 . 1 Action of vitamin D In terms of i t s metabolism and mode of action, vitamin D i s considered a steroi d pro-hormone rather than a vitamin. l,25(OH) 2D3 i s a highly potent hormone which a f f e c t s gene t r a n s c r i p t i o n (Norman, 19 68) and stimulates the synthesis of CaBP i n the int e s t i n e (Wasserman and Corradino, 1971). In the hypocalcemic state, l,25(OH)2D3 stimulates active and passive absorption of calcium and phosphorus along the entire i n t e s t i n a l t r a c t (Bar et a l . , 1978). CaBP has a yet 5 unresolved function i n t h i s increase i n i n t e s t i n a l absorption, but i t i s believed not to be involved i n the transfer of calcium across the i n t e s t i n a l wall into the bloodstream (DeLuca, 1980). In addition to the intestine, l,25(OH)2D3 acts with PTH on the osteoclasts of the s k e l e t a l system to release calcium and phosphorus into the c i r c u l a t i o n (Garabedien et a l . , 1974). 1.2 Parathyroid Hormone The parathyroid glands i n birds are discrete, paired glands s l i g h t l y caudal to the thyroid. These glands respond d i r e c t l y to the calcium ion concentration i n the plasma. During hypocalcemia, they become conspicuously enlarged and release PTH (Taylor et a l , 1962), a protein hormone consisting of 84 amino acids. PTH acts at the l e v e l of the bone, gut and kidney to increase plasma calcium concentrations. At the bone l e v e l , i t induces o s t e o c l a s t i c resorption of bone. This e f f e c t i s mediated i n concert with the action of l,25(OH)2D3 (Garabedien et a l . , 1974). At the renal l e v e l , i t induces excretion of phosphate and reabsorption of calcium ions by modifying the a c t i v i t y of the renal tubules (Martindale, 1969). In addition, i t enhances the i n t e s t i n a l absorption of calcium through the stimulation of the renal 25-OH-D3-la-hydroxylase system (Garabedien et a l . , 1972). 6 1 .3 Phosphorus Metabolism l,25(OH)2D3 stimulates the i n t e s t i n a l absorption of phosphorus (Tanaka and DeLuca, 1973). As well, phosphorus has a d i r e c t action on D 3 metabolism independent of calcium (Chen et a l . , 1974). When chicks were fed a d i e t d e f i c i e n t i n phosphorus, the rate of calcium absorption from the small i n t e s t i n e increased with an increased accumulation of l,25(OH)2D3 i n the i n t e s t i n a l mucosa (Edelstein et a l . , 1975; Friedlander et a l . , 1977) and an increase i n the a c t i v i t y of the renal la-hydroxylase (Sommerville et a l . , 1978). Dietary calcium and phosphorus deficiency a f f e c t s the metabolism of 2 5-OH - D 3 i n the chick; renal production of l,25(OH)2D3 increased e i g h t - f o l d i n calcium-deficient and three-fold i n phosphorus-deficient chicks compared with controls (Sommerville et a l . , 1978). The i n vivo accumulation of l,25(OH)2D3 i n the gut mucosa, and the CaBP concentration and rate of calcium absorption from the duodenum were increased by a s i m i l a r extent i n both experimental groups, but the accumulation of l,25(OH)2D3 i n bone increased three-fold i n the low-calcium chicks and showed no change i n the low phosphorus birds. These re s u l t s are consistent with the view that the adaptation to a low-phosphorus d i e t depends primarily on the a b i l i t y of the i n t e s t i n a l mucosa to accumulate l,25(OH)2D3 rather than on an increased renal production of the metabolite. 7 Whereas a low calcium d i e t stimulates PTH secretion, a low phosphorus , d i e t increases the concentration of ionized calcium i n the plasma and depresses PTH secretion. This depression would conserve phosphorus and permit the urinary excretion of the additional calcium absorbed by the gut during a low-phosphorus regimen. 1.4 The Ovulatory Cycle The events of the ovulatory cycle of the domestic hen have been extensively described (Kamiyoshi and Tanaka, 1972; Senior, 1974; Graber and Nalbandov, 1976; Wilson and Sharp, 1976; Hammond et a l , 1980; Johnson, 1984), yet the t r i g g e r f o r ovulation i s s t i l l unknown. The time i n t e r v a l from ovulation to o v i p o s i t i o n i s 24-28 hrs. Ovulation usually follows an o v i p o s i t i o n within 15-75 min, except when the l a s t o v i p o s i t i o n precedes a pause i n egg laying (Senior, 1974). The development of a series of s e n s i t i v e assays f o r the measurement of reproductive hormones i n chicken plasma has enabled the characterization of the ovulatory cycle's hormonal pattern (Hammond et a l . , 1980). Fundamentally, the concentrations of these hormones in the plasma r e f l e c t t h e i r r o l e . They are continually present i n small amounts and the basal concentrations are required for the maintenance of the reproductive state of the b i r d , but there are superimposed d a i l y and seasonal f l u c t u t a t i o n s which are concerned with s p e c i f i c events during the reproductive cycle. There i s now 8 good agreement that there i s a concomitant r i s e i n the plasma concentration of l u t e i n i z i n g hormone (LH) and the s t e r o i d hormones (progesterone, estrogen and testosterone), a l l of which peak 3 to 7 hrs before ovulation (Hammond et a l . , 1980). Although the precise roles of these hormones i n the ovulatory cycle are s t i l l uncertain, the t y p i c a l pre-ovulatory LH and st e r o i d peak i s causally linked with the process o f ovulation. Senior (1974) f i r s t suggested that estrogens may be involved i n the stimulatory mechanism for the release of LH required for ovulation. Observations by Wilson and Sharp (1976) suggested that LH release i s f a c i l i t a t e d by the combined actions of estrogen and progesterone i n a two-phase process. The f i r s t i s the priming phase, which depends on the presence of estrogen and progesterone i n the blood. The second i s the inductive phase which depends on the incremental changes i n the c i r c u l a t o r y l e v e l s of progesterone. Therefore, both estrogen and progesterone are necessary for priming the LH p o s i t i v e feedback mechanism, but progesterone induces the LH surge. Progesterone i s known to cause the release of gonadotrophins from the p i t u i t a r y gland of laying hens r e s u l t i n g i n ovulation (Tanaka et a l . , 1974). The release of gonadotrophins from the p i t u i t a r y gland i s mediated by hypothalamic gonadotrophin-releasing factors. Although ovulation i s caused by LH (Opel and Nalbandov, 1961), f o l l i c l e - s t i m u l a t i n g hormone (FSH) may also be involved 9 (Kamiyoshi and Tanaka, 1972). Plasma concentrations of progesterone are greater i n laying than non-laying hens (Furr, 1973). Lack of gonadotrophic a c t i v i t y i n non-layers may be due to diminished or d e f i c i e n t secretions of progesterone (Tanaka et a l . , 1974). Kawashima et a l . (1979) confirmed that the number of progesterone receptors i n the hypothalamus and p i t u i t a r y gland of laying hens are greater than i n non-laying hens. 2. CALCIUM METABOLISM AND REPRODUCTION A c t i v e l y laying hens have t o t a l plasma calcium concentrations of 20-35 mg/100 ml, while non-layer concentrations are 10-15 mg/ 100 ml (Thaeler, 1979). Estrogens play a major role i n these changes and administration of estrogens can stimulate high blood calcium concentrations i n non-laying females as well as i n males (Baksi and Kenny, 1977). With adequate dietary calcium, most of the elevated calcium demand i s met by increased i n t e s t i n a l absorption and secondarily by bone turnover (Simkiss, 1975). 2.1 l , 2 5 ( O H ) 2 D 3 The f i r s t report of a strong regulatory control of estrogen on vitamin D metabolism was by Kenny (1976). He observed that egg laying i n Japanese quail was accompanied by increases i n 2 5-OH - D 3-la-hydroxylase a c t i v i t y i n the kidney and suggested that gonadal hormones, which are elevated during 10 the ovulatory cycle, may be involved d i r e c t l y or i n d i r e c t l y as messengers i n the control of the renal 25-OH-D3~la-hydroxylase system. Elevation of plasma l,25(OH)2 n3 concentrations took place within the f i r s t s i x to seven hrs following o v i p o s i t i o n . I f o v i p o s i t i o n was followed by a pause, plasma l,25(OH)2 n3 concentrations declined. S i m i l a r l y , during periods of low gonadal a c t i v i t y i n the female, such as prepuberty and senescence, plasma concentrations of l,25(OH)2D3 were low. This was confirmed by both Baksi and Kenny (1977) and C a s t i l l o et a l . (1979) who showed that exogenous estradiol-17/3 administration had a strong stimulatory e f f e c t on the 25-OH-D3~la-hydroxylase. This was observed i n both laying chickens and Japanese quail (Paulson and Kenny, 1985). Likewise, exogenous estrogen w i l l stimulate the 25-OH-D3-la-hydroxylase system i n the kidney to produce l,25(OH)2D3 i n the immature b i r d of both sexes (Spanos et a l . , 1976). Pr o l a c t i n has a s t r i k i n g e f f e c t i n enhancing the production of l,25(OH)2 n3 i n the chick (Spanos et a l . , 1976). As well, i n l a c t a t i n g rats, whose plasma p r o l a c t i n concentrations are high, c i r c u l a t i n g l,25(OH)2D3 concentrations were four times higher than non-lactating rats (Boass et a l . , 1977). However, both estrogen and progesterone i n h i b i t e d l,25(OH)2D3 production i n the ovariectomized calcium-deprived female (Baski and Kenny, 1978). Bar et a l . (1978) suggested that vitamin D metabolism i s not d i r e c t l y affected by gonadal hormones but i s an adaptive mechanism to increases i n calcium needs for medullary bone formation and losses to the s h e l l during c a l c i f i c a t i o n . Studies by Tsang and Grunder (1984) found that estradiol-17/3 biosynthesis was interrupted i n vitamin D-deficient hens. They suggested a r o l e for vitamin D i n estrogen biosythesis. However, Tsang et a l . (1988) found that calcium deficiency, rather than vitamin D deficiency, was the more immediate cause of the interference. C a s t i l l o et a l . (1979) reported no c o r r e l a t i o n between plasma l,25(OH)2D3 concentration and the formation or resorption of medullary bone. However, Takahashi et a l . (1983) found that vitamin D deficiency i n hens caused a decrease i n the mineralized portion of medullary bone. Treatment with testosterone and e s t r a d i o l induced matrix formation of medullary bone i n vitamin D-deficient chicks, but i t s mineralization did not occur i n vitamin D deficiency i n spit e of marked hypercalcemia. Mineralization of medullary bone only occurred i n chicks supplemented with vitamin D 3 . This suggests that vitamin D 3 i s d i r e c t l y involved i n the mineralization of medullary bone rather than the increment i n plasma calcium l e v e l s . l,25(OH)2D3 may be involved i n the transfer of calcium from the plasma into the s h e l l gland during egg s h e l l formation. Bar and Norman (1981) found that i n t e s t i n a l and uterine l,25(OH)2D 3 receptor concentrations were s i g n i f i c a n t l y higher i n laying quail than mature non-laying female or 12 immature q u a i l . C i r c u l a t i n g plasma l,25(OH)2D 3 concentration reaches maximal l e v e l s when an egg i s undergoing rapid c a l c i f i c a t i o n i n the uterus (Abe et a l . , 1979). In addition, hens with low s h e l l q u a l i t y have s i g n i f i c a n t l y lower concentrations of c i r c u l a t i n g l,25(OH)2D3 than hens that produce w e l l - c a l c i f i e d s h e l l s (Soares et a l . , 1980). A vitamin D-dependent calcium binding protein, i d e n t i c a l to the i n t e s t i n a l vitamin D-dependent CaBP, has been found i n high concentrations i n the s h e l l gland of birds (Taylor and Wasserman, 1972). There i s also evidence of a s p e c i f i c binding protein for l,25(OH)2D3 i n the s h e l l gland of chicken and quail (Coty, 1980; Takahashi et a l . , 1980). However, no c o r r e l a t i o n was found between uterine CaBP concentrations and c i r c u l a t i n g concentrations of l,25(OH)2D 3. In fact, i n calcium-deficient hens, uterine CaBP concentrations were decreased despite the increase i n c i r c u l a t i n g plasma l,25(OH) 2D3 (Bar et a l . , 1984) and administration of l,25(OH)2D3 had no e f f e c t on CaBP concentrations i n the uterus (Nys and De Laage, 1984). This suggests that uterine CaBP i s regulated by factors other than l,25(OH)2D3. Neither was there any re l a t i o n s h i p found between plasma l,25(OH)2D3 concentration and s h e l l calcium deposition (Bar et a l . , 1984). However, uterine CaBP i s higher i n laying hens than i n non-laying birds (Bar and Hurwitz, 1973; Rosenberg et a l , 1986) or i n hens that lay s h e l l - l e s s eggs (Nys and De Laage, 1984). Spencer et a l . (1978) suggest that l,25(OH)2D3 promotes 13 calcium entry into the c e l l while CaBP acts as a calcium buffer without being regulated d i r e c t l y by l,25(OH)2D3, although the hormone may be e s s e n t i a l for i t s formation. 2.2 Parathyroid Hormone PTH i s concerned with the maintenance of adequate calcium concentration i n the blood and i t s a c t i v i t y appears to be d i r e c t l y related to the degree of plasma calcium deficiency (Mueller et a l . , 1970). In addition to i t s bone mobilizing e f f e c t s , i n v i t r o studies of dispersed chicken p i t u i t a r y c e l l s suggest that PTH may contribute to the suppression of p i t u i t a r y gonadotrophin a c t i v i t y i n the calcium-deficient b i r d (Luck and Scanes, 1980a). The mobilization of bone calcium for egg s h e l l formation i s a normal physiological process, even when di e t s are high i n calcium. van de Velde et a l . (1984) provide evidence that secretion of PTH i s consistently and s i g n i f i c a n t l y elevated during egg s h e l l formation. Likewise, parathyroid extract (PTE) induces a highly s i g n i f i c a n t increase i n 25-OH-D3-la-hydroxylase and l,25(OH)2D3 concentrations during egg s h e l l formation (Sedrani et a l . , 1981). Both osteoblasts and osteoclasts of medullary bone are highly active during s h e l l formation (van de Velde, 1984). This suggests that PTH may be involved i n the resorption of medullary bone, since a c t i v a t i o n of osteoclasts i s mediated by PTH (Taylor and Belanger, 1969). Sedrani et a l . (1981) suggest that the sex hormones are 14 responsible for the long-term calcium homeostasis and absorption from the gut and thus increase plasma l,25(OH)2D3 concentrations, while PTH i s involved i n short-term mobilization from the skeleton. 2.3 Calcium Deficiency Calcium status i s recognized as a major factor i n the regulation of reproductive a c t i v i t y i n the hen. R e s t r i c t i o n of dietary calcium has been observed to decrease egg production and s h e l l thickness (Buckner and Martin, 1920). Severe calcium r e s t r i c t i o n w i l l lead to the cessation of ovulation (Gilbert, 1971, 1973; Douglas et a l . , 1972; B l a i r and G i l b e r t , 1973; Roland et a l . , 1974; G i l b e r t and B l a i r , 1975) . Hens fed a calcium-deficient d i e t normally stop laying within 10 to 14 days (Roland et a l . , 1973; Luck and Scanes, 1979a). Hard-shell egg production drops within two days with a gradual decline i n s h e l l weight and s h e l l thickness. The f i r s t s o f t - s h e l l e d egg i s apparent within three or four days of the onset of the deficiency. Many s h e l l - l e s s eggs and eggs without membranes are produced before egg production ceases (Roland et a l . , 1973). Severe yolk mottling appears within 10 to 12 days, with many resembling regressed ova (Roland et a l . , 1973) . Total plasma calcium concentration begins to drop within three days and reaches a low of 15 mg/100 ml within f i v e days 15 (Roland et a l . , 1973). However, ionized calcium concentration drops s i g n i f i c a n t l y within 24 hours (Luck and Scanes, 1979a). With the cessation of ovulation, ionized calcium concentration drops to 70 per cent of normal values. As plasma calcium concentrations f a l l , plasma PTH concentrations increase and correspond to a decrease i n egg numbers and reduced c i r c u l a t i n g estrogen concentration (de Bernard et a l . , 1980). In chicks, calcium deficiency stimulates the a c t i v i t y of the 25-OH-la-hydroxylase d i r e c t l y or through stimulation of PTH secretion (Garabedien et a l . , 1972). Both increased l,25(OH)2D3 production and increased plasma phosphorus concentrations are observed i n calcium-deficient laying hens (Arnaud, 1978). The lack of calcium i n the die t s i g n i f i c a n t l y reduces feed consumption within 24 hours (Roland et a l . , 1973; Vohra et a l . , 1979). A drop i n feed consumption i s r e f l e c t e d i n reduced body weight of some hens, an increased mortality and i n a b i l i t y of some birds to stand (Roland et al.,1973). Apparently, some birds are able to stop or decrease egg production immediately and these have no v i s i b l e side e f f e c t s , while other birds are unable to adjust to the deficiency and a noticeable decrease i n comb siz e and body weight occurs. Roland et a l . (1973) found that calcium-deficient hens had s i g n i f i c a n t l y smaller ovaries and oviducts. P i t u i t a r y and 16 comb siz e were also reduced, suggesting a f a i l u r e i n gonadotrophin production or secretion. This would be r e f l e c t e d i n cessation of egg production. T i b i a ash i s also s i g n i f i c a n t l y lower i n calcium-deficient birds (Roland et a l . , 1973; Vohra et a l . , 1979). Hens tend to deplete t h e i r s k e l e t a l calcium s i g n i f i c a n t l y before egg production ceases. Both s k e l e t a l bone and medullary bone appear to be affected (de Bernard et a l . , 1980). 2 . 4 Vitamin D Deficiency The f i r s t sign of vitamin D deficiency i n the laying b i r d i s the production of t h i n or s o f t - s h e l l e d eggs within two or three weeks of being fed a vitamin D-deficient d i e t (Vohra et a l . , 1979; Shen et a l . , 1981). A sharp decline i n egg production follows. This i s consistent with the hypothesis that hens do not deplete t h e i r store of vitamin D u n t i l that time. Production returns to normal within seven to ten days of re-supplementation. Although there was a decline i n egg production, i t was not as severe as that observed with calcium deprivation (Vohra et a l . , 1979, Shen et a l . , 1981). Vitamin D and i t s metabolites were not measured i n these studies, thus i t could be suggested that these birds were not completely vitamin D-d e f i c i e n t , since Hart and DeLuca (1985) reported complete cessation of laying a f t e r four weeks of feeding hens a vitamin D-deficient d i e t . 17 Increased mortality i s observed i n vitamin D-deficient birds (Hart et a l . , 1925; Chang et a l . , 1969) but p a r a l y s i s was only associated with low l e v e l s of vitamin D (Shen et a l . , 1981). These birds had no decline i n egg production and i t appears that t h i s marginal l e v e l i s s u f f i c i e n t to prolong egg production at the expense of s k e l e t a l i n t e g r i t y . In addition, there was no reduction i n ovary and oviduct weight i n these bird s . This indicates the importance of ensuring complete vitamin D deficiency through impeccable housing conditions and measurement of plasma vitamin D metabolites. Tsang and Grunder (1984) found t o t a l plasma calcium concentrations to be 20 per cent lower i n vitamin D-deficient hens. As well, phosphorus l e v e l s are also s i g n i f i c a n t l y lower (Hart et a l . , 1925; Luck and Scanes, 1978). A s i g n i f i c a n t reduction i n bone ash i s evident (Chang et a l . , 1969; Vohra et a l . , 1979; Shen et a l . , 1981), with both medullary and s k e l e t a l bone being affected (Takahashi et a l . , 1983). 2.5 I o n i c Calcium C o n c e n t r a t i o n s Blood calcium exists i n two forms, as bound calcium ( i e . to proteins) and as i o n i c calcium. The i o n i c calcium i s i n equilibrium with the bound calcium. Ionic calcium appears to leave the blood by d i f f u s i o n and i s maintained by a d i s s o c i a t i o n of the organically bound f r a c t i o n . P r e c i p i t a t i o n of calcium carbonate i n the s h e l l gland acts as a favorable concentration gradient for the continued d i f f u s i o n of i o n i c 18 calcium from the blood to the developing s h e l l (Thaeler, 1979) . The i o n i c f r a c t i o n of blood calcium i s of the most physi o l o g i c a l s i g n i f i c a n c e with respect to calcium metabolism and homeostasis (Copp, 1969). A sinusoidal pattern of plasma ionized calcium concentrations i s observed r e l a t i v e to the po s i t i o n of the egg i n the oviduct (Luck and Scanes, 1979b; Parsons and Combs, 1981). Maximal concentrations of 1.8 mM were reached when the s h e l l gland was empty and minimal concentrations of 1.2 mM followed four hours of c a l c i f i c a t i o n of the s h e l l (Luck and Scanes, 1979b). This concentration remained constant u n t i l the f i n a l hour of c a l c i f i c a t i o n when concentrations started to increase. Total plasma calcium concentrations did not vary s i g n i f i c a n t l y during the egg laying cycle (Luck and Scanes, 1979b; Parsons and Combs, 1981). I t i s therefore stressed that estimates of t o t a l blood calcium concentration with no corresponding estimation of the degree of io n i z a t i o n may not be an accurate r e f l e c t i o n of calcium status. 2.5.1 Ionic Calcium Concentrations and Brain Interaction The p i t u i t a r y gland secretes hormones which are esse n t i a l to normal metabolism and co-ordinate other endocrine glands. The proximity of the p i t u i t a r y gland to the hypothalamus i s s i g n i f i c a n t with regard to the regulation of the secretion of the p i t u i t a r y hormones. The hypothalamo-pituitary system 19 integrates, processes and transduces information, r e s u l t i n g i n an appropriate hormonal secretion by the p i t u i t a r y gland. Studies by Taylor (1965) suggest that the cessation of egg production i s due to the f a i l u r e of gonadotrophin secretion by the anterior p i t u i t a r y , which causes the b i r d to stop ovulating. He speculated that the hypothalamic stimulation of the p i t u i t a r y i s reduced by the decrease i n the i o n i c calcium concentration i n the blood of calcium-depleted hens. Luck and Scanes (1979a) agreed that the hypothalamus i s an important s i t e of interaction between the brain and gonadal axis i n calcium homeostasis. They support the suggestion that hypothalamic a c t i v i t y i s regulated by calcium a c t i v i t y i n the blood as a means of preventing severe calcium stress and that there i s a threshold of a c t i v i t y below which the stimulation of ovulation i s prevented. Roland et a l . (1974) found an increase i n Periodic Acid S c h i f f (PAS) material i n the anterior p i t u i t a r y of calcium-d e f i c i e n t b i r d s . PAS-positive material i s associated with gonadotrophin secretory production of the anterior p i t u i t a r y . Gonadotrophic hormone concentrations of the anterior p i t u i t a r y have also been reported to be greater i n non-laying hens or hens with regressing ovarian f o l l i c l e s than i n laying hens (Nakajo and Imai, 1957; Imai et a l . , 1964). This suggests that the secretion of gonadotrophic hormones i s reduced with prolonged calcium deficiency r e s u l t i n g i n adverse e f f e c t s on the reproductive system of the laying hen. The a b i l i t y of 20 hypothalamic extracts to stimulate the reproductive system also suggests that calcium may be involved i n the production or release of gonadotrophin-releasing factors from the hypothalamus (Roland et a l . , 1974; Luck and Scanes, 1979a). The mechanism of action of LHRH at the l e v e l of the avian gonadotroph i s not f u l l y established. In v i t r o studies by Wakabayashi et a l . (1969) have suggested that hormone release i s voltage-dependent and that releasing factors act by combining with s p e c i f i c receptors on the c e l l surface to create depolarizing changes i n the pot e n t i a l across the c e l l membrane. This change i n potential causes an increase i n i n t r a c e l l u l a r calcium concentration. A calcium/magnesium dependence was further demonstrated for release of FSH and LH during the actions of releasing factors. However, the exact mechanism of action of calcium i n the production or release of releasing factors from the hypothalamus i s s t i l l not clear. There i s also evidence for the role of calcium ions i n the action of LHRH on LH secretion. Not only i s the presence of calcium required for LHRH to stimulate LH release i n v i t r o (Bonney and Cunningham, 1977), but also LH release i s stimulated by calcium ionophores (Luck and Scanes, 1980). In v i t r o studies with chicken p i t u i t a r y c e l l s suggest that the secretion of LH i n the domestic hen has an absolute dependence on the presence of calcium ions (Luck and Scanes, 1980a). The powerful i n t r a c e l l u l a r regulatory mechanisms that control the concentration of calcium i n the cytosol depend i n the l a s t 21 analysis on constancy of calcium i n the e x t r a c e l l u l a r f l u i d . Thus, with a calcium-restricted d i e t , which reduces ionized plasma concentrations i n the hen (Luck and Scanes, 1978, 1979a), low calcium concentrations i n the brain may have a d i r e c t e f f e c t on LH secretion by reducing the responsiveness of the hypothalamus to p o s i t i v e feedback by progesterone. The reduction i n LH output, without stimulation by LHRH, and lack of e f f e c t with LHRH observed i n v i t r o with low calcium concentrations s i m i l a r to concentrations found i n the plasma of hens which had stopped laying due to calcium-deficiency, supports the idea that ionized plasma calcium concentrations might have a d i r e c t e f f e c t on p i t u i t a r y function. These experiments suggest that calcium has a regulatory r o l e at the p i t u i t a r y i n addition to the hypothalamic e f f e c t demonstrated previously where LHRH acts by increasing the transport of calcium ions into the p i t u i t a r y c e l l s (Luck and Scanes, 1978, 1979a). Ef f e c t s of PTH and 1,25(0H)2D3 on LH secretion have also been studied as possible i n d i r e c t ways i n which reduced calcium a c t i v i t y could influence LH secretion (Luck and Scanes, 1980a). The production of both these hormones i s thought to be increased as a r e s u l t of a low plasma calcium concentration (Sommerville et a l . , 1978). The i n v i t r o e f f e c t s of PTE on chicken p i t u i t a r y c e l l s s i g n i f i c a n t l y reduced the basal LH secretion and blocked the enhancing e f f e c t of LHRH. l,25(OH) 2D3 had no e f f e c t on basal LH 22 secretion but prevented stimulation by LHRH (Luck and Scanes, 1980a). This suggests that i n addition to i t s bone mobilizing e f f e c t s , PTH may contribute to the suppression of p i t u i t a r y a c t i v i t y i n the calcium-deficient b i r d and l,25(OH)2D3 may have a regulatory role associated with the pre-ovulatory surge (Luck and Scanes, 1980a). 3. OBJECTIVES A. To determine how plasma ionized calcium concentration i s involved i n the process of ovulation by 1) characterizing the d a i l y hormonal and i o n i c pattern of calcium and vitamin D-deficient hens that have ceased ovulation. 2) Comparing these p r o f i l e s to the ovulatory p r o f i l e of normal laying hens. 3) Describing how these d i f f e r e n t hormonal and i o n i c patterns r e l a t e to one another and to ovulation. B. Secondarily, to determine the e f f e c t s of multiple blood sampling on the hen's ovulatory cycle. 23 Chapter 2 . M A T E R I A L S AND METHODS 24 1. Experimental Birds 1.1 Management. Two hundred Single Comb White Leghorn (SCWL) female chickens (DeKalb) were obtained from a commercial hatchery at one day of age and raised i n temperature-controlled (35°C) battery brooders with access to feed and water, ad libitum. Temperature was decreased each week by 2.8°C u n t i l the birds were 4 weeks of age. A 20% protein grower d i e t was fed u n t i l t h i s time. At 4 weeks of age, birds were transferred to p u l l e t cages, l i g h t i n g was reduced from 24 hrs of l i g h t to 8 hrs of l i g h t and 16 hrs of dark and birds received an 18% protein p u l l e t developer. Beginning at 16 wks of age, l i g h t i n g was increased by 1/2 hr each week u n t i l 16 hrs of l i g h t and 8 hrs of dark was reached. Birds were switched to an 18% protein layer crumble once they had reached 5% egg production. At 22 weeks of age, the birds were transferred to i n d i v i d u a l laying cages. Each cage was equipped with access to i n d i v i d u a l feed and water, both of which were provided ad  libitum. Lighting was maintained at 16 hrs of l i g h t and 8 hrs of dark with incandescent l i g h t s automatically c o n t r o l l e d by a time clock. Light i n t e n s i t y was 10 lux. A l l windows were covered with aluminum f o i l to block u l t r a v i o l e t l i g h t from entering the room. Temperature was controlled as close to 20°C as possible. Upon transfer to the i n d i v i d u a l cages, a l l hens were fed a control layer mash (corn/soyabean meal base) for 4 weeks 25 (Table 1). Daily egg production records were kept and t h i r t y hens that were laying between 90 and 100 % were selected for the experiment. Egg production (%) was defined as (number of eggs laid)/(number of hens x number of days) x 100. Blood c o l l e c t i o n cannulas were then s u r g i c a l l y implanted v i a the jugular vein to the opening of the r i g h t atrium of the heart. When a l l birds had recovered from the cannula placement and returned to t h e i r former l e v e l of egg production, the birds were randomly divided into three groups of 10 each - control, calcium-deficient (Ca-deficient) and vitamin D-deficient groups (D-deficient). 1.2 Diets. The control group continued to receive the control layer mash. The calcium-deficient group received a calcium-deficient diet, i d e n t i c a l to the control d i e t i n composition, except the calcium source was substituted with a non-nutritive c e l l u l o s e f i l l e r ( C e l u f i l , US Biochem Corp). The vitamin D-deficient group received a vitamin D-deficient d i e t , i d e n t i c a l to the control d i e t i n composition, except vitamin D was not added. A l l experimental diets and t h e i r calculated compositions are shown i n Table 1. The control d i e t alone was used for experiment 1, chapter 3. A l l three d i e t s were used for experiment 2, chapter 4. 2 . Experimental Protocol The experiment began when the birds were 32 weeks of age. A l l birds were weighed before and thereafter weekly during the 26 Table 1. Laying hen d i e t compositions 1. Ingredient Control Ca-Deficient D-Deficient (%) (%) (%) Corn, ground 67.8 67.8 67.8 Soyabean Meal (44%) 19.1 19.1 19.1 Limestone 7.5 7.5 Vegetable O i l 2.3 2.3 2.3 Multiphos 1.5 1.5 1.5 Iodized S a l t 0.4 0.4 0.4 Cellulose F i l l e r 7.5 Trace Minerals 2 1.11 1.11 1.11 Premix 3 0.27 0.27 0.27 Vitamin D 4 (in o i l ) 0.35 ml 0.35 ml Chemical Composition^ Crude protein, % 13.1 13.4 13.0 Total phosphorus, % 0.54 0.55 0.52 Calcium, % 3 . 4 0.36 3 . 3 1 C a l c u l a t e d nutrient content of a l l 3 d i e t s : protein, 14.5%; metabolizable energy, 2900 kcal/kg; l i n o l e i c acid, 2.9%; methionine, 0.36%; available phosphorus, 0.4%; calcium, 3.4% for control and D-deficient d i e t s ; 0.35% for Ca-deficient d i e t . 2Trace minerals supplied i n milligrams per kilogram of d i e t : magnesium, 1000; iron, 100; manganese, 60; zinc, 100; copper, 12; molybdenum, 2; iodine, 0.6; and selenium, 0.2. 3Premix supplied i n milligrams per kilogram of d i e t : DL-methionine, 1000; choline chloride, 1670; r i b o f l a v i n , 4.4; pyridoxine, 6; pantothenate, 4.4; menadione, 1; ni a c i n , 20; f o l a c i n , 0.5; vitamin Bi2/ 0.008; vitamin E, 10; thiamin, 1.6; b i o t i n , 0.2; and vitamin A, 2.8. 4 C r y s t a l l i n e vitamin D3 added per kilogram of d i e t : 70 uq per m i l l i l i t e r of o i l . 5By proximate analysis. 27 experiment. Feed intake was also monitored throughout the experiment by weighing feed at the beginning and end of each week. A b i r d was assessed d e f i c i e n t once egg production had ceased for 10 to 14 days. Birds d i f f e r e d i n t h e i r rate of depletion. In the calcium-deficient group, birds were d e f i c i e n t within two to three weeks. However, i n the vitamin D-deficient group, i t took from four to s i x weeks to deplete vitamin D i n the required number of bi r d s . Six birds/experimental group were selected for the experiment. Once birds were d e f i c i e n t of t h e i r respective nutrient, they were bled s e r i a l l y every two hours for 24-26 hrs. Blood sampling was achieved by s u r g i c a l l y implanting an indwelling Vascular-Access-Port Model SLA, 4 f r . , 24" S i l a s t i c catheter with two moveable retention rings (Access Technologies) v i a the jugular vein. Tubing had an OD of 1.2 mm (0.047 inches). This allowed for multiple sampling with minimal stress to the b i r d . Sampling began immediately a f t e r o v i p o s i t i o n and continued u n t i l the next ovipositon. Deficient birds were bled at the same time as the control birds. Six birds, two from each group, were bled at one time point. Af t e r the l a s t sample was taken, the d e f i c i e n t birds were s a c r i f i c e d by CO2 inhalation. This was immediately followed by the examination of the body cavity. Reproductive organs were weighed and examined for evidence of ovulation or 28 i n t e r n a l laying. Tibiae were removed and ashed fo r mineral content. Control birds were sampled a second time f o r 24 hrs, two weeks l a t e r . A f t e r t h e i r l a s t sample was taken, control birds were k i l l e d by C O 2 inhalation and treated the same as the d e f i c i e n t birds. 3. Surgical Procedure Birds were anesthetized with ketamine (25 mg/kg body weight) and xylazine (5 mg/kg body weight) im. A l l s u r g i c a l equipment was autoclaved before each procedure. Once the b i r d had l o s t consciousness, the neck area was prepped with iodine and covered with a s u r g i c a l drape. A small i n c i s i o n was made midway on the l e f t side of the neck, the jugular vein was found and the fat and muscle surrounding i t was teased away. When the jugular vein was i n f u l l view, 0000 s i l k was loosely t i e d around the vein above and below the s i t e t i g h t l y to cut o f f the i n c i s i o n . Once accomplished, the upper s i l k was t i e d t i g h t l y to cut o f f the blood flow. Then the jugular vein was nicked and the catheter of the access port inserted u n t i l the r i g h t atrium was reached (approximately 12 cm). The bottom s i l k was quickly t i e d around the catheter and the upper s i l k was t i e d around the upper part of the vein and catheter to ensure i t was stable. Blood was then withdrawn to ensure that the t i e s were not too t i g h t and that the r i g h t atrium was reached. The attached access port was then sutured into a 29 pocket of the neck made by clearing away ti s s u e . The access port was r i g h t next to the skin and e a s i l y observed and located from the outside. The i n c i s i o n was then sutured closed and swabbed with iodine. Heparinized s a l i n e (0.25 ml, 15 IU/ml) was injected to keep the catheter patent. A further 0.25 ml of heparinized saline was injected once/week for the duration of the experiment. 4 . Blood Sampling To c o l l e c t a sample, the area was swabbed with ethanol and a r i g h t angle Huber point needle was inserted into the access port. A heparinized syringe was attached to the needle and 4 ml of blood was withdrawn. Following each sampling, 0.25 ml of heparinized saline (15 IU/ml) was injected back to maintain patency of the l i n e . The red blood c e l l s were resuspended i n an equal volume of s t e r i l e s a l i n e and returned to the same donor b i r d p r i o r to the withdrawal of each subsequent sample. Blood volume was not greatly affected by blood sampling. There was no s i g n i f i c a n t difference (p>0.05) between i n i t i a l hematocrit values and hematocrit values a f t e r sampling for 24-26 hrs (Fig. 1). Hens were bled s e r i a l l y at two hour i n t e r v a l s for 24-26 hrs. Blood sampling began i n control birds immediately following o v i p o s i t i o n and continued u n t i l the next ov i p o s i t i o n . During the dark period, a f l a s h l i g h t covered with red cellophane was used to prevent stray l i g h t from 30 % hematocrit n = 6 for each treatment l l l l 1 L Control -TK - Ca-Def icient - B - D-Deficient 0 8 10 12 14 Time (hrs) 16 18 20 22 24 Fig 1. Mean hematocrit values of hens sampled over 24-hrs. No significant differences between time 0 and 24. 31 interrupting the hens' ovulatory pattern. Deficient birds were bled at the same time as the control b i r d s . This was to f a c i l i t a t e sampling, since the d e f i c i e n t birds did not have an ovi p o s i t i o n to use as a reference point. Excluded from the experiment were control birds that did not have a second ov i p o s i t i o n and d e f i c i e n t birds that appeared to be expelling s h e l l - l e s s eggs i n t e r n a l l y into t h e i r abdominal cavity. The f i n a l number of birds i n each group was s i x . Immediately following blood sampling, analysis of ionized calcium was performed on the whole blood. The blood was then centrifuged and the plasma separated and stored at -20°C i n aliquots suitable for assay. Each plasma sample was assayed for t o t a l calcium, inorganic phosphorus, l,25(OH)2D3, estradiol-17/3 and progesterone concentrations. 5. Hormone Assays 5.1 Determination of Plasma E s t r a d i o l Concentrations Analysis of ind i v i d u a l plasma samples was performed using a solid-phase radioimmunoassay with antibodies raised to human estradiol-17/? (Coat-A-Count E s t r a d i o l k i t , Intermedico) . The procedure i s based on antibody-coated tubes i n which 125j_ labeled e s t r a d i o l competes with e s t r a d i o l i n the sample for antibody s i t e s . Samples required no extraction. 32 5.1.1 Preparation of estradiol-17/9 standards i n hen plasma. A pool of hen plasma was obtained by bleeding a number of mature laying hens. The plasma was then stripped of a l l steroids by mixing i t with 50 mg of activated charcoal/ml plasma (Pharmacia Fine Chemicals) and s t i r r i n g for one hr at 20°C. The plasma was then centrifuged at 5000 g (Sorvall RC-5B) for 45 min at 4°C and vacuum f i l t e r e d through Whatman grade 934-AH glass f i b r e f i l t e r s . A stock solu t i o n containing 10 ng estradiol-17/3 (Steraloids, Inc.) per ml of phosphate-buffered s a l i n e with 0.1% g e l a t i n was prepared. The standards (0, 20, 50, 150, 500, 1800, and 3600 pg estradiol-17/0/ml plasma) were made by d i l u t i n g an appropriate amount of stock solution with stripped hen plasma. The k i t was equipped with human-based standards with analogous estradiol-17/3 concentrations. These standards and several i n t e r n a l control human plasma samples were run to ensure that estradiol-17/3 values f e l l within the expected range for the k i t . 5.1.2 Assay. One hundred /ul of standards and samples were added to e s t r a d i o l antibody-coated tubes. To each tube, 1 ml of buffered 1 2 5 I - e s t r a d i o l (30,000 cpm) was added. One ml of 1 2 5 I - e s t r a d i o l was added to non-antibody coated b o r o s i l i c a t e glass tubes for t o t a l counts. Non-specific binding tubes contained 100 ul of 0 standard and 1 ml buffered 1 2 5 I - e s t r a d i o l i n non-antibody coated tubes. Tubes were vortexed and incubated at room temperature for 3 hrs. After incubation, the supernatant containing the free hormone was 33 decanted. Dried tubes were then counted, with counts being inversely related to the amount of estradiol-17/3 i n the sample. Maximum binding was approximately 35-45% and s e n s i t i v i t y was 20 pg estradiol-17/?/ml plasma. 5.1.3 Calculations. The quantity of estradiol-17/3 per ml plasma was determined by using a l o g i t / l o g p l o t of the standards. 5.2 Determination of Plasma 1,25(OH)2^3 Concentrations Plasma samples were assayed for l,25(OH)2D3 using a competitive binding assay k i t (Incstar, Inc.) based on a bovine thymus receptor s p e c i f i c f or l , 2 5 ( O H ) 2 D 2 and l,25(OH)2D3. The assay involved a preliminary extraction and subsequent p u r i f i c a t i o n of vitamin D metabolites from plasma using a C^gOH Sep-pak cartridge. 5.2.1 Sep-Pak washing procedure. Both new and used Sep-Paks (Waters Associates, Inc.) were prepared as follows before use: C i 8 0 H Sep-Paks were washed by sequential additions of 5 ml isopropanol, 5 ml a c e t o n i t r i l e , 5 ml d i s t i l l e d water and 5 ml d i s t i l l e d water. This conditioning procedure allowed reuse of Sep-Pak cartridges at least three times without s i g n i f i c a n t loss of capacity or changes i n el u t i o n patterns. 5.2.2 Sample extraction. Plasma samples (0.75-1.0 ml) were added to 12 x 75 mm b o r o s i l i c a t e glass tubes and brought to 1 ml with s a l i n e . Twenty-two hundred counts per min (cpms) of [ 3H] 1,25 (OH) 2 D 3 i n 50 jLtl absolute ethanol were added to 34 each plasma sample and to a s c i n t i l l a t i o n v i a l for monitoring of recoveries. The samples were mixed thoroughly and allowed to stand for 15 min. The extraction of vitamin D metabolites was achieved by mixing plasma samples with 1 ml of a c e t o n i t r i l e and vortexing vigorously for 20-sec. Samples were allowed to stand for 10 min with several repeated vortexes, followed by centrifugation for 10 min at 1500 g (Beckman TJ-6). After centrifugation, the supernatant was decanted into a t e s t tube. The p e l l e t was resuspended i n 1 ml of a c e t o n i t r i l e and the f i r s t step was repeated. The supernatants were combined and mixed with 2 ml of 0.4 M K2HPO4, pH 10.6, and vortexed. This extract was applied d i r e c t l y to a pre-washed C i 8 0 H Sep-Pak. Excess s a l t was removed by washing the cartridge with 5 ml of d i s t i l l e d water (X2), and the polar l i p i d s were removed by washing the cartridge with 5 ml of methanol-water (5:95). The vitamin D metabolites were then eluted with 5 ml a c e t o n i t r i l e , and t h i s f r a c t i o n was dried under a gentle stream of nitrogen. The vitamin D metabolite extract was reconstituted i n 200 /Ltl of ice cold 0 standard, vortexed gently, capped and placed on i c e . A f t e r 5 min had passed, each sample was vortexed. After an additional 5 min, each sample was vortexed again. From t h i s volume, 50 / l l was used to determine the recovery of the sample (60-72%) , and two 50 fil aliquots were used f o r assay. 35 5.2.3 Assay. F i f t y /ii of standards (0, 25, 50, 100, 200, and 400 pg 1,25(OH)2D3/ml) and samples were added to 1.5 ml b o r o s i l i c a t e glass tubes on i c e . Total count and non-s p e c i f i c binding (NSB) tubes received 50 /xl of 0 standard. One hundred jul of NSB buffer was added to the t o t a l count tubes, while 400 /xl NSB buffer was added to each NSB tube. Then, 400 /xl of thymus receptor was added to the t o t a l count, standard and sample tubes. Tubes were gently vortexed and incubated for 60 min at 15-20°C. This was followed by the addition of 50 /xl of [ 3H] 1, 25 (OH) 2D 3 (11,500-13,500 dpms) . Tubes were vortexed and incubated for an additional 60 min at 15-20°C. After incubation, tubes were placed i n an ice bath for 10 min, and then 100 /xl of dextran-coated charcoal was added to each tube except the t o t a l count tubes. Tubes were vortexed and l e f t i n the ice bath for 30 min. Tubes were vortexed again and then centrifuged at 1800 g at 4°C for 15 min (Beckman TJ-6). After centrifugation, the supernatant containing the bound hormone was decanted into s c i n t i l l a t i o n v i a l s and counted for 5 min. Maximum binding was 30-40% and the minimum detectable concentration was 5 pg/ml. 5.2.4 Calculations. The pg of l,25(OH)2 n3 per tube was calculated using a l o g i t / l o g p l o t of the standards. The l,25(OH)2D3 concentrations/ml plasma were obtained by correcting the pg per tube data obtained from the graph, for recovery and sample volume. 36 5.3 Determination of Plasma Progesterone Concentrations Plasma progesterone concentrations were determined using a previously described radioimmunoassay (Rawlings et a l . , 1984) with minor modifications. 5.3.1 Assay. Progesterone was extracted from 200 /xl of plasma and buffer standards using 4 ml petroleum ether. One hundred u l of extracted standards (0.0313, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32 and 64 ng progesterone/ml) and 200 jul of each extracted sample were added to 1.5 ml b o r o s i l i c a t e glass tubes. Antiserum s p e c i f i c for progesterone (Dr. Frank Robinson, University of Alberta) was d i l u t e d to a working d i l u t i o n of 1:30,000 and 200 fj,l was added to each tube. Then, two hundred /xl of 1 2 5 I - l a b e l l e d progesterone was added at 10,000 cpm/tube and tubes were incubated for 24 hrs at 4°C. Two hundred ill of second antibody was then added to each tube at a working d i l u t i o n of 1:200. Af t e r incubating tubes for 24 hrs at 4°C, the supernatant was removed by as p i r a t i o n and the p e l l e t counted. Assay s e n s i t i v i t y was 0.1 ng/ml.. * 5.3.2. Calculations. The quantity of progesterone per tube was calculated using a l o g i t / l o g p l o t of the standards. The progesterone concentration/ml plasma was obtained by correcting the ng per tube data obtained from the graph, for sample volume. 37 6 . Bone A n a l y s i s Immediately a f t e r the b i r d was s a c r i f i c e d , the l e f t and r i g h t t i b i a e were removed and freed of attached muscle and connective ti s s u e s . The l e f t t i b i a e were fat-extracted for 48 hrs i n a Soxhlet apparatus, dried i n a drying oven at 95-100°C for 24 hrs to determine dry f a t - f r e e bone weight, and then ashed i n a muffle furnace at 600°C for 24 hrs. 7 . Plasma Ion A n a l y s i s Total plasma calcium concentration was measured by atomic absorption spectrophotometry (Perkin-Elmer 560). Aliquots of 50 Ml plasma were d i l u t e d 1:80 with 0.002% LaCl3 i n 0.01% HCI before measurement. Duplicate samples and standards (1.25, 2.5, 5.0 ppm) were measured at a wavelength of 422 nm. Plasma i o n i c calcium concentration was determined using a Radiometer Model CA1 calcium analyzer. This was c a r r i e d out immediately a f t e r blood c o l l e c t i o n on 125 /xl of whole blood. Standard conditions included c a l i b r a t i o n of the calcium analyzer with solutions of known ionized calcium content. Plasma inorganic phosphorus concentration was determined by the method described by Itaya and Ui (1966) using 10 /xl of plasma. Duplicate samples and standards (0-20 mg P/ 100 ml) were measured at a wavelength of 660 nm. A l l solvents and chemicals were obtained from commercial sources and were of a n a l y t i c a l grade. 38 Chapter 3 . THE EFFECTS OF MULTIPLE BLOOD SAMPLING ON THE HEN 1S OVULATORY CYCLE 39 1. INTRODUCTION The domestic hen has a d a i l y i o n i c and hormonal pattern that v a r i e s i n r e l a t i o n to ovulation and egg s h e l l c a l c i f i c a t i o n . Plasma ionized and t o t a l calcium concentrations decrease as calcium i s drawn from the blood for c a l c i f i c a t i o n of the egg s h e l l (Luck and Scanes, 1979b; Parsons and Combs, 1981). Plasma inorganic phosphorus concentrations increase as bone.is mobilized to meet the extra calcium needs for s h e l l c a l c i f i c a t i o n (Tanaka and DeLuca, 1973). The reproductive hormones follow a basal l e v e l u n t i l 7 to 10 hrs before ovulation, when they reach a peak concentration known as the ste r o i d peak (Hammond et a l . , 1980). This i s the signal for the next ovulation. Multiple blood sampling, at appropriate i n t e r v a l s , i s necessary to detect the variations of these d i f f e r e n t plasma hormones and ions over the course of the hen's ovulatory cycle and to determine how they r e l a t e to one another and to egg laying. The v a r i a t i o n i n the plasma concentrations of LH and the s t e r o i d hormones i s thought to be large between birds, whereas the v a r i a t i o n during the ovulatory cycle i s r e l a t i v e l y small. Small changes i n the concentrations of these parameters would be most e a s i l y detected using s e r i a l blood samples. This l o g i c would be accurate i f s e r i a l blood sampling does not i n i t s e l f a l t e r e i t h e r gonadotrophin secretion or the ovulatory cycle. There are no reports of what e f f e c t s e r i a l blood sampling 40 has on the ion concentration of the blood or calcium metabolism. However, several researchers have investigated the e f f e c t s of s e r i a l blood sampling on gonadotrophin and ste r o i d secretion. A study done by Johnson (1980) indicated that plasma LH concentrations declined when blood samples were removed s e r i a l l y and that laying was terminated. White and Etches (1984) found t h i s to be true i n some but not a l l cases. Many hens were unaffected by the s e r i a l blood sampling and continued to lay sequences of t h e i r predicted length. Furthermore, the mean concentration of LH was not s i g n i f i c a n t l y d i f f e r e n t from hens that were s e r i a l l y sampled every 2 hrs for 2 4 hrs or hens that were sampled once every 7 days. Nor was i t s i g n i f i c a n t l y d i f f e r e n t between the s e r i a l l y sampled hens that continued to lay and those that terminated laying. However, the p r o f i l e s of plasma LH concentrations between the two groups were d i f f e r e n t . Both groups always had peak LH concentrations p r i o r to ovulation. However, following the preovulatory surge, plasma LH concentrations declined r a p i d l y i n the hens that stopped laying a f t e r s e r i a l blood sampling, whereas a plateau i n plasma LH concentrations was maintained i n hens that continued to lay t h e i r normal sequence. These data (White and Etches, 1984) and that reported previously by Johnson (1980) suggests that both the plasma concentration of LH and the pattern of LH release can be affected by the frequency of removing blood samples. White 41 and Etches (1984) concluded that the changes were due to stress, since the concentration of plasma corticosterone was s i g n i f i c a n t l y higher i n hens that ceased laying compared with those that continued to lay. To eliminate the stress of repeated blood sampling and hemodilution, many researchers (Scanes et a l , 1977; Luck and Scanes, 1979b; Bedrak et a l , 1981) have sampled d i f f e r e n t birds at d i f f e r e n t hourly i n t e r v a l s . Data from several birds at d i f f e r e n t time points were pooled to obtain an ovulatory p r o f i l e . Recently, s e r i a l blood sampling from the same b i r d has resulted i n very s i m i l a r values and ovulatory p r o f i l e s as that of multiple b i r d samplings (Opel and Proudman, 1984; Proudman and Opel, 1989). Opel and Proudman (1984) found that s e r i a l bleeding of turkeys every 2 minutes for 2 0 min (which would be assumed to be extremely s t r e s s f u l ) did not a l t e r plasma p r o l a c t i n concentrations. As well, they sampled turkey hens hourly for 24 hrs with no s i g n i f i c a n t e f f e c t on plasma LH and p r o l a c t i n concentrations. Although i t would appear that s e r i a l blood sampling from one b i r d enables the researcher to obtain an accurate p r o f i l e of the hormones and ions during the ovulatory cycle, there i s no data on whether the time of sampling has an adverse e f f e c t on the cycle i t s e l f from the stress of blood sampling or large loss of blood volume. In addition, the patterns observed may be an a r t i f a c t of the blood sampling i t s e l f . 42 2. OBJECTIVES The objectives of t h i s study were 1) to compare two d i f f e r e n t time courses of s e r i a l blood sampling i n order to determine i f the stress of multiple blood sampling causes the ovulatory pattern of hormones and ions to change or influences the subsequent ovulatory cycle. 2) To determine i f the concentrations of the ions and hormones involved i n the reproductive process are affected by multiple blood sampling. 3. METHODS SCWL hens, 3 2 wks old, were bled every two hrs fo r 26 hrs, beginning at two d i f f e r e n t times. Management, di e t , s u r g i c a l and blood sampling techniques used were previously described i n chapter 2, Materials and Methods. 3.1. Sampling Time Courses. The i n i t i a l blood sampling was done every two hours immediately a f t e r one ovi p o s i t i o n u n t i l the next ov i p o s i t i o n (ovip-ovip). Oviposition was chosen as a reference point since i t i s a d i s t i n c t event and a l l birds would be sampled at the same time points during t h e i r ovulatory cycle. The birds were allowed to recover for 2 weeks a f t e r they l a i d t h e i r next egg before they were sampled again. The same b i r d was then s e r i a l l y bled every two hours from l a t e afternoon u n t i l the following day (aftn-aftn). The time selected was 1/2 hr before the l i g h t s went out (10 hrs before ovulation) u n t i l the following day at the same time. Late afternoon was chosen i n order to l i m i t the number 43 of samples taken p r i o r to the LH and s t e r o i d peaks, thus, lessening the inf luence of sampling on t h e i r concentrations and increas ing the chance of the hen l ay ing the next day. 4 . S T A T I S T I C A L ANALYSIS Changes i n plasma hormone and ion concentrations over the 2 6 hr sampling per iod and between treatments were analysed by ana lys i s of var iance , with hen, time, and treatment as main e f f ec t s . Treatment means at each time point were compared using Duncan's mul t ip le range t e s t . In a d d i t i o n , hen data was analyzed according to whether the hen l a i d the next day or not, regardless of the treatment. 5 . RESULTS The data presented here was c o l l e c t e d from s ix hens. In the ov ip-ov ip treatment, two b i rds continued to lay the day a f t er t h e i r second o v i p o s i t i o n , three b i rds had a one day pause and one b i r d had a three day pause before beginning to lay again. Five out of the s ix b i rds l a i d the day fo l lowing t h e i r second o v i p o s i t i o n i n the a f tn -a f tn treatment. The 2 6-hr cyc le of each hormone and ion for both treatments are shown i n F i g s . 2-7. In a l l cases, the ov ipos i t i ons of the two treatments (ovip-ovip and aftn-aftn) were sh i f t ed to l i e at the same time point on the graph. This would put t h e i r ovulatory cyc les i n sequence. Since ov ipos i t i ons f e l l between sampling times for many of the a f t n -aftn sampled hens, the time point closest to the actual o v i p o s i t i o n was chosen. When analysing the data, times 2, 4, 6, 8 and 10 hrs i n the ovip-ovip treatment were compared with times 28, 30, 32, 34 and 36 hrs i n the aftn-aftn treatment, respectively. Plasma ionized calcium concentrations (Fig 2) showed a s i g n i f i c a n t i n t e r a c t i o n (p<0.01) between treatment and ind i v i d u a l hen, in d i c a t i n g that some birds responded d i f f e r e n t l y between treatments. However, there was no o v e r a l l treatment e f f e c t . As well, when the data was analyzed comparing the birds that l a i d the following day (layers) with the birds that did not lay (non-layers), regardless of treatment, there was no s i g n i f i c a n t difference between the two groups. Variation within the same b i r d from day to day may account for the differences. Each b i r d had s i m i l a r ionized calcium p r o f i l e s over the 2 6 hrs. As well, both treatments followed the same ovulatory pattern. Total plasma calcium concentrations (Fig 3) also showed a s i g n i f i c a n t i n t e r a c t i o n (p<0.01) between treatment and hen. As well, there was a s i g n i f i c a n t o v e r a l l treatment e f f e c t . This can be seen i n the s i g n i f i c a n t l y higher concentrations observed i n the aftn-aftn treatment. Again, there was no s i g n i f i c a n t difference between the layers and non-layers, regardless of treatment. Furthermore, each b i r d had s i m i l a r t o t a l calcium p r o f i l e s over the 2 6 hrs and both treatments followed the same ovulatory pattern. Ionized calcium cone. (mM) ovip-ovip aftn-aftn oviposition n • 6 for each treatment 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Time (hrs) Fig 2. Mean ionized plasma calcium over two ovulatory cycles. Different letters differ significantly between treatments. 46 Total calcium cone, (mg/100 ml) 25 10 a 6 for each treatment j i i I I I I ovip-ovip aftn-aftn ^ oviposition a 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Time (hrs) Fig 3. Mean total plasma calcium over two ovulatory cycles. Different letters differ significantly between treatments. 47 Plasma inorganic phosphorus concentrations (Fig 4) showed a s i g n i f i c a n t i n t e r a c t i o n (p<0.001) between treatment and hen. However, the o v e r a l l treatment e f f e c t was not s i g n i f i c a n t and there was no s i g n i f i c a n t difference between the layers and non-layers, regardless of treatment. Again, the differences may be explained by v a r i a t i o n within birds from day to day. There was also a s i g n i f i c a n t i n t e r a c t i o n (p<0.05) between treatment and time, i n d i c a t i n g that the same ovulatory pattern was not followed by each treatment. The phosphorus concentrations between the two treatments were not s i g n i f i c a n t l y d i f f e r e n t at the time points where sampling overlapped and the same peak of phosphorus was observed i n both. Where the difference occurred was at the beginning of the ovulatory cycle. Hens that were bled from ovip-ovip began t h e i r ovulatory cycle at a much higher concentration of phosphorus than the hens sampled from aftn-aftn. However, the same general pattern was followed by each treatment. Furthermore, each hen had s i m i l a r inorganic phosphorus p r o f i l e s over time. Plasma l,25(OH)2D3 concentrations (Fig 5) showed a s i g n i f i c a n t i n t e r a c t i o n (p<0.001) between treatment and i n d i v i d u a l hen. Overall treatment e f f e c t was not s i g n i f i c a n t . This also was observed for the ionized and t o t a l calcium and inorganic phosphorus. Since a l l four parameters are c l o s e l y associated with calcium metabolism, i t seems reasonable that a l l four would respond i n a s i m i l a r manner. There was also a 48 Inorganic phosphorus cone, (mg/100 ml) n = 6 for each treatment 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Time (hrs) Fig 4. Mean plasma phosphorus over two ovulatory cycles. Different letters differ significantly between treatments. 49 300 1,25(OH)2D3 cone, (pg/ml) 250 200 150 100 6 for each treatment ovip-ovip aftn-aftn oviposition 0 2 4 6 8 10 12 14 16 18 2 0 2 2 2 4 2 6 2 8 3 0 3 2 3 4 3 6 Time (hrs) Fig 5. Mean plasma 1,25(OH)2D3 over two ovulatory cycles. Different letters differ significantly between treatments. 50 s i g n i f i c a n t i n t e r a c t i o n (p<0.001) between treatment and time. This was true only at four time points. These time points that were d i f f e r e n t were s i g n i f i c a n t l y lower i n the second treatment. However, the same general pattern was followed by each treatment. There was no s i g n i f i c a n t i n t e r a c t i o n between hen and time, in d i c a t i n g that each hen had s i m i l a r plasma l,25(OH)2D3 p r o f i l e s over the 26 hrs. Plasma e s t r a d i o l concentrations (Fig 6a) showed a s i g n i f i c a n t i n t e r a c t i o n among a l l three parameters. The s i g n i f i c a n t i n t e r a c t i o n (p<0.001) between treatment and ind i v i d u a l hen can be explained by differences between hens that l a i d the day a f t e r t h e i r second o v i p o s i t i o n (layers) i n one treatment but did not lay (non-layers) i n the other treatment. When p r o f i l e data for layers was compared to the non-layers, regardless of treatment, there was a s i g n i f i c a n t difference between the e s t r a d i o l concentrations of hens that l a i d the next day and those that did not lay (Fig 6b). Differences occurred at almost a l l time points. The hens that l a i d following t h e i r second o v i p o s i t i o n had several peaks of e s t r a d i o l throughout t h e i r ovulatory cycle, while those that did not lay had only one small peak at the beginning of t h e i r ovulatory cycle. This i s r e f l e c t e d i n the s i g n i f i c a n t i n t e r a c t i o n (p<0.05) between hen and time. Hence, hens had d i f f e r e n t ovulatory p r o f i l e s over the 26 hrs. In addition, there was a highly s i g n i f i c a n t i n t e r a c t i o n (p<0.01) between treatment and time, i n d i c a t i n g that the ovulatory Estradio l cone, (pg/ml) 250 r 200 150 100 50 0 ovip-ovip aftn-aftn oviposition n = 6 for each treatment j i i i i i i i 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Time (hrs) Fig 6a. Mean plasma estradiol over two ovulatory cycles. Different letters differ significantly between treatments. 52 Estradiol cone, (pg/ml) 250 r 0 Hens (layers) H - Hens (non-layers) oviposition 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs from oviposit ion) Fig 6b. Mean plasma estradiol cone, of layers vs. non-layers. Different letters differ significantly between treatments. 53 patterns between the two treatments were d i f f e r e n t . The ovip-ovip treatment had more hens that did not lay following the second o v i p o s i t i o n (2) than the aftn-aftn treatment (5). Mean concentrations were s i g n i f i c a n t l y d i f f e r e n t at several time points, as indicated. Plasma progesterone concentrations (Fig 7) showed no s i g n i f i c a n t i n t e r a c t i o n between treatment and time, hen and time or treatment and time. Blood volume was not greatly affected by the s e r i a l blood sampling regime, as r e f l e c t e d by hematocrit values (Fig 8). There was no s i g n i f i c a n t difference (p>0.05) between i n i t i a l hematocrit values and hematocrit values a f t e r sampling f o r 26 hrs. 54 Progesterone cone, (ng/ml) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 Time (hrs) Fig 7. Mean plasma progesterone over two ovulatory cycles. Different letters differ significantly between treatments. 55 % hematocrit 30 25 -20 15 10 0 n = 6 for each treatment 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs) Fig 8. Mean hematocrit values of control hens sampled over 24-hrs. No significant difference over time. 56 6 . D I S C U S S I O N The hormonal and i o n i c patterns and concentrations observed i n t h i s study were s i m i l a r to previous findings for ionized and t o t a l calcium, l,25(OH)2D3, e s t r a d i o l and progesterone (Senior, 1974; Graber and Nalbandov, 1976; C a s t i l l o et a l . , 1979; Luck and Scanes 1979a, b; Johnson, 1984), regardless of treatment. Furthermore, a c y c l i c pattern of plasma inorganic phosphorus was observed, related to ovulation and egg s h e l l c a l c i f i c a t i o n , not previously reported. Although i n d i v i d u a l b i r d e f f e c t s were noted between treatments, o v e r a l l treatment e f f e c t s were only s i g n i f i c a n t l y d i f f e r e n t i n regards to t o t a l calcium and e s t r a d i o l concentrations. These two parameters are very c l o s e l y associated i n the reproductive process. E s t r a d i o l i s necessary for the production of plasma proteins, notably v i t e l l o g e n i n , which binds a large portion of plasma calcium during yolk formation (Thaeler, 1979). The s i g n i f i c a n t e f f e c t s observed between treatments i n these two parameters are probably the r e s u l t of hemodilution. Approximately 3% of plasma constituents were removed with each blood sample and not returned with the red blood c e l l s . This resulted i n approximately 67% loss of t o t a l plasma constituents a f t e r the removal of the l a s t sample. Since proteins are synthesized at a r e l a t i v e l y slow rate, the d e f i c i t would probably not be made up between sampling times. 57 Thus, the birds sampled from aftn-aftn would i n i t i a l l y have higher t o t a l plasma calcium concentrations, whereas, by the same time i n the afternoon, the hens that were sampled ovip-ovip would have lower t o t a l plasma calcium concentrations due to the larger blood volume that was removed. In addition to the lowered concentration of plasma proteins, hens that were sampled from aftn-aftn would be i n i t i a l l y drawing calcium from bone into t h e i r plasma for s h e l l c a l c i f i c a t i o n (Gilbert, 1983) . I n i t i a t i n g sampling at t h i s time could have triggered the birds to overcompensate for loss of plasma calcium through the removal of blood and i n i t i a t e d increased bone turnover. Hence, higher plasma calcium concentrations may have been the r e s u l t . Regardless of treatment e f f e c t , plasma t o t a l calcium p r o f i l e s were s i m i l a r to e a r l i e r reports by Luck and Scanes (1979b). Both treatments produced r e s u l t s within the phys i o l o g i c a l range of a c t i v e l y laying hens (Soares, 1984). E s t r a d i o l concentration was the parameter most affected by s e r i a l bleeding. This would again a f f e c t t o t a l calcium concentrations. Hens that did not lay following t h e i r second ovi p o s i t i o n (non-layers), regardless of treatment, had lower peak concentrations of e s t r a d i o l p r i o r to ovulation compared with the birds that l a i d (layers). As well, i n birds that f a i l e d to lay, plasma concentrations of e s t r a d i o l declined throughout the rest of t h e i r ovulatory cycle, whereas, birds that continued to lay t h e i r normal sequence maintained a 58 higher e s t r a d i o l concentration u n t i l t h e i r next o v i p o s i t i o n . The birds that did lay following t h e i r second o v i p o s i t i o n had s i m i l a r plasma e s t r a d i o l p r o f i l e s to those observed by Senior (1974) and Graber and Nalbandov (1976). Furthermore, the mean es t r a d i o l concentration of each treatment was not s i g n i f i c a n t l y d i f f e r e n t from these researcher's findings, regardless of the number of layers and non-layers within the treatment. Plasma inorganic phosphorus concentrations have never been measured over an entire ovulatory cycle, however, the p r o f i l e observed followed a predicted pattern i n both treatments. Plasma phosphorus concentrations increased as plasma calcium concentrations decreased. This i s i n response to the increased bone turnover needed to meet the extra calcium needs during the egg s h e l l c a l c i f i c a t i o n stage (Tanaka and DeLuca, 1973). Although hens had s i m i l a r plasma phosphorus p r o f i l e s , there were differences observed between treatments at several time points. Hens that were bled from ovip-ovip began t h e i r ovulatory cycle at a much higher concentration of phosphorus that the hens sampled from aftn-aftn. This again may be attri b u t e d to a hemodilution e f f e c t , since the birds that were bled from aftn-aftn had a larger proportion of plasma removed by the time t h e i r cycle started again. Thus, these birds would begin t h e i r cycle at a much lower phosphorus concentration. 59 Plasma ionized calcium concentrations followed a c y c l i c pattern, related to ovulation and egg s h e l l c a l c i f i c a t i o n . This was reported previously by Luck and Scanes (1979b) and Parsons and Combs (1981). In addition, concentrations observed i n t h i s study were s i m i l a r to work done by Luck and Scanes (1979b) and Singh et a l . (1986). However, i n these studies, hen data was pooled for d i f f e r e n t time points to obtain a p r o f i l e over the ovulatory cycle. Therefore, the hens sampled for each time point were only sampled two to three times each and only those that continued to lay were included i n the data pool. In the present study, data from both hens that l a i d following t h e i r second oviposition and those that did not were used. This, however, does not explain the differences noted i n some hens between the d i f f e r e n t treatments. I t may be due to i n d i v i d u a l v a r i a t i o n within each b i r d , since large v a r i a t i o n s are known to occur between birds, as well as within birds because of variable calcium metabolism from day to day, as well as from week to week (Soares, 1984). Plasma l,25(OH)2D3 concentrations may also have been affected by hemodilution, since several time points d i f f e r e d between the two treatments. The l a s t two time points i n the aftn-aftn treatment are s i g n i f i c a n t l y lower than the ovip-ovip treatment. By the l a s t sampling times, the concentration of 60 the plasma vitamin D-binding proteins would presumably be lower due to the large removal of plasma. Fewer proteins are then avai l a b l e for transport of 25-OH-D3 to the kidney for conversion into i t s active metabolite, l,25(OH)2D3- Hence, lower plasma l,25(OH)2D3 concentrations are observed. The differences observed at e a r l i e r time points between the two treatments may be accounted for by the v a r i a t i o n that e x i s t s between birds and within the same b i r d , associated with variable calcium metabolism. Plasma progesterone concentrations were unaffected by the s e r i a l blood sampling. Concentrations and p r o f i l e s obtained i n t h i s study were si m i l a r to those obtained by Johnson (1984). Since peak concentrations of progesterone were observed i n both non-layers and layers, i t would appear that the i n h i b i t i o n of ovulation may occur at the l e v e l of the ovary with the release of the f o l l i c l e . A l l plasma hormones and ions, except e s t r a d i o l , followed s i m i l a r ovulatory patterns for the two d i f f e r e n t bleeding treatments. The i n i t i a l high peak of estrogen r i g h t a f t e r o v i p o s i t i o n was very t r a n s i t o r y i n nature and i t was not observed i n a l l ovip-ovip hens. I f sampling occurred ten minutes a f t e r an oviposition, the stero i d peak was missed. This phenomenon was also observed by Graber and Nalbandov (1976). Hens bled aftn-aftn always l a i d sometime between 61 sampling times. Sampling never occurred within seconds of an egg being l a i d , as i n the ovip-ovip group. This would account for the differences i n ovulatory patterns between treatments. One of the factors that was an important consideration i n t h i s study was the removal of a large percentage of plasma constituents, despite the return of saline-resuspended red blood c e l l s . Although the hormonal and i o n i c p r o f i l e s and concentrations observed here were s i m i l a r to previous studies, the concentrations of a l l hormones and ions may have been affected by hemodilution. This i s primarily because as more and more of the plasma proteins were removed, the b i r d could not p h y s i c a l l y manufacture them before the next sampling time. This i s p a r t i c u l a r l y true for plasma t o t a l calcium concentration, which i s most dependent on the plasma proteins for i t s plasma concentrations. Plasma t o t a l calcium concentration declined progressively over the 26 hrs and did not begin to increase at the beginning of the next ovulatory cycle, as did the other hormones and ions. Hemodilution may also explain the i n d i v i d u a l b i r d v a r i a t i o n between treatments. These birds may have responded d i f f e r e n t l y between treatments due to d i f f e r e n t concentrations of hormones and ions at d i f f e r e n t time points. Time points 12, 14 and 16 may have had higher concentrations i n the a f t n -aftn treatment because these were the f i r s t samples taken i n t h i s treatment, whereas, s i x or seven samples would have been taken at these same time points i n the ovip-ovip treatment. 62 Another important consideration of s e r i a l blood sampling was the disruption of the laying sequence. In some cases, birds did not continue to lay the following day a f t e r 14 blood samples had been taken. However, i n a l l cases the birds did return to t h e i r normal sequence within a few days of sampling. Usually birds that were sampled from aftn-aftn had a higher percentage of laying the following day (85%) than those birds that were sampled from ovip-ovip (33%). This i s probably due to the large removal of plasma i n the ovip-ovip group p r i o r to the s t e r o i d peak, thus decreasing the concentration of plasma LH, r e s u l t i n g i n cessation of ovulation. In addition, the stress of handling the b i r d a greater number of times before the occurrence of the s t e r o i d peak also may have prevented ovulation. Multiple blood sampling can i n t e r f e r e with the d a i l y ovulatory pattern, i f the birds are stressed enough to prevent gonadotrophin secretion and cause cessation of ovulation (Johnsom, 1980; White and Etches, 1984). However, i f the birds continue to lay, as observed here and i n the study by White and Etches (1984), the subsequent ovulatory cycle does not appear to be affected. The chance of a hen continuing to lay appears to be highly correlated with t h e i r egg laying sequence. White and Etches (1984) observed that i f the hens were sampled at the beginning of t h e i r egg laying sequence, there was a much greater chance of the hens continuing to lay 63 than hens that were sampled l a t e i n t h e i r laying sequence. As well, accustoming the birds to repeated handling w i l l reduce the stress that may i n h i b i t ovulation. In t h i s study, an indwelling catheter, with an attached access port, reduced the stress of blood sampling by decreasing the r i s k of i n f e c t i o n and i r r i t a t i o n . With s e r i a l blood sampling, method of blood sampling i s an important c r i t e r i a . The development of less s t r e s s f u l techniques, which involve unrestrained and undisturbed birds, have allowed for greater consistency and more accurate p r o f i l e s (Opel and Proudman, 1984) . Both methods of sampling had advantages and disadvantages. With the ovip-ovip treatment, the ov i p o s i t i o n could be used as a reference point. This allowed sampling times of a l l birds to f a l l at the same time within the ovulatory cycle. As well, i t allowed the capture of the peaks and troughs of hormones and ions that followed within a few seconds of an oviposition, such as the t r a n s i t o r y e s t r a d i o l peak. However, the chances of laying an egg following t h e i r second o v i p o s i t i o n was lower i n t h i s group than the aftn-aftn treatment. This may have been due to the greater number of times the birds were handled before the i n i t i a t i o n of the s t e r o i d peak or the removal of a large portion of the plasma, which may have lowered the concentration of gonadotrophins i n the blood necessary for ovulation to occur. 64 However, regardless of treatment, s e r i a l blood sampling can be used to obtain an accurate ovulatory p r o f i l e of plasma hormones and ions and t h e i r concentrations, i f the egg laying sequence i s not interrupted. Furthermore, since large v a r i a t i o n s e x i s t between birds, i t allows the determination of ovulatory p r o f i l e s of individual b i r d s . Since each b i r d has a unique ovulatory p r o f i l e for plasma hormone and ion concentrations, s e r i a l blood sampling, at appropriate i n t e r v a l s , i s a technique needed to observe these patterns. Small changes i n hormone and ion concentrations within an in d i v i d u a l b i r d cannot be obtained from bleeding several birds and pooling the data, since large fluctuations can e x i s t between birds. This i s important when determining what relationships e x i s t between the reproductive hormones and the hormones and ions involved i n calcium metabolism during ovulation and egg s h e l l c a l c i f i c a t i o n . 65 Chapter 4 . REPRODUCTIVE FAILURE IN CALCIUM-DEFICIENT AND VITAMIN D-DEFICIENT HENS 66 1. I N T R O D U C T I O N Egg laying i n the hen represents a challenge to the regulation of calcium metabolism. Because of the high rate of calcium transfer to the egg s h e l l , the reproductively active b i r d must maintain plasma calcium concentrations at higher l e v e l s that i t s non-laying counterpart. The increased need for calcium seems to be met primarily by increased i n t e s t i n a l absorption of calcium (Hurwitz et a l . , 1973). At le a s t part of t h i s adaptive mechanism i s mediated by the higher c i r c u l a t i n g l e v e l s of l,25(OH)2D3 observed during egg laying (Baski and Kenny, 1978). This hormone i s the most potent stimulator of calcium absorption known (Norman, 1974) and appears to be regulated according to calcium needs. 1,25(0H)2D3 has been c a l l e d the hormonal form of the vitamin because of i t s formation i n the kidney and the stringent control of i t s c i r c u l a t i n g l e v e l s (Fraser and Kodicek, 1970). Regulation of the plasma concentration of 1,2 5(0H)2D3 involves negative feedback of t h i s metabolite on i t s synthesis i n the kidney (Norman, 1974). PTH (Garabedian et a l . , 1972) and the calcium and phosphorus content of the die t (Boyle et a l . , 1971; Tanaka and DeLuca, 1973; Sommerville et a l . , 1978) are important factors that can stimulate renal la-hydroxylation of 25-OH-D3 and cause an elevation of l,25(OH)2D3 i n the blood. In addition, estrogen administration both i n vivo and i n v i t r o increases the 67 a c t i v i t y of the 25-OH-D3-la-hydroxylase i n the avian kidney (Tanaka et a l . , 1976, 1978; C a s t i l l o et a l . , 1977; Baksi and Kenny, 1977, 1978; Sendrani et a l . , 1981). However, the mechanism by which these factors influence the a c t i v i t y of the renal 25-OHD3-la-hydroxylase enzyme has not been f u l l y elucidated. During a calcium deprivation, ovulation ceases and as the ovaries and oviduct regress, steroidogenesis ceases (Roland et a l . , 1973). At t h i s time, the plasma concentrations of PTH (de Bernard et a l . , 1980) and l,25(OH) 2D 3 (Arnaud, 1978) increase. Total plasma calcium concentrations decline, as well as the ionized calcium concentrations (Luck and Scanes, 1979a). During vitamin D deprivation, ovulation also eventually ceases (Vohra et a l . , 1979). At t h i s time, the plasma concentration of PTH increases (Shen et a l . , 1981), however, plasma l,25(OH)2D3 concentration i s presumably absent or decreased. As well, plasma ionized (Singh et a l . , 1986) and t o t a l (Tsang and Grunder, 1984) calcium concentrations decline. Decreased concentrations of plasma calcium i s believed to be the cause of the f a i l u r e of ovulation during both calcium and vitamin D deprivation (Taylor, 1965). Depression of eith e r the t o t a l plasma calcium concentration or the ionized f r a c t i o n may be responsible for the i n h i b i t i o n of ovulation. The consensus i s that ionized plasma concentration i s the 68 c o n t r o l l i n g factor (Luck and Scanes, 1979a). There may be a threshold concentration of ionized calcium that i s necessary for ovulation to occur, which i s perceived at the l e v e l of the hypothalamus, p i t u i t a r y or ovary. Identifying how ionized calcium i s involved i n the process of ovulation can be an important step i n elucidating the mechanisms required for ovulation to occur. 2. OBJECTIVES To determine how plasma ionized calcium concentration i s involved i n the process of ovulation by 1) characterizing the d a i l y hormonal and i o n i c p r o f i l e of calcium and vitamin D d e f i c i e n t hens that ceased ovulation 2) Comparing these p r o f i l e s to the ovulatory p r o f i l e of normal laying hens. 3) Describing how these d i f f e r e n t i o n i c and hormonal patterns r e l a t e to one another and to ovulation. 3. METHODS Birds, s u r g i c a l and blood sampling techniques were ca r r i e d out as previously described i n Chapter 2, Materials and Methods. Blood sampling occurred only from ovip-ovip. 3.1 Calcium and Vitamin D-Deficient Diets. In order to produce calcium and vitamin D-deficient hens, s p e c i f i c d iets were developed and tested. A semi-purified control d i e t was formulated that would be able to support normal egg production 69 i n laying hens. As well, i t was necessary to ensure that manipulation of t h i s control d i e t to produce calcium-deficient and vitamin D-deficient diets would produce these required d e f i c i e n c i e s . 3.1.1 Methods. Sixty White Leghorn hens, 32 weeks of age, were fed a standard layer d i e t for 4 weeks to ensure that they were laying normally. Hens were at 97 per cent production when the t r i a l began. Egg production (%) was defined as (number of eggs laid)/(number of hens x number of days) x 100. Soft-shelled and s h e l l - l e s s eggs were included. Birds were randomly divided into three groups of 20 b i r d s . One group received the control d i e t , while the other two groups received either the calcium-deficient (Ca-deficient) or vitamin D-deficient (D-deficient) d i e t s (Table 1). Birds were housed i n i n d i v i d u a l laying cages i n a temperature-controlled (2 0°C) room and allowed access to feed and water, ad libitum. Incandescent l i g h t i n g provided a l i g h t cycle of 16 hrs of l i g h t and 8 hrs of dark per day. Windows were covered with aluminum f o i l to prevent exterior l i g h t from entering the room. Groups were well separated to prevent feed dust and/or p a r t i c l e s from cross-contaminating d i e t s . A l l birds were fed t h e i r respective diets for 8 weeks. During t h i s period, the weight change of each b i r d was monitored, as well as feed consumption and egg production. 70 3.1.2 S t a t i s t i c a l Analysis. Changes i n weight, feed consumption and egg production over the 8 weeks and between treatments were analysed using analysis of variance. Treatment means at each time point were compared using Duncan's multiple range t e s t . 3.1.3 Results and Discussion. I n i t i a l weight was not s i g n i f i c a n t l y d i f f e r e n t among the three groups (Fig 9a). The control group l o s t weight during the f i r s t week of the t r i a l , presumably as the birds became accustomed to the p u r i f i e d d i e t . Then, body weight s t a b i l i z e d and was not s i g n i f i c a n t l y d i f f e r e n t from the i n i t i a l weight for the remaining weeks of the t r i a l . Ca-deficient and D-deficient birds l o s t weight during the eight week t r i a l . Body weights were s i g n i f i c a n t l y d i f f e r e n t (p<0.001) from week 0 to week 8. As well, the d e f i c i e n t groups were s i g n i f i c a n t l y d i f f e r e n t from each other. The D-deficient birds had a huge weight loss u n t i l week 4, then they recovered somewhat and began to s t a b i l i z e by week 6. This i s when the majority of the birds had stopped laying hard-shelled eggs. The Ca-deficient birds l o s t a large amount of weight as they continued to lay hard-shelled eggs. By the time egg production had ceased i n most birds by week three, weight began to s t a b i l i z e . Feed consumption was s i g n i f i c a n t l y lower i n a l l three groups during the f i r s t week, as the birds adjusted to the di e t s (Fig 9b). In the control group, feed consumption increased dramatically i n week 2 and then gradually increased 71 Weight (kg) Control •^ K- Ca-Deficient - B - D-Deficient n » 20 for each treatment ! ! ! ! ! 2 3 4 5 6 Time (weeks) Fig 9a. Average weight change over 8 wks. Different letters differ significantly between treatments. 0 i 7 8 72 Feed consumption (g) 120 r 100 80 60 40 Control Ca-Deficient - H - D-Deficient n = 20 for each treatment 0 1 2 3 4 5 6 Time (weeks) Fig 9b. Average feed consumption over 8 wks. Different letters differ significantly between treatments. _i 8 73 Egg Product ion (%) H - D-Deficient 0 1 2 3 4 5 6 7 8 Time (weeks) Fig 9 c Average egg production over 8 wks. Different letters differ significantly between treatments. 74 over the next s i x weeks to be s i g n i f i c a n t l y d i f f e r e n t (p<0.001) from week 1. The D-deficient birds also dramatically increased t h e i r feed consumption i n week 2, however, the feed consumption over the next s i x weeks decreased and was not s i g n i f i c a n t l y d i f f e r e n t from the i n i t i a l low feed consumption. The Ca-deficient group showed a small increase i n feed consumption i n week 2, however, i t was not s i g n i f i c a n t l y d i f f e r e n t from the i n i t i a l low feed consumption. Thereafter, feed consumption decreased and was s i g n i f i c a n t l y lower (p<0.001) than the D-deficient group. Egg production was not s i g n i f i c a n t l y d i f f e r e n t among the three groups i n weeks 0 and week 1 (Fig 9c). The control group continued to lay at t h i s production l e v e l throughout the eight weeks. Egg production decreased i n both the D-deficient and Ca-deficient groups over the eight weeks and were s i g n i f i c a n t l y d i f f e r e n t (p<0.001) from week 0. However, egg production was not s i g n i f i c a n t l y d i f f e r e n t between the two d e f i c i e n t groups from week 4 to week 8. Birds were variable i n the time i t takes to deplete the system of calcium. However, most birds stopped laying within 10 to 14 days (Roland et a l . , 1973; Luck and Scanes, 1979a). It should also be noted that even though some birds were d e f i c i e n t i n plasma calcium, they were unable to adjust to the deficiency and continued to lay eggs at the expense of sk e l e t a l i n t e g r i t y (Roland et a l . , 1973). This could account for the 10 per cent egg production at week 8. 75 Complete vitamin D deficiency i s very d i f f i c u l t to achieve. Environmental conditions must be immaculate to avoid contamination of diets with vitamin D from other feed and/or birds . As well, residual vitamin D from the d i e t s fed before the depletion began can stay i n the system a long time and i s variable between birds. Because some birds would s t i l l have a small amount of vitamin D3 l e f t i n t h e i r system, they would s t i l l be able to produce eggs, again at the expense of t h e i r s k e l e t a l calcium. Although depletion of vitamin D3 was not achieved, a deficiency state was obtained. Although some birds i n both the d e f i c i e n t groups s t i l l continued to lay at 10 to 20 per cent by week 8, these birds were laying predominantly s o f t - s h e l l e d or s h e l l - l e s s eggs. Birds were very d i f f e r e n t i n t h e i r cessation of egg laying. Many birds stopped laying completely, while others continued to lay from d a i l y to every fourth or f i f t h day. However, a number of birds that were not laying for several weeks could be selected for experiment from a depleted group of birds. The hormonal and i o n i c p r o f i l e s of these d e f i c i e n t birds that had ceased ovulation could then be compared to the control birds that were laying normally. 4 . S T A T I S T I C A L A N A L Y S I S Changes i n plasma hormone and ion concentrations over the 24 hr sampling period and between treatments were analysed by analysis of variance, with time and treatment as main e f f e c t s . 76 Treatment means at each time point were compared using Duncan's multiple range t e s t . Duncan's multiple range t e s t was also used to compare hormone and ion concentrations within each treatment over the 24 hrs. Correlations between ion and hormone concentrations were calculated within each treatment. 5. RESULTS 5.1 Plasma Ionized Calcium Concentrations. Plasma ionized calcium concentration i n the control group was c y c l i c i n nature (Fig 10). A peak concentration of 1.57 mM was observed just p r i o r to or at ovulation, while a nadir concentration of 1.2 mM was observed 10-12 hrs post ovulation. No such pattern was observed i n either of the d e f i c i e n t groups (Fig 11, 12) . However, the mean concentration of the vitamin D-d e f i c i e n t group was not s i g n i f i c a n t l y d i f f e r e n t than the mean concentration of the control group. The peak concentration i n the cycle of the control group at time 2 was not s i g n i f i c a n t l y d i f f e r e n t from the vitamin D-deficient group, whereas, the nadir concentration at time 12 was s i g n i f i c a n t l y lower (Fig 13). The mean concentrations of both the control and vitamin D-deficient groups were s i g n i f i c a n t l y higher than the mean concentration of the calcium d e f i c i e n t group. S i g n i f i c a n t differences occurred from time 0 to time 10. 5.1.1 Correlations. A l l correlations are shown i n Table 2 (Page 82). In the control group, there was a s l i g h t 77 1.8 Ionized calcium cone. (mM) o v i p o s i t i o n P<0.0001 n = 6 at e a c h time point 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs from oviposit ion) Fig 10. M e a n ionized p lasma ca lc ium c o n e , of cont ro l hens . Di f ferent le t ters d i f fer s i g n i f i c a n t l y be tween time po in ts 78 Ionized calcium cone. (mM) P>0.05 n = 6 at each time point 0 8 10 12 14 Time (hrs) 16 18 20 22 24 Fig 11. Mean ionized plasma calcium concentration of calcium-deficient hens. No significant difference between times. 79 Ionized calcium cone. (mM) P>0.05 n = 6 at each time point 0 6 8 10 12 14 16 Time (hrs) 18 20 22 24 Fig 12. Mean ionized plasma calcium concentration of D-deficient hens. No significant difference between times. 80 Ionized calcium cone. (mM) a Control Ca-def icient - H - D-deficient n = 6 for each treatment X oviposition i i i i i i i i i i i i i 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs from oviposit ion) Fig 13. Mean plasma ionized calcium concentrations. Different letters differ significantly between treatments. 81 Table 2. Correlations between plasma hormones and ions for Control, Ca-deficient and D-deficient hens. Ca(i) Ca(t) P(i) E 2 P4 D3 Control + .481 -.32 + .25 -.19 + .36 Ca(i) — <.0001 <.01 <.05 >.05 <.001 + . 48 a + .13 + .26 -.15 + .04 Ca(t) <.0001b — >.05 <.05 >.05 >.05 -.32 + .13 -.24 + .31 -.35 P(i) <. 01 >.05 — <.05 <.01 <.001 + .25 + .26 -.24 -.01 + .23 E 2 <. 05 <.05 <.05 — >.05 <.05 -.19 -.15 + .31 -.01 -.34 P4 >. 05 >.05 <.01 >.05 <.01 Ca-Deficient + .22 -.04 + .22 -.05 -.18 Ca(i) — <.05 >.05 <.05 >.05 >.05 + .22 + .10 + .22 + .01 + .05 Ca(t) <.05 >.05 <.05 >.05 >.05 D-Deficient + .34 -.01 + .41 -.09 -.13 Ca(i) — >.01 >. 05 <.001 >.05 >.05 + .34 -.09 + .32 + .14 -.11 Ca(t) <. 01 >.05 <• 05 >.05 >.05 a R2 ^ p r o b a b i l i t i e s 82 s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between plasma ionized and t o t a l calcium concentrations. As well, plasma ionized calcium concentration had a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n with plasma e s t r a d i o l and l,25(OH)2D3 concentrations. Plasma ionized calcium concentration was also negatively correlated with plasma inorganic phosphorus concentration. In the calcium-deficient group, there was a s l i g h t p o s i t i v e c o r r e l a t i o n between plasma ionized, t o t a l calcium and es t r a d i o l concentrations. In the vitamin D-deficient group, plasma ionized calcium concentration had a po s i t i v e c o r r e l a t i o n with plasma t o t a l calcium and plasma e s t r a d i o l concentrations. 5.2 Other Plasma Hormones and Ions 5.2.1 T o t a l Calcium Concentrat ions . A l l groups exhibited s i g n i f i c a n t differences i n t o t a l plasma calcium concentration over the 24 hrs (Figs 14-16). However, the mean t o t a l plasma calcium concentration was s i g n i f i c a n t l y d i f f e r e n t among a l l three groups. S i g n i f i c a n t differences occurred at a l l time points (Fig 17). As well, the mean concentration of both d e f i c i e n t groups was s i g n i f i c a n t l y d i f f e r e n t from each other, with the calcium-deficient group having the lowest mean concentration. A s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n existed between plasma t o t a l calcium and plasma e s t r a d i o l concentrations i n a l l three groups (Table 2). 83 Total calcium cone, (mg/100 ml) oviposition -• 6 at each time point i i i i 0 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs from oviposit ion) Fig 14. Mean total plasma calcium cone, of control hens. Different letters differ significantly between time points 84 Total calcium cone, (mg/100 ml) 12 r abed 6 at each time point I I l I L 0 8 10 12 14 16 Time (hrs) 18 20 22 24 Fig 15. Mean total plasma calcium cone, of ca-deficient hens. Different letters differ significantly between time points 85 Total calcium cone, (mg/100 ml) 14 r a a 10 - b b 8 -P<0.05 n = 6 at each time point 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hrs) Fig 16. Mean total plasma calcium cone. of D-deficient hens. Different letters differ significantly between time points 86 25 Total calcium cone, (mg/100 ml) Control n = 6 for each treatment 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs from oviposit ion) Fig 17. Mean plasma total calcium concentrations. Different letters differ significantly between treatments. 87 5.2.2 Inorganic Phosphorus Concentrations. In both the control group (Fig 18) and the vitamin D-deficient group (Fig 19), s i g n i f i c a n t differences i n plasma inorganic phosphorus concentrations were observed over the 24-26 hrs. No s i g n i f i c a n t differences were observed i n the calcium-deficient group (Fig 20). Mean plasma inorganic phosphorus concentrations varied s i g n i f i c a n t l y among the three groups (Fig 21). Although there were s i g n i f i c a n t differences over the 24 hrs i n the vitamin D-d e f i c i e n t group, the mean concentration was s i g n i f i c a n t l y lower than both the control and calcium-deficient groups. In the control group, plasma inorganic phosphorus concentration had a s i g n i f i c a n t negative c o r r e l a t i o n with plasma e s t r a d i o l and plasma l,25(OH)2 D3 concentrations, while being p o s i t i v e l y correlated with plasma progesterone concentration (Table 2). 5.2.3 l,25(OH)2D3 Concentrations. S i g n i f i c a n t differences were observed i n both the control group (Fig 22) and the calcium-deficient group (Figs 23) over the 24-26 hrs. There were no s i g n i f i c a n t differences observed i n the vitamin D-deficient group (Fig 24) . The control and calcium-deficient groups were not s i g n i f i c a n t l y d i f f e r e n t at times 2, 4 and 12 (Fig 25). However, the mean concentration of a l l three groups was s i g n i f i c a n t l y d i f f e r e n t , with the calcium-deficient group having the highest mean concentration and the vitamin D-88 10 Inorganic phosphorus cone, (mg/100 ml) 0 oviposition abed P<0.0001 n • 6 at each time point J I I I I I I I L J I 0 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs from oviposit ion) Fig 18. Mean plasma phosphorus cone. of control hens. Different letters differ significantly between time points 89 Inorganic phosphorus cone, (mg/100 ml) n = 6 at each time point I I I I L 0 8 10 12 14 16 Time (hrs) 18 20 22 24 Fig 19. Mean plasma phosphorus cone, of D-deficient hens. Different letters differ significantly between time points 90 4 Inorganic phosphorus cone, (mg/100 ml) P>0.05 n = 6 at each time point 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hrs) Fig 20. Mean plasma inorganic phosphorus concentration of calcium-deficient hens. No significant difference between times. 91 10 Phosphorus cone, (mg/100 ml) 8 0 Control •^ K- Ca-def icient D-deficient J L n • 6 for each treatment _ J I I I L _ J I I I 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs from oviposit ion) Fig 21. Mean inorganic plasma phosphorus concentrations. Different letters differ significantly between treatments. 92 1,25(OH)2D3 cone, (pg/ml) ^ oviposition ibe ab cde icd cde def eU efc P<0.0001 n = 6 at each time point l I I I I I I I I L ab< 0 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs from oviposit ion) Fig 22. Mean plasma 1,25(OH)2D3 cone. of control hens. Different letters differ significantly between time points 93 1,25(OH)2D3 cone, (pg/ml) ab ,cde P<0.0001 n = 6 at each time point i i i i i i i i be de j i i i 0 8 10 12 14 16 Time (hrs) 18 20 22 24 Fig 23. Mean plasma 1,25(OH)2D3 cone, of ca-deficient hens. Different letters differ significantly between time points 94 60 1,25(OH)2D3 cone, (pg/ml) 50 40 P>0.05 n = 6 at each time point 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hrs) Fig 24. Mean plasma 1,25(OH)2D3 concentration of D-deficient hens. No significant difference between times. 95 1,25(OH)2D3 cone, (pg/ml) 400 -300 200 100 Control Ca-def icient D-deficient oviposition a— o n • 6 for each treatment J I I I I L 0 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs from oviposit ion) Fig 25. Mean plasma 1,25(OH)2D3 concentrations. Different letters differ significantly between treatments. 96 d e f i c i e n t group having the lowest mean concentration. A l l time points were s i g n i f i c a n t l y d i f f e r e n t between the vitamin D-deficient group and the two other groups. In the control group, plasma l,25(OH)2D3 concentration had a s i g n f i c a n t p o s i t i v e c o r r e l a t i o n with plasma e s t r a d i o l concentration and a s i g n i f i c a n t negative c o r r e l a t i o n with plasma progesterone concentration. 5.2.4 E s t r a d i o l Concentrations. S i g n i f i c a n t differences over the 24 hrs were observed i n a l l three groups for plasma e s t r a d i o l concentration (Figs 2 6-28). The mean concentration i n a l l three groups was s i g n i f i c a n t l y d i f f e r e n t from each other, with the control group having the highest mean concentration and the vitamin D-deficient group having the lowest mean concentration (Fig 29). A highly s i g n i f i c a n t difference between the control group and the d e f i c i e n t groups occurred at time 0 (oviposition) and time 18 (the ste r o i d peak). In addition, there was a s i g n i f i c a n t negative c o r r e l a t i o n between plasma e s t r a d i o l and progesterone concentrations i n the vitamin D-deficient group. 5.2.5 Progesterone Concentrations. A s i g n i f i c a n t pattern was observed i n the control group over the 26 hrs (Fig 30). There were no s i g n i f i c a n t differences between times 0 and 24 for either of the d e f i c i e n t groups (Fig 31, 32). A peak concentration for plasma progesterone was observed only in the control group (Fig 33). As well, the basal concentration was s i g n i f i c a n t l y higher than the d e f i c i e n t Estradio l cone, (pg/ml) oviposition be be be be be be be be P<0.01 J L _ n • 6 at each time point _ l I I I I L_ 0 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs from oviposit ion) Fig 26. Mean plasma estradiol cone, of control hens. Different letters differ significantly between time points 98 Estradio l cone, (pg/ml) be be be P<0.001 n = 6 at each time point J L J I I L ab be be 0 8 10 12 14 16 Time (hrs) 18 20 22 24 Fig 27. Mean plasma estradiol cone, of Ca-def icient hens. Different letters differ significantly between time points 99 Estradiol cone, (pg/ml) P<0.0001 n = 6 at each time point J I I I I I I L__ J I 0 2 4 6 8 10 12 14 16 Time (hrs) 18 20 22 24 Fig 28. Mean plasma estradiol cone, of D-deficient hens. Different letters differ significantly between time points 100 Estradio l cone, (pg/ml) 250 200 -Control Ca-def icient D-deficient oviposition n = 6 for each treatment 150 -100 b ~ % — a — • b b 0 4 6 8 10 12 14 16 18 20 22 24 26 Time (hrs from oviposit ion) Fig 29. Mean plasma estradiol concentrations. Different letters differ significantly between treatments. 101 a P r o g e s t e r o n e c o n e , (ng/ml) oviposition P<0.001 n = 6 at each time point 0 X 6 8 10 12 14 16 18 20 22 24 26 T ime (hrs f rom ov ipos i t i on ) Fig 30. Mean plasma progesterone cone. of control hens. Different letters differ significantly between time points 102 Progesterone cone, (ng/ml) 0.15 P>0.05 6 at each time point 0.05 0 8 10 12 14 16 Time 18 20 22 24 Fig 31. Mean plasma progesterone concentration of calcium-deficient hens. No significant difference between times. 103 0.5 Progesterone cone, (ng/ml) 0.4 0.3 0.2 0.1 0 0 be cd cd cd cd P<0.0001 n • 6 at each time point l I I I I I I L 8 10 12 14 16 Time (hrs) 18 20 22 24 Fig 32. Mean plasma progesterone concentration of D-deficient hens. Different letters differ significantly. 104 Progesterone cone, (ng/ml) 7 r - C o n t r o l Ca-def icient - B - D-deficient X oviposition n = 6 for each treatment ~n 1 1 i 20 22 24 26 0 — r 8 6  10 12 14 16 18 Time (hrs from oviposit ion) Fig 33. Mean plasma progesterone concentrations. Different letters differ significantly between treatments. 105 groups at a l l time points except time 22. The mean concentration of the d e f i c i e n t groups was not s i g n i f i c a n t l y d i f f e r e n t , nor did they d i f f e r s i g n i f i c a n t l y at any one time point between each other. 5.3 Bone analysis. Bone analysis to confirm calcium-deficiency i n the two d e f i c i e n t groups i s shown i n Figure 34. Percent bone ash was s i g n i f i c a n t l y higher (p<0.001) i n the control group. The calcium-deficient and vitamin D-deficient groups were not s i g n i f i c a n t l y d i f f e r e n t from each other. Body weight (Fig 35), % ovary weight (Fig 36) and % oviduct weight (Fig 37) were s i g n i f i c a n t l y higher (p<0.001) i n the control group. The calcium-deficient and vitamin D-d e f i c i e n t groups were not s i g n i f i c a n t l y d i f f e r e n t from each other i n these parameters. 106 % Bone ash G r o u p s wi th d i f f e ren t le t te rs d i f f e r s i g n i f i c a n t l y ( p <0 .001 ) Control Ca-deficient D-deficient Fig 34. Bone ash as a percentage of total bone weight. 107 2000 Body weight (g) 1500 1000 500 G r o u p s w i th d i f f e r e n t l e t t e r s d i f f e r s i g n i f i c a n t l y (P<0.001) Control l l l l Ca-def icient D-deficient Fig 35. Final mean body weights of all three groups. 108 Ovary weight (%) Groups with different letters differ significantly (P<0.001) Control Ca-deficient D-deficient Fig 36. Mean ovary weight as a percentage of mean body weight. 109 Oviduct weight (%) Groups with different letters differ signif icantly (P<0.001) Control i Ca-deficient D-deficient Fig 37. Mean oviduct weight as a percentage of mean body weight. n o 6 . DISCUSSION Daily hormonal and io n i c p r o f i l e s for the control hens were s i g n i f i c a n t l y d i f f e r e n t from both d e f i c i e n t groups. Both d e f i c i e n t groups had s i g n i f i c a n t l y lower mean plasma t o t a l calcium, inorganic phosphorus, e s t r a d i o l and progesterone concentrations than the control group. In addition, d i s t i n c t c y c l i c patterns of i o n i c calcium, inorganic phosphorus and progesterone concentrations were observed i n the control hens that were not observed i n the d e f i c i e n t hens. In the plasma of the domestic hen, calcium i s contained in two major compartments (Copp, 1969). The largest i s the t o t a l calcium f r a c t i o n , which i s bound to plasma proteins, mainly yolk lipoproteins. Concentrations of 20-30 mg/100 ml are considered normal for a reproductively active hen. In t h i s study, control hens had a mean value of 19 mg/100 ml, peak values ranging from 22-36 mg/100 ml. This was s i g n i f i c a n t l y higher than the mean for the calcium and vitamin D-deficient hens, which were 9.6 and 11 mg/100 ml, respectively. In addition, bone ash was s i g n i f i c a n t l y lower in both d e f i c i e n t groups. This observation was also made by Roland et a l . (1973), Vohra et a l . (1979) and Shen et a l . (1981). I t can therefore be concluded that both d e f i c i e n t groups were p h y s i o l o g i c a l l y d e f i c i e n t i n calcium. A f r a c t i o n of t h i s t o t a l calcium i s present as the free ion and the concentration of i o n i c calcium varies between 1.2-1.8 mM depending upon the age and stage of egg s h e l l 111 c a l c i f i c a t i o n (Luck and Scanes, 1979b; Singh et a l . , 1986). The concentrations of ionized plasma calcium presented here ranged from 1.25 mM - 1.57 mM for control hens. The changes during the ovulatory cycle were s i m i l a r to previous r e s u l t s . In t h i s study, mean plasma ionized calcium concentrations were not s i g n i f i c a n t l y d i f f e r e n t between the control group and vitamin D-deficient group. In addition, i t was only s l i g h t l y lower i n the calcium-deficient group despite the large decrease i n plasma t o t a l calcium concentration. This demonstrates that the body's primary physiological r o l e i s to maintain c i r c u l a t o r y l e v e l s of io n i c calcium to support basic functions. Singh et a l . (1986) observed a lower mean plasma io n i c calcium concentration for vitamin D-deficient hens than control hens. However, they observed the same mean concentration for control and calcium-deficient hens. Although the mean concentration of ionized calcium did not vary greatly among the three groups, the control group had a s i g n i f i c a n t c y c l i c pattern within the 24 hr time period, r e l a t i n g to ovulation and egg s h e l l c a l c i f i c a t i o n . No s i g n i f i c a n t fluctuations were observed i n eithe r the calcium-d e f i c i e n t or vitamin D-deficient groups within the 24 hrs. The peak concentration i n the control group was s i g n i f i c a n t l y higher than both d e f i c i e n t groups, whereas, the nadir at 10-12 hrs post ovulation i n the control group was as low as the calcium-deficient group. In the calcium and vitamin D-d e f i c i e n t groups, the concentrations ranged from 1.24-1.33 mM 112 and 1.35-1.45 mM, respectively. As c a l c i f i c a t i o n proceeded, the ionized calcium concentration decreased to a nadir j u s t p r i o r to the s t e r o i d peak, then i t gradually increased u n t i l i t reached a peak j u s t p r i o r to ovulation. Apart from i t s r o l e i n s h e l l formation, the i o n i c calcium component may be involved i n d i r e c t l y with ovarian function. Luck and Scanes (1979a) presented evidence that p i t u i t a r y function could be affected by plasma i o n i c calcium concentration i n v i t r o ; a low concentration of i o n i c calcium i n p i t u i t a r y cultures r e f l e c t e d a low output of LHRH and gonadotrophins. Since the ionized calcium concentration reached a peak j u s t p r i o r to ovulation, t h i s may be when a threshold concentration of ionized calcium i s necessary for ovulation. I t may also be that the c y c l i c nature of the ionized calcium f r a c t i o n i s an important mediator i n the process of ovulation. A l l three groups exhibited differences i n t o t a l plasma calcium concentrations over the 24-26 hr time period. The control group had a s i g n i f i c a n t steady decline from time 0 to time 26, whereas, the calcium and vitamin D-deficient groups had only s l i g h t observable fluctuations over the 24 hrs. This was presumably because the d e f i c i e n t hens were not t r y i n g to meet the demands of c a l c i f y i n g an eggshell. However, fluctuations would s t i l l e x i s t as active (calcium-deficient) and passive (vitamin D-deficient) i n t e s t i n a l absorption of 113 calcium occurred as the birds continued to eat to maintain plasma calcium concentrations. The ionized calcium f r a c t i o n had only a s l i g h t p o s i t i v e c o r r e l a t i o n with t o t a l calcium concentration i n the control group. A strong c o r r e l a t i o n would be expected since the two fractio n s e x i s t i n the plasma i n equilibrium (Copp, 1969). However, there was a steady decline i n the t o t a l calcium concentration over the 26 hrs, whereas, ionized calcium declined and returned to i t s i n i t i a l high concentration. Total calcium concentration i s very dependent upon the plasma proteins of the blood. Since a large proportion of these proteins were removed over the course of blood sampling, there probably was not a s u f f i c i e n t amount l e f t to return the t o t a l calcium concentration to i t s i n i t i a l high concentration. Blood ionized calcium concentration was correlated even less with the t o t a l plasma calcium concentration i n both d e f i c i e n t groups. Both groups would be t r y i n g to maintain plasma i o n i c calcium at the expense of plasma t o t a l calcium. Plasma t o t a l calcium would be lower i n the d e f i c i e n t groups as plasma e s t r a d i o l concentration decreased. This would decrease the number of plasma proteins which bind t o t a l calcium to maintain an equilibrium with the ionized calcium concentration. Plasma inorganic phosphorus concentration i n the control group had a strong s i g n i f i c a n t c y c l i c pattern that was related to ovulation and egg s h e l l c a l c i f i c a t i o n . In addition, the mean concentration of plasma phosphorus was s i g n i f i c a n t l y 114 higher i n the control group than both d e f i c i e n t groups. As calcium i s drawn from the blood for egg s h e l l c a l c i f i c a t i o n , bone i s mobilized to meet the extra calcium demand (Gilbert, 1983), r e s u l t i n g i n an increase i n plasma phosphorus concentration. However, calcium-deficient hens, who would be drawing on bone reserves to meet t h e i r calcium needs, had a s i g n i f i c a n t l y lower plasma inorganic phosphorus concentration than the control birds, although no fluctuations were observed over the 24 hr period. As plasma calcium concentrations decrease during egg s h e l l c a l c i f i c a t i o n , l,25(OH)2D3 concentrations increase i n response to PTH secretion (Garabedian et a l . , 1972). With the increase i n l,25(OH)2D3 concentration, there i s an increase i n absorption of phosphorus at the i n t e s t i n a l l e v e l , as well as an increase i n plasma phosphorus concentration from bone turnover. This increase i n phosphorus concentration i n the blood stimulates PTH to enhance phosphorus excretion at the kidney l e v e l (Clark and Sasayama, 1981). The birds i n t h i s study may have reached a point where the excretion of phosphorus at the kidney l e v e l exceeded the amount that was being absorbed by the i n t e s t i n e and released from the bone. The vitamin D-deficient group had s i g n i f i c a n t l y lower plasma inorganic phosphorus concentrations than even the calcium-deficient group, however, there was a small observable f l u c t u a t i o n . Low plasma inorganic phosphorus concentrations have been observed previously i n vitamin D-deficient birds 115 (Luck and Scanes, 1978). Birds d e f i c i e n t i n vitamin D would have only a small amount of the active metabolite, l,25(OH)2D3, available to break down bone to release phosphorus. Furthermore, active absorption of phosphorus from the i n t e s t i n e would be reduced. The fluctuations i n the phosphorus concentrations may be due to passive i n t e s t i n a l absorption of phosphorus as the birds ate when the l i g h t s were on. Plasma phosphorus concentration increased progressively from when the l i g h t s came on (time 0) u n t i l the l i g h t s went out (time 14). Plasma ionized calcium concentration had a strong negative c o r r e l a t i o n with plasma phosphorus concentration i n the control hens. As plasma calcium concentration decreases i n response to egg s h e l l c a l c i f i c a t i o n , plasma phosphorus concentration increases as bone i s broken down to meet the calcium needs of the b i r d (Gilbert, 1983). Since phosphorus metabolism i s intimately associated with calcium metabolism, i t would seem probable that phosphorus concentrations may be implicated i n disruption of ovulation. Since the mean plasma phosphorus concentration was s i g n i f i c a n t l y higher i n the control hens, the low phosphorus concentrations exhibited by the d e f i c i e n t hens may have been i n h i b i t o r y to ovulation. There may be a threshold concentration of plasma phosphorus that has a permissive r o l e i n the process of gonadotrophin release. Plasma l,25(OH)2D3 concentrations increased dramatically 116 i n calcium-deficient hens, presumably i n response to increased plasma PTH concentrations for maintenance of plasma calcium concentrations. S i g n i f i c a n t fluctuations were observed over the 24 hrs. In the vitamin D-deficient hens, plasma l,25(OH)2D3 concentrations were s t i l l present. No s i g n i f i c a n t fluctuations were notable, probably because there was not enough l,25(OH)2D3 present to e l i c i t t h i s response. Although the hens i n t h i s group were not completely d e f i c i e n t i n vitamin D, t h e i r concentrations of plasma l,25(OH)2D3 were s i g n i f i c a n t l y lower than the control group. As well, they were comparable i n calcium deficiency to the calcium-deficient hens. Thus, the vitamin D-deficient hens could be considered vitamin D-deficient, but not vitamin D-deplete. The concentrations and ovulatory p r o f i l e of plasma l,25(OH)2D3 observed i n the control hens are i n agreement with work done by C a s t i l l o et a l . (1979). A s i g n i f i c a n t c y c l i c pattern was observed i n the control hens. This would presumably be due to the increase i n calcium needed during egg s h e l l c a l c i f i c a t i o n . Although, plasma 1,25(0H)2D3 concentrations do not correlate well with s h e l l calcium deposition (Bar et a l . , 1984) or formation and resorption of medullary bone ( C a s t i l l o et a l . , 1979), renal 25-OH-la-hydroxylase does begin to r i s e i n almost exact c o r r e l a t i o n with the r i s e i n plasma calcium and the f a l l i n plasma phosphorus concentrations as the b i r d enters the egg s h e l l formation stage ( C a s t i l l o et a l . , 1979). This suggests that 117 the fluctuations i n t o t a l plasma calcium concentration cannot be explained by the plasma l e v e l s of l,25(OH)2D3. However, plasma l,25(OH)2D3 l e v e l s are highest immediatly before and during egg s h e l l c a l c i f i c a t i o n . In t h i s study, plasma l,25(OH)2D3 concentrations were highly correlated with estrogen and progesterone, as well as plasma phosphorus and ionized calcium concentrations. This would indicate that plasma l,25(OH)2D3 concentrations may have a regulatory role associated with phosphorus and ionized calcium i n the control of ovulation, consistent with i t s involvement i n calcium and phosphorus metabolism i n general. High c i r c u l a t i n g l,25(OH) 2D3 concentrations i n an environment low i n calcium, as observed i n calcium-deficient hens, may be i n h i b i t o r y to the release of gonadotrophic factors, as postulated by Luck and Scanes (1980a). Low concentrations of plasma l,25(OH)2 n3 as observed i n vitamin D-deficient hens may not be s u f f i c i e n t to increase plasma ionized calcium concentrations needed for ovulation. Therefore, there may be a window of plasma l,25(OH)2D3 concentration that maintains reproduction i n egg laying hens. Gonadal hormones have been implicated i n the regulation of calcium and phosphorus metabolism i n the laying hen through several modes of action. I t i s believed that estrogen i n i t i a t e s many of the changes i n calcium metabolism required for the reproductive process ( C a s t i l l o et a l . , 1977; Baski and Kenny, 1978). In preparation for the egg laying process, as 118 the female reaches maturity, there i s an increase i n calcium absorption (Bar et a l . , 1978), plasma calcium concentration (Riddle et and Reinhart, 1926) and deposition of medullary bone (Common et a l . , 1948). Estrogen might exert i t s control on calcium metabolism by regulating production of 1,25(0H)2D3 (Tanaka et a l . , 1976). I t i s of considerable i n t e r e s t that e s t r a d i o l can stimulate the conversion of vitamin D to i t s active form, l,25(OH)2D3, i n birds. Since there are renal receptors for estrogen (Concolino et a l . , 1976) and since estrogen increases renal weight i n vivo (Baski and Kenny, 1978), estrogen could d i r e c t l y e f f e c t the kidney la-hydroxylase. In addition, the laying hen's ovary c e l l s possess the 1,25(0H)2D3 receptor macromolecule i d e n t i c a l to those of l,25(OH)2D3 receptors i n other vitamin D target tissues (Dukoh et a l . , 1983). I t i s evident that the vitamin D hormone system i s intimately involved with the regulatory phenomena associated with egg laying i n birds. Although, the functions of 1,25(0H)2D3 and i t s receptor i n the ovary are not known, the need for a d e l i c a t e control of calcium and bone metabolism i n avian reproduction suggests an e f f e c t of 1,25(0H)2D3 on female sex hormones as a reasonable p o s s i b i l i t y . Moreover, l,25(OH)2D3 has an i n h i b i t o r y e f f e c t on the growth of ovarian c e l l s i n culture (Dukoh et a l . , 1983). The 1,25(0H)2D3 concentration required to e l i c i t t h i s growth i n h i b i t i o n corresponds to the exogenous 1,25(0H)2D3 concentrations 119 necessary to saturate the cytosol receptor i n v i t r o . One p o s s i b i l i t y of t h i s action i s that by reducing the number of estrogen hormone-secreting c e l l s , l,25(OH)2D3 action creates a negative feed back loop between the kidney and ovary, thereby regulating ovulation through s p e c i f i c concentrations of plasma l,25(OH) 2D 3. In considering the r o l e of estrogen and vitamin D metabolism i n the laying hen, i t seems possible that the stimulation of i n t e s t i n a l calcium absorption by estrogen i s mediated by a stimulation of l,25(OH)2D3 production. The r i s e i n plasma calcium concentration i s also, i n part, the r e s u l t of increased c i r c u l a t i n g l e v e l s of l,25(OH)2 n3. However, the estrogen induced c i r c u l a t i n g phosphoproteins are a s i g n i f i c a n t factor. Estrogen had a more dramatic e f f e c t on increasing l a -hydroxylase and plasma calcium concentration than did exogenous PTH ( C a s t i l l o et a l , 1977), although the e f f e c t s were additive. The action of estrogen on renal vitamin D metabolism could be i n d i r e c t , through PTH (DeLuca, 1980) or possibly through p r o l a c t i n , since estrogen i s known to increase p r o l a c t i n release (Hall et a l . , 1984). F i n a l l y , estrogen could a f f e c t bone mineral metabolism i n some other fashion independent of the kidney and la-hydroxylase system, with the r e s u l t i n g i o n i c changes being ultimately expressed at the renal l e v e l by the la-hydroxylase (Dukoh et a l , 1983). Regardless of the route whereby estrogen controls vitamin D b i o a c t i v a t i o n , the finding of l,25(OH)2D3 receptors and growth 120 i n h i b i t i o n of ovarian c e l l s reveals that l,25(OH)2D3 may modulate f o l l i c u l a r c e l l d i f f e r e n t i a t i o n or exert a negative influence on some ovarian function. These e f f e c t s are probably important to mineral metabolism during reproduction i n b i r d s . Disruption of estrogen catabolism i s also thought to be a factor i n cessation of ovulation. Tsang and Grunder (1984) reported interference with estrogen metabolism during vitamin D deprivation. Later, Tsang et a l . (1988) found that calcium deficiency rather that vitamin D deficiency was the more immediate cause of the interference. They did not know i f the cause was d i r e c t or i n d i r e c t , although none of the enzymes involved are calcium-dependent. However, hypocalcemia i s known to suppresss LH biosynthesis and to cause hyperparathyroidism through an increased secretion of PTH i n the laying hen (Luck and Scanes, 1979b). While plasma LH le v e l s influence estrogen synthesis, there i s no evidence that LH or PTH d i r e c t l y a f f e c t s estrogen metabolism. Although mean plasma e s t r a d i o l concentrations were s i g n i f i c a n t l y lower i n the d e f i c i e n t groups when compared with the control, a l l three groups exhibited s i g n i f i c a n t f l u c t u a t i o n s . These fluctuations may be necessary to increase l,25(OH)2D3 for maintenance of basal plasma calcium. Estrogen may play an important r o l e i n calcium metabolism by regulation of calcium l e v e l s necessary for ovulation. Decreased estrogen concentrations caused by increased l,25(OH)2D3 l e v e l s may reduce the enhancement of l,25(OH)2D3, and therefore, reduce the chance of ovulation. This i s supported by a report by Baksi and Kenny (1978), who found that gonadal hormones can influence the renal synthesis of l,25(OH)2D3 when dietary calcium i s adequate. However, these regulatory e f f e c t s of e s t r a d i o l are p a r t i a l l y or completely eliminated i n the calcium-deprived b i r d . As well, progesterone treatment had a sharp depresssive e f f e c t on l,25(OH)2 n3 concentration i n the calcium-deprived female. Plasma ionized calcium concentration was correlated with estrogen i n a l l three groups i n the present study. Onagbesan and Peddie (1989) observed that the thecal layers of hens' f o l l i c l e s secrete e s t r a d i o l stimulated by FSH, and that the stimulatory e f f e c t of FSH i s dependent upon external calcium. These researchers found that the stimulatory action of FSH was i n h i b i t e d i n a calcium-free medium or i n the presence of EGTA in a calcium-replete medium. The FSH-stimulated response was maximal i n 1.0 mM external calcium. This may indicate the unique importance of external calcium i n regulating gonadotrophin s e n s i t i v i t y of the f o l l i c u l a r c e l l s i n hens. During the egg laying cycle, plasma ionized calcium l e v e l s decrease from 1.8 mM i n the postlaying hours to 1-1.2 mM during s h e l l c a l c i f i c a t i o n (Singh et a l , 1986) so that t h e i r e f f e c t s may be of r e a l p h ysiological s i g n i f i c a n c e , contributing to the changes i n c i r c u l a t i n g estrogen during the cycle. The FSH-stimulated, androgen-primed estrogen secretion 122 i n rats was i n h i b i t e d by EGTA, suggesting that t h i s may be one s i t e of action of increased i n t r a c e l l u l a r calcium concentrations. LH-stimulated progesterone secretion from the granulosa c e l l s of hens i s p a r t i a l l y calcium-dependent (Asem and Hertelendy, 1986a). There i s some evidence that the action of LH i n promoting progesterone secretion by the granulosa c e l l s of the domestic hen i s mediated i n part by calcium (Asem and Hertelendy, 1986a, b; Asem et a l . , 1987). Direct evidence for a r o l e of calcium i n gonadotrophin-stimulated e s t r a d i o l secretion from avian c e l l s has not been reported, but i n rats, FSH-stimulated steroidogenesis by granulosa c e l l s i s a calcium-dependent event (Tsang and Carnegie, 1984). Since plasma inorganic phosphorus concentration may have a permissive role i n the process of ovulation, i t s e f f e c t s may also be mediated i n a fashion s i m i l a r to ionized calcium. However, the lack of either i n v i t r o or i n vivo studies i n t h i s area, has l e f t the role of inorganic phosphorus concentration in the process of ovulation undefined. Plasma progesterone concentrations were s i g n i f i c a n t l y higher i n the control birds and exhibited a peak 10 hrs p r i o r to ovulation. Both d e f i c i e n t groups had a low basal concentration of plasma progesterone and no peak was present over the 24 hr period. Sedrani et a l . (1981) suggested a cer t a i n minimum concentration of progesterone may need to be present for estrogen to exert i t s action. Previous studies 123 have indicated that an unusually high basal l e v e l of c i r c u l a t i n g estrogen and an usually low basal l e v e l of progesterone were associated with the f a i l u r e to lay eggs (Leszczynski et a l . , 1983). Generally, ovulation appears to be more strongly associated with c i r c u l a t i n g progesterone l e v e l s than with estrogen (Leszczynski et al.,1985). However, i t appears that basal c i r c u l a t i n g estrogen/progesterone concentrations correlate better with egg production than progesterone alone. The process of ovulation and i t s f a i l u r e i n calcium and vitamin D-deficient birds i s not simple. The c y c l i c nature of plasma ionized calcium may be an important mediator i n the event. As well, the peak concentration of ionized calcium exhibited j u s t p r i o r to ovulation may be a threshold concentration that i s necessary for ovulation to occur. Plasma phosphorus concentration may have a permissive r o l e i n the process of ovulation. A s p e c i f i c window of l,25(OH)2D3 concentration may also have a regulatory r o l e at the l e v e l of the ovary, with regard to estrogen production. In a decreased calcium environment, plasma ionized calcium and phosphorus concentrations are both low, which may i n h i b i t the s t e r o i d surge. Furthermore, high or low c i r c u l a t i n g plasma l,25(OH)2D3 concentrations may i n h i b i t e s t r a d i o l production at the ovarian l e v e l . Hence, an i n t e r a c t i o n of several hormonal and i o n i c factors may be responsible for the f a i l u r e of ovulation i n calcium and vitamin D-deficient hens. 124 Chapter 5 . CONCLUDING REMARKS 125 From the r e s u l t s presented i n t h i s t h esis, i t would appear that an i n t e r - r e l a t i o n s h i p e x i s t s among ionized calcium, phosphorus, l,25(OH)2D3 and the reproductive hormones. The c y c l i c nature of the ions and hormones during the ovulatory cycle i s also an important consideration. A threshold concentration of ionized calcium may be the t r i g g e r for ovulation to occur, perceived at the l e v e l of the p i t u i t a r y , hypothalamus or ovary. As well, the other plasma factors correlated with plasma ionized calcium concentrations cannot be discounted as having a role i n regulating ovulation. A precise r e l a t i o n s h i p may e x i s t among a l l these factors, such that i f one i s increased or decreased, an imbalance w i l l e x i s t i n the system and ovulation cannot proceed. Furthermore, i t can be shown from the r e s u l t s presented here that s e r i a l blood sampling, at appropriate i n t e r v a l s , can be used as a method for determining ovulatory p r o f i l e s i n i n d i v i d u a l hens without seriously compromising i o n i c and hormonal concentrations and p r o f i l e s . Furthermore, i t can be used to compare and define changes that occur between the ovulatory p r o f i l e s of a c t i v e l y laying hens and those that have ceased laying through a calcium or vitamin D deficiency. The research presented here leads to several experimental manipulations that may help i n elucidating the mechanisms of ovulation. The r o l e of ionized calcium can be further understood by infusing calcium and vitamin D-deficient birds with an ionized calcium solution. This may determine i f a 126 threshold concentration exists and what that concentration may be. The r o l e that plasma phosphorus may have can be understood more f u l l y by both i n v i t r o and i n vivo work. Low phosphorus concentrations used i n ovarian and p i t u i t a r y c e l l cultures may elucidate a minumum concentration needed, as well as where plasma phosphorus concentration exerts i t s action. Infusing calcium and vitamin D-deficient birds with phosphorus may also be revealing. These studies and others w i l l lead to a better understanding of the mechanisms of ovulation and i t s f a i l u r e during aberrant calcium metabolism. Understanding how the calcemic ions and hormones are involved i n ovulation i s important i n gaining further insight into the regulatory mechanisms of ovulation. Once regulatory mechanisms are elucidated, manipulation of the reproductive cycle may be possible, such that laying hens have a longer reproductive l i f e . 127 REFERENCES 128 Abe, E., Tanabe, R., Suda, T. and Yoshika, S. 1979. Circadian rhythmn of 1,25-dihydroxyvitamin D 3 production i n egg laying hens. Biochem Biophys Res Comm 88: 500-507. Arnaud, CD. 1978. Calcium homeostasis: regulatory elements and t h e i r integration. Fed Proc 37: 2557-2560. Asem, E.K. and Hertelendy, F. 1986a. Role of calcium i n l u t e i n i z i n g hormone-induced progesterone and c y c l i c AMP production i n granulosa c e l l s of the hen (Gallus  domesticus). Gen Comp Endocrinol 62: 120-128. Asem, E.K. and Hertelendy, F. 1986b. Trifluoperazine i n h i b i t s progesterone and c y c l i c AMP production i n granulosa c e l l s of the hen (Gallus domesticus). Gen Comp Endocrinol 64: 107-111. Asem, E.K., Molnar, M. and Hertelendy, F. 1987. Lutei n i z i n g hormone-induced i n t r a c e l l u l a r calcium mobilization i n granulosa c e l l s : comparison with f o r s k o l i n and 8-bromo-adenosine 3 1,5 1-monophosphate. Endocrinol 120: 853-859. Baksi, S.N. and Kenny, A.D. 1977. Vitamin D3 metabolism i n immature Japanese q u a i l : e f f e c t s of ovarian hormones. Endocrinol 101: 1216-1220. Baksi, S.N. and Kenny, A.D. 1978. Vitamin D metabolism i n Japanese q u a i l : gonadal hormones and dietary calcium e f f e c t s . Am J Physiol 234: E622-E628. Bar, A., Cohen, A., Edelstein, S., Shemesh, M., Montecuccoli, G. and Hurwitz, S. 1978. Involvement of c h o l e c a l c i f e r o l metabolism i n birds i n the adaptation of calcium absorption to the needs during reproduction. Comp Biochem Physiol 59B: 245-249. Bar, A. and Hurwitz, S. 1972. Relationship of duodenal calcium-binding protein to calcium absorption i n laying fowl. Comp Biochem Physiol 41B: 735-744, Bar, A. and Hurwitz, S. 1973. Uterine calcium-binding protein i n the laying fowl. Comp Biochem Physiol 45A: 579-586. Bar, A., Dubrov, D., Eisner, U. and Hurwitz, S. 1976. Calcium-binding protein and calcium absorption i n the laying q u a i l . Poult Sci 55: 622-628. 129 Bar, A. and Norman, A.W. 1981. Studies on the mode of action of c a l c i f e r o l . XXXIV. Relationship of the d i s t r i b u t i o n of 25-hydroxyvitamin D 3 metabolites to gonadal a c t i v i t y and egg s h e l l formation i n the q u a i l . Endocrinol 109: 950-955. Bar, A., Rosenberg, J . and Hurwitz, S. 1984. The lack of relationships between vitamin D 3 metabolites and calcium-binding protein i n the eggshell gland of laying birds. Comp Biochem Physiol 78B: 75-79. B l a i r , R. and Gil b e r t , A.B. 1973. The influence of supplemental phosphorus i n a low-calcium d i e t designed to induce a resting phase i n laying hens. B r i t Poult S c i 14: 1431-1435. Bedrak, E., Harvey, S. and Chadwick, A. 1981. Concentrations of p i t u i t a r y , gonadal and adrenal hormones i n serum of laying and broody white rock hens (Gallus domesticus). J Endocrinol 89: 197-204. Boass, A., Toverud, S.U., McCain, T.A., Pike, J.W. and Haussler, M.R. 1977. Elevated serum l e v e l s of 1, 25-dihydroxycholecalciferol i n l a c t a t i n g r a t s . Nature 267: 630-632. Bonney, R.C. and Cunningham, F.J. 1977. E f f e c t of i o n i c environment on the release of LH from chicken anterior p i t u i t a r y c e l l s . Mol C e l l Endocrinol 7: 245-251. Buckner, G.D. and Martin, J.H. 1920. E f f e c t of calcium on the composition of the eggs of laying hens. J B i o l Chem 41: 195-203. C a s t i l l o , L., Tanaka, Y., DeLuca, H.F. and Sunde, M.L. 1977. The stimulation of 2 5-hydroxyvitamin D3~la-hydroxylase by estrogen. Arch Biochem Biopys 179: 211-217. C a s t i l l o , L. , Tanaka, Y., Wineland, M.J., Jowsey, J.O. and DeLuca, H.F. 1979. Production of l,25(OH)2D3 and formation of medullary bone i n the laying hen. Endocrinol 104: 1598-1601. Chang, S.I., McGinnis, J . and Pubols, M.H. 1969. Influences of an anti-ovulatory compound on the expression of vitamin D deficiency signs i n the laying hen. Poult Sci 48: 154-159. Chen, T.C, C a s t i l l o , L. , Korycka-Dahl, M. and DeLuca, H.F. 1974. Role of vitmin D metabolites i n phosphate transport of rat i n t e s t i n e . J Nutr. 104: 1056-1060. 130 Clark, N.B. and Sasayama, Y. 1981. The rol e of parathyroid hormone on renal excretion of phosphate i n the Japanese q u a i l . Gen Comp Endocrinol 45: 234-241. Common, R.H., Rutledge, N.A. and Hale, R.W. 1948. Observations on the mineral metabolism of p u l l e t s . 8. The influence of gonadal hormones on the retention of calcium and phosphorus. J Agric S c i 38: 64-80. Concolino, G., Marocchi, A., Concolino, F., Sciarra , F., S i l v e r i o , F. and Conti, C. 1976. Human kidney s t e r o i d receptors. J Steroid Biochem 7: 831-834. Copp, D.H., 1969. Review: Endocrine control of calcium homeostasis. J Endocrinol 43: 137-161. Coty, W.A. 1980. A s p e c i f i c , high a f f i n i t y binding protein for la-25-dihydroxyvitamin D i n the chick oviduct s h e l l gland. Biochem Biophys Res Comm 93: 285-292. Dacke, C.G., Musacchia, X.J., Volkert, W.A. and Kenny., A.D. 1973. C y c l i c fluctuations i n the l e v e l s of blood calcium, pH and pC0 2 i n Japanese q u a i l . Comp Biochem Physiol 44A: 1267-1275. de Bernard, B., Stagni, N., Camerotto, R., V i t t u r , F., Zanetti, M., Zamboni Zallone, A and T e t i , A. 1980. Influence of calcium depletion on medullary bone of laying hens. C a l c i f Tissue Int 32: 221-228. DeLuca, H.F. 1980. Some new concepts emanating from a study of the metabolism and function of vitamin D. Nutr Rev 38: 169-182. Douglas, CR., Harms, R.H. and Wilson, H.R. 1972. The use of extremely low dietary calcium to a l t e r the production pattern of laying hens. Poult S c i 51: 2015-2020. Dukoh, S., Donaldson, CA., Marion, S.L., Pike, J.W. and Haussler, M.R. 1983. The ovary: a target for 1,25-dihydroxyvitamin D 3 . Endocrinol 112: 200-206. Edelstein, S.A., Ha r e l l , A., Bar, A. and Hurwitz, S. 1975. The functional metabolism of vitamin D i n chicks fed low calcium and low phosphorus d i e t s . Biochem Biophys Acta 385: 438-442. Fraser, D.R., and Kodicek, E. 1970. Unique biosynthesis by kidney of a b i o l o g i c a l l y active vitamin D metabolite. Nature 228: 764-766. 131 Friedlander, E.J., Henry, H.L. and Norman, A.W. 1977. Studies on the mode of action of c a l c i f e r o l . E f f e c t s of dietary calcium and phosphorus on the rel a t i o n s h i p between the 25-hydroxyvitamin D3-la-hydroxylase and production of chick i n t e s t i n a l calcium-binding protein. J B i o l Chem 252: 8677-8683. Furr, B.J.A. 1973. Radioimmunoassay of progesterone i n peripheral plasma of the domestic fowl i n various phy s i o l o g i c a l states i n f o l l i c u l a r venous plasma. Acta Endocrinol 72: 89-100. Garabedien, M., Holick, M.F., DeLuca, H.F. and Boyle, I . T . 1972. Control of 25-hydroxycholecalciferol metabolism by parathyroid glands. Proc Nat Acad S c i USA 69: 1673-1676. Garabedien, M., Tanaka, Y., Holick, M.F. and DeLuca, H.F. 1974. Response of i n t e s t i n a l calcium transport and bone calcium mobilization of l,25(OH)2D3 i n thyropara-thyroidectomized rats. Endocrinol 94: 1022-1027. Gi l b e r t , A.B. 1971. A review of the rol e of calcium i n regulating egg production i n the domestic fowl. Proc 14th World's Poultry Cong 4: 781-785. Gi l b e r t , A.B. 1983. Calcium and reproductive function i n the hen. Proc Nutr Soc 42: 195-212. G i l b e r t , A.B. and B l a i r , R. 1975. A comparison of the ef f e c t s of two low-calcium diets on egg production i n the domestic fowl. B r i t Poult Sci 16: 547-552. Graber, J.W. and Nalbandov, A.V. 1976. Peripheral estrogen l e v e l s during the laying cycle of the hen. B i o l Reprod 14: 109-14. H a l l , T.R., Harvey, S. and Chadwick, A. 1984. Oestradiol-17/3 modifies fowl p i t u i t a r y p r o l a c t i n and growth hormone secretion i n v i t r o . Gen Comp Endocrinol 56: 299-307. Hammond, R.W., Olson, D.M., Frenkel, R.B., B i e l l i e r , H.V. and Hertelendy, F. 1980. Prostaglandins and st e r o i d hormones i n plasma and ovarian f o l l i c l e s during the ovulation cycle of the domestic hen. Gen Comp Endocrinol 42: 195-202. Hart. L.E. and DeLuca, H.F. 1985. E f f e c t of vitamin D3 metabolites on calcium and phosphorus metabolism i n chick embryos. Am J Physiol 248: E281-285. 132 Hart, E.B., Steenbock, H., Lepkovsky, S., Kletzien, S.W.F., Halpen, J.G. and Johnson, O.N. 1925. The n u t r i t i o n a l requirement of the chick. V. Influence of u l t r a - v i o l e t l i g h t on the production, h a t c h a b i l i t y and f e r t i l i t y of the egg. J B i o l Chem 65: 579-595. Holick, M.F., Schnoes, H.K., DeLuca, H.F., Gray, R.W., Boyle, I.T. and Suda, T. 1972. Is o l a t i o n and i d e n t i f i c a t i o n of 24,25-Dihydroxycholecalciferol: a metabolite of vitamin D3 made i n the kidney. Biochem 11: 4251-4255. Holick, M.F., Schnoes, H.K., DeLuca, H.F., Suda, T. and Cousins, R.J. 1971. Is o l a t i o n and i d e n t i f i c a t i o n of 1,25-Dihydroxycholecalciferol: a metabolite of vitamin D active i n the in t e s t i n e . Biochem 10: 2799-2804. Hurwitz, S., Bar, A. and Cohen, I. 1973. Regulation of calcium absorption by fowl i n t e s t i n e . Am J Physiol 225: 150-154. Imai, K., Yamashita, K. and Nakajo, S. 1964. Gonadotrophin i n the anterior p i t u i t a r y and i n blood plasma of molting hens. Proc Annual Meetings of W.P.S.A.-Japan, Nov. 1964, pp. 34-36. Johnson, A.L. 1981. Comparison of three s e r i a l blood sampling techniques on plasma hormone concentrations i n the laying hen. Poult S c i 60: 2322-2327. Johnson, A.L. 1984. Interactions of progesterone and l u t e i n i s i n g hormone leading to ovulation i n the domestic hen. Reprod B i o l Poult 17: 133-143. Kamiyoshi, M. and Tanaka, K. 1972. Augmentative e f f e c t of f o l l i c l e stimulating hormone on LH-induced ovulation i n the hen. J Reprod Fert 29: 141-143. Kawashima, M., Kamiyoshi, M. and Tanaka, K. 1979. Cytoplasmic progesterone receptor concentration i n the hen hypothalamus and p i t u i t a r y : difference between laying and non-laying hens and changes during the ovulatory cycle. B i o l Reprod 20: 581-585. Kenny, A.D. 1976. Vitamin D metabolism: physiological regulation i n egg laying Japanese q u a i l . Am J Physiol 230: 1609-1615. Leszczynski, D.E., Hagan, R.C, Bitgood, J . J . and Kummerow, F.A. 1985. Relationship of plasma e s t r a d i o l and progesterone l e v e l s i n egg productivity i n domestic chickens. Poult S ci 64: 545-549. 133 Leszczynski, D.E., Pikul, J . and Kummerow, F.A. 1983. Relationship of c i r c u l a t i n g estrogen and progesterone to plasma l i p i d s and egg production i n chicken hens. Poult S c i 62: 1457 (Abstr). Luck, M.R. and Scanes, C.G. 1978. Gonadotrophin secretion i n the domestic fowl during calcium deficiency. Gen Comp Endocrinol 34: 80-81. Luck, M.R. and Scanes, C.G. 1979a. The re l a t i o n s h i p between reproductive a c t i v i t y and blood calcium i n the calcium-d e f i c i e n t hen. Br Poult S c i 20: 559-564. Luck, M.R. and Scanes, C.G. 1979b. Plasma l e v e l s of ionized calcium i n the laying hen (Gallus domesticus). Comp Biochem Physiol 63A: 177-181. Luck, M.R. and Scanes, C.G. 198 0a. Ionic and endocrine factors influencing the secretion of l u t e n i z i n g hormone by chicken anterior p i t u i t a r y c e l l s i n v i t r o . Gen Comp Endocrinol 41: 260-265. Luck, M.R. and Scanes, C.G. 1980b. The e f f e c t of egg-shell c a l c i f i c a t i o n on the response of plasma calcium a c t i v i t y to parathyroid hormone and c a l c i t o n i n i n the domestic fowl (Gallus domesticus). Comp Biochem Physiol 65A: 151-154. Lund, J . and DeLuca, H.F. 1966. B i o l o g i c a l l y active metabolite of vitamin D from bone, l i v e r and blood serum. J L i p i d Res 7: 739-744. Martindale, L. 1969. Phosphate excretion i n the laying hen. J Physiol 203: 82-83. Mueller, G.L., Anast, C S . and Breitenbach, R.P. 1970. Dietary calcium and ultimobranchial body and parathyroid gland i n the chicken. Am J Physiol 218: 1718-1722. Nakajo, S. and Imai, K. 1957. Studies on the gonadotrophin i n the anterior p i t u i t a r y i n domestic fowl. I I . Gonadotrophin content i n hens at various stages of reproduction. Jap J Anim Reprod 3: 49-51. Nys, Y. and De Laage, X. 1984. Ef f e c t s of suppression of eggshell c a l c i f i c a t i o n and of 1,25(0H)2D3 on Mg 2 +, C a 2 + and Mg2+HC03~ ATPase, a l k a l i n e phosphatase, carbonic anhydrase and CaBP l e v e l s . I. The laying hen uterus. Comp Biochem Physiol 78A: 833-838. Norman, A.W. 1968. The mode of action of vitamin D. B i o l Rev 43: 97-137. 134 Norman, A.W. 1974. The hormone-like action of 1,25(OH)2cholecalciferol (a metabolite of the f a t soluble vitamin D) i n the in t e s t i n e . Vitam Horm 32: 325-384. Onagebesan, O.M. and Peddie, M.J. 1989. Calcium-dependent stimulation of estrogen secretion by FSH from theca c e l l s of the domestic hen (Gallus domesticus). Gen Comp Endocrinol 75: 177-186. Opel, H. and Nalbandov, A.V. 1961. F o l l i c u l a r growth and ovulation i n hypophysectomized hens. Endocrinol 69: 1016-1028. Opel, H. and Proudman, J.A. 1984. Two methods for s e r i a l blood sampling from unrestrained, undisturbed turkeys with notes on the e f f e c t s of acute stressors on plasma l e v e l s of p r o l a c t i n . Poult S c i 63: 1644-1652. Parsons, A.H. and Combs, G.F. 1981. Blood ionized calcium cycles i n the chicken. Poult S c i 60: 1520-1522. Paulson, S.K. and Kenny, A.D. 1985. Survey of vitamin D metabolite l e v e l s during growth and development of Japanese q u a i l . Poult S c i 64: 2004-2006. Proudman, J.A. and Opel, H. 1989. Daily changes i n plasma p r o l a c t i n , corticosterone, and l u t e i n i z i n g hormone i n the unrestrained, ovariectomized turkey hen. Poult S c i 68: 177-184. Rawlings, N.C., Jeffcoate, I.A. and Reiger, D.L. 1984. The influence of estradiol-17/3 and progesterone on peripheral serum concentrations of l u t e i n i z i n g hormone and f o l l i c l e stimulating hormone i n the ovariectomized ewe. Theriogenol 22: 473-488. Riddle, O. 1942. C y c l i c changes i n blood calcium, phosphorus and f a t i n r e l a t i o n to egg laying and estrogen production. Endocrinol 63: 177-181. Riddle, O. and Reinhart, W.H. 1926. Studies on the physiological reproduction i n birds. XXI. Blood calcium changes i n the reproductive cycle. Am J Physiol 76: 660-666. Roland, D.A., Sr., Sloan, D.R., Wilson, H.R. and Harms, R.H. 1973. Influence of dietary calcium deficiency on yolk and serum calcium, yolk and organ weights and other selected production c r i t e r i a of the p u l l e t . Poult S c i 52: 2220-2225. 135 Roland, D.A., Sr., Sloan, D.R., Wilson, H.R. and Harms, R.H. 1974. Relationship of calcium to reproductive abnormalities i n the laying hen (Gallus domesticus). J Nutr 104: 1079-1085. Rosenberg, J . , Hurwitz, S. and Bar, A. 1986. Regulation of kidney calcium-binding protein i n the b i r d (Gallus  domesticus). Comp Biochem Physiol 83A: 227-281. Scanes, C.G., Sharp, P.J. and Chadwick, A. 1977. Changes i n plasma p r o l a c t i n concentration during the ovulatory cycle of the chicken. J Endocrinol 72: 401-402. Sendrani, S.H., Taylor, T.G. and Akhtar, M. 1981. The regulation of 1,25(0H)2D3 i n the kidney of Japanese quail by sex hormones and parathyroid hormone. Gen Comp Endocrinol 44: 514-523. Senior, B.E. 1974. Changes i n the concentration of estrone and e s t r a d i o l i n the peripheral plasma of the domestic hen during the ovulatory cycle. Acta Endocrinol 77: 588-596. Shen, H., Summers, J.D. and Leeson, S. 1981. Egg production and s h e l l q u a l i t y of layers fed various l e v e l s of vitamin D 3. Poult S c i 60: 1485-1490. Simkiss, K. 1975. Calcium and avian reproduction. Symp zool Soc Lond 35: 307-337. Singh, R., Joyner, C.J., Peddie, M.J. and Taylor, T.G. 1986. Changes i n the concentrations of parathyroid hormone and io n i c calcium i n the plasma of laying hens during the egg cycle i n r e l a t i o n to dietary d e f i c i e n c i e s of calcium and vitamin D. Gen Comp Endocrinol 61: 20-28. Soares, J.H., J r . 1984. Calcium metabolism and i t s c o n t r o l -a Review. Poult S c i 63:2075-2083. Soares, J.H., J r . , Ottinger, M.A., Buss, E.G. and Kaetzel, D.M., J r . 1980. Plasma concentrations of 1,25(0H)2D3, e s t r a d i o l and calcium i n laying hens selected for d i f f e r e n t i a l egg s h e l l q uality. Poult S c i 59: 1663 (Abstr). Sommerville, B.A., Swaminathan, R. and Care, A.D. 1978. A comparison of the ef f e c t s of dietary calcium and phosphorus deficiency on the i n v i t r o and i n vivo metabolism of 25-hydroxycholecalciferol i n the chick. Br J Nutr 39: 411-414. 136 Spanos, E., Pike, J.W., Haussler, M.R., Colston, K.W. and Evans, I.M.A. 1976. C i r c u l a t i n g l,25(OH)2D3 i n the chicken: enhancement by i n j e c t i o n of p r o l a c t i n and during egg laying. L i f e S c i 19: 1751-1756. Spencer, R. Charman, M., Wilson, P. and Lawson, D.E.M. 1978. The r e l a t i o n s h i p between vitamin D-stimulated calcium transport and i n t e s t i n a l calcium-binding protein i n the chicken. Biochem J 170: 93-101. Taher, A.I., Gleaves, E.W. and Beck, M.M. 1986. E f f e c t of s e r i a l blood sampling on interpretation of estradiol-17/3 involvement i n laying hen calcium metabolism. Comp Biochem Physiol 84A: 715-718. Takahashi, N., Abe, E., Tanabe, R. and Suda, T. 1980. A h i g h - a f f i n i t y cytosol binding protein f o r la-25-dihydroxycholecalciferol i n the uterus of Japanese q u a i l . Biochem J 190: 513-518. Takahashi, N., Shinki, T., Abe, E., Horiuchi, N., Yamaguchi, A., Yoshiki, S. and Suda, T. 1983. The r o l e of vitamin D i n the medullary bone formation i n egg-laying Japanese qua i l and i n immature male chicks treated with sex hormones. C a l c i f Tissue Int 35: 465-471. Tanaka, Y., C a s t i l l o , L. and DeLuca, H.F. 1976. Control of renal vitamin D hydroxylase i n birds by sex hormones. Proc Natl Acad Sci 73: 2701-2705. Tanaka, Y. and DeLuca, H.F. 1973. The control of 25-hydroxyvitamin D metabolism by inorganic phosphorus. Arch Biochem Biophys 154: 566-574. Tanaka, K., Kamiyoshi, M. and Sakaida, M. 1974. E f f e c t s of progesterone on the hypothalamic gonadotrophin-releasing a c t i v i t y and on the p i t u i t a r y gonadotrophic a c t i v i t y i n hens and cocks. Poult Sci 53: 1772-1776. Taylor, A.N. and Wasserman, R.H. 1972. Vitamin D-induced calcium-binding protein: comparative aspects i n kidney and i n t e s t i n e . Am J Physiol 223: 110-114. Taylor, T.G. 1965. Calcium-endocrine relationships i n the laying hen. Proc Nutr Soc 24: 49-54. Taylor, T.G. and Belanger, L.F. 1969. The mechanism of bone resorption i n laying hens. C a l c i f Tissue Res 4: 162-173. Taylor, T.G., Morris, T.R. and Hertendy, F. 1962. The e f f e c t of p i t u i t a r y hormones on ovulation i n calcium-deficient p u l l e t s . Vet Rec 74: 123-125. 137 Thaeler, D.A. 1979. The egg-laying b i r d : Major physiological changes i n calcium associated with oestrogen control. World Poult S c i J 35: 32-42. Tsang, B.K. and Carnegie, J.A. 1984. Calcium-dependent regulation of progesterone production by is o l a t e d rat granulosa c e l l s : E f f e c t s of the calcium ionophore A23187, prostaglandin E 2 , dl-Isoproterenol and cholera toxin. B i o l Reprod 30: 787-794. Tsang, C.P.W. and Grunder, A.A. 1984. Ef f e c t s of vitamin D3 deficiency on estradiol-17/3 metabolism i n the laying hen. Endocrinol 115: 2170-2175. Tsang, C.P.W., Grunder, A.A., Soares, H. and Narbaitz, R. 1988. E f f e c t s of c h o l e c a l c i f e r o l or calcium metabolism i n the laying hen. Br Poult S c i 29: 753-759. Tucker, G., Gagnor, R.E. and Haussler, M.R. 1973. Vitamin D-25-hydroxylase: tissue occurrence and apparent lack of regulation. Arch Biochem Biophys 155: 47-57. van de Velde, J-P., Loveridge, N. and Vermeiden, J . 1984. Parathyroid hormone responses to calcium stresses during eggshell c a l c i f i c a t i o n . Endocrinol 115: 1901-1904. Vohra, P., Siopes, T.D. and Wilson, W.C 1979. Egg production and body weight changes of Japanese qu a i l and leghorn hens following deprivation of either supplementary calcium or vitamin D 3 . Poult S c i 58: 432-440. Wakabayashi, K., Kamberi, A.I. and McCann, S.H. 1969. In v i t r o response of the rat p i t u i t a r y to gonadotrophin-releasing factors and ions. Endocrinol 85: 1046-1056. Wasserman, R.H. and Corradino, R.A. 1971. Metabolic r o l e of vitamins A and D. Ann Rev Biochem 40: 501-532. White, J.M. and Etches, R.J. 1984. The e f f e c t of s e r i a l removal of blood on plasma concentrations of l u t e i n i z i n g hormone during the ovulatory cycle of the hen. Poult S c i 63: 822-824. Wilson, S.C. and Sharp, P.J. 1976. Induction of LH release by gonadal steroids i n the ovariectomized domestic hen. J Endocrinol 71: 87-98. 138 

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