UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Studies on egg shell calcification Grant, Elisabeth Anne 1972

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1972_A6_7 G73.pdf [ 5.19MB ]
Metadata
JSON: 831-1.0101928.json
JSON-LD: 831-1.0101928-ld.json
RDF/XML (Pretty): 831-1.0101928-rdf.xml
RDF/JSON: 831-1.0101928-rdf.json
Turtle: 831-1.0101928-turtle.txt
N-Triples: 831-1.0101928-rdf-ntriples.txt
Original Record: 831-1.0101928-source.json
Full Text
831-1.0101928-fulltext.txt
Citation
831-1.0101928.ris

Full Text

S T U D I E S ON E G G S H E L L C A L C I F I C A T I O N BY E L I S A B E T H ANNE GRANT B . S c , MCMASTER U N I V E R S I T Y , 1968 T H E S I S SUBMITTED IN P A R T I A L F U L F I L M E N T T H E REQUIREMENTS FOR THE DEGREE OF MASTER OF S C I E N C E IN THE DEPARTMENT OF PHYSIOLOGY WE ACCEPT THIS T H E S I S AS CONFORMING TO THE REQUIRED STANDARD T H E U N I V E R S I T Y OF B R I T I S H COLUMBIA F E B R U A R Y , 1972 In presenting t h i s thesis i n p a r t i a l f ulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t freely available for reference and study. I further agree that permission for extensive copying of th i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of PKYS ( .OL06-V The University of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT. THE TRANSPORT OF CALCIUM FROM THE BLOOD OF THE L A Y I N G HEN ( G A L L U S OOMESTICUS) TO THE SHELL OF THE D E V E L O P I N G EGG IS A VERY RAPID BUT DISCONTINUOUS P R O C E S S . THESE S T U O I E S WERE CARRIED OUT IN ORDER TO EXAMINE THE WAY IN WHICH THIS PROCESS IS C O N T R O L L E D . IN THE I N I T I A L EXPERIMENTS CALCIUM TRANSLOCATION ACROSS THE SHELL GLAND WAS STUDIED USING AN IN VITRO SYSTEM S IMILAR TO THAT DESCRIBED BY EHRENSPECK EJ_ AL ( 1 9 6 7 ) . THE MAGNITUDE OF THE OBSERVED F L U X E S WAS S IMILAR TO THOSE REPORTED BY EHRENSPECK EJ_ A_L, B U T , IN CONTRAST TO THEIR R E S U L T S , NO CLEAR CORRELAT ION COULD BE FOUND BETWEEN THE FLUX AND THE P H Y S I O L O G I C -AL CONDITION OF THE S H E L L GLAND. THE TWO HORMONES OF CALCIUM H O M E O S T A S I S , PARATHYROID HORMONE AND C A L C I T O N I N , WERE ALSO WITHOUT INFLUENCE ON THE S Y S T E M . HOWEVER THE DIBUTYRYL D E R I V A T I V E OF C Y C L I C AMP INCREASED THE CALCIUM FLUX FROM THE SHELL GLAND LUMEN TO THE BLOOD BUT OIO NOT A F F E C T FLUX IN THE NORMAL P H Y S I O L O G I C A L D I R E C T I O N , I . E . BLOOD - * LUMEN. ONE FUNDAMENTAL C R I T I C I S M OF THE IN VITRO SYSTEM IS THAT THE F L U X E S MEASUREO ARE EXTREMELY LOW. C A L C U L A T I O N OF THE FLUX WHICH MUST OCCUR IN VIVO SHOWED THAT THIS WAS 5 0 0 - 1 0 0 0 T IMES LARGER THAN THE F L U X E S OBTAINED IN V I T R O . IN VIEW OF THE D E F I C I E N C I E S OF THE IN VITRO SYSTEM A L L SUBSEQUENT S T U D I E S WERE CARRIED OUT ON THE INTACT H E N . TWO OF THE MOST L I K E L Y MECHANISMS FOR CONTROL OF S H E L L GLAND FUNCTION INVOLVE E I T H E R THE ENDOCRINE S Y S T E M , WHICH THE I I IN VITRO S T U O I E S IMPL IED WAS NOT I N V O L V E D , OR THE AUTONOMIC NERVOUS S Y S T E M . THE NEURAL INFLUENCE UPON SHELL FORMATION WAS STUOIED USING NEURAL BLOCKING AGENTS AS THE COMPLEX ANATOMY OF THE SHELL GLAND NERVOUS SUPPLY PRECLUDED NEURAL S E C T I O N . AN E X T E N S I V E L I T E R A T U R E SURVEY F A I L E D TO PROVIDE S U F F I C I E N T DATA ON BLOCKING DRUGS IN CHICKENS SO THAT AN E X T E N S I V E S E R I E S OF EXPERIMENTS WAS PERFORMED IN ORDER TO D E F I N E A P P R O P R I A T E DOSES TO PROOUCE 70-80$ BLOCKADE IN THE ANESTHATI ZED HEN FOR PERIODS UP TO TWO HOURS. U S I N G THE DRUG DOSAGES SO OBTAINED THE E F F E C T OF BLOCKAOE OF VARIOUS BRANCHES OF THE AUTONOMIC NERVOUS SYSTEM ON THE INCORPORATION OF INTRAVENOUSLY I N J E C T E D 45CA INTO THE EGG S H E L L , AND ON THE D I S A P P E A R A N C E OF THIS L A B E L FROM THE P L A S M A , WAS STUDIED IN A N E S T H A T I Z E D H E N S . THE DATA FROM BOTH T Y P E S OF EXPERIMENT SHOWED T H A T , UNDER THE E X P E R I M E N T A L C O N D I T I O N S , THE RATE OF CALCIUM TRANSPORT ACROSS THE SHELL GLAND IS INDEPENDENT OF THE NERVOUS S U P P L Y . I I I T A 3 L E O F C O N T E N T S P A G E G E N E R A L I N T R O D U C T I O N 1 C H A P T E R I : C A L C I U M T R A N S P O R T B Y T H E S H E L L G L A N D _m V I T R O . I N T R O D U C T I O N 15 M E T H O D S 17 R E S U L T S A N D D I S C U S S I O N . . . . . . 20 C H A P T E R I I : O B S E R V A T I O N S O N T H E E F F E C T O F C E R T A I N D R U G S U P O N T H E A N E S T H A T I Z E D C H I C K E N . I N T R O D U C T I O N 37 M E T H O D S 4-6 R E S U L T S A N D D I S C U S S I O N 49 S Y N O P S I S 60 C H A P T E R Ml : C A L C I U M T R A N S P O R T B Y T H E S H E L L G L A N D J_N V I V O . I N T R O D U C T I O N 61 M E T H O O S 64 R E S U L T S A N D D I S C U S S I O N 67 A P P E N D I X : A D E S C R I P T I O N O F HOW A U N I F O R M P O P U L A T I O N O F H E N S WAS O B T A I N E D WHOSE E G G S W E R E I N T H E L I N E A R P O R T I O N O F E G G S H E L L C A L C I F I -C A T I O N 91 3 I B L I O G R A P H Y 79 I V L I S T O F T A B L E S FAC I NG PAGE T A B L E I THE COMPOSIT ION OF S O L U T I O N S USED IN THE I N Vi TRO S H E L L GLAND E X P E R I M E N T S 17 T A B L E II SUMMARY OF THE IN V ITRO E X P E R I M E N T A L ARRANGEMENTS 19 T A B L E I I I 45CA F L U X E S IN THE J_N Vi TRO S H E L L GLAND SYSTEM WITH T I S S U E O B T A I N E D FROM HENS IN VARIOUS P H Y S I O L O G I C A L C O N D I T I O N S . 20 T A B L E IV T H E E F F E C T OF MAGNESIUM CHLORIDE AND GLUCOSE ON THE J_N VITRO 45CA F L U X E S ACROSS A S H E L L GLAND E X C I S E D DURING A C T I V E EGG S H E L L C A L -C I F I C A T I O N 24 T A B L E V THE E F F E C T OF CHANGING THE INCUBATION BUFFER ANO AERAT ION GAS ON _I_N VI TRO 45CA F L U X E S IN THE S H E L L GLAND 26 T A 3 L E VI THE E F F E C T OF P T H AND C T ON THE INFLUX ( 3 L O O D - » LUMEN) OF 45CA IN THE S H E L L GLAND I N V ITRO P R E P A R A T I O N „ „ „ 0 * = = * » = o , o o o . . . 27 T A B L E V I I THE E F F E C T OF D IBUTYRYL C Y C L I C AMP ON 45CA F L U X E S IN THE J_N Vi TRO S H E L L GLAND 32 T A B L E V I I I CHEMICALS USED IN THE DRUG E X P E R I M E N T S 46 T A 3 L E IX T A B L E OF AGONIST DRUGS USED TO C H A L L E N G E BLOCKADE 47 T A B L E X A SUMMARY OF ALL THE DRUG E X P E R I M E N T S . THE E F F E C T OF D I F F E R E N T DOSES OF BLOCKING DRUGS ON THE R E S P O N S E S TO VARIOUS AGONISTS 50 T A B L E XI A SUMMARY OF THE CARDIOVASCULAR R E S P O N S E S IN THE PROPRANALOL E X P E R I M E N T S 55 T A B L E X I I THE E F F E C T OF VARIOUS P H Y S I O L O G I C A L S T A T E S OF THE HEN ON THE H A L F L I V E S OF THE E X P O -NENTIAL COMPONENTS C A L C U L A T E D FROM THE A R T E R I A L 45CA D I S A P P E A R A N C E CURVE 70 T A B L E X I I I THE E F F E C T OF BLOCKAOE OF VARIOUS BRANCHES OF THE AUTONOMIC NERVOUS SYSTEM ON THE I N -CORPORATION OF 45CA INTO THE EGG S H E L L 76 T A B L E X I V THE E F F E C T OF VARIOUS NEURAL BLOCKING DRUGS ON THE H A L F L I V E S OF THE E X P O N E N T I A L C O M -PONENTS C A L C U L A T E D FROM THE A R T E R I A L PLASMA 45CA D I S A P P E A R A N C E CURVE IN HENS A C T I V E L Y C A L C I F Y I N G AN EGG 77 V T A B L E XV A SUMMARY OF THE MEAN HALF L I V E S OF THE THREE E X P O N E N T I A L COMPONENTS C A L C U L A T E D FROM THE A R T E R I A L PLASMA 4 5 C A D I S A P P E A R -ANCE CURVE IN HENS UNDER VARIOUS C O N -DI T I ONS , F A C I N G PAGE 7 8 T A B L E XV I SUMMARY OF THE T I M I N G OF HENS ON A R E V E R S E D L I G H T FROM 7S30PM TO 9 S 3 0 A M ) . . , O v I P O S I T I ON 8Y SCHEOULE (L I G H T 93 VI L I S T OF F I G U R E S F A C I N G PAGE F IGURE 1 . THE APPARATUS USED IN THE _^N VITRO S H E L L GLAND EXPERIMENTS 1 8 F IGURE 2 . A GRAPH SHOWING NET E F F L U X OF 45CA (LUMEN -> BLOOD) IN A SHELL GLAND E X C I S E D DURING A C T I V E SHELL C A L C I F I C A T I O N . L A B E L L E D CALCIUM WAS PRESENT ON BOTH S I D E S OF THE MEMBRANE 2 2 F IGURE 3 . E F F L U X ( L U M E N S BLOOD) OF 45CA IN A S H E L L GLAND PREPARATION TAKEN DURING A C T I V E SHELL C A L C I F I C A T I O N . (THE MATERIAL WAS FROM THE SAME GLAND AS THAT USED IN F I GURE 2 . ) 2 3 F IGURE 4 . THE E F F E C T OF ADDIT ION OF MAGNESIUM CHLORIDE AND GLUCOSE TO THE INCUBATION MEDIUM ON 45CA F L U X E S IN A SHELL GLAND E X C I S E D DURING A C T I V E EGG SHELL C A L -C I F I CAT I ON 2 5 F I G U R E . 5 . THE E F F E C T OF PTH ON 45CA INFLUX (BLOOD -> LUMEN) IN A SHELL GLAND E X C I S E D JUST A F T E R THE EGG HAD BEEN L A I D 2 8 F IGURE 6 . THE E F F E C T OF PTH AND C T ON CALCIUM INFLUX (BLOOD -* LUMEN) IN A SHELL GL ANO E X C I S E D DURING A C T I V E EGG SHELL C A L -C I F I C A T I O N 2 9 F IGURE 7 . THE E F F E C T OF DIBUTYRYL C Y C L I C AMP ON 45CA F L U X E S IN A SHELL GLANO E X C I S E D WHILE THE EGG WAS IN THE ISTHMUS 3 0 FIGURE 8 . L O G - D O S E RESPONSE CURVES TO P H E N Y L E P H R I N E ( T O P ) AND ISOPROTERENOL (BOTTOM) 4 9 F IGURE 9 . THE E F F E C T OF T IME ON ^ S L O C K A D E OF THE HYPOTENSIVE RESPONSE TO A C E T Y L C H O L I N E BY A T R O P I N E . ( T O P S S I N G L E D O S E S , BOTTOM t CONTINUOUS INTRAVENOUS I N F U S I O N ) . 51 F IGURE 1 0 . THE E F F E C T OF TIME ON ^ B L O C K A D E OF THE H Y P E R T E N S I V E RESPONSE TO NOREPINEPHRINE ( T O P ) OR P H E N Y L E P H R I N E (BOTTOM) BY S I N G L E DOSES OF D I 8 E N Z Y L I N E 52 VII F l G U R E 1 1 . F IGURE 1 2 . F IGURE 1 3 . F IGURE 14 . F i G U R E 1 5 . F IGURE 1 6 . F IGURE 1 7 . F IGURE 1 8 . THE E F F E C T OF A S INGLE DOSE OF P R O -PRANALOL ON THE 8 L 0 0 0 PRESSURE ( L E F T ) AND HEART RATE ( R I G H T ) OF AN ANESTHATI ZED CHICKEN F A C I N G PAGE 56 THE E F F E C T OF A CONTINUOUS INTRAVENOUS INFUSION OF PROPRANALOL ON THE BLOOD PRESSURE ( L E F T ) AND HEART RATE ( R I G H T ) OF AN ANESTHAT I ZED H E N . , 57 L E F T : - THE E F F E C T OF HEART RATE ON THE HEART RATE RESPONSE TO ISOPROTERENOL IN AN ANESTHATI ZED C H I C K E N . R I G H T : - THE E F F E C T OF TIME ON ^BLOCKADE OF THE HEART RATE RESPONSE TO I S O P R O -TERENOL BY A CONTINUOUS INTRAVENOUS INFUSION OF PROPRANALOL IN THE SAME C H I C K E N . . . . A GRAPH TO SHOW THE TIME COURSE OF INCORPORATION OF 45CA INTO THE EGG S H E L L . . 58 67 GRAPH TO SHOW THE RATE OF D I S A P P E A R A N C E OF 45CA TRACER FROM THE A R T E R I A L PLASMA OF A HEN WHICH IS A C T I V E L Y C A L C I F Y I N G AN EGG 68 GRAPH TO SHOW THE RATE OF D I S A P P E A R A N C E OF 45CA FROM THE A R T E R I A L PLASMA OF A HEN WHICH IS OUT OF LAY 69 A DIAGRAMMATIC R E P R E S E N T A T I O N OF THE P O S S I B L E FATE OF INTRAVENOUSLY I N J E C T E D 45CA , SHOWING P H Y S I O L O G I C A L COMPARTMENTS WHICH COULD BE INVOLVED 71 DIAGRAM OF THE C I R C U I T USED TO MONITOR THE TIME OF O V I P O S I T I O N IN THE E X P E R I M E N T A L HENS 92 VIII ACKNOWLEDGEMENTS I AM VERY GRATEFUL TO DR. C O . pARKES FOR HIS ENCOURAGE-MENT IN TH IS PROJECT AND ALSO FOR HIS HAVING KEPT THE F A I T H THROUGHOUT ITS MANY MEANDER IN G S » I WOULD ALSO L I K E TO THANK DR. J . R . LEDSOME AND DR. F . L L O Y FOR THEIR H E L P F U L D I S C U S S I O N S ON THE DRUG E X P E R I M E N T S , S P E C I A L THANKS ARE EXTENDED TO MR. KURT HENZE WHO PREPARED ALL THE OIAGRAMS, AND TO MLSS DLANE HARTMAN WHO TYPED THE M A N U S C R I P T . IX " E G G S A S P E R A T I N G E G G S P E R I M E N T S " X GENERAL INTRODUCTION. IN THE FEMALE DOMESTIC CHICKEN ( G A L L U S D O M E S T I C A ) ONLY THE L E F T OVARY AND OVIOUCT BECOME F U N C T I O N A L , WHILE THE RIGHT ONES A T R O P Y , SO THAT ONLY ONE EGG CAN BE PRODUCED AT A T I M E . THERE IS NO READY E X P L A N A T I O N FOR THIS PHENOMENON. TAYLOR ( 1 9 7 0 A ) WROTE "ONE CAN ONLY S P E C U L A T E ON THE EVOLUTIONARY REASON FOR THE D ISAPPEARANCE OF THE RIGHT OVARY AND O V I D U C T . A REASONABLE GUESS IS THAT TWO OVARIES WERE DISADVANTAGEOUS BECAUSE OF THE PROBLEM OF PROVIDING ENOUGH CALCIUM FOR THE S H E L L S OF TWO EGGS AT O N C E 1 ' . , P O S T - E M B R Y O N I C DEVELOPMENT OF THE OVUM CAN BE O I V I D E O INTO THREE S T A G E S . THE TWO MAJOR EVENTS WHICH OCCUR DURING THE LONG I N I T I A L PHASE ARE A SLOW D E P O S I T I O N OF YOLK (MAINLY NEUTRAL F A T S ) , WHICH MAY CONTINUE FOR MANY MONTHS, AND FORMATION OF THE F O L L I C L E AROUND THE V I T E L L I N E MEMBRANE. DURING THE FOLLOWING 6 0 DAYS THE YOLK P R O T E I N S ARE L A I O OOWN IN VACUOLES WITHIN THE OVUM. THE D E V E L O P I N G F O L L I C L E IS NOT THE S I T E OF YOLK FORMATION. YOLK PRECURSORS ARE SYNTHES I SED IN THE L I V E R ANO TRANSPORTED TO THE OVARY IN THE P L A S M A . BECAUSE OF TH IS THE BLOOD OF THE L A Y I N G HEN IS C H A R A C T E R I Z E D BY A HIGH CONTENT OF L I P I D , FREE FATTY A C I D S AND P H 0 S P H O P R 0 T E I N , G I L B E R T ( 1 9 6 7 ) . ABOUT 9 - 1 4 DAYS BEFORE O V U L A T I O N , THE D E P O S I T I O N OF P H O S P H O L I P I D S C A U S E S THE OVUM TO ENLARGE R A P I O L Y , - 2 -SO T H A T , T Y P I C A L L Y T H E W E I G H T R I S E S FROM 1 G TO 1 8 G . C H A R A C T E R I S T I C OF T H I S P E R I O D I S A H I G H P L A S M A C A L C I U M L E V E L W H I C H O C C U R S I N R E S P O N S E TO E S T R O G E N , U R I S T ( 1 9 5 9 ) • T H I S E L E V A T E O P L A S M A C A L C I U M I S N O T I N V O L V E O I N EGG S H E L L C A L C I F I C A T I O N AS I T O C C U R S I N M A N Y V E R T E B R A T E S W H I C H L A Y Y O L K Y E G G S W I T H U N C A L C I F I E D S H E L L S . S L M K I S S ( 1 9 6 1 ) S U G G E S T E D T H A T I T I S C O N C E R N E D W I T H T H E T R A N S P O R T O F Y O L K P R E C U R S O R S A N D T H A T T H E P H 0 S P H 0 P R 0 T E I N A N D L I P O P R O T E I N C O M P O N E N T S OF T H E Y O L K A R E S O L U B L E I N P L A S M A O N L Y AS L O N G AS T H E R E I S S U F F I C I E N T C A L C I U M P R E S E N T . T H E G O N A D O T R O P I N D I R E C T L Y R E S P O N S I B L E F O R O V U L A T I O N I S A P P A R E N T L Y L E U T I N I Z I N G H O R M O N E (LH), F R A P S ( 1 9 5 5 ) . T H E M A T U R E OVUM I S S H E D FROM T H E F O L L I C L E BY R U P T U R E OF T H E S T I G M A , B U T T H E P R E C I S E E V E N T S L E A D I N G UP TO T H E D I S R U P T I O N OF T H E T I S S U E S I N R E S P O N S E TO L H H A V E N O T Y E T B E E N E L U C I D A T E D , G I L B E R T ( 1 9 6 7 ) . T H E O V I D U C T , W H I C H C O N D U C T S T H E R E L E A S E D OVUM TO T H E E X T E R I O R , CAN SE D I V I D E D I N T O F I V E R E G I O N S ; T H E I N F U N D I B U L U M J, M A G N U M , I S T H M U S , S H E L L G L A N D ( U T E R U S ) A N D V A G I N A . P A S S A G E O F T H E EGG DOWN T H E O V I D U C T T A K E S A P P R O X I M A T E L Y 2 5 H O U R S . T H E OVUM I S E N G U L F E D B Y T H E F U N N E L - L I K E I N F U N O I B U L U M V E R Y SOON A F T E R O V U L A T I O N , A N D S P E N D S A B O U T 3 0 M I N I N T H I S R E G I O N . IT I S H E R E T H A T T H E OVUM I S F E R T I L I Z E D I N AN I N S E M I N A T E D H E N . T H E OVUM P A S S E S I N T O T H E M A G N U M WHERE T H E E G G - W H I T E P R O T E I N S A R E L A I D D O W N . IT R E M A I N S I N T H E MAGNUM AN A V E R A G E OF A L M O S T T H R E E H O U R S , SO T H A T , A L T H O U G H T H E M A G N U M I S T H E L O N G E S T P A R T O F T H E O V I O U C T , T H E EGG T R A V E R S E S T H I S P O R T I O N R E L A T I V E L Y Q U I C K L Y . T H E P E R I S T A L T I C M O V E M E N T S OF T H E M A G N U M F O R C E T H E -3-OVUM INTO THE ISTHMUS WHERE, DURING THE NEXT 7 5 M I N , THE S H E L L MEMBRANES ARE L A I D OOWN. THE REGION FOLLOWING THE ISTHMUS IS THE S H E L L GLAND. HERE THE EGG REMAINS FOR ABOUT 2 0 HOURS OURING WHICH TIME IT IS ENCLOSED IN A C A L C I F I E D S H E L L , WARREN & SCOTT ( 1 9 3 5 ) . THUS THE EGG SPENDS SOME 8 0 $ OF THE T IME OF EGG FORMATION IN THE SHELL GLAND. DURING ITS F I R S T F I V E HOURS IN THE SHELL GLAND THE EGG IMBIBES WATER SO THAT THE WEIGHT OF ITS A L8U M E N APPROXIMATELY D O U B L E S . T H I S PROCESS HAS BEEN C A L L E D " P L U M P I N G " , BURMESTER ( 1 9 4 0 ) . THE RATE OF CALCIUM D E P O S I T I O N IS I N I T I A L L Y LOW, BUT INCREASES GRADUALLY DURING THE F I R S T FEW HOURS OF THE E G G ' S STAY IN THE U T E R U S . FROM ABOUT THE F I F T H OR S I X T H HOUR CALCIUM D E P O S I T I O N PROCEEDS AT A F A I R L Y CONSTANT RATE UNT IL JUST BEFORE THE EGG IS L A I D , BURMESTER E_T M . ( I 9 3 9 ) F B R A D F I E L D ( 1 9 5 1 ) . THUS THERE IS A O I S C R E T E BEGINNING TO S H E L L C A L C I F I C A T I O N A F T E R THE " P L U M P I N G " STAGE AND A POINT AT WHICH C A L C I F I C A T I O N C E A S E S , ABOUT 1 - 2 HOURS BEFORE THE EGG IS L A I D . IN A L A Y I N G HEN THE E S T R O G E N S , S E C R E T E D BY THE OVARIAN F O L L I C L E S , A C T I N G IN SYNERGISM WITH ANDROGENS, INDUCE THE FORMA-TION OF MEDULLARY BONE IN THE MARROW C A V I T I E S OF MOST BONES IN THE S K E L E T O N . T H I S MEDULLARY B O N E , WHICH IS IN THE FORM OF F I N E I N T E R L A C I N G S P I C U L E S GROWING OUT FROM THE ENDOSTEAL L I N I N G OF THE BONE AND INVAOING THE MARROW S P A C E , D E V E L O P S IN P U L L E T S 1 0 - 1 4 DAYS BEFORE THE F I R S T EGG IS L A I D AND P E R S I S T S THROUGHOUT THE WHOLE OF THE L A Y I N G S E A S O N . IT ACTS AS A L A B I L E S K E L E T A L RESERVOIR FOR EGG SHELL C A L C I F I C A T I O N , TAYLOR ( 1 9 ^ 5 » 1 9 7 0 B ) . TAYLOR ( 1 9 6 1 ) CONCLUDED THAT SOME DEGREE OF S K E L E T A L M O B I L -IZAT ION WAS AN E S S E N T I A L FEATURE OF EGG SHELL FORMATION, EVEN - 4 -IN HENS ON AN ADEQUATE CALCIUM D I E T , BECAUSE THE RATE OF ABSORPTION OF CALCIUM FROM THE GUT WAS NOT S U F F I C I E N T TO PROVIDE ALL THE CALCIUM FOR THE EGG S H E L L . HOWEVER, HURWITZ ( 1 9 7 0 ) CLA IMS T H A T , IN HENS GIVEN A NORMAL CALCIUM D I E T ( 3 . 5 - 4 . 0 $ C A ) , THE ABSORPTION OF CALCIUM FROM THE GUT IS S U F F I C I E N T TO ACCOUNT FOR A L L THE CALCIUM IN THE EGG S H E L L . IN THE P I G E O N , WHICH LAYS TWO EGGS 4 8 HOURS A P A R T , THE ROLE OF MEDULLARY BONE SEEMS F A I R L Y C L E A R C U T . C A L C I F I C A T I O N OF EACH SHELL IS ACCOMPANIED BY MEDULLARY BONE RESORPTION A S S O C I A T E D WITH INTENSE O S T E O C L A S T I C A C T I V I T Y . SOON A F T E R THE F I R S T EGG IS L A I O THE BONE RESORBING PHASE G I V E S WAY TO ONE DOM INATEO BY BONE FORMATION, BLOOM E T AL ( 1 9 4 1 ) . IN THE DOMESTIC HEN IN WHICH THE TIME INTERVAL BETWEEN C A L C I F I C A T I O N OF S U C C E S S I V E EGGS MAY BE ONLY 5~6 HOURS THE C E L L U L A R CHANGES IN MEDULLARY BONE ARE L E S S WELL D E F I N E D . BLOOM ET M. ( 1 9 5 8 ) WERE ABLE TO DEMONSTRATE TWO W E L L - D E F I N E D H I S T O L O G I C A L P A T T E R N S , AT THE START OF SHELL FORMATION BOTH O S T E O B L A S T S AND O S T E O C L A S T S WERE P R E S E N T , WHEREAS WHEN S H E L L C A L C I F I C A T I O N WAS WELL AOVANCEO THE S P I C U L E S OF MEDULLARY BONE WERE COVEREO WITH HUGE NUMBERS OF O S T E O C L A S T S ANO THERE WAS A P R E C I P I T O U S DROP IN THE NUMBER OF O S T E O B L A S T S . THE H I S T O L O G I C A L S T U D I E S OF BELANGER AND TAYLOR ( 1 9 6 7 ) ON MEDULLARY BONE DURING THE EGG CYCLE IN HENS FED A LOW CALCIUM D IET R E V E A L E D THAT MEDULLARY BONE RESORPTION IS APPARENTLY UNDER THE INFLUENCE OF ENLARGED O S T E O C Y T E S . THE DATA ON THE L A Y I N G HEN HAS BEEN SUMMARIZED BY TAYLOR ( 1 9 7 0 S ) AS F O L L O W S : -" 1 . GROWTH OF MEDULLARY BONE IS CONTINUOUS, ALTHOUGH THE - 5 -ACTUAL RATE OF GROWTH MAY VARY DEPENDING UPON THE C IRCULATORY L E V E L OF ESTROGENS AND ANDROGENS. 2 . GROWTH IS FROM THE SURFACE OF THE S P I C U L E S , WHICH PENETRATE FURTHER INTO THE MARROW CAVITY THE LONGER THE BIRD IS IN L A Y . 3. R E S O R P T I O N , AS A RESULT OF O S T E O C Y T I C O S T E O L Y S I S , ACCOMPANIED OR FOLLOWED BY O S T E O C L A S I S , IS INTERMITTANT AND A S S O C I A T E D WITH C A L C I F I C A T I O N OF THE S H E L L . AT THE START OF S H E L L FORMATION RESORPTION OCCURS MAINLY IN THE S P I C U L E S NEAREST THE CENTRE OF THE MARROW C A V I T Y , BUT AS SHELL FORMATION PROCEEDS RESORPTION EXTENDS TOWARDS THE CORT ICAL BONE. AT ANY ONE TIME SOME S U R F A C E S OF THE MEDULLARY BONE MAY BE UNDERGOING A C T I V E RESORPTION WHILE A D J A C E N T S U R F A C E S ARE GROWING EQUALLY A C T I V E L Y . " D I R E C T E V I D E N C E OF THE ROLE OF MEDULLARY BONE IN EGG SHELL C A L C I F I C A T I O N WAS OBTAINED BY MUELLER EJ_ AJ. ( 1 9 6 4 ) , WHO FE 0 L A Y I N G HENS A CONSTANT S P E C I F I C A C T I V I T Y RATION FOR 2 6 D A Y S , A F T E R WHICH AN IDENT ICAL BUT 45CA - FREE RATION WAS FED FOR A FURTHER 2 6 D A Y S . AT THE END OF THE E X P E R I M E N T IT WAS FOUND THAT THE PERCENTAGE OF 45CA IN THE LAST EGG L A I D WAS VERY S IMILAR TO THAT FOUND IN THE MEDULLARY B O N E . THESE BIRDS WERE ON A 1 . 9 5 $ CALCIUM D I E T AND HENCE MAY HAVE BEEN SOMEWHAT CALCIUM D E F I C I E N T THUS E M P H A S I S I N G THE ROLE OF MEDULLARY BONE . N E V E R -T H E L E S S THE DATA C L E A R L Y SHOWS THAT MEDULLARY BONE CAN SERVE AS A L A B I L E RESERVOIR FOR EGG S H E L L C A L C I U M . TWO MAJOR T H E O R I E S ON THE MECHANISM OF MEDULLARY B ONE RESORPTION HAVE BEEN A D V A N C E D . URIST ( 1 9 5 9 ) PROPOSED THAT RESORPTION IS BROUGHT ABOUT BY A REDUCTION IN THE L E V E L OF ESTROGEN C I R C U L A T I N G IN THE BLOOD A F T E R O V U L A T I O N . T H I S THEORY WOULD E X P L A I N THE EVENTS OCCURING IN THE BONES OF P IGEONS IN WHICH THERE IS L I K E L Y TO BE A S U B S T A N T I A L REDUCTION OF ESTROGEN AFTER THE OVULATION OF THE F I R S T F O L L I C L E , ANO IN WHICH THE F A L L IN ESTROGEN FOLLOWING OVULATION OF THE SECOND F O L L I C L E - 6 -MUST BE P R E C I P I T O U S . HOWEVER, AS TAYLOR ( 1 9 7 0 S ) POINTS O U T , IN THE L A Y I N G HEN THERE ARE A LARGE NUMBER OF F O L L I C L E S D E V -E L O P I N G IN THE OVARY AND THESE ARE A L L PRESUMEABLY S E C R E T I N G ESTROGENS SO THAT IT IS PROBABLE THAT THE F A L L IN PLASMA E S T R O -GEN OCCURRING A F T E R OVULATION IS R E L A T I V E L Y S L I G H T . TAYLOR & HERTELENOY ( 1 9 6 1 ) SHOWED THAT THE L E V E L OF D I F F U S I B L E CALCIUM IN THE PLASMA OF L A Y I N G HENS F E L L S I G N I F -ICANTLY DURING EGG SHELL C A L C I F I C A T I O N . THEY POINTED OUT THAT D I F F U S I B L E CALCIUM IS A F A I R L Y GOOD APPROXIMATION OF IONIC C A L C I U M , AND SUGGESTED THAT TH IS F A L L IN IONIC CALCIUM COULD ACT AS A ST IMULUS TO THE PARATHYROID GLAND. THEY PROPOSED THAT THE CONSEQUENT R I S E IN THE L E V E L OF PARATHYR0 ID HORMONE (PTH) COULD ACT AS THE ST IMULUS FOR MEDULLARY BONE R E S O R P T I O N . THERE IS SOME E V I D E N C E THAT PTH L E V E L S MAY BE HIGH DURING EGG S H E L L C A L C I F I C A T I O N . TAYLOR ( 1 9 6 5 ) AND URIST ( 1 9 5 9 ) STATE THAT THE PARATHYROID GLANDS ARE ENLARGED IN L A Y I N G H E N S . MUELLER E_T AL_ ( 1 9 6 9 ) REPORTED THAT CULTURES OF BONES REMOVEO FROM HENS WHILST THEY WERE A C T I V E L Y C A L C I F Y I N G AN EGG SHOWED INCREASED L A C T A T E PRODUCT ION . T H I S IS S IMILAR TO THE E F F E C T OF ADOIT ION OF PTH TO MAMMALIAN 80NE C U L T U R E S . TAYLOR & BELANGER ( 1 9 6 9 ) LOOKED AT THE E F F E C T OF PTH ON MEDULLARY BONE H I S T O L O G Y . THEY I N J E C T E D HENS WITH 1 0 0 U.S.P. UNITS OF L I L L Y PARATHYROID E X T R A C T AT THE T IME AN EGG ENTERED THE S H E L L GLAND AND K I L L E D THEM 2 , 4 » 6 AND 8 HOURS L A T E R . WHEN THE BONE HISTOLOGY OF THESE BIRDS WAS COMPARED WITH BIRDS UNDERGOING NATURAL MEDULLARY BONE RESORPTION DUE TO EGG S H E L L C A L C I F I C A T I O N THE HORMONE WAS FOUND TO ENHANCE THE NATURALLY OCCURING CHANGES - 7 -IN BONE C E L L S , THE MINERAL FRACTION AND THE ORGANIC M A T R I X . O S T E O L Y S I S WAS ALSO S T I M U L A T E D IN C O R T I C A L B O N E . T H E S E DATA GIVE D IRECT SUPPORT TO THE H Y P O T H E S I S THAT THE NATURAL R E S O R P -TION OF MEDULLARY BONE OURING EGG S H E L L FORMATION IN HENS IS INDUCED BY P T H . L A Y I N G HENS ALSO RESPOND TO THE S T R E S S OF EGG SHELL C A L -C I F I C A T I O N BY INCREASING CALCIUM ABSORPTION FROM THE GUT , BRONSCH EJ_ A_L ( 1 9 7 0 ) , AND BY D E C R E A S I N G URINARY CALCIUM E X -C R E T I O N , TAYLOR & K I R K L E Y ( 1 9 6 7 ) . HURWITZ & BAR ( 1 9 6 9 ) MEASURED CALCIUM ABSORPTION DURING VARIOUS PHASES OF EGG SHELL C A L C I F I C A T I O N USING Y T T R I U M - 9 1 AS A N 0 N - A B S 0 R B A B L E MARKER AND FOUND T H A T , ON A NORMAL CALCIUM D IET ( 3 * 5 6 $ C A ) , HENS ABSORBED 4 0 $ OF THE DIETARY CALCIUM WHEN NOT C A L C I F Y I N G AN E G G , 6 8 $ OF THE DIETARY CALCIUM DURING THE EARLY PHASE OF EGG SHELL C A L C I F I C A T I O N AND 7 2 $ OF THE DIETARY CALCIUM DURING L A T E EGG S H E L L C A L C I F I C A T I O N . THUS UNDER THE S T R E S S OF EGG S H E L L C A L C I F I C A T I O N THE RATE OF A8S0RPT I ON OF CALCIUM ALMOST D 0 U 8 L E 0 . HURWITZ ( 1 9 7 0 ) USED THESE RESULTS TO C A L C U L A T E THE RATE OF ABSORPTION DURING EGG SHELL C A L C I F I C A T I O N FOR HENS ON A NORMAL CALCIUM O I E T , AND CONCLUDED THAT INCREASED I N T E S T I N A L ABSORPTION COULD ACCOUNT FOR ALL THE CALCIUM D E P O S I T E D IN THE S H E L L . HOWEVER BURMESTER _ET_ A_L ( 1 9 3 9 ) » BURMESTER ( 1 9 4 0 ) AND B R A D F I E L O ( 1 9 5 1 ) HAVE SHOWN THAT THE RATE OF CALCIUM D E P O S I T I O N IS LOW FOR THE F I R S T F IVE HOURS THE EGG SPENDS IN THE S H E L L GLANO SO THAT THE MAIN PERIOD OF SHELL C A L C I F I C A T I O N ONLY L A S T S ABOUT 1 5 HOURS. T H I S MAKES THE RATE OF CALCIUM D E P O S I T I O N IN THE EGG S H E L L GREATER THAN THE INCREASED RATE OF CALCIUM ABSORPTION FOUND - 8 -DURING EGG SHELL C A L C I F I C A T I O N . THEREFORE CALCIUM ABSORPTION IS U N L I K E L Y TO BE THE SOLE FACTOR IN PROVIDING CALCIUM FOR THE EGG S H E L L . FURTHERMORE AS THE MAXIMAL PERIOD OF EGG SHELL C A L C I F I C A T I O N OCCURS IN THE EARLY HOURS OF THE MORNING, AT WHICH TIME THE HENS ARE NOT E A T I N G , THE SUPPLY OF A B S 0 R 8 E D CALCIUM IS DECREASED AT TH IS T IME AS THE CONTENTS OF THE GUT ARE USEO U P . T H E R E F O R E , EVEN IN BIROS ON AN AOEQUATE CALCIUM D I E T , THERE IS A VERY STRONG P O S S I B I L I T Y THAT BONE M O B I L I Z A T I O N IS IMPORTANT DURING EGG S H E L L C A L C I F I C A T I O N . ALTHOUGH HURWITZ & BAR HAVE SUGGESTED THAT ABSORPTION OF CALCIUM IS A P A S S I V E PROCESS ( HURWITZ & 3AR ( 1 9 6 8 , 1 9 6 9 ) , AND BAR & HURWITZ ( 1 9 6 9 ) ) , THE BULK OF THE E V I D E N C E , BOTH FROM THEIR OWN S T U D I E S ( HURWITZ & BAR ( 1 9 6 9 ) AND BAR & HURWITZ ( 1 9 6 9 ) ) » AND FROM OTHER L A B O R A T O R I E S , WOULD INDICATE THAT IN THE HEN IT IS A CARRIER MED IATED PROCESS WHICH IS UNDER THE INFLUENCE OF V ITAMIN 0. S T U D I E S ON THE CELLUAR ANO S U B C E L L U L A R L O C A L I Z A T I O N OF V ITAMIN D AND ITS M E T A B O L I T E S SHOW THAT THE V ITAMIN IS F I R S T M E T A B O L I Z E D TO A MORE POLAR COMPOUND WHICH A S S O C I A T E S S T E R E O -S P E C I F I C A L L Y WITH A RECEPTOR IN THE NUCLEUS ANO, BY CONTROLL ING GENE E X P R E S S I O N , U L T I M A T E L Y R E S U L T S IN THE P H Y S I O L O G I C A L RESPONSES C H A R A C T E R I S T I C OF V ITAMIN D , NORMAN E_T AL^  ( 1 9 6 9 , 1 9 7 1 ) ANO DELUCA ( 1 9 7 1 ) . WASSERMAN & TAYLOR ( 1 9 6 6 ) AND TAYLOR & WASSERMAN ( 1 9 6 7 ) SHOWEO THAT ADMINISTRAT ION OF V ITAMIN 0 3 TO R A C H I T I C CHICKS R E S U L T E D IN THE APPEARANCE OF A CALCIUM BINDING PROTEIN ( C A S P ) IN THE I N T E S T I N A L T I S S U E WHICH WAS NOT PRESENT IN UNTREATED - 9 -R A C H I T I C C H I C K S . THE V ITAMIN D ENHANCEO DUODENAL ABSORPTION OF 47CA IN R A C H I T I C CHICKS OCCURRED ALMOST S IMULTANEOUSLY WITH THE APPEARANCE OF THE V ITAMIN D3~INDUCE D F A C T O R , AND THERE WAS GOOD CORRELAT ION 8ETWEEN THE CONCENTRATION OF B INDING FACTOR AND THE RATE OF ABSORPTION OF 47C-A , V/ASSERMAN & TAYLOR ( 1 9 6 9 ) . THE V ITAMIN D3 - I N D U C E D PRODUCTION OF CABP COULD BE PREVENTED WITH ACT INOMYCIN D , CORRADINO & WASSERMAN ( 1 9 6 8 , 1 9 7 1 ) , S U G G E S T -ING THAT V ITAMIN D ACTS VIA GENE D E R E P R E S S I O N TO CAUSE THE FORMATION OF A S P E C I F I C CALCIUM BINDING P R O T E I N (CABP), WHICH IS I N I T I M A T E L Y INVOLVED IN CALCIUM TRANSPORT ACROSS THE CHICK I N T E S T I N A L WALL . IF TH IS IS CORRECT THEN THE AMOUNT OF CABP IN THE I N T E S T I N A L MUCOSA SHOULD VARY WITH THE CALCIUM A B S O R P -T I V E C A P A C I T Y OF THE GUT , AND THE CALCIUM NEED OF THE ANIMAL UNDER D I F F E R E N T S I T U A T I O N S . THE F INDINGS OF WASSERMAN C O -WORKERS S U B S T A N T I A T E T H I S , S E E WASSERMAN & TAYLOR ( 1 9 6 8 ) FOR R E F E R E N C E S , AND MAY BE SUMMARIZED AS F O L L O W S * -1 . THE CABP CONTENT AND CALCIUM A B S O R P T I V E E F F I C I E N C Y OF VARIOUS SEGMENTS OF THE CHICK SMALL I N T E S T I N E VARY IN THE SAME D I R E C T I O N I . E . DUODENUM > J E J U N U M > I L E U M . 2. YOUNG RAPIDLY -GROWING CHICKS WHICH NEED TO ABSORB MORE CALCIUM HAVE MORE MUCOSAL CABP THAN OLOER I N D I V I D U A L S . 3 . L A Y I N G HENS , WHICH INCREASE THEIR I N T E S T I N A L ABSORPTION OF CALCIUM IN RESPONSE TO THE S T R E S S OF EGG SHELL FORMATION, HAVE MORE MUCOSAL CABP THAN N O N - L A Y I N G HENS OF THE SAME A G E . TH I S D I F F E R E N C E IS NOT DUE TO ESTROGENS S I N C E THE L A T T E R WERE NOT C A P A B L E OF I N C R E A S -ING CALCIUM ABSORPTION IN THE L I G A T E D DUODENUM OF - 1 0 -R A C H I T I C C H I C K S , NOR DID THEY CAUSE INDUCTION OF CA8P. 4. CHICKS THAT HAVE ADAPTED TO LOW CALCIUM INTAKES ABS0R8 MORE 47CA AND HAVE MORE I N T E S T I N A L CABP THAN CHICKS MAINTAINED ON A NORMAL CALCIUM D I E T . 5 . TWO OTHER T I S S U E S WHICH CONTAIN E P I T H E L I A L C E L L S ACROSS WHICH CALCIUM IS TRANSFERRED HAVE ALSO SEEN SHOWN TO CONTAIN CABP. THE KIDNEYS OF V I T A M I N 0 3 TREATED R A C H I T I C CHICKS CONTAIN CABP, WHILE THEIR COLON, L I V E R AND MUSCLE DO NOT, AND THE UTERUS OF THE L A Y I N G HEN PRODUCES CABP IN RESPONSE TO V I T A M I N 0 3 . IT I S OF S P E C I A L INTEREST THAT CABP IS ONLY FOUND IN THOSE T I S S U E S HAVING A MECHANISM FOR MOVING CALCIUM ACROSS THE TOTAL E P I T H E L I A L C E L L , AND HENCE ONE CAN CONCLUOE THAT IT IS S P E C I F I C A L L Y INVOLVED IN THE TRANSMURAL FLUX OF CALCIUM. THERE ARE TWO SCHOOLS OF THOUGHT ABOUT THE ROLE OF CABP IN CALCIUM TRANSLOCATION. ONE IDEA I S THAT IT I S INVOLVED IN ACTIVE TRANSPORT, THE OTHER I S THAT IT IS INVOLVEO IN F A C I L -ITATED TRANSPORT. I N VI TRO STUDIES BY DOWOLE JET_ J^L ( I 9 6 0 ) AND SCHACHTER E_T AL. 0 9 6 1 ) SUGGEST THAT CALCIUM TRANSPORT IN THE RAT GUT IS AN A C T I V E PROCESS. HOWEVER I N T E S T I N A L TRANSPORT IN THE CHICKEN APPEARS TO BE A P A S S I V E PROCESS, HURWITZ & BAR ( 1 9 6 8 , 1 9 6 9 ) AND BAR & HURWITZ ( 1 9 6 9 ) . INOEED WASSERMAN & K A L L F E L Z ( 1 9 6 2 ) ANO WASSERMAN EJ_ .AL ( 1 9 6 6 ) HAVE SHOWN THAT IN CHICKS THE EFFECT OF V I T A M I N D I S TO INCREASE BOTH E F F L U X (LUMEN TO PLASMA) ANO INFLUX (PLASMA TO LUMEN) OF CALCIUM. - 1 1 -THEY CONCLUDE FROM THIS THAT THE E F F E C T OF V ITAMIN D IS TO INCREASE THE P A S S I V E P E R M E A B I L I T Y OF THE I N T E S T I N E TO C A L C I U M , WHICH IS CONSISTANT WITH THE IDEA THAT C A B P IS A CARRIER INVOLVED IN F A C I L I T A T E D D I F F U S I O N OF CALCIUM ACROSS THE I N T E S T I N A L WALL. V I T A M I N D HAS OTHER E F F E C T S ON THE L A Y I N G HEN B E S I O E S INCREASING CALCIUM ABSORPTION FROM THE GUT . As POINTED OUT A B O V E , IT CAUSES INDUCTION OF C A B P IN THE SHELL GLAND AND HENCE INCREASES CALCIUM TRANSLOCATION IN TH IS ORGAN. E V E N IF THE ROLE OF C A B P HERE IS TO AID IN F A C I L I T A T E D D I F F U S I O N THE CALCIUM TRANSPORTED IS D E P O S I T E D AS INSOLUBLE CALCIUM CARBONATE ON THE EGG S H E L L SO THAT BACK D I F F U S I O N IS N E G L I G I B L E . THUS THE E F F E C T OF V ITAMIN D IS TO INCREASE CALCIUM TRANSPORT TO THE EGG S H E L L . B E C A U S E , IN THE ABSENCE OF V ITAMIN D , L A Y I N G HENS PRODUCE VERY FEW EGGS (THE EGGS WHICH THEY OO PRODUCE ARE S O F T - S H E L L E O ) , BUT NO OTHER CHANGES OCCUR IN THE SECONOARY SEX C H A R A C T E R I S T I C S OF THE B I R D S , TURK & MCGLNNIS ( 1 9 6 4 ) CONCLUDED THAT V ITAMIN D IS INVOLVED IN THE REGULATION OF OVULATION ANO/OR F O L L I C U L A R GROWTH. T H I S E F F E C T OF V ITAMIN D MAY BE MEDIATED BY CHANGES IN PLASMA CALCIUM L E V E L S INFLUENCING THE S E C R E T I O N OF P I T U I T A R Y GONADOTROPINS, WHICH, IN T U R N , REGULATE OVARIAN A C T I V I T Y , TAYLOR ( 1 9 6 5 , 1 9 7 0 B ) . HOWEVER NOT ALL DATA IS C O N -S I S T A N T WITH THIS V IEW, SEE G I L B E R T ( 1 9 6 7 ) , SO T H A T , UNT IL FURTHER 0ATA IS O B T A I N E D , THE R E L A T I O N S H I P BETWEEN V ITAMIN D , PLASMA CALCIUM L E V E L S AND OVARIAN FUNCTION MUST REMAIN O B S C U R E . THE AVERAGE EGG SHELL WEIGHS 5 G AND CONSISTS ALMOST E N T I R E L Y OF CALCIUM C A R B O N A T E , AS CAL C I T E C R Y S T A L S , S L M K I S S - 1 2 -( 1 9 6 8 ) . As THESE C R Y S T A L S ARE NOT HYDRATEO CALCIUM IONS ONLY MAKE UP 4 0 $ OF THE EGG S H E L L , THE REMAINING 6 0 $ C O N S I S T I N G OF CARBONATE IONS. S l N C E THE B I C A R B O N A T E - C A R B O N I C ACIO SYSTEM IS AN IMPORTANT BUFFER SYSTEM IN THE BODY THE L A Y I N G HEN HAS TO MAKE ADJUSTMENTS IN HER A C I D - B A S E B A L A N C E . AS SOON AS THE EGG ENTERS THE SHELL GLAND THE PH OF THE BLOOO B E G I N S TO F A L L CAUSING A C I D O S I S . T H I S IS P A R T I A L L Y COMPENSATED BY A F A L L IN PC02 DUE TO INCREASEO R E S P I R A T I O N , MONGIN & L A C A S S A G N E ( 1 9 6 6 A ) , AND A DECREASE IN B ICARBONATE ION E X C R E T I O N BY THE K I D N E Y S , ANDERSON ( 1 9 6 7 ) . HOWEVER THE BICARBONATE ION L E V E L OF THE BLOOD S T I L L F A L L S 3 0 $ ANO THE PH REMAINS LOW UNTIL JUST BEFORE O V I P O S I T I O N , MONGIN & L A C A S S A G N E ( 1 9 6 6 B ) , INDICAT ING THAT THE BIRD IS UNABLE TO ADJUST C O M P L E T E L Y . SEVERAL S T U O I E S ON THE E F F E C T OF P E T U R B A T I O N S IN THE A C I D - B A S E S T A T U S OF L A Y I N G HENS ON S H E L L FORMATION HAVE SHOWN THAT A C I D O S I S CAUSES AN INCREASE IN S H E L L STRENGTH WHILE A L K A L O S I S CAUSES A DECREASE IN SHELL S T R E N G T H , HALL & HELBACKA • ( 1 9 5 9 ) » FRANK & BURGER ( 1 9 6 5 ) AND MUELLER ( 1 9 6 6 ) . A C E R T A I N AMOUNT OF CONTROVERSY E X I S T S IN THE L I T E R A T U R E ON THE MECHANISM OF FORMATION OF THE CARBONATE FRACT ION OF THE EGG S H E L L , BUT IT IS GENERALLY A C C E P T E D THAT CARBONIC ANHYDRASE is I N V O L V E D . COMMON ( 1 9 4 1 ) DEMONSTRATED THE P R E S E N C E OF CARBONIC ANHYDRASE (C.A.) IN S H E L L GLAND T I S S U E 8Y 8 IOCHEMICAL MEANS, WHILST D l A M A N S T E I N & SCHLU*NS ( 1 9 6 4 ) HAVE H I S T O C H E M I C A L L Y L O C A L I Z E D ITS P O S I T I O N TO THE A P I C E S OF THE U T E R I N E TUBULAR G L A N D S . - 1 3 -GUTOWSKI & M I T C H E L L ( 1 9 4 5 ) SUGGESTED THAT TWO BICARBONATE IONS REACTED TO FORM A CARBONIC ACID MOLECULE AND A C A R B O N -ATE R A D I C L E . THE L A T T E R WOULD BE USED IN THE C A L C I F I C A T I O N REACTION WHILE C A . WOULD CAUSE THE ACID TO D I S S O C I A T E INTO CARBON DIOXIOE ANO WATER AND SO SERVE TO S H I F T THE E Q U I L I B R I U M IN FAVOR OF THE FORMATION OF MORE CARBONATE IONS. GUTOWSKI AND M I T C H E L L ' S THEORY OF THE A C T I O N OF C . A . HAS BEEN REFUTED BY D l A M A N T S T E I N & SCHLUNS ( 1 9 6 4 ) , WHO CALCULATED T H A T , I F ALL THE CARBON D I O X I D E OF THE SHELL GLAND S E C R E T I O N WAS PRESENT AS C A R B O N A T E , THE S O L U B I L I T Y PRODUCT OF C A L C I U M CARBONATE WOULD BE EXCEEDED AND C R Y S T A L L I N E C A L C I U M CARBONATE WOULD BE P R E C I P I T A T E D W I T H I N THE TUBULAR GLANDS OF THE SHELL GLAND E P I T H E L I U M . S I N C E T H I S DOES NOT OCCUR THEY CONCLUDED THAT THE A N I O N SECRETED INTO THE SHELL GLAND LUMEN WAS B I C A R B O N A T E NOT CARBONATE I O N . D l A M A N T S T E I N ( 1 9 6 6 ) SUGGESTED THAT THE SHELL GLAND D E R I V E S THE SHELL CARBONATE FROM I T S OWN M E T A B O L I C CARBON D I O X I D E . I . E . BLOOD S H E L L GLAND LUMEN I . E . 2 H C 0 3 - = H2C03 + C 0 3 — C A . H20 + C02 METABOL I C C 0 2 + H20 C A . H2C03 H+ INTO BLOOD * H+ + + C 0 3 — D E P O S I T E D AS C A C 0 3 ?? - 1 4 -THE IDEA THAT THE FREE H+ L I B E R A T E D IN THE LUMEN IN SOME WAY RETURNS TO THE BLOOD IS SUPPORTED BY THE F INDING OF EL JA C K & LA K E (1967) THAT THE PH OF U T E R I N E F L U I D IS ABOUT 7 . 7 . TH I S MECHANISM WOULD ACCOUNT FOR THE PRESENCE OF C.A. IN THE GLANDULAR C E L L S AND A L S O , BY S E C R E T I O N OF H+ IONS INTO THE BLOOO S T R E A M , WOULD ACCOUNT FOR THE DROP IN BLOOO PH DURING SHELL FORMATION. FURTHERMORE D IAMANTSTE IN SUGGESTED THAT THE FALL IN THE PH OF THE BLOOD COULD CAUSE THE D I S S O C I A T I O N OF CALCIUM FROM ITS PROTEIN COMPLEX IN THE BLOOO AND HENCE F A C I L I T A T E CALCIUM S E C R E T I O N BY THE S H E L L GLAND. DlA M A N T S T E I N ' S THEORY IS SUPPORTED BY THE OATA OF LbRcHER & HODGES ( 1 9 6 9 ) , WHO SHOWED THAT 57$ OF A 45CA TRACER E N T E R I N G THE SHELL GLAND DID NOT EMERGE IN THE VENOUS E F F L U E N T , WHILE NONE OF A 1 4C—L A B E L L E D SAMPLE OF SOOIUM BICARBONATE WAS L O S T . FROM THE ABOVE D I S C U S S I O N ONE CAN S E E THAT THE L A Y I N G HEN MAKES MANY ADAPTAT IONS TO THE PROCESS OF EGG L A Y I N G , AND SHOWS CHANGES NOT ONLY IN HER CALCIUM M E T A B O L I S M , BUT ALSO IN HER A C I D - B A S E BALANCE IN ORDER TO F A C I L I T A T E EGG S H E L L FORM-A T I O N . THIS STUDY WAS UNDERTAKEN TO EXAMINE THE MANNER IN WHICH THESE METABOLIC CHANGES ARE REGULATED AND COORDINATED TO ALLOW THE REGULAR PRODUCTION OF F U L L Y - F O R M E D , C A L C I F I E D E G G S . - 1 5 -CHAPTER I : C A L C I U M TRANSPORT BY THE S H E L L GLAND J_N V I T R O . INTRODUCTI ON. THE AVIAN SHELL GLAND PROVIDES A MODEL SYSTEM IN WHICH TO STUDY THE TRANSLOCATION OF R E L A T I V E L Y LARGE AMOUNTS OF CALCIUM BY B I O L O G I C A L MEMBRANES, AND P O S S E S S E S SEVERAL F E A T U R E S WHICH F A C I L I T A T E THE STUOY OF THIS PHENOMENON. THEY CAN BE SUMMARIZED AS F O L L O W S f -1 . IT S E C R E T E S LARGE Q U A N T I T I E S OF CALCIUM (2G IN A PERIOO OF APPROXIMATELY 15 H O U R S ) . 2 . IT HAS REGULAR PERIODS OF SHELL D E P O S I T I O N , IMPLYING THAT THE " P U M P " MUST BE TURNED ON AND OFF F A I R L Y SPEC I F I C A L L Y . 3 . THERE IS A D I S T I N C T PHYSICAL S E P A R A T I O N OF THE C E L L U L A R S I T E OF CALCIUM TRANSLOCATION FROM THE S I T E OF MINERAL D E P O S I T I O N . EHRENSPECK £JT AJ_ ( 1 9 6 7 ) DEVELOPED AN J[_N VITRO P R E P A R A T I O N OF THE CHICKEN S H E L L GLAND IN WHICH THEY COULD SHOW CALCIUM O TRANSFER TO THE MUCOSAL OR LUMEN S I D E OF THE T I S S U E , AS TRACED WITH 45CA, WITHOUT AN EXTERNAL TRANSMURAL CONCENTRATION G R A D I E N T . THE TRANSFER WAS DEPENDENT ON THE PRESENCE OF AN EGG IN THE S H E L L GLAND AT THE TIME OF ITS E X C I S I O N AND REQUIRED ENERGY DERIVED FROM O X I D A T I V E M E T A B O L I S M . AS O I S C U S S E D IN THE GENERAL INTRODUCTION, THE L E V E L S OF PARATHYROID HORMONE ( P T H ) ARE THOUGHT TO INCREASE DURING EGG S H E L L C A L C I F I C A T I O N . URI ST ( 1 9 6 7 ) POINTS OUT THAT THE U L T I -MOBRANCHIAL BODIES OF L A Y I N G HENS ARE ENLARGEO AND CONTAIN - 1 6 -E A S I L Y E X T R A C T A B L E LARGE AMOUNTS OF C A L C I T O N I N ( C T ) , SUGGEST ING THAT THIS HORMONE MIGHT ALSO BE E X P E C T E D TO INFLUENCE EGG SHELL FORMATION IN SOME WAY. TH I S F I R S T S E R I E S OF EXPERIMENTS WAS UNDERTAKEN TO EXAMINE THE MANNER IN WHICH THE TWO CALCIUM R E G U L A T I N G HORMONES MIGHT INFLUENCE MOVEMENT OF CALCIUM ACROSS THE SHELL GLAND IN V I T R O . TABLE I THE COMPOSITION OF SOLUTIONS USED IN THE IN VITRO S H E L L GLAND EXPERIMENTS SOLUT1 ON NACL T R I S CL KCL C A C L 2 MGCL2 GLUCOSE PH (MM) (MM) (MM) (MM) (MM) (MM) STOCK BUFFER NO. 1 1 3 0 2 0 5 2 . 5 7 . 2 STOCK BUFFER NO. 2 1 3 0 2 0 2 0 2 . 0 1 . 0 1 5 . 0 7 . 2 - 1 7 -METHODS. WHITE LEGHORN H E N S , WEIGHING APPROXIMATELY 2 K G , OBTAINED FROM THE POULTRY FARM OF THE U N I V E R S I T Y OF 3 R I T I S H C O L U M B I A , WERE USED IN THESE E X P E R I M E N T S . THE HEN WAS ANESTHATI ZED WITH CARBON DIOXIDE AND K I L L E D BY D E C A P I T A T I O N . 3EFORE THE SHELL GLAND WAS REMOVED A NOTE WAS MADE OF THE STAGE OF THE OVULATORY CYCLE AT D E A T H , I . E . THE P O S I T I O N OF THE EGG IN THE OVIDUCT E T C . THE E X C I S E D SHELL GLAND WAS IMMEDIATELY SUBMERGED IN WHICH EVER STOCK BUFFER WAS TO BE USED IN THE E X P E R I M E N T ( E I T H E R STOCK BUFFER NO. 1 OR NO. 2 , SEE T A B L E L ) . TWO T Y P E S OF E X P E R I M E N T , S I N G L E AND D U P L I C A T E , WERE PERFORMED. THE SHELL GLAND WAS T IED ONTO THE END OF A GLASS TUBE (D IAMETER 2 . 1 CM) USING TWO P O L Y E T H Y L E N E STRAPS A P P L I E D WITH A PRESSURE GUN (TYPE G S - 2 B , PANDUIT CO R P . , T I N L E Y PA R K , I I I . ) , IN ORDER TO MIN IMIZE V A R I A T I O N S IN THE S T R E T C H OF THE MEMBRANES. IN THE S I N G L E EXPERIMENTS THE SEROSAL S IDE OF THE GLAND FACED THE INTERIOR OF THE TUBE WHILE THE V I L L I ON THE MUCOSAL S I D E FACED OUTWAROS, SEE FIGURE 1 . E X C E S S T I S S U E AROUNO THE EDGES WAS TRIMMED AWAY. IN THE D U P L I C A T E EXPERIMENTS THE SHELL GLAND WAS CUT IN HALF L O N G I T U D I N A L L Y AND EACH HALF WAS MOUNTED ON A SEPARATE GLASS T U B E . TWO B A S I C T Y P E S OF D U P L I C A T E E X P E R I M E N T WERE PERFORMED, SEE T A B L E I I . IN TYPES A AND C BOTH SAMPLES WERE MOUNTED AS IN THE S INGLE E X P E R I M E N T S , I . E . MUCOSAL S IDE FACING OUTWARDS. I N TYPE B ONE MEMBRANE WAS MOUNTED IN THE O P P O S I T E MANNER, I . E . MUCOSAL S IDE FACING INWARDS. THE APPARATUS USED IS SHOWN IN FIGURE 1 . EACH TUBE WAS CLAMPED SO THAT THE ENO CLOSED BY THE SHELL GLAND COULD BE LOWERED INTO A 1 0 0 ML BEAKER CONTAINING 5 0 ML OF BUFFER (THE I n n e r S o l u t i o n S a m p l i n g S y r i n g e 9 5 % 0 , , 5 % C O , 37 C W a f e r O u t e r S o l u t i o n S a m p l i n g S y r i n g e 9 5 % 0 2 , 5 % C 0 2 3 - W a y S t o p c o c k I n n e r S o l u t i o n 1 0 0 m l B e a k e r O u t e r S o l u t i o n 37 C W a t e r FIGURE 1 THE APPARATUS USEO IN THE J_N V I TRO SHELL GLANO EXPERIMENTS. -18-OUTER S O L U T I O N ) . THE SOLUTION WITHIN T H E T U B E (THE INNER S O L U T I O N ) WAS 1 0 ML OF THE SAME BUFFER AS WAS USED FOR THE OUTER S O L U T I O N . HYOROSTATIC PRESSURE GRADIENTS WERE AVOIDED BY A O J U S T I N G THE P O S I T I O N OF THE TUBE TO K E E P THE INNER AND OUTER L I Q U I D SURFACE L E V E L S THE S A M E . BOTH THE INNER AND OUTER SOLUT IONS WERE AERATED WITH 9 5 $ 0 2 , 5 $ C 0 2 ; THE C O N -TINUOUS STREAM OF B U B B L E S ALSO SERVED TO ST IR THE S O L U T I O N S , T H E WHOLE APPARATUS WAS MAINTAINED AT 3 7 . 0 ± 0 . 5 ° C WITH A C I R C U L A T I N G WATER B A T H . E I T H E R INNER OR OUTER SOLUTION COULO BE L A B E L L E D BY ADDING 4 5 C A (OBTAINED FROM NEW ENGLAND N U C L E A R , BOSTON M A S S . ) TO THE STOCK BUFFER? WHILE SAMPLES COULD BE REMOVED FROM E I T H E R CHAMBER AND COUNTED TO DETERMINE CALCIUM F L U X E S . SAMPLE S I Z E WAS 5 0 yUL. THE OUTER CHAMBER SAMPLING S Y S T E M , AS SHOWN IN F IGURE 1 , COMPRISED A 1 0 ML SYRINGE INTO WHICH 1 - 2 ML OF SOLUTION WERE DRAWN V IA THE BENT GLASS T U B E . A 5 0 / J L AL IQUOT WAS REMOVED FOR COUNTING AND THE REST OF THE SAMPLE WAS RETURNED TO THE OUTER CHAMBER. A SUMMARY OF THE 0 I F F E R E N T T Y P E S OF EXPERIMENTAL A R R A N G E -MENT IS PRESENTED IN T A B L E I I . AN INCUBATION WAS STARTEO WITH ADDIT ION OF THE L A 8 E L AND THE SAMPLES WERE MAINTAINED AT 3 7 . 0 ± 0 . 5 ° C FOR APPROXIMATELY SEVEN HOURS. 5 0 / JL AL IQUOTS OF THE A P P R O P R I A T E SOLUTION WERE COUNTED EVERY HALF HOUR. EACH 5 0 ^JL SAMPLE WAS A P P L I E D TO ONE EDGE OF 4 X 5 CM P I E C E OF WHATMAN NO. 1 F I L T E R PAPER FOLDEO INTO A C O N C E R T I N A . THE M I C R O P I P E T IN WHICH THE SAMPLE WAS MEASURED WAS RINSED WITH D I S T I L L E D WATER AND THE WASHING A P P L I E D TO THE SUMMARY OF THE IN V ITRO EXPERIMENTAL ARRANGEMENTS TYPE OF EXPER 1 MENT S U B -GROUP SAMPLE No. SHELL GLAND MOUNTING -MUCOSA OF SHELL GLAND PO I NTED STOCK BUFFER USED S O L N . TO WHICH 4 5CA WAS ADOED VO L . OF SO L N . (ML) VO L . OF 4 5CA ADDED A C T i v i T Y OF THE Fl NAL SOLUT1 ON JJCUR 1 ES/ML SOLUT1 ON SAMPLED FLUX MEASUREO * Si NGLE OUTWARDS 1 I NNER 1 0 5 0 3 . 6 5 OUTER 1 NFLUX DUPL1CATE A 1 OUTWARDS 1 INNER 1 0 100/JL 1 N 2 5 ML 1 0ML 1 N EACH 1 NNER OUTER 1 NFLUX 2 OUTWARDS 1 I NNER 1 0 2 . 9 2 OUTER 1 NFLUX B 1 1NWARDS 1 OR 2 OUTER 5 0 1 0 0 1 . 4 6 1 NNER 1 NFLUX 2 OUTWARDS OUTER 5.0 1 0 0 1 . 4 6 INNER E F F L U X . c 1 OUTWARDS 1 INNER & OUTER 1 0 5 0 5 0 2 5 0 3 . 6 5 3 . 6 5 1 NNER NET EF F L U X 2 OUTWARDS 1 OUTER 5 0 2 5 0 3 . 6 5 INNER EF F L U X * INFLUX » BLOOD - » LUMEN EF F L U X = LUMEN BLOOD - 1 9 -F I L T E R P A P E R , WHICH WAS THEN ORIED UNOER A HEAT LAMP AND P L A C E D , SPOTTED EDGE DOWN, IN A GLASS V IAL CONTAINING 1 5 ML OF S C I N T I L L A T I O N F L U I O (4G PPO, 5 0 MG POPOP*/ L I T R E OF T O L U E N E ) . COUNTING WAS DONE ON A BECKMAN L S - 2 3 3 S C I N T I L L A T I O N COUNTER. A PLOT OF T IME VERSUS CPM IN 5 0 JJL OF THE SOLUTION GAVE A MEASURE OF CALCIUM FLUX AND S L O P E S OF THE L I N E S SO OBTAINED WERE USED TO C A L C U L A T E THE F L U X E S OF CALCIUM IN NMOLES OF CA/ CM2 HR. AL L DRUGS AND HORMONES WERE ADDED TO THE INNER SOLUT ION OF THE SAMPLE A F T E R S U F F I C I E N T TIME HAD E L A P S E D FOR A CONTROL FLUX TO BE E S T A B L I S H E D . IN THE CASE OF PTH THE V E H I C L E WAS ALSO T E S T E D FOR P O S S I B L E E F F E C T S . THE VOLUME OF SOLUT ION ADDED WAS S M A L L , GENERALLY BETWEEN 2 0 AND 1 0 0 JJL, TO MIN IMIZE VOLUME CHANGES IN THE INNER CHAMBER. THE HORMONES USED IN THESE EXPERIMENTS WERE P U R I F I E O P O L Y P E P T I D E S . THE PTH HAD BEEN E X T R A C T E D FROM B E E F PARATHYROIDS BY THE METHOD OF POTTS E_T AJL ( 1 9 6 6 ) , WHILE THE CT HAD BEEN E X T R A C T E D FROM SALMON ULTIM0BRANCHI AL GLANDS BY THE METHOD OF O 'DOR ET AL. ( 1 9 6 9 ) . THE DIBUTYRYL C Y C L I C AMP WAS OBTAINED FROM S IGMA CHEMICAL C o . , L o s AN G E L E S , C A L . ; IT WAS THE ANHYDROUS FORM OF MONOSOOIUM DIBUTYRYL C Y C L I C 3 ' , 5 ' - AOENOSINE MONOPHOSPHATE. *PPO 2 , 5 - 0 IPHENYLOXAZOLE POPOP 1 , 4 - B L S - 2 ( 5 —PHEN0XAZOLYI )—BENZENE BOTH S C I N T I L L A T I O N GRADE OBTAINED FROM FRAZER MEDICAL S U P P L I E S , VANCOUVER, B . C . TABLE I I I 4 5CA FL U X E S IN THE IN V ITRO SHELL GLAND SYSTEM WITH T I S S U E OBTAINED FROM HENS IN VARIOUS PHYSIOLOGICAL CONDITIONS PHYS1OLOG1CAL STATE OF HEN CALCIUM FL U X E S * IN NMOLES CA/CM2 HR TYPE OF EXPERIMENT INFLUX EF F L U X EGG IN ISTHMUS 2 1 . 7 9 t 1 . 0 3 2 . 2 2 ± 1 . 4 DU P L I C A T E TYPE B PLUMPING EGG IN SHELL GLAND 8 1 . 5 8 t 1 . 6 5 7 7 . 8 1 ± 0 . 9 9 DU P L I C A T E TYPE A ACT IVE CA L C I F I C A T I O N OF EGG SHELL 5 3 . 6 9 ± 2 . 0 6 9 4 . 4 5 ± 3 . 2 0 DU P L I C A T E TYPE A 2 3 . 1 6 * 0 . 6 8 4 0 . 8 1 ± 0 . 6 2 DU P L I C A T E TYPE B EGG JU S T LA I D 3 8 . 2 ± 4 . 9 S I N G L E 5 7 . 8 ± 2 . 3 S I N G L E 1 7 8 . 2 * 4 . 0 S INGLE 5 9 . 0 0 + 1 . 7 5 6 5 . 5 5 ± 0 . 4 1 DU P L I C A T E TYPE A * INFLUX = BLOOD -> LUMEN EF F L U X = LUMEN -* BLOOD - 2 0 -RE S U L T S & D I S C U S S I O N . CH A R A C T E R I Z A T I O N OF THE IN VITRO S Y S T E M . THE DRAMATIC CHANGES IN S E C R E T I O N L E V E L S E X H I B I T E D BY EACH PART OF THE OVIDUCT AS THE EGG P A S S E S FROM THE OVARY TO THE EXTERIOR SUGGEST THAT THE TRANSLOCATION OF CALCIUM BY THE SHELL GLAND IS A DISCONTINUOUS P R O C E S S . THE CALCIUM TRANSPORTING C A P A C I T Y OF THE T I S S U E MIGHT WELL BE LOW WHEN THE EGG IS NOT IN THE GLANO AND BECOME MUCH GREATER DURING S H E L L FORMATION. ALTHOUGH THESE V A R I A T I O N S MAY BE E X H I B I T E D IN VITRO THERE IS NO A PRIORI REASON WHY THEY S H 0 U L 0 S T I L L BE E V I D E N T IN E X C I S E D S T R I P S OF S H E L L GLANO T I S S U E . THE I N I T I A L EXPERIMENTS IN TH IS S E C T I O N WERE PLANNED TO EXAMINE THE R E L A T I O N S H I P BETWEEN THE P H Y S I O L O G I C A L STATE OF THE HEN AND THE CALCIUM T R A N S L O C A T I N G C A P A C I T Y OF E X C I S E D S H E L L G L A N O . THE VALUES FOR INFLUX OF CALCIUM INTO THE LUMEN OF THE S H E L L GLAND WHICH WERE OBTAINED IN THE VARIOUS P H Y S I O L O G I C A L S T A T E S ( L . E . EGG IN D I F F E R E N T PARTS OF THE OVIDUCT) O V E R L A P P E D , WITH LOW V A L U E S BEING OBTAINED BOTH DURING A C T I V E S H E L L C A L -C I F I C A T I O N AND WHEN THE EGG WAS IN THE ISTHMUS, WHILST THE HIGHEST V A L U E WAS OBTAINED IN A PREPARAT ION TAKEN A F T E R THE EGG WAS L A I O . FOR EXAMPLE ( S E E TABLE I I I ) , WHEN THE EGG WAS IN THE ISTHMUS, WHERE EGG WHITE P R O T E I N S WERE BEING INCORPORATED, THE INFLUX MEASUREO WAS 2 2 NMOLES CA/CM2 HR, WHEREAS WHEN THE EGG WAS IN THE PLUMPING STAGE THE INFLUX WAS APPROXIMATELY 8 0 NMOLES CA/CM2 HR. A WIDE V A R I A T I O N IN F L U X E S WAS OBTAINED EVEN WITH BIRDS IN THE SAME P H Y S I O L O G I C A L C O N D I T I O N . THE I N F L U X E S - 2 1 MEASURED DURING A C T I V E SHELL C A L C I F I C A T I O N WERE 9 4 . 5 4 ANO 2 3 NMOLES CA/CM2 HR, WITH THE F I R S T TWO VALUES BE ING 0 8 T A I N E 0 FROM THE TWO HALVES OF THE SAME S H E L L GLAND. GLAND P R E P A R A -T IONS TAKEN AFTER THE EGG HAD BEEN L A I D SHOWED INFLUX RATES OF BETWEEN 3 8 AND 1 7 8 NMOLES CA/CM2 HR. T H U S , UNDER THE E X P E R I M E N T A L CONDIT IONS U S E D , THE ABSOLUTE S I Z E OF THE INFLUX WAS INDEPENDENT OF THE P H Y S I O L O G I C A L STATE OF THE S H E L L GLAND. THE DATA OBTAINED HERE DO NOT AGREE WITH THAT OF EHRENSPECK _E_T _AL ( 1 9 6 7 ) * WHO FOUND THE FLUX TO BE 3 0 NMOLES CA/ C M 2 HR IN BOTH D I R E C T I O N S WHEN NO EGG WAS P R E S E N T , WHILE DURING A C T I V E C A L C I F I C A T I O N INFLUX INTO THE SHELL GLAND LUMEN WAS 5 0 NMOLES CA/CM2 HR ANO E F F L U X WAS 2 0 NMOLES CA/CM2 HR. T H U S , ALTHOUGH THE F L U X E S MEASURED IN THIS STUDY AND THOSE OF EHRENSPECK EJ_ A_L WERE OF A S I M I L A R MAGNITUDE , THE R E S U L T S OBTAINED HERE SHOWED THAT INFLUX WAS INDEPENDENT OF THE P H Y S I O L O G I C A L STATE OF THE SHELL GLAND AND WAS VERY V A R I A B L E FROM PREPARATION TO P R E P A R A T I O N , WHILST THE RESULTS OF EHRENSPECK EJ_ AJ- ( 1 9 6 7 ) SHOWED THAT INFLUX AND E F F L U X WERE EQUAL WHEN NO EGG WAS P R E S E N T IN THE O V I D U C T , BUT THAT INFLUX WAS TWICE E F F L U X WHEN THE SHELL GLANO WAS E X C I S E D DURING A C T I V E SHELL C A L C I F I C A T I O N . NO MATTER WHAT THE P H Y S I O L O G I C A L STATE OF THE GLANO THE E F F L U X OBTAINED WAS ALWAYS GREATER THAN THE INFLUX IN THESE E X P E R I M E N T S , S E E TABLE I N . WHEN THE EGG WAS IN THE ISTHMUS THE E F F L U X WAS APPROXIMATELY 1 . 5 T IMES AS LARGE AS I N F L U X , WHEREAS DURING A C T I V E C A L C I F I C A T I O N IT WAS TWICE AS LARGE AS I N F L U X . T H I S DATA IS THUS IN O IRECT C O N F L I C T WITH THAT OF 1 280000-275000-c o 3 £ 270000 Counts in outer Solution at Time Zero Counts expected in inner Solution if the System had reached Equilibrium o -< o i n 265000 a. o 26OO00-3 4 5 6 Time in Hours F IGURE 2. A GRAPH SHOWING NET E F F L U X OF 45CA (LUMEN-* BLOOD) IN A SHELL GLANO E X C I S E O DURING A C T I V E S H E L L C A L C I F I C A T I O N . L A B E L L E D CALCIUM WAS P R E S E N T ON BOTH S I D E S OF THE MEMBRANE. - 2 2 -EHRENSPECK J E T A J - ( 1 9 6 7 ) . IN ORDER TO CHECK THAT THESE RESULTS WERE NOT OUE TO P H Y S I C A L DRAWBACKS IN THE EXPERIMENTAL SYSTEM A OOUBLE LA B E L EXPERIMENT WAS PERFORMED ON A SHELL GLAND E X C I S E D DURING ACTIVE SHELL C A L C I F I C A T I O N ( D U P L I C A T E TYPE C SAMPLE NO. 1 ) . T H I S EXPERIMENT E L I M I N A T E D ANY SPURIOUS RESULTS DUE TO E X -CHANGE D I F F U S I O N OF NON-LABELLED CALCIUM FOR 4 5 C A , SINCE THE S P E C I F I C A C T I V I T Y OF THE SOLUTIONS ON EITHER SIOE OF THE MEM8RANE WAS THE SAME. THE RESULTS ARE SHOWN IN FIGURE 2. THE ACTUAL PARAMETER MEASURED HERE WAS NET E F F L U X . FROM ZERO TO THREE HOURS THE NET E F F L U X WAS ~ 7 5 . 3 ± 2 1 . 7 NMOLES CA/ CM2 HR. THUS IN FACT A NET INFLUX OF + 7 5 . 3 ^ 2 1 . 7 NMOLES CA/ CM2 HR WAS TAKING P L A C E . HOWEVER FROM 3 . 5 TO 7 HOURS THE NET E F F L U X BECAME P R A C T I C A L L Y N I L , I . E . - 1 . 7 7 ± 5 . 2 NMOLES CA/ CM2 HR, I M P L Y I N G THAT THE TWO FLUXES HAD COME TO E Q U I L I B R I U M AND INFLUX WAS APPROXIMATELY EQUAL TO E F F L U X . WHEN ONE CONSIDERS SAMPLE NO. 2 IN T H I S EXPERIMENT WHICH MEASURED E F F L U X IN THE OTHER HALF OF THE SHELL GLAND, SEE FIGURE 3 , ONE FINDS THAT FROM ZERO TO THREE HOURS E F F L U X WAS I N C R E A S I N G NON-UN IFORMLY AND AT T H I S TIME WAS NOT VERY GREAT. HOWEVER FROM 3 . 5 TO 7 HOURS THE E F F L U X WAS LINEAR ANO WAS EQUAL TO 1 2 6 . 9 + 4 . 5 NMOLES CA/ CM2 HR. DURING 3 . 5 TO 7 HOURS THE NET E F F L U X WAS FROM - 7 TO + 3 NMOLES CA/CM2 HR. THEREFORE THE INFLUX MUST HAVE 8EEN SOMEWHERE FROM 1 1 6 TO 1 3 6 NMOLES CA/CM2 HR, I . E . 1 2 6 ± 1 0 NMOLES CA/CM2 HR. THUS THE DUPLICATE L A B E L EXPERIMENT INDICATED THAT THE INFLUX WAS ONLY GREATER THAN THE E F F L U X DURING THE F I R S T THREE HOURS OF THE EXPERIMENT WHEN THE I N D I V I D U A L FLUXES WERE SMALL AND NON-LINEAR SO THAT THEY WERE 3 0 0 0 0 n i . i i i i i i i 0 1 2 3 4 5 6 7 Time in Hours GU RE 3. E F F L U X (LUMEN BLOOD ) OF 45CA IN A SHELL GLANO PREPARAT ION TAKEN OURING A C T I V E SHELL C A L C I F I C A T I O N (THE MATERIAL WAS FROM THE SAME GLAND AS THAT USEO IN F IGURE 2.) - 2 3 -U N M E A S U R E A B L E . ONCE THE F L U X E S BECAME L INEAR INFLUX ANO E F F L U X HAD COME TO E Q U I L I B R I U M ANO NO NET EXCHANGE OF CALC IUM WAS TAKING P L A C E . AGAIN THIS DOES NOT AGREE WITH E H R E N S P E C K E_T AL_ ( 1 9 6 7 ) » B U T , AS THIS GROUP CARRIED OUT NO E X P E R I M E N T S WITH L A B E L ON BOTH S I D E S OF THE MEM8RANE, A D I R E C T COMPARISON OF DATA IS D I F F I C U L T TO M A K E . IN THE D U P L I C A T E TYPE B EXPERIMENTS E F F L U X WAS ALWAYS FOUNO TO BE GREATER THAN I N F L U X , SEE T A B L E S II I AND V , WHILE THE DOUBLE L A B E L E X P E R I M E N T IMPLIEO THAT THE F L U X E S WERE EQUAL AFTER THE I N I T I A L THREE HOURS. T H I S APPARENT PARADOX MAY BE E X P L A I N E D IN THE FOLLOWING MANNERT — IN THE E F F L U X S E T U P THE MUCOSAL SURFACE OF THE SHELL GLANO WAS POINT ING OUTWARDS, WHEREAS IN THE INFLUX SETUP IT WAS P O I N T I N G INWAROS; 8 U T , IN BOTH C A S E S , THE L A B E L WAS ADDED TO THE OUTER SOLUT ION WHILE THE INNER SOLUTION WAS S A M P L E D . S I N C E THE MUCOSAL SURFACE OF THE S H E L L GLANO HAS MANY INVAGINATIONS ANO M I C R O V I L L I IT HAS A LARGER TOTAL SURFACE AREA THAN THE SEROSAL S U R F A C E . T H E R E F O R E , IN THE E F F L U X C A S E , THERE WAS A LARGER S U R F A C E AREA OF THE S H E L L GLAND IN CONTACT WITH THE 45CA S O L U T I O N , G I V I N G R I S E TO A LARGER MEASURED E F F L U X THAN I N F L U X . HOWEVER, THE D I F F E R E N C E IS A RESULT OF THE GEOMETRY OF THE E X P E R I M E N T A L ARRANGEMENT RATHER THAN A P H Y S I O L O G I C A L E F F E C T . E H R E N S P E C K E_T A_L ( 1 9 6 7 ) USED A S IMILAR EXPERIMENTAL DESIGN TO THE ONE USED IN THE D U P L I C A T E TYPE B E X P E R I M E N T S , SO THAT IT IS D I F F I C U L T TO E X P L A I N WHY THEY DIO NOT S E E THIS E F F E C T . QU I TE P O S S I B L Y , AS THE SURFACE AREA OF THE MEMBRANE USED IN THEIR EXPERIMENTS ( 2 . 9 6 CM2) WAS SMALLER THAN THAT USED HERE TABLE IV THE E F F E C T OF MAGNESIUM CHLORIDE AND GLUCOSE ON THE IN V ITRO 45CA FL U X E S ACROSS A SHELL GLAND EX C I S E O DURING ACT IVE EGG SHELL CA L C I F I C A T I O N MEDIUM* FLUXES (NMOLES CA/CM2 HR) FLUX D IRECTION io RESPONSE 0 FLUX RAT I o tfo CHANGE IN FLUX RATIO 1 NFLUX EF F L U X STOCK BUFFER No. 1 23.16 ±0.68 40.81 ±0.62 0.5675 +MGCL2 (IMM) 27.44 ±0.55 52.88 ±4.0 I NFLUX EF F L U X +18.48 +29.60 0.5189 -8.56 +GLUCOSE (1MM) 28.40 ±0.70 60.39 ±1.33 INFLUX EF F L U X +3.49 +14.2 0.4703 -9.366 * REAGENTS WERE ADDED TO BOTH INNER AND OUTER S O L U T I O N S . * $ CHANGE IN THE SLOPE OVER THAT IMMEDIATELY PRIOR TO THE ADDITION OF THE REAGENT. " FLux RATIO - i N F L U X . BLOOD -> LUMEN E F F L U X LUMEN -* BLOOD - 2 4 -( 3 . 4 7 C M 2 ) , THE GEOMETRY E F F E C T IN THEIR EXPERIMENTS COULO HAVE BEEN REDUCED. THERE WERE A NUMBER OF OTHER MINOR EXPERIMENTAL D I F F E R -ENCES BETWEEN THE SYSTEM USED HERE AND THAT EMPLOYED BY EHRENSPECK EJ_ A J L ( 1 9 6 7 ) . FOR E X A M P L E , STOCK BUFFER NO. 1, WHICH WAS USED AS THE MEDIUM IN MOST OF THE E X P E R I M E N T S , D I F F E R E D FROM THE MEOIUM OF EHRENSPECK E_T AL IN THAT IT L A C K E D BOTH MAGNESIUM AND G L U C O S E , SEE T A B L E I . ALTHOUGH IT APPEARED U N L I K E L Y THAT E I T H E R WAS HAVING A MAJOR E F F E C T ON THE S Y S T E M , AS THE OVERALL F L U X E S OBSERVED HERE AND THOSE OF EHRENSPECK EJ_ A I L WERE S I M I L A R , THE INFLUENCE OF BOTH CHEMICALS ON THE F L U X E S WAS T E S T E D . A s E X P E C T E D THERE WAS NO MAJOR CHANGE IN FLUX R A T E S , SEE T A B L E IV ANO FIGURE 4. 1 MM MGCL2 INCREASED INFLUX 1 8 $ AND E F F L U X 3 0 $ , WHILE 1 MM GLUCOSE ONLY INCREASED INFLUX 3 $ ANO E F F L U X 1 4 $ . DE S P I T E THESE R E S U L T S S E V E R A L MORE D U P L I C A T E TYPE B EXPERIMENTS WERE CARRIED OUT USING STOCK BUFFER NO. 2, WHICH WAS VERY S IMILAR TO THE MEDIUM USED BY EHRENSPECK E_T AL ( 1 9 6 7 ) . AS CAN BE S E E N FROM T A B L E V, THE ONLY CHANGE IN THE RESULTS WAS THAT THE INFLUX WAS LOWER THAN WHEN BUFFER NO. 1 WAS U S E O , IT OROPPED FROM APPROXIMATELY 2 2 TO 9 NMOLES CA/ CM2 HR. IN A RECENT P A P E R , P U B L I S H E D A F T E R TH IS S E R I E S OF EXPERIMENTS WAS C O M P L E T E D , EHRENSPECK E_T A_L ( 1 9 7 1 ) ALSO FOUNO THAT OMISSION OF GLUCOSE FROM THEIR MEDIUM HAD NO E F F E C T ON THEIR F L U X E S . IN THESE EXPERIMENTS THE BUFFERS WERE AERATED WITH 9 5 $ 0 2 , 5 $ C 0 2 . THE CARBON DIOXIDE WAS INCLUOED BECAUSE LBRCHER & HODGES ( 1 9 6 9 ) SHOWED THAT THE CARBONATE FRACT ION OF THE EGG x - Sample # I -o - II II 2 -Influx Efflux 6 0 0 0 - 1 0 1 2 3 4 5 6 7 8 Time in Hours F IGURE 4. THE E F F E C T OF ADDIT ION OF MAGNESIUM CHLORIDE AND GLUCOSE TO THE INCUBATION MEDIUM ON 45CA F L U X E S IN A SHELL GLANO E X C I S E D DURING A C T I V E S H E L L C A L C I F I CAT I ON. - 2 5 -SHELL WAS FORMEO FROM CARBON O I O X I D E , ANO IF THE CALCIUM PUMP WAS L I N K E D IN SOME WAY TO THE CARBONATE T R A N S F E R , LACK OF CARBON DIOXIDE COULD BE I N H I B I T O R Y . IN ONE E X P E R I M E N T AERATION WITH 1 0 0 $ 0 2 WAS T R I E D , SEE TABLE V. THERE WAS NO CHANGE IN INFLUX 8UT E F F L U X DROPPED FROM ABOUT 4 8 TO 2 0 NMOLES CA/CM2 HR. AN INCREASE IN E F F L U X WOULD TEND TO DECREASE EGG SHELL C A L C I F I C A T I O N . HOWEVER, IN V I V O , ANY CALCIUM TRANSPORTED INTO THE S H E L L GLANO IS D E P O S I T E D AS INSOLUBLE CALCIUM CARBONATE SO THAT ANY INCREASE IN E F F L U X WOULD HAVE N E G L I G I B L E E F F E C T S ON EGG S H E L L C A L C I F I C A T I O N . EHRENSPECK JET AL ( 1 9 6 7 ) USEO 1 0 0 $ 02 TO AERATE THEIR S Y S T E M . HOWEVER IN THEIR ( 1 9 7 1 ) PAPER THEY REPORT THAT S U B S T I T U T I O N OF B ICARBONATE FOR T R I S BUFFER AND AERAT ION WITH 9 5 $ 0 2 , 5 $ C02 HAD NO E F F E C T ON THEIR J_N V ITRO F L U X E S . FURTHERMORE, INH IB IT ION OF CARBONIC ANHYDRASE WITH 5 0 MM ACETAZOLAM I OE INC RE A SE 0 BOTH INFLUX AND E F F L U X IN THEIR S Y S T E M , BUT HAD NO S I G N I F I C A N T E F F E C T ON THE FLUX R A T I O . THEY INTERPRETED THESE RESULTS AS AN INDICATION THAT CALCIUM AND CARBONATE TRANSLOCATION WERE NOT T IGHTLY C 0 U P L E 0 . THUS THESE R E S U L T S TEND TO AGREE WITH THE DATA FOUNO H E R E . IN THEIR 1971 PAPER EHRENSPECK E_T AL. R E P E A T E D THE FLUX RATIO EXPERIMENTS WHICH THEY HAD DONE IN 1 9 6 7 AND 0 8 T A I N E D E S S E N T I A L L Y S IMILAR D A T A . THEY ALSO S U B S T A N T I A T E D THEIR L A B E L EXPERIMENTS BY SHOWING THAT TOTAL CALCIUM CHANGEO. THEY FOUND THAT WHEN PAIRED MEMBRANES WERE INCUBATED IN SOLUT IONS WHICH CONTAINED 7 5 ± 1 /JG CA/ ML AFTER FOUR HOURS OF INCUBATION THE CALCIUM CONCENTRATIONS OF THE SOLUT IONS TABLE V THE E F F E C T OF CHANGING THE INCUBATION BUFFER AND AERATION GAS ON IN V ITRO 45CA FLUXES IN THE SH E L L GLANO PHYS1OLOG1CAL ST A T E OF GLANO STOCK BUFFER AERAT1 ON CALCIUM FL U X E S NMOLES CA/CM2 HR I NFLUX EF F L U X FLUX RATIO* EGG IN ISTHMUS 1 95$ 0 2, 5$ C02 21.79 i 1.0 32.22 t 1.4 0.6763 AC T I V E SHELL CA L C I F I C A T I O N i 95$ 021 5$ C02 23.16 ± 0.68 40.81 4 0.62 0.5675 ACT IVE SHELL CALC I F ICAT ION 2 95$ 0 2 t 5$ C02 8.029 i0.108 48.67± 1.23 0.1649 ACT IVE SHELL CA L C I F I C A T I O N 2 100$ 0 2 9.99 t 0.13 20.04± 0.35 0.4988 * FLUX _ i N F L U X _ BLOOD - » LUMEN E F F L U X LUMEN -* BLOOD - 2 6 -H A 0 INCREASED TO 81 "t 1 jjQ CA/ ML ON THE MUCOSAL S IDE ANO DECREASED TO 6 6 ± 2 yG CA/ ML ON THE SEROSAL S I D E . AT PRESENT THERE SEEMS NO EXPLANAT ION FOR THE D I S A G R E E -MENT BETWEEN THE RESULTS FOUND HERE AND THOSE OF EHRENSPECK EJ_ A X . THEIR F L U X E S WERE OF THE SAME ORDER OF MAGNITUDE AS THOSE REPORTED H E R E . HOWEVER THEY FOUND THAT INFLUX AND E F F L U X WERE EQUAL WHEN NO EGG WAS PRESENT IN THE OVIDUCT BUT THAT INFLUX WAS TWICE E F F L U X WHEN THE SHELL GLAND WAS E X C I S E D DURING A C T I V E SHELL C A L C I F I C A T I O N , WHILE THESE EXPERIMENTS WERE UNABLE TO SHOW ANY R E L A T I O N S H I P BETWEEN EGG SHELL C A L -C I F I C A T I O N AND THE S I Z E S OF THE F L U X E S . ALTHOUGH THE F L U X E S OBTAINED HERE WERE VERY V A R I A B L E , SEE TABLE I I I , THE DOUBLE L A B E L EXPERIMENT IMPLIED THAT INFLUX ANO E F F L U X WERE EQUAL EVEN IN A SHELL GLANO REMOVED FROM A HEN OURING A C T I V E C A L -C I FI CAT I ON. HORMONES. DE S P I T E THE OBVIOUS DRAWBACKS TO THE EXPERIMENTAL S Y S T E M , THE E F F E C T S OF PARATHYROIO HORMONE (PTH) AND C A L C I T O N I N (CT) ON THE J_N VITRO F L U X E S WAS T E S T E D . A SUMMARY OF THE RESULTS IS SHOWN IN TABLE VI. IN THE F I R S T S I N G L E EXPERIMENT REPORTED THE PTH APPEARED TO CAUSE A LARGE INCREASE IN I N F L U X , BUT TH IS EARLY RESULT WAS NEVER R E P E A T E D AND MAY WELL HAVE 8 E E N DUE TO A LEAK IN THE MEMBRANE. THE SECOND S INGLE E X P E R I M E N T IN T A B L E VI IS SHOWN IN FIGURE 5 . ALTHOUGH THE HORMONE APPEARS TO HAVE BEEN R E S -P O N S I B L E FOR A MARKED INCREASE IN INFLUX THE CONTROL A P P L I C A T I O N TABLE V« " T H E E F F E C T OF PTH ANO CT ON THE INFLUX (BL O O D - » LUMEN ) OF '4~5'CA IN THE SHELL GLAND JN V ITRO PR E P A R A T I O N TYPE OF EXPERIMENT PHYS1OL O G1C AL STATE OF HEN PTH VEH1 CLE PTH ( L I L L Y U.S.P. U N I T S ) CT (M.R.C. INFLUX NMOLES CA/CM2 HR TREATMENT io * RESPONSE M 1 LL 1 -UN 1 TS ) CONTROL AFTER PTH VEH 1 CLE AFTER PTH AFTER CT S INGLE EGG JUST L A I D O.OIM NH4COOCH3 0 .501M M E R C A P T O -ETHANOL PH 5 . 3 350 3 8 . 2 1 4 . 9 821 .91 ± 3 3 . 3 PTH +2051 . 5 S INGLE EGG JUST L A I D O.OIM N H 4 C O O C H 3 PH 5 . 3 25 5 7 . 8 ± 2 . 3 112 . 1 ±1 .97 2 0 3 . 8 ± 4 . 5 PTH • VEH 1 CLE +82 +94 OUPL1CATE TYPE A PLUMPING EGG IN SH E L L GLANO STOCK BUFFER NO. 1 40 8 1 . 5 8 ± 1 . 6 5 77.81 ± 0 . 9 9 132 . 7 9 ± 2 . 9 4 101 . 8 5 ± 2 . 8 3 PTH VE H I C L E +24 . 8 +70 . 7 O U P L1C A T E TYPE A EGG JUST L A I D STOCK BUFFER No. 1 , 0 .125M IN C Y S T E 1 N E HCL 40 5 9 . 0 0 ± 1 . 7 5 6 5 . 5 5 ±0.41 6 5 . 9 7 ± 2 . 3 5 73.01 ± 2 .36 PTH VEH1 CLE +23.7 +0.6 DUPL1CATE TYPE A CA L C 1 FY ING EGG IN SHELL GLAND STOCK BUFFER No. 1 , 0 .125M IN C Y S T E I N E HCL 200 400 5 3 . 6 9 ± 2 . 06 9 4 . 4 5 ± 3 .20 90 .42 ± 0 . 3 2 67 .41 ± 0 . 9 2 9 8 . 32 PTH VEH1 CLE CT +25 . 6 - 5 . 2 7 +8.74 * io INCREASE OF THE SLOPE OVER THAT IMMEDIATELY PRIOR TO THE ADOITION OF THE CHEMICAL L I S T E D IN THE TREATMENT COLUMN. - 2 7 -OF V E H I C L E INOICATES THAT THE E F F E C T IS M I N I M A L . IN FACT THE INCREASE IN INFLUX OUE TO V E H I C L E ALONE IS S L I G H T L Y GREATER THAN THAT CAUSEO BY PTH; I . E . $ RESPONSE TO V E H I C L E WAS + 95$ WHILE $ RESPONSE TO PTH WAS ONLY +84$. THEREFORE FROM THIS EXPERIMENT ONE WOULD CONCLUDE THAT PTH HAS NO E F F E C T ON CALCIUM I N F L U X . THE D U P L I C A T E TYPE A E X P E R I M E N T S , TABLE V I , IN GENERAL SUPPORT THIS C O N C L U S I O N , ALTHOUGH THE L A S T TWO SHOW A S L I G H T TENDANCY FOR PTH TO INCREASE CALCIUM I N F L U X , S E E FIGURE 6. IN VIEW OF THE VARIAT ION IN THE S I Z E OF F L U X E S FROM MEM8RANE PREPARAT ION TO P R E P A R A T I O N , AND BETWEEN THE TWO HALVES OF THE SAME MEMBRANE, THIS TENDANCY CAN HARDLY BE CONSIDERED S I G N I F I C A N T . THE IN VIVO S T U D I E S OF THE E F F E C T S OF PTH D E P R I V A T I O N OR E X C E S S IN L A Y I N G HENS ARE NOT D E F I N I T I V E . WHILE TOTAL P A R A -THYROIDECTOMY IN HENS LEADS TO DEATH WITHIN 4 8 HOURS MANY ANIMALS HAVE ACCESSORY PARATHYROIO T I S S U E EMBEDDED IN THE ULT I M08RANCHIAL OR THYROID GLANDS AND THESE ARE ABLE TO S U R V I V E , URIST ( 1 9 6 7 ) . THE PRESENCE OF THIS ACCESSORY T I S S U E C O M P L I C A T E S THE I N T E R -P R E T A T I O N OF THE EXPERIMENTAL R E S U L T S . POL IN & S T U R K I E (1957) FOUND THAT PARATHYROIDECTOMY OF L A Y I N G HENS CAUSED PREMATURE E X P U L S I O N OF THE EGG 3-7 HOURS AFTER THE O P E R A T I O N , WHICH THEY SUGGESTED COULD PROTECT THE HEN AGAINST HYPOCALCEMIA IN THE ABSENCE OF THE P A R A T H Y R O I D S , S INCE HENS WHICH DID NOT E X P E L THEIR EGGS PREMATURELY GENERALLY O I E D . THE HENS STOPPED L A Y I N G FOR ABOUT S I X DAYS ANO THEN RESUMED L A Y I N G NORMAL E G G S . THEY SHOWEO A MARKED DROP IN BOTH THE D I F F U S I B L E AND NON-0 I F F U S I B L E CALCIUM L E V E L S IN PLASMA 1 8 TO 2 4 HOURS AFTER THE O P E R A T I O N . THE OROP IN N O N - D I F F U S I B L E PLASMA CALCIUM WAS PROBABLY DUE TO Time in Hours F IGURE 5. THE E F F E C T OF PTH ON 45CA INFLUX ( B L O O D - » L U M E N ) IN A SHELL GLAND E X C I S E D JUST AFTER THE EGG HAD BEEN L A I D . -28-S TARVATI ON AND SURGICAL TRAUMA S INCE S H A M - O P E R A T E D AND STARVED HENS SHOVED A S IMILAR DECREASE IN THIS PLASMA CALCIUM F R A C T I O N . A FEW OF THE PARATHYR0 IDECTOMIZED HENS IN THIS STUDY SHOWED A MUCH LONGER PAUSE BEFORE RESUMING EGG PRODUCT ION, AND OTHERS DIED A FEW DAYS AFTER THE O P E R A T I O N ; THESE MAY HAVE BEEN BIRDS WHICH DIO NOT P O S S E S S ACCESSORY PARATHYROID T I S S U E . THUS PARATHYROIDECTOMY IN BIRDS D E C R E A S E S THE D I F F U S I B L E PLASMA CALCIUM L E V E L AND MAY ALSO CAUSE AN INTERRUPTION IN EGG P R O D U C -T I O N , ALTHOUGH THE R E S U L T S ARE NOT D E F I N A T I V E BECAUSE OF THE P R E S E N C E OF ACCESSORY GLANO T I S S U E . S T U D I E S OF THE E F F E C T OF EXOGENOUS PTH ON L A Y I N G HENS ALSO OO NOT GIVE VERY C L E A R C U T R E S U L T S . HENS REQUIRE A VERY LARGE DOSE OF MAMMALIAN PTH AND E F F E C T S ARE ONLY PRODUCED IN VERY YOUNG ANIMALS OR HENS IN A C T I V E LAY WITH VERY A C T I V E B O N E S . CANDLISH & TAYLOR ( 1 9 7 0 ) FOUND THAT THE HYPERCALCEMIC E F F E C T OF L I L L Y PARATHYROID EXTRACT WAS VERY RAPID IN L A Y I N G H E N S , THE MEAN RESPONSE TIME BEING 7~8 M I N U T E S . URIST _E_T M. ( 1 9 ^ 0 ) FOUND THAT PARATHYROID EXTRACT I N J E C T I O N S CAUSED A MODERATE DISTURBANCE OF EGG PRODUCTION IN L A Y I N G H E N S . THUS THE RESULTS WITH EXOGENOUS PTH SHOW THE SAME GENERAL E F F E C T S OF PTH ON L A Y I N G HEN PHYSIOLOGY AS THE PARATHYROIO A B L A T I O N EXPERIMENTS D I S C U S S E D A B O V E . THE ABSENCE OF A RESPONSE TO PTH J_N V I TRO MAY NOT N E C E S S A R I L Y MEAN THAT PTH HAS NO ACTION J^N V I V O . L l F S H I T Z E_T AJL (19&9) WERE UNABLE TO SHOW ANY RESPONSE OF I N T E S T I N A L LOOPS FROM NORMAL RATS TO PTH, BUT FOUND THAT WHEN THE LOOPS WERE TAKEN FROM PARA— THYROIDECTOMI ZED RATS THEY 010 GET AN INCREASED ABSORPTION OF CALCIUM AND P H O S P H A T E . THEREFORE RESPONSES IN AN IN V ITRO 4 0 0 0 -I Time in Hours F IGURE 6 THE E F F E C T OF PTH AND CT ON CALCIUM INFLUX (BLOOD -> LUMEN ) IN A SHELL GLANO E X C I S E D DURING A C T I V E EGG SHELL C A L C I F I C A T I O N . - 2 9 -SYSTEM MAY DEPEND UPON THE PARATHYROID S T A T U S OF THE ANIMAL FROM WHICH AN ORGAN IS E X C I S E D . AS THE L A Y I N G HEN IS KNOWN TO HAVE HYPERTROPHI ED PARATHYR0 I 0S , URIST ( 1 9 ^ 7 ) , TAYLOR ( 1 9 6 5 ) , IT IS P O S S I B L E THAT THE PRESENCE OF A LARGE AMOUNT OF ENDO-GENOUS P T H , P O S S I B L Y BOUND TO C E L L U L A R R E C E P T O R S , P R E C L U D E S THE ACTION OF EXOGENOUS PTH JI_N V I TRO . WHEN THE E F F E C T OF SALMON C A L C I T O N I N ( C T ) WAS T E S T E D IN THE IN VITRO SYSTEM IT WAS FOUNO TO HAVE VERY L I T T L E E F F E C T ON CALCIUM I N F L U X ; THE INCREASE IN INFLUX WAS ONLY 8 . 7 $ , SEE FIGURE 6 , ANO TABLE V I . TH IS ABSENCE OF A RESPONSE TO C T IN VITRO WAS NOT UNEXPECTED S I N C E ATTEMPTS TO SHOW AN IN VIVO E F F E C T OF C T IN B IRDS HAVE BEEN LARGELY U N S U C C E S S F U L . URIST ( 1 9 6 7 ) POINTS OUT THAT THE ULT IMOBRANCHIAL GLANDS IN THE L A Y I N G HEN ARE H Y P E R P L A S T I C ANO CONTAIN EAS I L Y - E X T R A C T A B L E LARGE AMOUNTS OF C T . HOWEVER WlTTERMAN JE_T _AJL ( 1 9 6 9 ) , USING THE RAT B I O A S S A Y , SHOWED THAT THE CONCENTRATION OF CT IN THE ULT IMOBRANCHIAL T I S S U E OF NINE MONTH OLD L A Y I N G HENS WAS NO D I F F E R E N T FROM THAT IN THREE WEEK OLD CHICKS OR THREE MONTH OLD P U L L E T S , WHICH HAD A NORMAL PLASMA CALCIUM AND NOT THE E S T R 0 G E N - I N D U C E 0 HYPERCALCEMIA FOUNO IN L A Y I N G H E N S . CANDLISH & TAYLOR ( 1 9 7 0 ) WERE UNABLE TO SHOW AN E F F E C T OF PORCINE C T ON THE PLASMA CALCIUM OF THE L A Y I N G H E N . S P E E R S E_T AL. ( 1 9 7 0 ) ULT I M08RANCH I ALECTOM I ZED CHICKS AT ONE TO TWO OAYS OF AGE AND REARED THEM TO MATURITY THROUGH S E V E R A L MONTHS OF NORMAL EGG PRODUCT ION. THE U L T I M 0 B R A N C H I A L E C T O M I ZED CHICKS ATE L E S S AND FORMED SMALLER EGGS WITH A TREND TOWARDS REDUCED SHELL T H I C K N E S S IN THE EARLY L A Y I N G PERIOD BUT NOT DURING SUBSEQUENT WEEKS. THUS NO VERY GREAT DISTURBANCE OF CALCIUM 5 0 0 0 - . THE EFFECT OF DIBUTYRYL C Y C L I C A M P ON 45CA FLUXES IN A SHELL GLANO E X C I S E D WHILE THE EGG WAS IN THE ISTHMUS. - 3 0 -META80L I SM WAS OBSERVED IN THE ABSENCE OF CT. THE ONLY P O S I T I V E RESPONSE OF CHICKENS TO CT TO DATE IS THAT OF KRAINTZ & INTSHER ( 1 9 6 9 ) » WHO SHOWED THAT PORCINE CT HAD NO E F F E C T ON F IVE DAY OLD CHICKS OR INTACT C O C K S , BUT BOTH PORCINE AND AVIAN CT CAUSED A DROP IN PLASMA CALCIUM IN P A R T I A L L Y PARATHYR0 IDECTOMIZED C O C K S . TH I S I M P L I E S THAT CT MAY HAVE A ROLE IN NORMAL CALCIUM HOMEOSTASIS IN THE HEN BUT IT I S , AT B E S T , A MINOR ONE. CY C L I C AMP. THE CONCEPT THAT MANY HORMONES ACT BY WAY OF A TWO MESSENGER SYSTEM IS NOW GENERALLY A C C E P T E D . SUCH HORMONES MAY BE REGARDED AS F I R S T MESSENGERS WHICH TRAVEL FROM THEIR C E L L S OF ORIGIN TO THE C E L L S OF THEIR TARGET T I S S U E S TO ST IMULATE THE FORMATION THEREIN OF A SECOND M E S S E N G E R . THE ONLY SECONO MESSENGER I D E N T I F I E D TO DATE IS C Y C L I C 3'5' - ADENOSINE MONOPHOSPHATE ( C Y C L I C AMP), AND MANY HORMONES ARE THOUGHT TO ACT VIA T H I S M E S S E N G E R , ROBISON _ET AL ( 1 9 6 8 , 1 9 7 1 ) . THE DIBUTYRYL D E R I V A T I V E WAS USED S INCE MANY WORKERS HAVE SHOWN T H A T , IN TH IS FORM THE C Y C L I C AMP IS MORE A C T I V E THAN THE PARENT COMPOUNO IN MIMIC ING HORMONE A C T I O N S , AND IN SEVERAL C A S E S IT HAS BEEN E F F E C T I V E WHERE C Y C L I C AMP I T S E L F WAS V I R T U A L L Y I N A C T I V E , ROBISON ET AL ( 1 9 6 8 ) . POSTERNAK EJ_ AL ( 1 9 6 2 ) SHOWED THAT DIBUTYRYL C Y C L I C AMP IS R E S I S T A N T TO THE I N A C T I V A T I O N CAUSED BY PHOSPHODIESTERASE C A T A L Y S I N G THE OPENING OF THE C Y C L I C PHOSPHATE R I N G . T H I S LED TO THE SUGGESTION THAT THE INCREASED POTENCY OF THE DIBUTYRYL D E R I V A T I V E COULD BE DUE TO ITS R E S I S T A N C E TO DEGRADATION BY PHOSPHODI E S T E R A S E . -31-LN THE PRESENT S E R I E S OF EXPERIMENTS DIBUTYRYL C Y C L I C AMP HAD NO E F F E C T ON INFLUX BUT INCREASED E F F L U X , SEE F IGURE 7. INFLUX REMAINED AT ABOUT 20 NMOLES CA/CM2 HR, WHILE E F F L U X INCREASED FROM 30 TO 75 NMOLES CA/CM2 HR, SEE TABLE V I I . WHEN STOCK BUFFER NO. 2 WAS USED AS THE INCUBATION MEDIUM THE INFLUX REMAINED A80UT 8 NMOLES CA/CM2 HR; THE E F F E C T ON E F F L U X WAS NOT T E S T E D IN THIS E X P E R I M E N T . THE LACK OF RESPONSE OF INFLUX TO DIBUTYRYL C Y C L I C AMP DOES NOT COMPLETELY RULE OUT THE INVOLVEMENT OF ADENYL C Y C L A S E IN TH IS CALCIUM T R A N S L O C A T I O N . LACK OF REACT ION COULD 8E DUE TO POOR ENTRY INTO C E L L S , R08IS0N E_T A_L (1967), OR IT COULD BE DUE TO THE I N A B I L I T Y OF THE C E L L S TO REMOVE THE 2'-0-ACYL GROUP OF DIBUTYRYL C Y C L I C AMP WHICH POSTERNAK E_T AJ_ (1 962) HAVE SHOWN IS NECESSARY BEFORE THE DIBUTYRYL D E R I V A T I V E CAN SHOW B I O L O G I C A L A C T I V I T Y . THE P H Y S I O L O G I C A L MEANING OF THE INCREASE IN E F F L U X IS OBSCURE S INCE THE IN VIVO CALCIUM FLUX IS OBVIOUSLY FROM SEROSAL TO MUCOSAL S I D E , I . E . I N F L U X . IT WOULD THEREFORE SEEM S U P E R -FLUOUS TO HAVE A HORMONALLY MEDIATED FLUX IN THE O P P O S I T E D I R E C T I O N . HOWEVER THERE IS SOME IN VIVO DATA WHICH LENDS SUPPORT TO THE INCREASE IN E F F L U X FOUND H E R E . MUELLER ET AL (1969) ADDED AM I NOPHYLL I NE TO THE D I E T OF L A Y I N G H E N S , AT A CONCENTRATION OF 0.05$, AND FOUND THAT IT CAUSED THE HENS TO LAY EGGS WITH S H E L L S OF REDUCED T H I C K N E S S . AM INOPHYLL INE IS THE SOLUS I L I Z E D FORM OF T H E O P H Y L L I N E WHICH INHIB ITS THE CYTOPLASMIC P H O S P H O D I E S T E R A S E AND HENCE CAUSES AN INTRACELLULAR BU ILDUP OF C Y C L I C AMP. THEY ALSO FOUND A S I G N I F I C A N T INCREASE IN TABLE V I I THE EF F E C T OF D IBUTYRYL CY C L I C AMP ON 45CA FL U X E S IN THE IN V ITRO SH E L L GLAND ST A T E OF GLAND STOCK BUFFER No. CALCIUM FL U X E S IN NMOLES CA/CM2 HR CONTROL AFTER OI B U T Y R Y LCY C L I C AMP (5X10-4M IN INNER S O L N . ) INFLUX EF F L U X FLUX RATIO* INFLUX EF F L U X FLUX RATIO* EGG IN ISTHMUS 1 2 1 . 7 9 ± 1.0 3 2 . 2 2 ± 1 . 4 0 . 6 7 6 3 2 1 . 4 5 ± 0 . 7 6 7 6 . 3 4 ± 1 . 8 0 . 2 8 1 0 AC T I V E EGG SH E L L CA L C I F I C A T I O N 2 8.029 ± 1 . 0 8 4 8 . 6 7 ± 1 . 2 3 0 . 1 6 4 9 9.58 ± 0 . 3 7 * FLUX RATIO = » NFLUX = BLOOD - » LUMEN E F F L U X LUMEN BLOOD -32-T HE PH ANO BICARBONATE CONCENTRATION OF THE SHELL GLAND F L U I D . IF THE CALCIUM WAS TRANSPORTED AWAY FROM THE MUCOSAL S IDE OF THE SHELL GLAND DUE TO THE INCREASE IN C Y C L I C AMP, AS THE IN VITRO DATA TEND TO S U G G E S T , THEN THE BICARBONATE C O N C E N T R A T I O N , AND HENCE P H , WOULD R ISE BECAUSE THE REACT ION OF CALCIUM IONS WITH THE BICARBONATE IONS TO PRODUCE THE SOLID CALCIUM CARBONATE OF THE EGG SHELL WOULD BE REOUCEO DUE TO LACK OF C A L C I U M . I F IT IS A C C E P T E D THAT AN INCREASE IN INTRACELLULAR C Y C L I C AMP CAUSES AN INCREASE IN E F F L U X THEN ONE MUST ASK WHICH HORMONE, IF ANY , COULD BE THE EXTERNAL MEDIATOR IN T H I S S Y S T E M ; I . E . WHICH HORMONE BY R E A C T I N G WITH A S P E C I F I C RECEPTOR S I T E ON THE C E L L MEMBRANE COULD A C T I V A T E A MEMBRANE-BOUND AOENYL C Y C L A S E AND HENCE INCREASE THE C Y C L I C AMP C O N C E N T R A T I O N . IT IS NOW WELL E S T A B L I S H E D THAT PTH ACTS VIA C Y C L I C AMP IN BONE AND K I D N E Y , CHASE & AURBACH ( 1 9 6 8 , 1 9 6 9 ) , CHASE E_T AJ_ ( 1 9 6 9 ) ANO RASMUSSEN & TENENHOUSE ( 1 9 6 8 ) . HOWEVER PTH L E V E L S ARE THOUGHT TO BE HIGH DURING EGG S H E L L C A L C I F I C A T I O N BECAUSE OF THE P O S T U L A T E D ROLE OF PTH IN THE BREAKDOWN OF MEDULLARY BONE , TAYLOR ( 1 9 6 5 , 1 9 7 0 B ) . IF PTH WERE THE HORMONE INVOLVED TH IS WOULO CREATE A P H Y S I O L O G I C A L P A R A 0 0 X S INCE A HORMONE WHOSE L E V E L WAS HIGH DURING EGG S H E L L C A L C I F I C A T I O N WOULD TEND TO INH IB IT EGG S H E L L C A L C I F I C A T I O N . MURAD EJT_ AJ. ( 1 9 7 0 ) PRESENT E V I D E N C E THAT CT INCREASES C Y C L I C AMP IN KIDNEY AND BONE C E L L S . THUS THE EXTERNAL MEOIATOR IN THE SHELL GLAND SYSTEM COULD BE CT. T H I S WOULD SEEM A MORE L I K E L Y CANDIDATE S INCE AT THE END OF EGG S H E L L C A L C I F I C A T I O N ONE COULO E N V I S A G E A TRANSIENT INCREASE IN PLASMA CALCIUM WHICH - 3 3 -COULO TRIGGER THE R E L E A S E OF CT, AND ANY E X C E S S CALCIUM WHICH HAD BEEN PUMPED INTO THE SHELL GLAND LUMEN WOULD BE RETURNEO TO THE BLOOD. IN THIS REGARD IT WOULD BE OF INTEREST TO TEST THE E F F E C T OF CT ON E F F L U X , AN E X P E R I M E N T WHICH WAS NOT DONE H E R E . COMPARISON BETWEEN THE J_N V I TRO AND J_N V I VO S Y S T E M . IT IS P O S S I B L E TO C A L C U L A T E THE APPROXIMATE FLUX WHICH MUST TAKE P L A C E IN VIVO IN ORDER TO C A L C I F Y THE SHELL AT THE OBSERVEO R A T E . IN ORDER TO OO TH IS ONE MUST KNOW THE AREA OF THE SHELL GLAND IN CONTACT WITH THE S H E L L . TH IS CAN BE APPROXIMATED F A I R L Y WELL BY ASSUMING THAT IT IS EQUAL TO THE SURFACE AREA OF THE EGG S H E L L . MUELLER & SCOTT ( 1 9 4 0 ) C A L C U L A T E D THE SURFACE AREA OF 5 0 WHITE LEGHORN P U L L E T E G G S , USING THREE D I F F E R E N T METHODS, AND FOUND THAT THE AVERAGE SURFACE AREA WAS ABOUT 7 0 CM2. TAYLOR ( 1 9 6 5 ) S T A T E S T H A T , ON A LOW CALCIUM O I E T , THE HEN M O B I L I Z E S APPROXIMATELY 1 2 0 MG CA/ HR FROM THE S K E L E T O N FOR EGG SHELL C A L C I F I C A T I O N . T H E R E F O R E O N E CAN C A L C U L A T E THAT THE J^ N VIVO FLUX SHOULD BE 4 2 . 8 ^ M O L E S CA/CM2 HR. THE IN VITRO F L U X E S OBTAINED IN THESE EXPERIMENTS RANGED FROM 8 TO 1 8 0 NMOLES CA/CM2 HR, WHILE THOSE OF EHRENSPECK E_T AL ( 1 9 6 7 ) RANGED FROM 2 0 TO 5 0 NMOLES CA/CM2 HR. ON AVERAGE THE IN VITRO R A T E S WERE ABOUT 5 0 0 - 1 0 0 0 T IMES SLOWER THAN THE C A L C U L A T E O IN vivo R A T E . OBVIOUSLY THERE IS SOME FACTOR PRESENT IN THE INTACT HEN WHICH IS ABSENT IN THE IN VITRO S Y S T E M . ONE OF THE MOST OBVIOUS D I F F E R E N C E S BETWEEN THE IN VIVO SYSTEM ANO T H I S EXPERIMENTAL ONE IS LACK OF BLOOD FLOW. G I L B E R T - 3 4 -(19^7) C A L C U L A T E O THAT DURING EGG SHELL C A L C I F I C A T I O N ABOUT 1/6TH TO 1/8TH OF THE CARDIAC OUTPUT MUST GO THROUGH THE S H E L L GLAND FOR THE AMOUNT OF CALCIUM FOUND IN THE EGG S H E L L TO BE D E P O S I T E D IN THE F I F T E E N HOURS OF A C T I V E SHELL C A L C I F I C A T I O N . S I N C E THE BLOOD IS CONSTANTLY RENEWED A CONCENTRATION GRAOIENT ( E . G . FOR C A L C I U M ) CAN ALWAYS 8E MAINTAINED BETWEEN THE SEROSAL AND MUCOSAL S I D E S OF THE SHELL GLAND. B U T , IN THE IN VITRO S Y S T E M , THE BLOOD IS R E P R E S E N T E D BY A STAGNANT CHAMBER SO THAT THE CONCENTRATION OF CALCIUM ON THE SEROSAL S IDE CAN F A L L , THEREBY ALLOWING BACK D I F F U S I O N TO OCCUR. FURTHERMORE, IN VIVO THE CALCIUM IS P R E C I P I T A T E D AS THE C A R B O N A T E , MAKING THE PUMP FUNCT IONALLY I R R E V E R S I B L E . OBVIOUSLY THE IN VITRO SYSTEM DOES NOT APPROXIMATE THIS S I T U A T I O N . THE S H E L L GLAND IS AN EXTREMELY THICK AND MUSCULAR ORGAN WHICH MUST PRESENT A VERY GREAT BARRIER TO GAS D I F F U S I O N . ALTHOUGH THE _I_N V I TRO SYSTEM WAS CONSTANTLY BUBBLED WITH 9 5 $ 02, 5 $ C02, THERE IS THE P O S S I B I L I T Y THAT THESE GASES DID NOT D I F F U S E FAST ENOUGH THROUGH THE SHELL GLAND TO REACH THE VALUES THEY WOULD HAVE A T T A I N E D IN THE INTACT H E N . T H I S MIGHT WELL L I M I T THE RATE AT WHICH AN ENERGY DEPENDENT PUMP COULO O P E R A T E . BEFORE THE A C T I V E PERIOD OF EGG S H E L L C A L C I F I C A T I O N , DURING THE F I R S T F I V E HOURS IN THE SHELL GLANO, THE EGG IMBIBES WATER AND S W E L L S , A PROCESS KNOWN AS P L U M P I N G , BURMESTER ( 1 9 4 0 ) . TH I S GREATLY INCREASES THE S I Z E OF THE EGG AND THE WALLS OF THE SHELL GLANO ARE THEREFORE S T R E T C H E D . TH IS S T R E T C H I N G PROCESS COULD BE NECESSARY FOR THE FUNCTIONING OF THE PUMP, E S P E C I A L L Y - 3 5 -SINCE GILBERT & LAKE ( 1 9 6 3 ) HAVE SHOWN THAT THE SHELL GLANO CONTAINS AN E X T E N S I V E I N T R I N S I C NERVOUS S U P P L Y , SO THAT STRETCH COULD SET UP LOCAL R E F L E X A C T I V I T Y WHICH WOULD THEN ACTIVATE THE PUMP. IN THE EXPERIMENTS PERFORMED HERE AN ATTEMPT WAS MADE TO SUBJECT ALL THE PREPARATIONS TO THE SAME AMOUNT OF STRETCH, BUT THERE WAS NO WAY TO A S C E R T A I N HOW CLOSELY THIS STRETCH APPROXIMATED THE IN VIVO S I T U A T I O N . ALSO THE IN VITRO PREPARATION USED SMALL P I E C E S OF THE SHELL GLAND AND THE WHOLE I N T R I N S I C NERVOUS SYSTEM COULD BE NECESSARY FOR A C T I V I T Y . HOWEVER, GILBERT ( 1 9 6 7 ) HAS POINTEO OUT THAT THE F A I R L Y COMMON PRODUCTION OF MINIATURE EGGS (USUALLY SMALL Q U A N T I T I E S OF ALBUMEN SURROUNOED BY A S H E L L ) DOES NOT SUPPORT THE HYPOTHESIS THAT D I S T E N S I O N OF THE SHELL GLAND PER SE IS NECESSARY FOR C A L C I F I C A T I O N . HE SUGGESTS THAT THE STIMULUS FOR SHELL FORMATION MAY OCCUR IN THE ISTHMUS WITH NEURAL COORDINATION BETWEEN THIS REGION AND THE SHELL GLAND. THE IN VITRO PREPARATION OBVIOUSLY LACKS AN E X T R I N S I C NERVOUS SUPPLY AND, SINCE F R E E D M A N & S T U R K I E ( 1 9 6 3A) HAVE SHOWN THAT THE SHELL GLAND HAS AN E X T E N S I V E E X T R I N S I C NERVOUS SUPPLY CONTAINING BOTH SYMPATHETIC ANO PARASYMPATHETIC NERVES, IT I S REASONABLE TO POSTULATE THAT THESE COULD BE INVOLVED. T H I S APPEARS EVEN MORE PROBABLE WHEN ONE CONSIDERS THAT BIRDS POSSESS AN ION PUMP WHICH I S DEPENDENT ON AN INTACT NERVE S U P P L Y , V IZ THE SALT GLAND , S CHMIDT-NIELSON ( 1 9 6 0 ) . THE DEPENDENCE OF THE SHELL GLANO CALCIUM PUMP ON AN INTACT NERVE SUPPLY COULO E X P L A I N THE VERY LOW FLUXES OBTAINED IN VITRO COMPARED WITH THE IN vivo FLUX. THEREFORE IT WAS DECIDED THAT THE IN VITRO -36-P R E P A R A T I ON WOULD BE ABANDONED ANO P O S S I B L E NERVOUS EFFECTS ON THE SHELL GLAND PUMP I N V I V O WOULD BE E X A M I N E D . CHAPTER I I O B S E R V A T I O N S ON THE E F F E C T OF C E R T A I N DRUGS UPON T H E A N E S T H A T I Z E D C H I C K E N . I NTRODUCTION. AS AN ADAPTATION OF E L I M I N A T I O N OF E X C E S S S A L T INGESTEO IN THEIR FOOD AND DRINK MARINE BIRDS HAVE A HIGHLY DEVELOPED SALT GLANO, SCHM I D T - N l ELSON ( i 9 6 0 ) . TH I S GLAND ACTS TO REMOVE SOOIUM CHLORIDE FROM THE ANIMAL WHEN IT IS S U B J E C T E D TO AN OSMOTIC LOAO OF ANY K I N D . F A N G E ET AL ( 1 9 5 8 ) SHOWEO THAT S E C R E -TION COULD BE PROOUCEO BY S T I M U L A T I O N OF A NERVE TO THE GLANO, AND THAT THIS NERVE WAS C H O L I N E R G I C BECAUSE THE RESPONSE TO THE NERVE ST IMULAT ION WAS BLOCKED BY ATROPINE ANO THE GLAND WAS S T I M U L A T E D BY A C E T Y L C H O L I N E ANO M E C H O L Y L . S I N C E THE RESPONSE DUE TO AN OSMOTIC LOAO WAS ALSO BLOCKEO ALMOST COMPLETELY BY ATROPINE THEY CONCLUDED THAT THE S A L T GLAND S E C R E T I O N WAS DEPENDENT UPON P A R A S Y M P A T H E T I C NERVOUS A C T I V I T Y . T H I S IDEA IS NOW GENERALLY A C C E P T E D . THE LOW FLUX RATES OBSERVED WITH THE IN VITRO SYSTEM SUGGESTED THAT SOME FACTOR WHICH IS NORMALLY PRESENT IN THE INTACT ANIMAL HAS BEEN REMOVEO. THE HOST 0 8 V I 0 U S D I F F E R E N C E BETWEEN THE J_N VIVO AND J_N VITRO S I T U A T I O N S IS T H A T , IN THE L A T T E R , THE BLOOD ANO NERVOUS S U P P L I E S HAVE BEEN S E V E R E D . I N VIEW OF THE NATURE OF THE CONTROL OF THE S A L T GLAND D E S C R I B E D A B O V E , A S E R I E S OF EXPERIMENTS WERE CONDUCTED TO I N V E S T I G A T E THE R E L A T I O N S H I P , IF A N Y , BETWEEN VARIOUS BRANCHES OF THE AUTONOM IC NERVOUS SUPPLY AND CALCIUM TRANSLOCATION IN THE SHELL GLAND. FREEOMAN & S T U R K I E ( 1 9 6 3 A ) HAVE SHOWN THAT THE SHELL GLANO HAS BOTH SYMPATHET IC ANO P A R A S Y M P A T H E T I C INNERVAT ION . A L L NERVES - 3 7 -- 3 8 -S U P P L Y I N G THE UTERUS WERE FOUNO TO ORIGINATE IN THE L E F T S IOE OF THE BODY, WHICH IS NOT S U R P R I S I N G S INCE ONLY THE L E F T OVIDUCT D E V E L O P S IN H E N S . THEY FOUND THAT THE S Y M P A T H E T I C INNERVATION IS S U P P L I E D BY THE HYPOGASTRIC N E R V E , A D IRECT CONTINUATION OF THE AORTIC P L E X U S , WHICH COURSES ALONG THE HYPOGASTRIC ARTERY TO THE U T E R U S . THE P A R A S Y M P A T H E T I C P E L V I C NERVES ORIGINATE FROM THE P E L V I C V I S C E R A L RAMI OF S P I N A L NERVES 3 0 - 3 3 AND J O I N TOGETHER IN A COMPLEX FASHION TO FORM THE L E F T P E L V I C N E R V E . THE L E F T P E L V I C NERVE HAS NUMEROUS GANGLIA FROM WHICH MANY NERVES RAMIFY AND THE WHOLE COMPLEX IS KNOWN AS THE P E L V I C P L E X U S . NERVES EMANATING FROM THIS P L E X U S COURSE ALONG THE MIDDLE U T E R I N E AND POSTERIOR UTERINE A R T E R -IES TO THEIR TERMINATION ON THE U T E R U S . THESE P A R A S Y M P A T H E T I C NERVES ARE EMBEDDED IN THE URETERS AND BLOOD V E S S E L S , MAKING THEIR E X I S I O N D I F F I C U L T WITHOUT OAMAGING THE BLOOD SUPPLY TO THE U T E R U S . THE HYPOGASTRIC NERVE IS F A I R L Y D I S C R E T E ANO CAN BE R E A D I L Y S E E N ON THE SURFACE OF THE HYPOGASTRIC A R T E R Y . HOWEVER THIS ARTERY ENTERS THE DORSAL SIDE OF THE SHELL GLAND, FREEDMAN & ST U R K I E ( 1 9 6 3 B ) , AND THEREFORE RETRACT ION OF THE SHELL GLAND IS REQUIRED IN ORDER TO F IND THE N E R V E . PREL IMINARY EXPERIMENTS SHOWEO THAT SUCH MANIPULAT ION OF THE SHELL GLANO CAUSED PREMATURE E X P U L S I O N OF THE EGG AND HENCE PRECLUOEO STUOY OF THE C A L C I F I C A T I O N P R O C E S S . ALSO THERE IS S T I L L UNCERTAINTY AS TO WHETHER THERE IS S Y M P A T H E T I C R E P R E S E N T A T I O N IN THE P E L V I C P L E X U S OF THE H E N , FREEDMAN & S T U R K I E ( 1 9 6 3 A ) . THEREFORE PHARMACOLOGICAL BLOCKERS WERE CHOSEN AS THE P R E F E R R E D METHOD FOR STUDYING THE E F F E C T OF THE VARIOUS BRANCHES OF THE - 3 9 -AUTONOMIC NERVOUS SUPPLY ON THE C A L C I F I C A T I O N OF THE EGG SHELL BY THE INTACT HEN'S UTERUS. THERE I S NOW GENERAL ACCEPTANCE OF THE THEORY OF NEURO-HUMORAL TRANSMISSION, I . E . NERVES TRANSMIT THEIR IMPULSES ACROSS MOST SYNAPSES ANO NEUROEFFECTOR JUNCTIONS BY MEANS OF S P E C I F I C CHEMICAL AGENTS. THE NEUROHUMORAL TRANSMITTOR OF ALL PREGANGLIONIC AUTONOMIC F I B R E S , ALL POSTGANGLIONIC PARASYM-P A T H E T I C F I B R E S AND A FEW POSTGANGLIONIC SYMPATHETIC F I B R E S I S A C E T Y L C H O L I N E ( C H O L I N E R G I C F I B R E S ) ; WHILE IN THE MAJORITY OF POSTGANGLIONIC SYMPATHETIC F I B R E S THE TRANSMITTOR IS NOREPINEPHRINE (ADRENERGIC F I B R E S ) , GOODMAN & GLLMAN ( 1 9 6 5 ) . CHOLINERGIC JUNCTIONS CAN BE FURTHER D I F F E R E N T I A T E D INTO TWO RECEPTOR TYPES ON THE B A S I S OF THE E F F E C T S OF THE A L K A L O I D S MUSCARINE AND N I C O T I N E . MUSCARINE IS A PARASYMPATHOMIMETIC WHICH A C T I V A T E S THE CHOLINERGIC RECEPTORS AT THE AUTONOMIC EFFECTOR C E L L S AND THEREFORE, BY CONVENTION, THESE ARE CALLEO MUSCARINIC. N I C O T I N E IN LOW OOSES S T I M U L A T E S , AND IN HIGHER DOSES PARALYSES AUTONOMIC GANGLIA. THE SAME SEQUENCE OCCURS AT AT THE MOTOR END P L A T E S OF CERTAIN TYPES OF SK E L E T A L MUSCLE. HENCE THESE JUNCTIONS ARE KNOWN AS N I C O T I N I C JUNCTIONS. THESE TWO TYPES OF CHOLINERGIC RECEPTOR RESPOND D I F F E R E N T L Y TO CE R T A I N BLOCKING AGENTS. GANGLIONIC N I C O T I N I C RECEPTORS ARE BLOCKED BY HE XAMETH0NIUM SALTS WHILE ATROPINE AND OTHER RELATED BELLADONNA A L K A L O I D S S E L E C T I V E L Y BLOCK MUSCARINIC RECEPTORS, GOODMAN AND GILMAN ( 1 9 6 5 ) . BLOCKAOE OF N I C O T I N I C RECEPTORS BLOCKS ALL THE AUTONOMIC GANGLIA AND HENCE PARALYSES BOTH THE SYMPATHETIC AND PARASYMPATHETIC NERVOUS S U P P L I E S , WHEREAS MUSCARINIC BLOCKADE S E L E C T I V E L Y I N H I B I T S THE PARASYMPATHETIC - 4 0 -NERVOUS SUPPLY• THEREFORE ATROPINE WAS CHOSEN AS THE P A R A -SYMPATHET IC BLOCKING A G E N T . ADRENERGIC RECEPTORS HAVE ALSO BEEN D I F F E R E N T I A T E D INTO TWO T Y P E S . AHLQUIST ( 1 9 4 8 ) STUDIED THE POTENCY OF F I V E SYMPATHOMIMETIC AMINES ON A VARIETY OF T I S S U E RESPONSES BOTH IN ISOLATED ORGAN BATHS AND INTACT A N I M A L S . HE WAS ABLE TO C L A S S I F Y SYMPATHET IC RECEPTORS INTO TWO GROUPS WHICH HE C A L L E D ALPHA ANO B E T A . THE ORDER OF POTENCY FOR S T I M U L A T I O N OF ALPHA RECEPTORS WAS E P I N E P H R I N E , N O R E P I N E P H R I N E , M E T H Y L N O R E P I N E P H R I N E , M E T H Y L E P I N E P H R I N E AND I S O P R O T E R E N O L ; WHILE FOR THE BETA RECEPTORS IT WAS I S O P R O T E R E N O L , E P I N E P H R I N E , METHYLEP I NEPHRINE , METHYLNOREPINEPHRINE AND N O R E P I N E P H R I N E . IN GENERAL ALPHA RECEPTOR ST IMULAT ION RESULTED IN EXCITATORY RESPONSES ANO BETA RECEPTOR S T I M U L A T I O N IN INHIBITORY R E S P O N S E S . THERE WERE TWO E X C E P T I O N S , CARDIAC E X C I T A T I O N , WHICH WAS C L A S S I F I E D CHEMICALLY AS A BETA RECEPTOR R E S P O N S E , AND I N H I B I T I O N OF THE GUT , WHICH APPEARED TO BE AN ALPHA R E S P O N S E . IT WAS SHOWN SUBSEQUENTLY THAT THE GUT RESPONSE WAS MEDIATEO BY BOTH ALPHA AND BETA R E C E P T O R S , AHL QUI ST & LEVY ( 1 9 5 9 ) . AHLQUI ST* S CLASS I FI CAT I ON HAS S INCE BEEN V I N D I C A T E D BY S T U O I E S WITH ALPHA AND BETA BLOCKING DRUGS. BEFORE HIS PROPOSAL THE ADRENERGIC BLOCKING ACTION OF THE ERGOT A L K A L O I D S HAD BEEN DEMONSTRATED BY DALE ( 1 9 0 6 ) , BUT THESE WERE ALL ALPHA B L O C K E R S . I T WAS NOT UNT IL 1 9 5 8 THAT POWELL & S L A T E R D E S C R I B E D D I C H L O R O I S O P R O T E R E N O L , THE F I R S T BETA ADRENERGIC RECEPTOR BLOCKING DRUG. S l N C E THEN MANY D I F F E R E N T ADRENERGIC BLOCKING DRUGS HAVE BEEN D E S C R I B E D . ONE OF THE MOST E F F E C T I V E ANO - 4 1 -S P E C I F I C ALPHA BLOCKERS IS THE HALOALKAMINE PHEN0XYBEN2AM INE (o I BENZYL I NE ) , Nil CKERSON & HOLLENBERG ( 1 9 6 7 ) . T H I S IS AN I R R E V E R S I B L E BLOCKER AND IT TAKES SOME TIME BEFORE ITS E F F E C T S BECOME MAXIMAL . THE FAVOURED BETA BLOCKER TO DATE APPEARS TO BE PROPRANALOL ( I N D E R A L ) , F l T Z G E R A L D ( 1 9 ^ 9 ) • THE EXPERIMENTAL PROTOCOL WHICH WAS TO BE ADOPTED FOR THE C A L C I F I C A T I O N EXPERIMENTS REQUIRED A CONTINUOUS BLOCK FOR UP TO TWO HOURS SO THAT DRUG DOSE L E V E L S WHICH GAVE A UNIFORM MAXIMAL BLOCKADE OVER T H I S TIME PERIOD WERE R E Q U I R E D . THE L I T E R A T U R E ON THE PHARMACOLOGY OF THE C H I C K E N IS VERY SCANTY ANO HENCE IT WAS D I F F I C U L T TO FIND THE REQUIRED DOSES IN THE L I T E R A T U R E . IN THEIR 1 9 6 2 REVIEW A R T I C L E BUNAG & WALASZEK STATE T H A T , " T H E ONLY CHOL INERGIC BLOCKING AGENT WHICH HAS BEEN I N V E S T I G A T E D IN THE AVIAN CARDIOVASCULAR SYSTEM IS A T R O P I N E 1 1 . THERE IS SOME CONTROVERSY ABOUT THE DOSAGE REQUIRED FOR COMPLETE C H O L I N E R G I C B L O C K A D E . G l B 8 S ( 1 9 2 6 ) SHOWED THAT INTRAVENOUS A D M I N I S T R A T I O N OF A T R O P I N E SULPHATE 0 . 5 - 1 . 0 MG/ KG IN THE CHICKEN COMPLETELY BLOCKED VAGAL SLOWING OF THE H E A R T . LLOYD & PICKFORD ( 1 9 6 1 ) WERE ABLE TO BLOCK OEPRESSOR RESPONSES TO A C E T Y L C H O L I N E WITH 1 - 2 MG OF ATROPINE GIVEN INTRAVENOUSLY . HOWEVER HARVEY E_T A_L ( 1 9 5 4 ) FOUND THAT INTRAVENOUS DOSES OF ATROPINE BELOW 5 MG/KG NEVER COMPLETELY BLOCKED A C E T Y L C H O L I N E -INDUCED VASODEPRESSI ON, ANO THAT DOSES OF 9—12 MG/ KG WERE GENERALLY R E Q U I R E D . WlTH SUCH DOSES THE BLOCK L A S T E D FOR 1 - 3 HOURS. BUNAG & WALASZEK ( 1 9 6 2 ) CONFIRMED THAT 5 ~ 1 0 MG/KG OF ATROPINE GIVEN INTRAVENOUSLY BLOCKED THE OEPRESSOR RESPONSE - 4 2 -TO A C E T Y L C H O L I N E BUT LOWER DOSES ( 0 . 5 - 2 . 0 MG / K G) ONLY A C H I E V E D P A R T I A L B L O C K A D E . THUS I T APPEARS THAT THE HEN REQUIRES MORE A T R O P I N E TO BLOCK THE EFFECTS OF A C E T Y L C H O L I N E THAN I T DOES TO BLOCK P A R A S Y M P A T H E T I C N E U R O T R A N S M I S S I O N , I N APPARENT C O N T R A D I C T I O N TO THE USUAL F I N D I N G S , GOODMAN & G l L M A N ( 1 9 6 5 ) . THE USUAL INTRAVENOUS DOSE OF A T R O P I N E FOR C H O L I N E R G I C BLOCKADE I N DOGS I S 1.0 MG / KG , BARNES & E L T H E R I N G T O N ( 1 9 6 4 ) , SO THAT THE DOSE REQUIRED BY THE CHICKEN APPEARS TO BE VERY L A R G E . LARGE DOSES OF ATROPINE CAN HAVE UNDESIRABLE NON-S P E C I F I C EFFECTS SUCH AS G A N G L I O N I C BLOCKADE AND I N H I B I T I O N OF THE RESPONSES TO OTHER T R A N S M I T T O R S , NAMELY H I S T A M I N E , SEROTONIN AND N O R E P I N E P H R I N E , GOODMAN & GLLMAN ( 1 9 6 5 ) . THE LARGE BLOCKING DOSES REQUIRED IN THE HEN MAY BE DUE TO E I T H E R THE PRESENCE OF AN A T R O P I N A S E , AS HAS BEEN D E S C R I B E D IN THE R A B B I T , AMBACHE ( 1 9 5 5 ) , OR BECAUSE OF RAPID DESTRUCT ION OF THE DRUG AS A RESULT OF THE HIGH METABOLIC RATE IN B I R D S , ST U R K I E ( 1 9 6 5 ) . IN E I T H E R C A S E , A CONTINUOUS INTRAVENOUS INFUSION WOULD OBVIATE THE N E C E S S I T Y FOR LARGE N O N S P E C I F I C D O S E S . AS P R E L I M I N A R Y E X P E R I M E N T S ON THE BLOCKADE OF THE DEPRESSOR RESPONSE TO 4 JJG/KG OF A C E T Y L C H O L I N E BY 0.5 MG/KG OF A T R O P I N E SHOWED THAT THE BLOCK D I O INDEED DECAY VERY Q U I C K L Y E X P E R I M E N T S WERE CONDUCTED TO F I N D THE L E V E L OF A T R O P I N E NEEDED FOR A CONTINUOUS INTRAVENOUS I N F U S I O N . CAMPOS « U R Q U I L L A ( 1 9 6 9 ) HAD DEVELOPED AN INTRAVENOUS I N F U S I O N OF A T R O P I N E FOR THE DOG I N WHICH THEY USED A P R I M I N G DOSE OF 0.25 MG / KG AND GAVE A CONTINUOUS I N F U S I O N OF 5 ^ G / K G / M I N . TH I S WAS USED AS A - 4 3 -START IN G POINT FOR THE EXPERIMENTS TO FIND A S U I T A B L E INFUSION L E V E L OF ATROPINE FOR APPROXIMATELY 1 OOF4 BLOCKADE IN THE C H I C K E N . THE CONTINUOUS INFUSION OF ATROPINE ALSO MEANT THAT BLOCKADE COULO BE MAOE TO LAST FOR AS LONG AS REQUIRED AND HENCE THE REQUIREMENTS OF THE PROTOCOL FOR THE C A L C I F I C A -TION EXPERIMENTS COULD BE M E T . DATA ON ADRENERGIC BLOCKERS IN C H I C K E N S IS NOT AS SCARCE AS THAT ON PARASYMPATHET IC B L O C K E R S . AS WITH ATROPINE THE DOSES REQUIRED IN THE CHICKEN ARE QUITE LARGE COMPARED WITH THOSE IN MAMMALS. HARVEY E_T AJ_ ( 1 9 5 4 ) WERE THE F I R S T WORKERS TO MAKE A R E A L L Y S Y S T E M A T I C STUDY OF ADRENERGIC BLOCKADE IN THE C H I C K E N . AT THAT T IME ONLY ALPHA BLOCKERS WERE A V A I L A B L E . THEY FOUND THAT 2 0 MG/KG OF DIBENAMINE F A I L E D TO INDUCE ADRENERGIC B L O C K A D E , BUT THE MORE POTENT S U B S T I T U T E D H A L O -AL KAM INE D I B E N Z Y L I N E (PHENOXYBENZAM INE ) , AT L E V E L S OF 2 0 - 6 0 MG/KG, DID PRODUCE A VASOMOTOR REVERSAL OF THE RESPONSE TO E P I N E P H R I N E INDICAT ING ALPHA B L O C K A O E . BUNAG & WALASZEK ( 1 9 6 2 ) REPORT THAT THEY WERE UNABLE TO KEEP THEIR CHICKENS A L I V E LONG ENOUGH TO ALLOW T E S T I N G OF THE OEGREE OF BLOCKAOE WHEN THEY GAVE SUCH HIGH DOSES OF D I B E N Z Y L I N E . THEY- SHOW QUITE MARKED REOUCTION OF THE PRESSOR RESPONSE TO E P I N E P H R I N E ANO NOREPINEPHRINE WITH 2 0 M G / K G OF D I B E N Z Y L I N E BUT NO R E V E R S A L . BOLTON <S 3OWMAN ( 1 9 6 9 ) ALSO FOUND VASOMOTOR REVERSAL WITH E P I N E P H R I N E WITH 2 0 - 6 0 M G / K G OF D I B E N Z Y L I N E . T H E S E EXPERIMENTS WERE ALL HAMPERED BY THE LACK OF A S P E C I F I C ALPHA AGONIST SO THAT COMPLETE BLOCK HAD TO R E S U L T IN VASOMOTOR REVERSAL AS THE BETA E F F E C T S WERE UNMASKED. WITH THESE N O N S P E C I F I C AGONISTS -44-AN ABSOLUTE MEASURE OF THE L E V E L OF BLOCKADE WAS NOT P O S S I B L E . P H E N Y L E P H R I N E IS A S P E C I F I C ALPHA ADRENERGIC A G O N I S T , GOODMAN & GILMAN ( 1 9 6 5 ) . USING TH IS IT WAS P O S S I B L E TO OETERMINE ABSOLUTE L E V E L S OF ADRENERGIC BLOCKADE AND HENCE S E L E C T A S U I T A B L E DOSE OF D I B E N Z Y L I N E TO GIVE THE REQUIRED B L O C K A D E . A CONTINUOUS INFUSION WAS NOT NECESSARY WITH THIS DRUG BECAUSE IT IS AN I R R E V E R S I B L E B L O C K E R , GOODMAN & GLLMAN ( 1 9 6 5 ) . THE F I R S T REPORT OF THE USE OF A BETA ADRENERGIC BLOCKER IN C H I C K E N S WAS BY BUNAG & WALASZEK ( 1 9 6 2 ) , WHO USED D I C H L O R O I S O P R O T E R E N O L , THE F I R S T BETA BLOCKER D I S C O V E R E D . THEY FOUND THAT A DOSE OF 1 0 MG/KG A B O L I S E D THE DEPRESSOR RESPONSE TO 1 0 JJQ/KG OF THE S P E C I F I C BETA AGONIST I S O P R O T E R E N O L . THE BLOCKADE WAS OF F A I R L Y LONG DURATION AND WAS P R E S E N T THREE HOURS A F T E R THE DRUG A D M I N I S T R A T I O N . BOLTON & BOWMAN ( 1 9 6 9 ) ALSO FOUND THAT A DOSE OF 5 ~ 1 0 M G / K G D I C H L 0 R 0 I S 0 P R 0 T E R E N O L ABOL ISHED THE DEPRESSOR RESPONSE TO I S O P R O T E R E N O L . A DOSE OF 0 . 2 MG/KG OF PROPRANALOL A B O L I S H E D THE DEPRESSOR RESPONSE TO I S O P R O T E R E N O L , ALTHOUGH SOME HENS REQUIRED AS MUCH AS 1 MG/KG. HOWEVER THESE WORKERS DID NOT STATE HOW LONG THE PROPRANALOL BLOCKADE L A S T E D , AND THEREFORE IT WAS NECESSARY TO CONDUCT BLOCKING EXPERIMENTS TO FIND THE OPTIMAL L E V E L OF PROPRANALOL TO PRODUCE A GOOD BLOCKADE FOR AS LONG AS TWO HOURS AS THE PROTOCOL OF THE C A L C I F I C A T I O N EXPERIMENTS DEMANDED. THE EXPERIMENTS D E S C R I B E O IN THIS CHAPTER ARE THE R E S U L T OF A STUDY TO F IND S U I T A B L E BLOCKING DOSES OF THE VARIOUS DRUGS D I S C U S S E D ABOVE IN ORDER TO HAVE MAXIMAL AUTONOMIC BLOCKADE OVER PERIOOS AS LONG AS TWO HOURS, SO THAT THE E F F E C T OF -45-V A R I O U S BRANCHES OF THE AUTONOMIC NERVOUS SYSTEM ON EGG SHELL C A L C I F I C A T I O N COULD BE E V A L U A T E D . T A B L E V I I I CHEMICALS USED IN THE DRUG EXPERIMENTS CHEMICAL DRUG NAME S U P P L 1 E R USE SOOIUM PENTOBARBITOL SOMNOPENTYL (PARENTERAL S O L N . ) PiTTMAN—MOORE * ONTAR 1 0 ANESTHE TIc ACETYLCHOL1NE CHLOR1OE MERCK & Co., N . J . PARASYMPATHET1c AGON 1ST ATROPINE SULPHATE B.D . H . , T O R O N T O PARASYMPATHET1c BLOCKER NOREP1NEPHR1NE H C L . D.L. ARTERENOL B GRADE C A L B 1 O C H E M . , Los ANGELES A L P H A - S Y M P A T H E T 1c AGON 1 ST PHENYLEPHR1NE H C L . WINTHROP L A B S . , AURORA, ONTARIO A L P H A - S Y M P A T H E T1c AGON 1 ST PHENOXYBENZAM1NE H C L . DlB E N Z Y L 1NE SMITH C L I N E & FRENCH I . A . C , MONTREAL A L P H A - S Y M P A T H E T 1 c BLOCKER ISOPROTERENOL SULPHATE K&K L A B S . 1 N C . , HOLLYWOOD, C A L . B E T A - S Y M P A T H E T 1 C AGON 1 ST PROPRANALOL . H C L . 1NDERAL A Y E R S T , MONTREAL BETA -SYMPATHE T 1 C BLOCKER -46-METHOOS. WHITE LEGHORN H E N S , E I T H E R IN LAY OR IN MOULT , WEIGHING APPROXIMATELY 2 KG WERE USED IN THESE E X P E R I M E N T S . THEY WERE OBTAINED FROM THE POULTRY FARM OF THE U N I V E R S I T Y OF B R I T I S H C O L U M B I A . THE BIRDS WERE A N E S T H A T I Z E D WITH 0 . 7 ML OF SOOIUM PENTOBARBITOL ( S O M N 0 P E N T Y L , PARENTERAL S O L U T I O N , P L T T M A N -MOORE, O N T . ) , INJECTEO INTO THE SAPHENOUS VE IN IN THE L E G , AND A N E S T H E S I A WAS MAINTAINED WITH 0 . 1 5 ML DOSES GIVEN AS REQUIRED ( U S U A L L Y AT 1 5 - 2 0 MIN I N T E R V A L S ) . A P O L Y E T H Y L E N E CANNULA ( INTRAMEDIC P . E . 5 0 , CLAY ADAMS, D I V I S I O N OF 3E C T O N , DICKENSON & Co., P A R S I P P A N Y , N . J . ) WAS INSERTED INTO THE RIGHT MEDIAL WING VE IN FOR ADMINISTRAT ION OF A N E S T H E T I C ANO DRUGS. IN EXPERIMENTS WHERE A CONTINUOUS I N T R A -VENOUS INFUSION OF A BLOCKING DRUG WAS USED A SECONO P . E . 5 0 CANNULA WAS INSERTED INTO THE MEDIAL VE IN ON THE OTHER WING. T H I S CANNULA WAS ONLY USEO FOR THE INFUSION TO ENSURE THAT NO BLOCKING AGENT WAS ACC I OENTLY FL U S HE 0 INTO THE A N I M A L . AN INTRAMEOIC P . E . 1 0 CANNULA WAS PLACEO IN THE RIGHT BRACHIAL ARTERY FOR MEASUREMENT OF BLOOD P R E S S U R E , WHICH WAS RECOROEO USING A PRESSURE TRANSDUCER (STATHAM MODEL P23AA, HATO R E Y , PUERTO R I C O ) ATTACHED TO A PEN RECORDER (GILSON MEDICAL E L E C T R O N I C S , MIDDLETON W I S . ) . IN EXPERIMENTS WHERE HEART RATE WAS MEASUREO TH IS WAS DONE, E ITHER BY COUNTING THE P U L S E S IN THE BLOOO PRESSURE T R A C E , OR BY USE OF A HEART RATE METRE ( E K E G E L E C T R O N I C C O . , VANCOUVER, B.C.) A T T A T C H E D TO THE CUMULATIVE CHANNEL OF THE GLLSON RECORDER. A L L CANNULAE WERE F I L L E O WITH H E P A R I N I Z E D S A L I N E ( 1 0 MG T A B L E IX TABLE OF AGONIST DRUGS USED TO CHALLENGE BLOCKADE DRUG TYPE OF AGON I ST DOSE USEO FOR CHALLENGE DETERM1 NAT 1 ON OF DOSE L E V E L RESPONSE MEASURED A C E T Y L C H O L1N E PARASYMPATHET I c 4 ^IGM/KG LOW RANGE USED BY BUNAG & WALASZEK 0 9 6 2 ) ( B . P . 1 54MM H G . ) DECREASE IN B . P . NOREP1NEPHR1NE A L P H - S Y M P A T H E T I c 4 ^JGM/KG S E E BOLTON & BOWMAN ( 1 9 6 9 ) ( B . P . t 80MM H G . ) I NCREASE IN B . P . PHENYLEPHR1NE A L P H A - S Y M P A T H E T 1 c 4 0 ^JGM/KG FROM L O G - D O S E RESPONSE CURVE ( S E E FIGURE 8 ) I NCREASE IN B . P . 1 SOPROTERENOL B E T A - S Y M P A T H E T 1 c 0 . 4 ^JGM/KG FROM L O G - D O S E RESPONSE CURVE ( S E E FIGURE 8 ) I NCREASE IN H . R . ABBREVI AT IONS B . P . BLOOD PRESSURE H . R . HEART RATE - 4 7 -HEPARIN / 1 0 0 ML OF 0 . 9 $ S A L I N E ) . TABLE V I I I L I S T S ALL THE DRUGS USED IN THESE EXPERIMENTS AND THEIR S U P P L I E R S . DRUGS WERE ROUTINELY D I S S O L V E D IN 0 . 9 $ S A L I N E . THE ONLY E X C E P T I O N WAS D I B E N Z Y L I N E WHICH PROOUCEO A MILKY SUSPENSION IN S A L I N E ; IT WAS MADE UP AS FOLLOWS! 5 0 MG OF D I B E N Z Y L I N E WERE D I S S O L V E D IN 0.8 ML OF PROPYLENE GLYCOL AND THEN A C I D I F I E D WITH 0.2 ML OF 0.1 N HCL AND 4 ML OF 0 . 9 $ S A L I N E WERE ADDED. THE R E S U L T I N G CLEAR SOLUTION WAS USED FOR I N J E C -T I O N . THE AGONIST DRUGS AND THE RESPONSES MEASURED ARE G IVEN IN THE ACCOMPANYING TABLE ( T A B L E I X ) . THE DOSE L E V E L S OF A C E T Y L C H O L I N E AND NOREPINEPHRINE WERE BASED ON DOSES USEO IN THE L I T E R A T U R E ; EXAMPLES OF THE ACTUAL RESPONSES OBTAINED IN THESE EXPERIMENTS ARE SHOWN IN P A R E N T H E S E S . THE P H E N Y L E P H R I N E AND ISOPROTERENOL DOSE L E V E L S WERE TAKEN AS THE MIDPOINT OF THE L INEAR PORTION OF THEIR R E S P E C T I V E L O G - D O S E RESPONSE C U R V E S . THE L A T T E R WERE CONSTRUCTED BY F INDING THE RESPONSES TO VARIOUS DOSES OF THE DRUGS IN A CHICKEN AND P L O T T I N G THE LOG OF THE DOSE VERSUS THE RESPONSE AS SHOWN IN THE R E S U L T S . SE V E R A L CHALLENGE DOSES OF THE AGONIST DRUG WERE GIVEN TO THE HEN PRIOR TO THE ADDIT ION OF THE BLOCKING AGENT IN ORDER TO E S T A B L I S H THE RESPONSE WHEN NO BLOCKAOE WAS P R E S E N T . AFTER THE BLOCKING AGENT HAD BEEN GIVEN THE RESPONSE TO THE AGONIST DRUG WAS T E S T E D EVERY 1 0 M I N U T E S . IN THE INFUSION EXPERIMENTS A PRIMING DOSE WAS GIVEN ANO THEN THE INFUSION WAS STARTED IMMEDIATELY AND CONTINUED FOR TWO HOURS. THE BLOCKAOE WAS CHALLENGED EVERY 1 0 MINUTES DURING THE INFUSION ANO FOR A FURTHER 9 0 MINUTES A F T E R THE I N F U S I O N . A L L INFUSIONS WERE CARRIED OUT USING A MULT I SPEED TRANSMISSION INFUSION PUMP (HARVARD APPARATUS CO . I N C . , DOVER, MA S S . ) AT A RATE OF 0 . 0 3 5 ML/MIN FOR THE ATROPINE INFUSIONS AND A RATE OF 0.02 ML/MIN FOR THE PROPRANALOL I N F U S I O N S . FIGURE 8. L O G - D O S E CURVES TO PHENYLEPHRINE ( T O P ) AND ISOPROTERENOL ( B O T T O M ) . REGRESSION L I N E FOR EXPERIMENTAL P O I N T S . 95$ CONFIDENCE L I M I T S ON REGRESSION L I N E . CHALLENGE OOSE USEO IN THE BLOCKING DRUG E X P E R I M E N T S . - 4 9 -RE S U L T S ANO D I S C U S S I O N . F IGURE 8 SHOWS THE LOG DOSE RESPONSE CURVES TO P H E N Y L -EPHRINE AND ISOPROTERENOL IN AN ANESTHATI ZED C H I C K E N . THE ARROWS INDICATE THE DOSE L E V E L S E L E C T E D FOR USE AS A C H A L L E N G E DOSE IN THE EXPERIMENTS WITH THE BLOCKING DRUGS. IN THE BLOCKING EXPERIMENTS $ BLOCKADE AT ANY TIME T WAS C A L C U L A T E D AS F O L L O W S : -c,o BLOCKAOE = 1 0 0 -OBSERVED RESPONSE TO AGONIST DRUG AT T IME T X 1 0 0 RESPONSE WHEN NO BLOCKING AGENT WAS P R E S E N T AL L S L O P E S WERE C A L C U L A T E D USING THE L E A S T MEAN SQUARES METHOD AS DESCRI BED BY S T E E L E & T 0 R R I E ( 1 9 6 0 ) , AND ALL C A L C U L A -T IONS WERE DONE ON A DESK COMPUTER (OL I VETT I -UN0ERWOOD PROGRAMMA 1 0 1 , NEW YORK) TA B L E X G I V E S A SUMMARY OF ALL THE TYPES OF DRUG E X P E R I M E N T S PERFORMED. A T R O P I N E . FROM TABLE X IT CAN BE SEEN THAT A S INGLE DOSE OF ATROPINE ( 0 . 5 MG/KG) GAVE A VERY SHORT BLOCKADE WHICH WOULD HAVE DECAYED TO 3 0 $ IN AN HOUR. THE ACTUAL EXPERIMENT IS D E P I C T E O AT THE TOP OF FIGURE 9. THE PROTOCOL USED IN THIS EXPERIMENT WAS BASED ON T A B L E X A SUMMARY OF ALL THE DRUG E X P E R I M E N T S . THE E F F E C T OF D I F F E R E N T DOSES OF BLOCKING DRUGS ON THE RESPONSES TO VARIOUS AGONISTS DRUG AGONIST PRIMING DOSE MG/KG I NFUS1 ON DOSE ^JG/KG/M 1 N $ BLOCKADE DURING INFUSION A F T E R INFUSION (ONE HOUR) ATROPINE ACETYLCHOL1NE 0 . 5 1 0 0 $ 00WN TO 3 0 $ 0 . 2 5 8 . 7 5 7 5 $ 1 0 . 5 8 0 $ 7 0 $ DOWN TO 1 5 $ 1 7 . 5 9 5 $ 9 5 $ DOWN TO 2 5 $ Dl BENZYL 1 NE NOREPINEPHRINE 1 0 o 7 0 MIN LATER AD0E0 ANOTHER 1 0 5 5 $ 6 5 $ PHENYLEPHR1NE 5 7 0 $ PROPRANALOL 1SOPROTERENOL 0 . 5 9 0 $ DOWN TO 5 0 $ 0 . 2 5 6 9 5 $ 9 5 $ DOWN TO 0 $ 5 1 0 0 $ 9 0 $ DOWN TO 4 0 $ 1 0 9 5 $ 9 5 $ DOWN TO 9 0 $ 1 0 9 0 $ 8 5 $ OOWN TO 7 0 $ - 5 0 -THAT USUALLY USED IN A 0 0 G , WHERE A DOSE OF 0.5 MG/KG IS GIVEN ANO V L O T H OF TH IS 0 0 S E IS ADDED EVERY HOUR (DR. F . L L O Y , PERSONAL COMMUNICAT ION) . ONE CAN READILY S E E THAT IN A CHICKEN T H I S WOULD GIVE A VERY POOR L E V E L OF BLOCKADE BECAUSE THE BLOCK DECAYS SO F A S T . CAMPOS & URQUILLA ( 1 9 6 9 ) USED A PRIMING DOSE OF 0.25 MG/KG AND A INFUSION L E V E L OF 5 G/K G/MIN IN DOGS FOR P A R A S Y M P A T H E T I C B L O C K A O E . OBVIOUSLY A LARGER DOSE WOULD BE REQUIRED IN A C H I C K E N . THE PRIMING DOSE WAS KEPT AT 0.25 MG/KG BUT THE L E V E L OF THE INFUSION WAS I N C R E A S E D . THE F I R S T INFUSION L E V E L TR IED WAS 8.5 yu G / K G / M I N . TH I S GAVE A BLOCKADE OF 7 5 $ , S E E TABLE X . THE DECAY OF BLOCKADE AFTER S T O P P I N G THE INFUSION WAS NOT T E S T E O IN TH IS E X P E R I M E N T . INCREASING THE INFUSION L E V E L TO 1 0 . 5 ^ G / K G / M I N GAVE A BLOCKAOE OF 8 0 $ . A s SOON AS THE INFUSION WAS STOPPEO THE BLOCK STARTED TO DECAY AND WAS ONLY 1 5 $ A F T E R ONE HOUR. WHEN THE INFUSION L E V E L WAS 1 7 . 5 ^ J G / K G / M I N AN E S S E N T I A L L Y 1 0 0 $ BLOCK WAS O B T A I N E D . AGAIN AS SOON AS THE INFUSION WAS STOPPED THE BLOCK RAPIDLY O E C A Y E D . TH IS E X P E R I M E N T IS SHOWN AT THE BOTTOM OF F IGURE 9. S I N C E THE BLOCK WAS CONSTANT AND COMPLETE WITH T H I S DOSAGE T H I S WAS THE OOSE CHOSEN FOR USE IN THE C A L C I F I C A T I O N E X P E R I M E N T S . A 1 7 . 5 ^JG/KG/MIN INFUSION WOULD ONLY ADMINISTER 1.08 MG/KG OVER A PERIOD OF ONE HOUR, SO THAT THE TOTAL DOSE OF ATROPINE GIVEN TO THE CHICKEN IN THIS TIME WOULO ONLY BE 1.33 MG / KG. THE OECAY OF THE BLOCKADE IMMEDIATELY AFTER STOPPING THE INFUSION SHOWED THAT IN FACT THE ATROPINE WAS NOT REMAINING IN AN A C T I V E FORM FOR VERY LONG SO THAT THE TOTAL DOSE A C T U A L L Y IN THE BODY A F T E R ONE HOUR WAS MUCH L E S S THAN T H I S . THEREFORE THE REPORTS CD c X X • U l m o X o m O X H o CO o r n X z (0 c o c o m • n — m X — o w o o z «• z • n m CO - n z r m r n o — CO o m z o H H m H O r H O — • n O > - n o X o — X m -i H — c o m O -< X X o r m o X z o z m X o X — o z X — Z r m z C — o m o z CD X z c m r r n H CO o c n > CO o CO r — •< — z > o X H > o z o X H m —• > X r Z < O o — •H m X •n z CO z — m • o " z H • c m X w • m _ X z -< " n - i X c o o CO X -t — m o z z CO CO — — z < m X B l o c k a d e of B lood Pressure Response to A c e t y l Chol ine % Blockade of Blood Pressure Response to Acetyl Choline 3 r m - 5 1 -IN THE L I T E R A T U R E THAT VERY HIGH OOSES OF ATROPINE ARE NECESSARY TO BLOCK THE VASODEPRESSOR RESPONSE TO A C E T Y L C H O L I N E WERE NOT BORNE OUT BY THESE R E S U L T S . BUNAG & WALASZEK ( 1 9 6 2 ) S T A T E D THAT DEPRESSOR RESPONSES TO 5 - 5 0 ^JG DOSES OF A C E T Y L C H O L I N E WERE ONLY P A R T I A L L Y BLOCKEO BY 0 . 5 - 2 . 0 MG/KG OOSES OF ATROPINE AND THAT 5—10 M G / K G OF A T R O P I N E WERE REQUIRED BEFORE COMPLETE 8 L 0 C K A D E WAS A T T A I N E D . HARVEY £ 1 AL ( 1 9 5 4 ) REPORTED THAT IT REQUIRED 9 - 1 2 MG/KG OF ATROPINE TO BLOCK A C E T Y L C H O L I N E - I N D U C E 0 V A S 0 D E P R E S S I 0 N , ANO THAT DOSES BELOW 5 M G / K G NEVER COMPLETELY BLOCKEO THIS R E S P O N S E . T H E S E WORKERS 0 0 NOT SAY WHAT DOSE OF A C E T Y L C H O L I N E THEY USED AS AN AGONIST OR HOW LONG THEY WAITED BEFORE THEY T E S T E D THE B L O C K . IT IS C O N C E I V A B L E THAT IF THEY WAITED MORE THAN 1 5 MIN THE BLOCK HAD ALREADY D E C A Y E D . THEY DO POINT OUT THAT WITH 9 - 1 2 MG/KG OOSES THE BLOCK L A S T E D 1 - 3 HOURS. IN VIEW OF THE RAPID DECAY OF BLOCKADE WITH SMALL DOSES OF ATROPINE WHICH HAS BEEN DEMONSTRATED H E R E , F IGURE 9 , IT APPEARS THAT THESE WORKERS WERE G I V I N G VAST Q U A N T I T I E S OF ATROPINE IN ORDER TO HAVE A LONG L A S T I N G B L O C K . AS POINTED OUT IN THE INTRODUCTION THESE CAN BE N O N - S P E C I F I C ANO HENCE ARE U N D E S I R A B L E . T H E RESULTS PRESENTED HERE ARE MORE IN ACCORD WITH THE EARLY REPORT OF G l B B S ( 1 9 2 6 ) THAT 0 . 5 - 1 . 0 M G / K G OF ATROPINE WAS S U F F I C I E N T TO PREVENT CARDIAC SLOWING DUE TO VAGAL S T I M U L A T I O N C O M P L E T E L Y . LLOYD & P l C K F O R D ( 1 9 6 1 ) REPORTED THAT IN THREE HENS THEY WERE ABLE TO BLOCK DEPRESSOR RESPONSES TO A C E T Y L C H O L I N E WITH 1 - 2 MG OF ATROPINE ADMINISTERED INTRAVENOUSLY . THEY 010 NOT GIVE THE EXACT DOSE OF A C E T Y L C H O L I N E USED TO TEST THE B L O C K , I w 41 3 C m jz £ g •o 5. O »> o 5 co z o o I s l l 100 Dibenzyline 10 mg./kg. I 80-6 0 40 20 0 H 1 1 1 1 1 1 1 1 1 — I — I — I — I — I — I 0 30 60 90 120 150 Time in Minutes Dibenzyline 5 mg./kg. I 100-i O H — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — i — n 0 30 60 90 120 150 180 Time in Minutes F IGURE 1 0 . THE E F F E C T OF TIME ON ^BLOCKADE OF THE HYPER TENSIVE RESPONSE TO NOREPINEPHRINE ( T O P ) OR P H E N Y L E P H R I N E (BOTTOM) BY S INGLE DOSES OF D I B E N Z Y L I N E . REGRESSION L I N E FOR THE EXPERIMENTAL P O I N T S . 95/o CONFIDENCE L I M I T S ON REGRESSION L I N E . - 5 2 -HOWEVER S INCE THEY WERE USING HENS APPROXIMATELY 2 KG IN WEIGHT THE DOSE OF ATROPINE WAS 0 . 5 - 1 . 0 M G / K G , WHICH IS IN GOOD A G R E E -MENT WITH THE RESULTS FOUND H E R E . D I B E N Z Y L I N E ( P H E N O X Y B E N Z A M I N E ) . TWO L E V E L S OF D I B E N Z Y L I N E WERE T E S T E D IN THE C H I C K E N . BOTH E X P E R I M E N T S ARE I L L U S T R A T E D IN FIGURE 1 0 AND SUMMARIZED IN T A B L E X. THE F I R S T DOSE USED WAS 1 0 MG/KG, D I S S O L V E D IN S A L I N E . EVEN WHEN WARMEO TH IS DID NOT D I S S O L V E VERY WELL BUT GAVE A MILKY S U S P E N S I O N , SO THAT IT IS P O S S I B L E THAT NOT A L L THE DRUG WAS A S S I M I L A T E O BY THE C H I C K E N . THE AGONIST USED TO TEST 8 L 0 C K A 0 E IN THIS E X P E R I M E N T WAS N O R E P I N E P H R I N E , WHICH IS NOT A S P E C I F I C ALPHA A G O N I S T . FOR TRUE 1 0 0 $ BLOCKADE ALL THE ALPHA RECEPTORS WOULD BE BLOCKED AND A V A S 0 D E P R E S S I 0N WOULD R E S U L T AS THE BETA RECEPTORS BECAME UNMASKED. |F ONE R E A L L Y A C H I E V E D THIS ABSOLUTE L E V E L OF BLOCKADE THE BLOCKING DRUG WOULD GENERALLY BE P R E S E N T IN E X C E S S , THEREBY PROBABLY CAUSING N O N S P E C I F I C E F F E C T S . THEREFORE AS A T E S T SUBSTANCE FOR ALPHA ADRENERGIC BLOCKADE NOREPINEPHRINE WAS NOT A GOOD C H O I C E . IT WAS USE0 BECAUSE IT WAS ONE OF THE T E S T SUBSTANCES USED BY OTHER WORKERS IN E X P E R I -MENTS ON C H I C K E N S , S E E HARVEY EJ. AL. 0954) AND BUNAG & WALASZEK ( 1 9 6 2 ) . $ 8 L 0 C K A 0 E IN TH IS E X P E R I M E N T WAS C A L C U L A T E D ASSUMING THAT AT 1 0 0 $ BLOCK NOREPINEPHRINE PRODUCED NO E F F E C T ON BLOOD P R E S S U R E , I . E . D ISREGARDING VASOMOTOR R E V E R S A L . T H I S WOULD GIVE V A L U E S OF BLOCKADE WHICH WOULD BE TOO H I G H . EVEN WITH THIS ERROR THE - 5 3 -BLOCKAOE OBTAINED WAS ONLY 5 5 $ AND IT ONLY INCREASED TO 6 5 $ WHEN ANOTHER 1 0 M G / K G DOSE WAS A D D E D , SEE TABLE X . T H I S EXPERIMENT TENDS TO AGREE WITH THE DATA IN THE L I T E R A T U R E , NAMELY THAT IT TAKES 2 0 - 6 0 M G / K G OF D I B E N Z Y L I N E TO PRODUCE VASOMOTOR REVERSAL WITH E P I N E P H R I N E OR NOREPINEPHRINE ANO HENCE PRESUMEABLY TO CAUSE COMPLETE ADRENERGIC BLOCKADE IN THE C H I C K E N , HARVEY £ j r _AL ( 1 9 5 4 ) AND BOLTON & BOWMAN ( 1 9 6 9 ) . WHEN P H E N Y L E P H R I N E WAS USED AS THE AGONIST A D I F F E R E N T RESULT WAS 0 8 T A I N E D . P H E N Y L E P H R I N E IS A S P E C I F I C ALPHA AGONIST WITH ALMOST NO BETA E F F E C T S , GOODMAN & GLLMAN ( 1 9 6 5 ) . HENCE 1 0 0 $ BLOCK IS 0 8 TA I NED WHEN THE VASOPRESSOR RESPONSE IS A B O L I S H E D , ALLOWING MEANINGFUL C A L C U L A T I O N S OF $ B L O C K A O E . IN TH IS EXPERIMENT THE D I B E N Z Y L I N E WAS D I S S O L V E D IN A C I D I F I E D PROPYLENE GLYCOL AND D I L U T E D WITH S A L I N E IN A MANNER S I M I L A R TO THAT DESCRIBEO BY HARVEY E_T A_L ( 1 9 5 4 ) . NO S 0 L U 8 I L I T Y PROBLEMS WERE ENCOUNTERED. THE DOSE USED WAS 5 M G / K G , HALF THE DOSE GIVEN IN THE PREV IOUS E X P E R I M E N T . THE R E S U L T S ARE SHOWN AT THE BOTTOM OF FIGURE 1 0 . THERE WAS INCOMPLETE BLOCKADE FOR THE F I R S T THIRTY MINUTES BUT AFTER THAT A STEADY BLOCK WAS OBSERVED WHICH D E C A Y -ED FROM 8 0 $ TO 7 0 $ OVER A PERIOO OF TWO HOURS. THE INCOMPLETE BLOCK FOR THE F I R S T HALF HOUR WAS NOT S U R P R I S I N G S INCE D I B E N Z Y L I N E IS AN I R R E V E R S I B L E BLOCKER AND IT TAKES TIME FOR THE REACT ION WITH THE RECEPTOR S I T E TO OCCUR. THE MOLECULAR CONFIGURATION D I R E C T L Y R E S P O N S I B L E FOR BLOCKADE IS PROBABLY A HIGHLY R E A C T I V E CARSON IUM ION FORMED WHEN THE T H R E E - M E M 8 E R E 0 RING B R E A K S , ANO THE R E L A T I V E L Y SLOW ONSET OF ACTION IS PROBABLY - 5 4 -DUE TO THE TIME FOR THE FORMATION OF TH IS R E A C T I V E INTERMEDIATE WHICH THEN ACTS AS AN A L K Y L A T I N G A G E N T , GOODMAN & GLLMAN ( 1 9 6 5 ) . TH E S E WRITERS ALSO POINT OUT THAT THE P R E S E N C E OF A CATECHOLAMINE DURING THE DEVELOPMENT OF BLOCKADE BY A H A L O -ALKAMINE CAN DECREASE THE DEGREE OF BLOCKADE A T T A I N E D . THEY E X P L A I N TH IS AS DUE TO COMPETIT ION BETWEEN THE TWO DRUGS FOR THE SAME POPULATION OF R E C E P T O R S . ONCE BLOCKAOE IS COMPLETE IT IS UNALTERED BY THE PRESENCE OF A SYMPATHETOMIMETIC A M I N E , I N D I C A T I N G THAT THE DRUG IS NO LONGER IN E Q U I L I B R I U M WITH THE R E C E P T O R S . THUS THE FACT THAT BLOCKADE WAS T E S T E D WITH THE AGONIST DURING THE DEVELOPMENT OF BLOCKADE PROBABLY MEANT THAT THE OBSERVEO BLOCKADE WAS L E S S THAN THAT WHICH WOULD HAVE DEVELOPED IF NO T E S T I N G HAD BEEN CARRIED OUT FOR THE F I R S T HOUR. IT WAS THEREFORE DECIDED THAT DURING THE C A L C I F I C A T I O N EXPERIMENTS THE D I B E N Z Y L I N E WOULD BE ADDED AN HOUR BEFORE THE 45CA SO THAT FULL BLOCKADE WOULD HAVE TIME TO D E V E L O P . AS THE 5 MG/KG DOSE OF D I B E N Z Y L I N E GAVE S U F F I C I E N T BLOCKADE IT WAS CHOSEN AS THE DOSE FOR USE IN THE C A L C I F I C A T I O N E X P E R I -M E N T S . IT APPEARS AS THOUGH THE LARGE DOSES OF O I B E N Z Y L I N E REQUIRED FOR VASOMOTOR REVERSAL TO E P I N E P H R I N E AND N O R E P I N E -PHRINE IN THE CHICKEN WERE OUE TO THE NATURE OF THE AGONIST U S E D . GOODMAN & GILMAN ( 1 9 6 5 ) POINT OUT THAT S I N C E THESE TWO AGONISTS HAVE BOTH ALPHA ANO BETA E F F E C T S THEIR E F F E C T S ON BLOOD PRESSURE ARE C O M P L E X , ANO FA ILURE TO A P P R E C I A T E T H I S HAS LEO TO FREQUENT M I S I N T E R P R E T A T I O N S OF THE P R O P E R T I E S OF ADRENERGIC BLOCKING A G E N T S . FROM THE EXPERIMENTS REPORTEO HERE IT IS CLEAR THAT A TABLE XI A SUMMARY OF THE CARDIOVASCULAR RESPONSES IN THE PROPRANALOL EXPERIMENTS PROPRANALOL DOSE SLOPE OF H.R. vs RESPONSE TO 0.4 /IG/KG OF I SOPROTERENOL (BEFORE PROPRANALOL) SLOPE OF H.R. vs T IME (BEFORE PROPRANALOL) MEAN BLOOD PR E S S U R E * PRIMING MG/KG 1 NFUS1 ON ^JG/KG/M 1 N BEFORE PROPRANALOL AFTER PROPRANALOL 0.25 6 - 0 . 6 9 9 ± 0 . 0 7 2 0.592 ± 0 . 0 5 7 1 4 1 . 7 6 ± 1.46 1 1 6 . 1 8 ± 1.42 5 - 1 . 8 7 7 ± 0 . 3 5 9 0.277 ± 0 . 0 6 0 1 3 5 . 7 7 ± 1 .28 1 1 1 . 3 6 ± 0 . 8 7 1 0 - 0 . 9 6 3 ± 0 . 1 6 9 0.757 ± 0 . 0 7 5 1 3 3 . 5 0 ± 1 . 1 6 97.71 ± 2.44 - 0 . 4 7 9 ± 0 . 1 7 6 0 . 1 3 7 5 ± 0 . 0 7 8 1 5 6 . 7 4 ± 1 . 1 3 118.71 ± 1 . 1 9 0.5 - 0 . 8 9 7 ± 0 . 1 2 5 - 0 . 3 3 1 2 ± 0 . 0 9 1 125.21 ± 1 . 8 5 1 1 5 . 0 0 ± 1 . 5 3 * IN THE INFUSION EXPERIMENTS A PAIRED T - T E S T ON THE MEAN BLOOD PRESSURES BEFORE AND AFTER THE ADDITION OF PROPRANALOL, REGARDLESS OF THE L E V E L OF INFUSION, SHOWED THAT THE DROP IN BLOOD PRESSURE WAS S I G N I F I C A N T AT THE 0 . 5 $ L E V E L (P < 0 . 0 0 5 ) . - 5 5 -OOSE OF 5 MG/KG OF D I B E N Z Y L I N E G IVES 7 0 - 8 0 $ BLOCKADE IN THE CHICKEN WHICH IN MOST CASES IS S U F F I C I E N T TO E L U C I D A T E THE E F F E C T OF ALPHA ADRENERGIC BLOCKADE ON VARIOUS P H Y S I O L O G I C A L P R O C E S S E S . P R O P R A N A L O L . ALTHOUGH A S I N G L E DOSE OF 0.5 MG/KG OF PROPRANALOL GAVE A 9 0 $ BLOCK OF THE INCREASE IN HEART RATE CAUSED BY 0.4 ^ G / K G OF I S O P R O T E R E N O L , THE L E V E L OF BLOCKADE WAS NOT CONSTANT , SO T H A T , AT THE END OF ONE HOUR, THE BLOCK HAD DECAYEO TO 5 0 $ ( T A B L E X ) . A CONTINUOUS INFUSION WAS CHOSEN IN ORDER TO MAINTAIN A HIGH L E V E L OF BLOCKADE FOR UP TO TWO HOURS AS REQUIRED IN THE C A L C I F I C A T I O N E X P E R I M E N T S . IN COMBINATION WITH A PRIMING DOSE OF 0.25 M G / K G , TWO INFUSION L E V E L S WERE T R I E D , 5~6 ^ G / K G / M I N AND 1 0 ^ J G / K G / M I N . BOTH L E V E L S GAVE 9 0 - 9 5 $ BLOCKADE DURING THE I N F U S I O N , B U T , WHEN THE INFUSIONS WERE S T 0 P P E 0 , THE LOWER INFUSION L E V E L SHOWED A FASTER OECAY OF BLOCKADE ( T A B L E X ) . THE 1 0 ^ J G / K G / M I N I N -FUSION L E V E L WAS S E L E C T E D FOR USE IN THE C A L C I F I C A T I O N E X P E R I -MENTS AS IT GAVE A GOOD BLOCK FOR AT L E A S T TWO HOURS. A SUMMARY OF THE CARDIOVASCULAR CHANGES OCCURRING IN THE PROPRANALOL EXPERIMENTS IS GIVEN IN TABLE X I . FROM THIS TABLE ONE CAN SEE THAT IN FOUR OUT OF F I V E EXPERIMENTS THE HEART RATE INCREASED WITH TIME BEFORE THE PROPRANALOL WAS G I V E N . T H I S WAS PROBABLY DUE TO AN INCREASE IN SYMPATHET IC A C T I V I T Y . AN ANESTHATI ZED ANIMAL IS NO LONGER ABLE TO MAINTAIN A PROPER H O M E O S T A S I S . IMMOBIL IZATION OF AN ANIMAL IN THE S U P I N E P O S I T I O N F IGURE 1 1 . THE E F F E C T OF A S INGLE DOSE OF PROPRANALOL ON THE BLOOO PRESSURE ( L E F T ) ANO HEART RATE ( R I G H T ) OF AN A N E S T H A T I Z E O C H I C K E N . REGRESSION L I N E FOR THE EXPERIMENTAL P O I N T S . 95$ CONFIDENCE L I M I T S ON REGRESSION L I N E . CAN CAUSE POOLING OF BLOOD IN THE E X T R E M I T I E S ANO THE ANIMAL CAN ALSO LOSE BODY H E A T . SURGICAL TRAUMA CAN ALSO CAUSE INCREASEO SYMPATHET IC F I R I N G . BARBITURATE A N E S T H E S I A TENDS TO DEPRESS R E S P I R A T I O N AND THIS COULD CAUSE A C I O O S I S WHICH WOULD A C T I V A T E THE C H E M 0 R E C E P T 0 R S . ON THE OTHER HAND THE BLOOD PRESSURE IS GENERALLY STEADY UNDER BARBITURATE A N E S T H E S I A DUE TO INCREASES IN P E R I P H E R A L R E S I S T A N C E SO THAT BARORECEPTOR RESPONSES ARE PROBABLY NOT IMPORTANT, CHENWETH & VAN DYKE ( 1 9 6 9 ) . IN THE EXPERIMENT WHERE THE HEART RATE DID NOT INCREASE WITH T I M E , BUT D E C R E A S E D , THERE WAS S T I L L A CHANGE IN HEART RATE ANO THIS ALLOWED THE T E S T I N G OF THE E F F E C T OF THE SAME DOSE OF ISOPROTERENOL AT D I F F E R E N T HEART R A T E S . IT WAS FOUND T H A T , WHETHER THE HEART RATE INCREASED OR DECREASED WITH T I M E , THE PLOT OF HEART RATE VERSUS HEART RATE RESPONSE TO 0.4 JJG/KG OF ISOPROTERENOL WAS ALWAYS L INEAR AND ALWAYS HAD A N E G A T I V E SLOPE ( T A B L E X I ) . TH I S I M P L I E S THAT AT HIGHER HEART RATES FEWER BETA RECEPTORS WERE A V A I L A B L E FOR REACT ION WITH I S O P R O T E R E N O L . IF THE CHANGE IN HEART RATE WAS DUE TO A CHANGE IN SYMPATHET IC A C T I V I T Y AN INCREASE IN HEART RATE WOULO INDEED MEAN THAT FEWER BETA RECEPTORS WERE A V A I L A B L E FOR REACT ION WITH EXOGENOUS ISOPROTERENOL S INCE THEY WOULD ALREADY BE A C T I V A T E D 8Y ENDOGENOUS TRANSMITTOR. THE NEGAT IVE L INEAR RESPONSE OF HEART RATE VERSUS HEART RATE RESPONSE TO 0.4 ^JG / KG OF ISOPROTERENOL OCCURRED IN EVERY E X P E R I M E N T . THE S L O P E S VARIEO MARKEDLY ( T A B L E X l ) 8UT THIS WOULD BE E X P E C T E O S I N C E THE RECEPTOR POPULAT IONS IN THE HEART AND THE SYMPATHET IC F IGURE 1 2 . THE E F F E C T OF A CONTINUOUS INTRAVENOUS INFUSION OF PROPRANALOL ON THE BLOOO PRESSURE ( L E F T ) AND HEART RATE ( R I G H T ) OF AN ANESTHATI ZED H E N . REGRESSION L I N E FOR THE EXPERIMENTAL P O I N T S . 9 5 $ CONFIDENCE L I M I T S ON REGRESSION L I N E . - 5 7 -A C T I V I T Y NECESSARY TO MAINTAIN A GIVEN HEART RATE WOULD VARY FROM INDIVIDUAL TO I N D I V I D U A L . TH I S V A R I A T I O N OF RESPONSE TO ISOPROTERENOL WITH HEART RATE WAS IMPORTANT BECAUSE ADDIT ION OF PROPRANALOL INVARIABLY OROPPED THE HEART RATE ( S E E F IGURES 1 1 AND 1 2 FOR E X A M P L E S ) AS THE ENDOGENOUS SYMPATHET IC A C T I V I T Y ON THE HEART WAS B L O C K E D . THEREFORE IT WAS NECESSARY TO USE THIS L I N E AS A CORRECTION FACTOR WHEN C A L C U L A T I N G THE $ BLOCKADE BY P R O P R A N A L O L . FROM THE PLOT IT WAS P O S S I B L E TO PREOICT WHAT THE RESPONSE TO ISOPROTERENOL WOULD BE AT ANY PARTICULAR HEART R A T E , SO THAT THE UNBLOCKED RESPONSE TO ISOPROTERENOL COULD BE FOUND BY TAKING THE HEART RATE J U S T BEFORE THE CHALLENGE DOSE OF ISOPROTERENOL WAS G IVEN AND READING THE E X P E C T E D INCREASE IN HEART RATE FROM THE G R A P H . S L N C E THE ACTUAL RESPONSE TO THE C H A L L E N G E DOSE OF ISOPROTERENOL WAS KNOWN $ BLOCKADE COULD BE C A L C U L A T E D . AN EXAMPLE OF THE L INEAR PLOT OF HEART RATE VERSUS HEART RATE RESPONSE TO ISOPROTERENOL IS G IVEN IN F IGURE 1 3 ANO B E S I D E THIS IS THE E F F E C T OF AN INTRAVENOUS INFUSION OF PROPRANALOL ON BLOCKING THE RESPONSE TO ISOPROTERENOL IN THE SAME H E N . THE HEART RATE VERSUS HEART RATE RESPONSE L I N E WAS USED AS A CORRECTION FACTOR TO C A L C U L A T E THE FO BLOCKADE IN THE SECOND GRAPH. PROPRANALOL NOT ONLY HAS A PROFOUND E F F E C T ON HEART RATE BUT ALSO CAUSES A MARKED DROP IN BLOOD P R E S S U R E , S E E F I G U R E S 1 1 AND 1 2 ANO TABLE X I . TH I S DROP IN BLOOD PRESSURE IS PROBABLY PRIMARILY DUE TO THE OROP IN HEART RATE S I N C E THE BETA V A S O -DILATOR RECEPTORS IN THE P E R I P H E R A L C I R C U L A T I O N ARE OF OOUBT -FUL P H Y S I O L O G I C A L S I G N I F I C A N C E , GOODMAN & G l L M A N ( 1 9 ^ 5 ) , ANO I I VO X vji m V* « x ro CO co o o z o z o n z r o — ro z ro r — n z o — x co o z X m o x X m m x •o rn x CO 2 Co m — z O H z > r r — T O z o m — • z H CO o o — X CO > o i-c r co m co z — o O rn z • C D -< C CO « H > x co m o m o x Response Increase in Heart Rate (Isoproterenol) m o O - 4 -i m m o x m co x o z C O > CO m x X r > z m o m m x H m o > x — - » co o X X > X H O rn H m o x C D m H Z > o - r z — * z x o rn z > - I z o z o H X x m ro co m z o X o m z — X z > X > < m z o c co z x c CO o z o x x x o x X > z > r o X) CO X X m CO x o z co rn co o x x o H ro x m z o r a> •< — o z o z H - H X — m z c CO o > c X CO m m rn m o o •n x m o z CD r o o > a m o x m x ro > x X rn co x o z co ro co o x x o H ro x ro z o r > z > z ro co H X > M ro o o x o ro z C X ro f -ro x ro ro • n • n ro o o X X ro > x X > ro o z X ro x ro > x x > ro JO a <? CD ro 2 % Blockade ( Heart Rate Response to Isoproterenol ) o 1 1 o 1 1 o o *1 o ro oi 3 o n a 3 Q O -58-PRESUMEA8LY THE ALPHA RECEPTORS ARE NOT A F F E C T E D BY P R O P R A N A L O L . IF TH IS POSTULATE IS CORRECT IT MIGHT BE A P P R O P R I A T E TO ENQUIRE WHY THE BLOOD PRESSURE DOES NOT R I S E AGAIN WHEN THE HEART RATE R I S E S AS THE BLOCK WEARS O F F . IN BOTH THE E X P E R I -MENTS D E P I C T E D , F I G U R E S 11 AND 12, THE BLOOD PRESSURE REMAINS A P P R O X I M A T E L Y THE SAME AND MAY EVEN D E C L I N E A L I T T L E . IN THE MAJORITY OF PROPRANALOL EXPERIMENTS THIS WAS THE CASE ALTHOUGH IN ONE EXPERIMENT THE BLOOD PRESSURE OID R I S E AGAIN AS THE BLOCK WORE O F F . IT IS P O S S I B L E THAT THE FA ILURE OF THE BLOOD PRESSURE TO RETURN TO NORMAL L E V E L S IS DUE TO V A S O D I L A T A T I O N CAUSED BY AN A C I D O S I S A R I S I N G FROM THE RESPIRATORY D E P R E S S A N T E F F E C T S OF THE A N E S T H E T I C . PROPRANALOL ALSO HAS LOCAL A N E S T H E T I C P R O P E R T I E S , DOLLERY EJ_ AL_ (1969), AND IT IS C O N C E I V A B L E THAT THIS LOCAL A N E S T H E T I C A C T I V I T Y COULD ACT LONGER ON THE P E R I P H E R A L VASCULAR SYSTEM THAN IT DOES ON THE H E A R T . THE P O S S I B I L I T Y ALSO A R I S E S THAT PROPRANALOL HAS A GREATER E F F E C T ON THE P E R I -PHERAL VASCULAR BETA RECEPTORS THAN IT DOES ON THE H E A R T . THERE IS A GROWING AMOUNT OF E V I D E N C E THAT THESE TWO T Y P E S OF BETA RECEPTOR ARE NOT THE S A M E , DOLLERY ET AJ. (1969), WHICH IS S U B S T A N T I A T E D BY THE DEVELOPMENT OF ICI 50172, A S E L E C T I V E BLOCKER FOR THE HEART BETA R E C E P T O R S , OUNLOP & SHANKS (1968). I N D E E D , IN ORDER TO ACCOUNT FOR SOME OF THEIR R E S U L T S , BOLTON & BOWMAN (1969) HAVE SUGGESTEO THAT BETA RECEPTOR BLOCKAOE IN THE CHICKEN COULD BE MORE E F F E C T I V E P E R I P H E R A L L Y THAN IN THE H E A R T . HOWEVER, AS POINTED OUT A B O V E , THE P E R I P H E R A L E F F E C T S ON BLOOD PRESSURE ARE PROBABLY SMALL COMPARED WITH THE HEART RATE E F F E C T S , SO THAT THIS IS U N L I K E L Y TO BE THE E N T I R E E X P L A N A T I O N . PROBABLY GENERAL HOMEOSTATIC DEGENERATION UNOER - 5 9 -T HE INFLUENCE OF PROLONGED A N E S T H E S I A IS AN IMPORTANT FACTOR IN THE F A I L U R E OF THE BLOOD PRESSURE TO RETURN TO THE L E V E L OBSERVEO BEFORE PROPRANALOL WAS AODED. THE BLOCKING DOSE OF PROPRANALOL FOUND HERE FOR MAXIMAL BLOCKADE WAS IN GOOD AGREEMENT WITH THE V A L U E REPORTED BY BOLTON & BOWMAN (1969)» WHO FOUND THAT 0.2-1 MG/KG DOSES OF PROPRANALOL BLOCKED THE DEPRESSOR RESPONSE TO 0.5Y U G OF ISOPROTERENOL IN A H E N . THEY WERE USING THE DEPRESSOR E F F E C T OF ISOPROTERENOL AS A T E S T , WHILE THE E F F E C T OF ISOPROTERENOL ON HEART RATE WAS USED IN THE ABOVE E X P E R I M E N T S . HOWEVER AS O ISCUSSED A B O V E , THE BLOOD PRESSURE RESPONSE WAS P R 0 B A 8 L Y MAINLY DUE TO CHANGES IN HEART RATE AND THEREFORE THE TWO PARAMETERS ARE C O M P A R A B L E . -60-SY N O P S I S OF ORUG E X P E R I M E N T S . IN E S S E N C E THIS S E R I E S OF EXPERIMENTS WAS CONOUCTEO TO FI NO S U I T A B L E DOSES OF AUTONOMIC NERVOUS SYSTEM BLOCKERS IN THE CHICKEN TO GIVE MAXIMAL BLOCKADE OVER PERIODS OF UP TO TWO HOURS. THE EXTENDED PERIODS OF BLOCKADE WERE REQUIRED FOR THE PROTOCOL OF THE EGG SHELL C A L C I F I C A T I O N EXPERIMENTS D I S C U S S E D IN THE NEXT C H A P T E R . THE DRUGS AND DOSES FOUND WERE AS F O L L O W S : -TYPE OF BLOCKADE DRUG DOSE P R I M I N G MG/KG INFUSION /JG/KG/M 1 N PA R A S Y M P A T H E T I C ATROP1NE 0.25 17.5 AL P H A - S Y M P A T H E T I C D1 BENZYL 1NE 5.0 — BE T A - S Y M P A T H E T I c PROPRANALOL 0.25 10.0 CHAPTER I I I : C A L C I U M TRANSPORT BY THE SHELL GLAND IN VIVO. I NTROOUCTION. AS THE CALCIUM F L U X E S OBSERVED IN THE IN VITRO SYSTEM WERE VERY MUCH LOWER THAN THOSE WHICH ARE KNOWN TO OCCUR IN THE INTACT HEN THE REMAINDER OF THESE S T U D I E S WERE CARRIED OUT ON THE WHOLE A N I M A L . S I N C E BIRDS P O S S E S S AN ION PUMP WHICH IS OEPENDENT ON P A R A S Y M P A T H E T I C NERVOUS A C T I V I T Y , I . E . THE S A L T GLAND, S C H M I O T -NLE L S O N ( 1 9 6 0 ) , THE I N I T I A L EXPERIMENTS WERE UNDERTAKEN TO DETERMINE THE NERVOUS INVOLVEMENT IN SHELL GLAND F U N C T I O N . TH I S WAS DONE BY STUOYING THE E F F E C T OF BLOCKADE OF VARIOUS BRANCHES OF THE AUTONOMIC NERVOUS SYSTEM ON THE C A L C I F I C A T I O N OF THE EGG S H E L L . TWO PARAMETERS MAY BE USED AS A MEASURE OF EGG SHELL C A L C I F I C A T I O N . THE S I M P L E S T SYSTEM IS TO STUDY THE INCORPORATION OF L A B E L INTO THE EGG S H E L L . LURCHER & HOOGES ( 1 9 6 9 ) SHOWED THAT 1 1 0 - 1 7 0 MIN A F T E R INTRAVENOUS 45CA I N J E C T I O N INTO ANESTHATI ZE D HENS 4 5 ~ 5 3 $ OF THE 45CA DOSE APPEARED IN THE EGG S H E L L . WHEN THEY R E P E A T E D THE E X P E R I M E N T IN UNANESTHATI ZED BIRDS THEY FOUND THAT 3 8 - 4 5 $ APPEARED IN THE EGG SHELL A F T E R TWO HOURS. THESE WORKERS DID NOT LOOK AT INCORPORATION AT SHORTER T IME PERIODS IN ANY D E T A I L ALTHOUGH THEY 010 MENTION THAT 1 2 $ OF THE DOSE APPEARED IN THE SHELL IN L E S S THAN 2 0 M I N . AS THESE MINIMAL DATA DID NOT ADEQUATELY D E S C R I B E THE T IME COURSE FOR THE REACTION A PRELIMINARY STUDY WAS UNDERTAKEN TO DETERMINE THE INCORPORATION OF L A B E L L E D CALCIUM INTO THE EGG SHELL OVER THE PERIOO FROM ZERO TO TWO HOURS. T H I S WOULD GIVE SOME MEASURE OF THE RATE OF INCORPORATION OF. L A B E L INTO - 6 1 --62-THE EGG S H E L L . ANOTHER WAY IN WHICH A P ICTURE OF THE FACTORS INVOLVED IN EGG SHELL C A L C I F I C A T I O N CAN BE 0 8 T A I NED IS BY STUDYING THE D I S A P P E A R A N C E OF 45CA FROM THE BLOOD. I T IS GENERALLY A C C E P T E D THAT THE DECREASE WITH TIME OF THE TRACER CONCENTRATION IN THE BLOOD CAN BE E X P R E S S E O MATHEMATICALLY BY A S E R I E S OF EXPONENT IAL TERMS. GENERALLY THESE ARE FOUND BY CURVE A N A L Y S I S . THE ORIGINAL CURVE IS PLOTTED ON S E M I L 0 G A R I T H M I C C O O R D I N A T E S , A STRAIGHT L I N E IS F I T T E D THROUGH THE T A I L OF THE C U R V E , THE ABSOLUTE VALUES OF THE D I F F E R E N C E S BETWEEN THE ORDINATE VALUES OF THE ORIGINAL CURVE AND THE STRAIGHT L I N E ARE P L O T T E D , AND THE PROCEOURE IS REPEATED UNTIL ONLY A F INAL STRAIGHT L I N E R E M A I N S . THE STRAIGHT L I N E S SO OBTAINED ARE THE E X P O N E N T I A L COMPONENTS OF THE C U R V E . IN G E N E R A L , A CLOSED SYSTEM OF N COMPARTMENTS IN STEADY STATE G IVES R I S E TO N-1 EXPONENTIAL TERMS IN THE D I S A P P E A R A N C E C U R V E , ROBERTSON ( 1 9 5 7 ) . A N A L Y S I S OF THE PLASMA 45CA D I S A P P E A R A N C E CURVE WOULD GIVE SOME IDEA OF THE NUMBER OF P H Y S I O L O G I C A L PARAMETERS WHICH ARE INVOLVEO IN THE D I S A P P E A R A N C E OF CALCIUM FROM THE BLOOD DURING EGG SHELL CALCI FI CAT I O N . -COMPARTMENTAL ANALYS I S I M P L I E S THAT IT IS P O S S I B L E TO RESOLVE THE CURVE INTO ALL ITS EXPONENTIAL COMPONENTS. T H I S IS NOT ALWAYS F E A S I B L E , E S P E C I A L L Y IN THE CASE OF VERY FAST R E A C T I O N S , S INCE ONE NEEDS AT L E A S T THREE POINTS TO D E F I N E A STRAIGHT L I N E AND TH IS MIGHT REQUIRE SAMPLING AT TIME PERIODS WHICH ARE TOO CLOSE TOGETHER TO BE P R A C T I C A L . I T ALSO I M P L I E S STEADY STATE C O N D I T I O N S , I . E . IN ANY COMPARTMENT THE RATE OF -63-REMOVAL OF THE SUBSTANCE B E I N G S T U D I E O I S EQUALLED BY I T S RATE OF REPLACEMENT SO THAT THE CONCENTRATION AND AMOUNT OF THE SUBSTANCE B E I N G S T U D I E D I S C O N S T A N T . T H I S C O N D I T I O N I S SELDOM S T R I C T L Y TRUE SO THAT ANY COMPARTMENTAL A N A L Y S I S I S N E C E S S A R I L Y AN A P P R O X I M A T I O N , NEVERTHELESS I T DOES G I V E AN OVERALL P I C T U R E OF THE SYSTEM UNDER S T U D Y . 3Y COMPARISON OF THE PLASMA 4 5 C A D I S A P P E A R A N C E CURVES I N HENS WITH AND WITHOUT AN EGG IN THE SHELL GLANO I T SHOULD BE P O S S I B L E TO OETERMINE WHICH E X P O N E N T I A L COMPONENT CORRESPONDS WITH EGG SHELL C A L C I F I C A T I O N * , WHILE COMPARISON OF THESE CURVES I N HENS W I T H A C T I V E AND I N A C T I V E O V A R I E S SHOULD G I V E SOME I N S I G H T INTO THE C O N T R I B U T I O N OF OTHER PARAMETERS OF C A L C I U M M E T A B O L I S M W H I C H CHANGE I N THE L A Y I N G HEN (SUCH AS 80NE R E M O D E L L I N G , GUT A B S O R P T I O N ANO RENAL E X C R E T I O N ) . TWO TYPES OF E X P E R I M E N T WERE PERFORMED. THE P R E L I M I N A R Y E X P E R I M E N T S WERE DESIGNED TO E X A M I N E THE EFFECT OF NEURAL BLOCKADE ON I N C O R P O R A T I O N OF TRACER INTO THE EGG S H E L L , WHILE A N A L Y S I S OF THE PLASMA 45CA D I S A P P E A R A N C E CURVE I N B I R D S UNOER V A R I O U S P H Y S I O L O G I C A L C O N D I T I O N S GAVE SOME I N S I G H T INTO THE P H Y S I O L O G I C A L PARAMETERS CORRESPONDING TO THE E X P O N E N T I A L COMPONENTS O B T A I N E D FROM THE C U R V E . T H I S P E R M I T T E D I N T E R -P R E T A T I O N OF ANY E F F E C T S OF THE NEURAL B L O C K I N G DRUGS ON THE D I S A P P E A R A N C E CURVE I N TERMS OF P H Y S I O L O G I C A L CHANGES IN THE H E N . -64-METHOOS. THE EXPERIMENTAL ANIMALS WERE WHITE LEGHORN L A Y I N G H E N S , OBTAINED FROM THE POULTRY FARM OF THE U N I V E R S I T Y OF B R I T I S H CO L U M B I A , WEIGHING 1.5-2 K G . THEY WERE MAINTAINED ON A D IET OF L A Y I N G P E L L E T S (B U C K E R F I E L D S , VANCOUVER, B . C . ) , CONTAINING 2 . 5 $ C A L C I U M , SUPPLEMENTED WITH FLAKED OYSTER S H E L L S TO ENSURE MAXIMAL CALCIUM I N T A K E . CARE WAS TAKEN TO S E L E C T ANIMALS WHICH WERE L A Y I N G DOWN EGG S H E L L AT A STEAOY R A T E . THE METHOD OF DETERMINING THE PHASE OF THE L A Y I N G CYCLE IS D E S C R I B E D IN THE AP P E N D I X . THE BIRDS WERE A N E S T H A T I Z E D WITH 0.6 ML OF SOMNOPENTYL , GIVEN INTRAVENOUSLY , AND A N E S T H E S I A WAS MAINTAINED WITH 0 . 1 5 ML AL IQUOTS OF TH IS A N E S T H E T I C , GIVEN AS RE QUI RE 0. A P.E. 50 CANNULA WAS INSERTED INTO THE RIGHT MEDIAL WING VE IN FOR ADMINISTRAT ION OF A N E S T H E T I C , 45CA AND DRUGS. IN EXPERIMENTS WHERE A CONTINUOUS INTRAVENOUS INFUSION OF A BLOCKING DRUG WAS USED A SECOND P.E. 5 0 CANNULA WAS INSERTEO INTO THE MEDIAL VE IN ON THE OTHER WING. A P.E. 1 0 CANNULA WAS P L A C E D IN THE RIGHT BRACHIAL ARTERY FOR BLOOD PRESSURE MEASUREMENT, WHICH WAS RECORDED USING A STATHAM P 2 3 AA PRESSURE TRANSDUCER ATTATCHED TO A GLLSON RECORDER. IN EXPERIMENTS IN WHICH THE A R T E R I A L PLASMA 45CA L E V E L S WERE MONITORED A SECOND P.E. 1 0 CANNULA WAS INSERTEO IN TO THE L E F T BRACHIAL ARTERY AND USED FOR C O L L E C T I O N OF BLOOO S A M P L E S . AL L CANNULAE WERE F I L L E D WITH H E P A R I N I Z E D S A L I N E ( 1 0 MG H E P A R I N/100 ML OF 0 . 9 $ S A L I N E ) . IN THE PREL IMINARY S E R I E S OF EXPERIMENTS EACH HEN WAS - 6 5 -G I V E N , INTRAVENOUSLY , 0.2 MCURIES 0 F 45CA ( S P E C I F I C A C T I V I T Y 0.5 yucuRiE OF 45CA / M G OF C A ) , D I S S O L V E D IN 0.5 ML OF 0 . 9 $ S A L I N E . THE BIRDS WERE K I L L E D WITH AN OVERDOSE OF A N E S T H E T I C AT VARIOUS TIME INTERVALS AFTER INTRODUCTION OF THE T R A C E R , THE EGG REMOVED FROM THE O V I O U C T , ANO THE S H E L L I S O L A T E D . THE EGG SHELL WAS D I S S O L V E D IN 5 0 ML OF 1 1 . 5 N H C L , THE SHELL MEMBRANE BEING D I S C A R O E O . THE 45CA IN A 50 ^ JL AL IQUOT OF THE R E S U L T I N G SOLUTION WAS COUNTED AS D E S C R I B E D IN CHAPTER I. A PLOT OF TIME VERSUS $ 45CA DOSE ADOED TO THE HEN A P P E A R I N G IN THE EGG SHELL WAS C O N S T R U C T E D , S E E F IGURE 1 4 , AND FROM THIS TWO TIME INTERVALS WERE S E L E C T E D FOR FURTHER S T U D Y . THE EARLY TIME PERIOD ( 3 0 MIN) ON THE R I S I N G PORTION OF THE CURVE WAS TAKEN AS A MEASURE OF THE RATE OF EGG S H E L L C A L C I F I C A T I O N , WHILE THE LATE TIME PERIOD ( 1 2 0 M IN ) ON THE P L A T E A U PORTION OF THE CURVE WAS USED AS A MEASURE OF THE TOTAL AMOUNT OF L A B E L INCORPORATED INTO THE EGG 8 Y ANY PART ICULAR B I R D . USING THE DRUG DOSAGES WORKED OUT IN THE P R E C E E D I N G C H A P T E R , THE E F F E C T OF BLOCKAOE OF VARIOUS BRANCHES OF THE AUTONOMIC NERVOUS SYSTEM ON THE INCORPORATION OF 45CA INTO THE EGG SHELL AT THESE TWO TIME PERIODS WAS S T U D I E D . T H E E X P E R I -MENTAL PROTOCOL USED WAS THE SAME AS IN THE PREL IMINARY E X -P E R I M E N T S . IN THE TWO HOUR INCORPORATION S T U D I E S THE D I S A P P E A R A N C E OF 45CA FROM THE A R T E R I A L PLASMA WAS ALSO MONITORED BY TAKING 0.4 ML BLOOO SAMPLES FROM THE L E F T BRACHIAL A R T E R I A L CANNULA AT FREQUENT I N T E R V A L S . THE BLOOD WAS C E N T R I F U G E D IN A -66-C L I N I C A L C E N T R I F U G E TO REMOVE THE C E L L S ANO A 50 JJL ALIQUOT OF THE A R T E R I A L PLASMA WAS COUNTED AS D E S C R I B E D IN CHAPTER I . TH E RATE OF D I S A P P E A R A N C E OF COUNTS FORM THE PLASMA WAS ANALYSED IN AN ATTEMPT TO D E S C R I B E THE INDIVIDUAL EVENTS CONTRIBUT ING TO THE OVERALL RATE OF TRACER MOVEMENT. THE METHOD S E L E C T E D WAS THAT OF ROBERTSON ( 1 9 5 7 ) , WHICH I S , E S S E N T I A L L Y , THE GRAPHICAL F I T T I N G OF STRAIGHT L I N E S TO THE EXPERIMENTAL C U R V E , START ING WITH THE SLOWEST R A T E . F IGURE 15 I L L U S T R A T E S THE P R O C E D U R E . TH E HALF L I V E S OF EACH EXPONENT IAL RATE WERE C A L C U L A T E D FROM THE S L O P E S OF THE SEMILOGARITHMIC PLOTS IN THE USUAL MANNER. THE A R T E R I A L PLASMA 45CA D I S A P P E A R A N C E CURVE WAS ALSO EXAMINED IN HENS WHICH HAD NO EGG P R E S E N T IN THE SHELL GLAND AND COMPARED WITH THE CURVE OB T A I N E 0 DURING A C T I V E S H E L L C A L C I F I C A T I O N WHEN NO DRUGS WERE P R E S E N T . TWO GROUPS OF HENS WERE U S E D . IN ONE GROUP THE HENS WERE IN A C T I V E LAY BUT WERE ON THE PAUSE DAY OF THEIR C Y C L E , WHILE THE HENS IN THE OTHER GROUP WERE OUT OF LAY ANO HAD INACT IVE O V A R I E S . THE EXPERIMENTAL DESIGN REMAINED THE SAME AS D E S C R I B E D E A R L I E R . 6 0 - t O 30 60 90 120 T i m e in M i n u t e s IGURE 1 4 . A GRAPH TO SHOW THE T I M E COURSE OF" I N C O R P O R A T I O N OF 4 5 C A INTO THE EGG S H E L L . - 6 7 -R E S U L T S AND D I S C U S S I O N . F IGURE 1 4 SHOWS THE E F F E C T OF T IME ON THE INCORPORATION OF 45CA INTO THE EGG S H E L L . WITH INCREASING T IME MORE OF THE 45CA DOSE APPEARED IN THE EGG S H E L L . HOWEVER THE GRAPH WAS APPROACHING A P L A T E A U I N D I C A T I N G THAT AT LONGER TIME PERIODS THAN TWO HOURS VERY L I T T L E MORE THAN 5 0 $ OF THE 45CA DOSE WOULD APPEAR IN THE EGG S H E L L . THE DATA OF L B R C H E R & HOOGES ( 1 9 6 9 ) F I T S WELL ONTO TH IS C U R V E . THESE WORKERS SHOWED THAT AFTER 1 0 0 MINUTES 5 1 . 0 $ OF AN INTRAVENOUS 45CA DOSE GIVEN TO AN ANESTHATI ZE D HEN HAD BEEN INCORPORATED INTO THE EGG S H E L L . A F T E R LONGER TIME P E R I O D S , 1 5 5 - 1 8 5 M I N , THEY FOUND THAT 4 5 - 5 3 $ OF THE 45CA DOSE HA0 BEEN INCORPORATED, INDICAT ING THAT THE PLOT OF TIME VERSUS $ 45CA DOSE A P P E A R I N G IN THE EGG S H E L L DIO INDEED P L A T E A U AS THE R E S U L T S PRESENTED HERE I N D I C A T E D . T H I S WAS NOT S U R P R I S I N G S I N C E THE BLOOD D I S A P P E A R A N C E CURVE SHOWS THAT THE AMOUNT OF L A B E L IN THE BLOOD DROPS P R E C I P I T O U S L Y IN TWO HOURS (F IGURE 1 5 ) SO T H A T , AS TIME WEARS ON, L E S S AND L E S S OF THE CALCIUM D E P O S I T E D IN THE S H E L L WILL BE L A B E L L E D . LBRCHER & HODGES ( 1 9 6 9 ) ALSO LOOKED AT THE INCORPORATION OF INTRAVENOUSLY I N J E C T E D 45CA INTO EGG S H E L L S BY UNANESTHATI ZED H E N S , AND FOUND THAT 3 8 - 4 5 $ OF THE DOSE WAS PRESENT IN THE EGG SHELL AFTER 1 2 0 M I N U T E S . THEREFORE THE ANESTHATI ZED HEN APPEARS TO C A L C I F Y EGGS AT THE SAME RATE AS AN U N A N E S T H A T I Z E 0 B I R D . E X A M P L E S OF THE D I S A P P E A R A N C E OF 45CA FROM THE A R T E R I A L PLASMA IN A BIRO C A L C I F Y I N G AN EGG SHELL AND IN A BIRO WITH e CO o 0. CD o 10 0-O 40 60 80 Time in Minutes FIGURE 15. GRAPH TO SHOW THE RATE OF D I S A P P E A R A N C E OF 45CA TRACER FROM THE A R T E R I A L PLASMA OF A HEN WHICH IS A C T I V E L Y C A L C I F Y I N G AN E G G . THE CURVE HAS BEEN ANALYSED K I N E T I C A L L Y STRAIGHT L I N E S , ROBERTSON (1957). x — O -Q -BY MANUAL F I T T I N G OF K 3 -K2 -K1 -E X P E R I M E N T A L P O I N T S . P O I N T S O B T A I N E D BY S U B T R A C T I O N OF THE SLOWEST E X P O N E N T I A L COMPONENT FROM THE C U R V E . P O I N T S O B T A I N E D BY S U B T R A C T I N G THE I N T E R M E D I A T E E X P O N E N T I A L COMPONENT FROM THE O E R I V E D CURVE (O ). S l N C E THESE P O I N T S D E F I N E A S T R A I G H T L I N E T H I S L I N E I S THE FASTEST E X P O N E N T I A L COMPONENT OF THE C U R V E . RATE CONSTANT FOR THE SLOWEST E X P O N E N T I A L COMPONENT. RATE CONSTANT FOR THE I N T E R M E D I A T E E X P O N E N T I A L COMPONENT. RATE CONSTANT FOR THE FASTEST E X P O N E N T I A L COMPONENT. - 6 8 -INACT IVE OVARIES ARE SHOWN IN F I G U R E S 1 5 AND 1 6 . IT MAY BE NOTED THAT THE P O S I T I O N OF THE CURVE R E L A T I V E TO THE X - A X I S WAS D I F F E R E N T IN THESE TWO B I R D S . IN GENERAL IT VARIED FROM BIRD TO BIRD PROBABLY BECAUSE OF D I F F E R E N C E S IN BODY WEIGHT AND HENCE BLOOD VOLUME. HOWEVER IN THE EXAMPLES SHOWN BOTH BIRDS WEIGHED 1.5 KG SO THAT THIS COULD NOT BE THE E X P L A N A -TION H E R E . SE V E R A L WORKERS REPORT THAT I N J E C T I O N OF ESTROGEN INTO CHICKENS INCREASES PLASMA AND BLOOD VOLUME, G I L B E R T ( 1 9 6 7 ) » THUS ONE MIGHT E X P E C T L A Y I N G HENS TO HAVE A LARGER 8 L 0 0 D VOLUME THAN BIRDS OF THE SAME AGE AND WEIGHT WHICH WERE OUT OF L A Y . COMPARISON OF THE BLOOO CURVES IN THE TWO TYPES OF BIRD SHOWS THAT THE CURVE IN THE BIRD OUT OF LAY WAS VERY MUCH SHALLOWER AND F L A T T E R THAN THAT IN THE BIRO C A L C I F Y I N G AN EGG S H E L L . T H I S WAS BORNE OUT BY THE K I N E T I C PARAMETERS OBTAINED BY CURVE A N A L Y S I S , S E E TABLE X I I . THREE EXPONENT IAL COMPONENTS WERE OBTAINEO FROM THE TWO HOUR PLASMA 45C A D I S A P P E A R A N C E C U R V E . THE SLOWEST COMPONENT HAD A HALF L I F E WHICH WAS TWICE AS LONG WHEN THE BIRD WAS OUT OF LAY AS WHEN IT WAS C A L C I F Y I N G AN EGG ( 3 0 0 MIN AS COMPAREO TO 1 5 0 M I N ) . THE BIRDS IN A C T I V E LAY ON A PAUSE DAY SHOWED A LARGE V A R I A -TION IN THIS K I N E T I C COMPONENT. IN ALL C A S E S THE HALF L I F E WAS LONGER THAN WHEN AN EGG WAS B E I N G C A L C I F I E D AND IN GENERAL IT WAS ABOUT 2 4 0 MIN AS OPPOSED TO 1 5 0 M I N , A80UT A 6 0 $ I N C R E A S E . HOWEVER IN ONE CASE THE HALF L I F E WAS EVEN LONGER THAN THAT IN BIROS OUT OF L A Y , I N D I C A T I N G THAT INDIVIDUAL V A R I A T I O N S WITHIN THE TWO GROUPS OF BIRDS NOT C A L C I F Y I N G AN EGG WERE SO IOO - | — r — i — i — i — r — i — i — i — i — i — i — i 0 20 40 60 80 100 120 Time in Minutes GRAPH TO SHOW THE RATE OF D I S A P P E A R A N C E OF 45CA FROM THE A R T E R I A L PLASMA OF A HEN WHICH IS OUT OF L A Y . ALL LEGENDS AS IN F IGURE 15 . - 6 9 -LARGE THAT THESE TWO GROUPS COULO PERHAPS BE CONSIDERED AS O N E . THERE WAS A S L I G H T INDICAT ION THAT THE INTERMEDIATE E X P O N E N T I A L COMPONENT HAD A LONGER HALF L I F E IN THE BIRDS OUT OF LAY THAN IN THOSE WITH A C T I V E O V A R I E S . THERE WAS INDIVIDUAL VARIAT ION IN THE HALF L I F E OF THE F A S T E S T EXPONENTIAL COMPONENT BUT S INCE THIS K I N E T I C PARAMETER IS THE ONE MOST S U B J E C T TO ERROR DUE TO THE NATURE OF THE CURVE A N A L Y S I S , ROBERTSON ( 1 9 5 7 ) , NO REAL D I F F E R E N C E S BETWEEN THE GROUPS OF BIROS COULD BE D I S C E R N E D . THE K I N E T I C A N A L Y S I S OF THE TWO HOUR PLASMA 45CA D I S -APPEARANCE CURVE GAVE THREE E X P O N E N T I A L S SO THAT THE BLOOD CAN BE CONSIDERED AS EXCHANGING WITH THREE OTHER COMPARTMENTS. S I N C E CHANGES COULD BE SEEN IN THESE COMPONENTS IN HENS UNDER D I F F E R E N T PHYS IOLOGICAL CONDIT IONS SOME IDEA OF WHICH P H Y S I O L O G I C A L PARAMETERS THESE MIGHT R E P R E S E N T COULD BE O B T A I N E D . F IGURE 1 7 G IVES A DIAGRAMMATIC R E P R E S E N T A T I O N OF P O S S I B L E P H Y S I O L O G I C A L COMPARTMENTS WHICH COULD EXCHANGE WITH PLASMA C A L C I U M . THE PLASMA CALCIUM IS IN E Q U I L I B R I U M WITH CALCIUM IN THE E X T R A C E L L U L A R F L U I D S AND THE SOFT T I S S U E S . SLNCE THE H E N ' S E X T R A C E L L U L A R F L U I D VOLUME IS APPROXIMATELY F I V E T IMES THE VOLUME OF THE P L A S M A , S T U R K I E ( 1 9 6 5 ) , ONE WOULD E X P E C T RAPID LOSS OF CALCIUM INTO THIS COMPARTMENT. TH IS PROBABLY GAVE R I S E TO THE F A S T E S T COMPONENT IN THE BLOOO CURVE WITH A HALF L I F E OF 2-4 M I N . EXCHANGE WITH THE C E L L S WOULD BE COMPLEX AND DEPEND UPON A C T I V E P R O C E S S E S ; PETURBATI ON OF THE BLOOD TABLE X I I THE E F F E C T OF VARIOUS PHYSIOLOGICAL ST A T E S OF THE HEN ON THE HALF L I V E S OF THE EXPONENTIAL COMPONENTS CALCULATED FROM THE ARTERIAL 45CA D ISAPPEARANCE CURVE EGG I N SH E L L GLAND PHYSIOLOGICAL STATE OF HEN HALF L I F E IN M INUTES $ CA45 DOSE IN EGG SHELL AFTER 2HRS FAST ( K I ) INTERMED1 ATE ( K 2 ) SLOW ( K 3 ) YES ACT IVE LAY — 1 6 5 . 4 7 ± 2 0 . 7 2 * 5 3 . 0 7 — — 1 6 1 . 7 9 ± U . 0 2 4 6 . 0 4 — — 1 3 9 . 7 9 ± 6.01 5 1 . 7 7 2 . 8 5 * 0.19 1 7 . 7 9 ± 0.41 1 3 3 . 7 8 ± 7.49 5 6 . 1 3 3.27 ± 0.06 1 8 . 9 7 ± 0.46 138.01 ± 6.32 4 8 . 1 9 3.65 ^ 0.01 18 . 0 4 ± 0.34 1 5 7 . 8 4 ± 7.66 5 3 . 2 3 No ACT IVE LAY 2.44*- 0.02 18.11 ± 1.32 2 1 0 . 4 8 ± 4.41 — 3.99 * 0.01 2 0 . 7 2 ± 0.89 3 2 6 . 7 8 + 2 0 . 3 4 — 1.74 ± 0 . 0 2 1 3 . 6 8 ± 0.48 2 7 4 . 6 0 ± 1 4 . 3 6 — 2.28 ± 0 . 0 1 1 8 . 3 8 ± 0.77 2 2 7 . 0 2 ± 8.70 — No OUT OF LAY 4.19 ± 0 . 1 8 2 0 . 6 6 ± 0.62 3 0 2 . 1 4 ± 1 9 . 1 0 — 3 . 4 8 ± 0.08 2 4 . 4 4 ± 1.55 3 0 3 . 9 9 ± 2.11 — * * S.E. ON THE L E A S T MEAN SQUARES S L O P E . - 7 0 -CURVE DUE TO THIS WOULD PROBABLY BE MINOR. ABSORPTION FROM THE GUT WOULD D I L U T E THE PLASMA CALCIUM POOL WITH U N L A B E L L E D C A L C I U M . THE L E V E L OF CALCIUM IN THE D IET A F F E C T S THE AMOUNT OF CALCIUM A B S O R B E D , HURWITZ & BAR ( 1 9 6 9 ) * S I N C E THE HENS USED IN THIS STUDY HAD A C C E S S TO GRIT IT SEEMS A P P R O P R I A T E TO ASSUME THAT THEY ABSORBED CALCIUM AT A RATE COMPARABLE TO THE HENS ON THE HIGHEST CALCIUM D I E T IN THE S T U O I E S OF HURWITZ & BA R , I . E . 2 . 4 3 G/HEN/DAY. TH I S WOULD MEAN THAT DURING EGG SHELL C A L C I F I C A T I O N EACH HEN ABSORBED ON AVERAGE 1 .68 M G / M I N . THE PLASMA CALCIUM L E V E L IN A L A Y I N G HEN IS GENERALLY APPROXIMATELY 2 5 MG;1 AND THE PLASMA VOLUME OF A 2 KG HEN IS ABOUT 100 M L , S T U R K I E ( 1 9 6 5 ) ; THEREFORE THE TOTAL AMOUNT OF CALCIUM IN THE PLASMA IS ROUGHLY 2 5 MG. THUS IT FOLLOWS THAT APPROXIMATELY 7 $ OF THE TOTAL AMOUNT OF CALCIUM IN THE PLASMA IS ABSORBED EVERY M I N U T E . THE BIRDS USED IN THESE EXPERIMENTS WERE TAKEN DURING THEIR N I G H T , S E E THE A P P E N D I X . S I N C E ABSORPTION D E C R E A S E S DURING THE N I G H T , AS THE CONTENTS OF THE GUT ARE USED U P , THE ABSORPTION RATE C A L C U L A T E D ABOVE ( MAY BE SOMEWHAT H I G H , BUT IT G I V E S SOME I0EA OF MAGNITUDE AND SHOWS THAT ABSORPTION FROM THE GUT WOULD BE AN IMPORTANT CONTRIBUTOR TO THE PLASMA 45CA D I S A P P E A R A N C E C U R V E . HURWITZ ( 1964) HAS SHOWN THAT BONE CAN BE D IV IDED INTO TWO COMPARTMENTS, EXCHANGABLE AND S T A B L E BONE . HE CONCLUDES FROM HIS DATA THAT THE MAJOR PORTION OF EXCHANGE IS DUE TO PHYSICOCHEMICAL P R O C E S S E S RATHER THAN A C T I V E BONE R E S O R P T I O N . MUELLER EJ_ A_L (1964 ) STATE THAT FROM 4 . 3 TO 4 . 9 G OF THE S K E L E T A L CALCIUM P A R T I C I P A T E S IN EGG SHELL FORMATION, OF WHICH Cells Egg Shell F IGURE 1 7 . A DIAGRAMMATIC R E P R E S E N T A T I O N OF THE P O S S I B L E FATE OF INTRAVENOUSLY I N J E C T E O 4 5 C A , SHOWING P H Y S I O L O G I C A L COMPARTMENTS WHICH COULO BE I N V O L V E D . - 7 1 -1 G IS TURNED OVER D A I L Y . T H I S WOULD MEAN THAT 0.7 MG OF CA /MIN WAS BEING EXCHANGED WITH THE BONE. USING THE SAME ASSUMPTIONS AS A B O V E , THIS MEANS THAT THE BONE TURNOVER RATE IS E Q U I V A L E N T TO ABOUT 2 . 8 $ OF THE TOTAL PLASMA CALCIUM BE ING EXCHANGED PER M I N U T E . EXCHANGE WITH BONE CAN THEREFORE BE E X P E C T E O TO CONTRIBUTE S I G N I F I C A N T L Y TO THE PLASMA 45C A D I S A P P E A R A N C E C U R V E . THE L A Y I N G HEN ALSO S E C R E T E S CALCIUM IN ITS U R I N E . ALTHOUGH THE L E V E L OF CALCIUM S E C R E T I O N D E C R E A S E S ON L A Y I N G DAYS THE HEN S T I L L S E C R E T E S 7.8 MEQ OF CA / O AY ( E Q U I V A L E N T TO 0 . 3 1 2 G / D A Y), TAYLOR & K I R K L E Y ( 1 9 6 7 ) . HURWITZ & GRIMINGER ( 1 9 6 1 ) REPORT A S L I G H T L Y LOWER VALUE OF 0.22 G/HEN/DAY. THUS CALCIUM IS LOST IN THE URINE AT A RATE OF 0 . 1 5 - 0 . 2 2 M G / M I N , OR WITH THE SAME ASSUMPTIONS AS B E F O R E , ABOUT 0 . 6 1 - 0 . 8 6 $ OF THE TOTAL PLASMA CALCIUM IS LOST IN THE URINE EVERY M I N U T E . THUS SOME CONTRIBUTION TO THE PLASMA 45CA D I S A P P E A R A N C E CURVE CAN BE E X P E C T E D . ENDOGENOUS FECAL E X C R E T I O N COULD ALSO CAUSE L O S S OF CALCIUM FROM THE BLOOD. ACCORDING TO MUELLER EJ_ A_L ( 1 9 6 4 ) ENDOGENOUS E X C R E T I O N IS R E S P O N S I B L E FOR THE LOSS OF 7 $ OF THE INTAKE ON L A Y I N G DAYS AND 1 2 $ ON NONLAYING D A Y S , WHICH IS ABOUT 0 . 1 6 - 0 . 2 8 G/DAY. HOWEVER, S INCE THE HENS IN THIS STUDY WERE NOT C 0 L 0 S T 0 M I ZED , ENDOGENOUS E X C R E T I O N INCLUDED URINE AS WELL AS F E C E S , AND AS THE L E V E L S OF ENDOGENOUS E X C R E T I O N ARE VERY CLOSE TO THOSE FOR URINE E X C R E T I O N A L O N E , ONE MIGHT INFER THAT ENDOGENOUS FECAL E X C R E T I O N IS N E G L I G I B L E . THE OTHER COMPARTMENT INTO WHICH THE PLASMA L O S E S CALCIUM - 7 2 -IN THE L A Y I N G HEN IS THE EGG S H E L L . FROM THE R E S U L T S REPORTEO HERE (F IGURE 1 4 ) IT IS E V I D E N T THAT 5 0 $ OF THE BLOOO CALCIUM L A B E L IS LOST TO THE SHELL IN THE SPACE OF TWO HOURS, SO THAT THIS MUST MAKE A VERY IMPORTANT CONTRIBUTION TO THE D I S A P P E A R -ANCE C U R V E . IF THERE WAS NO R E C Y C L I N G OF TRACER ONE WOULO E X P E C T EGG SHELL C A L C I F I C A T I O N TO PRODUCE AN EXPONENT IAL COMPONENT IN THE BLOOD CURVE WITH A HALF L I F E OF ROUGHLY 1 2 0 M I N . DURING A C T I V E SHELL C A L C I F I C A T I O N THE HALF L I F E OF THE SLOWEST C O M -PONENT WAS 1 5 0 MINUTES ANO THIS GREATLY INCREASED WHEN NO EGG WAS PRESENT IN THE SHELL GLAND. THE E X T R A C E L L U L A R F L U I O VOLUME IS F I V E T IMES AS LARGE AS THE PLASMA VOLUME AND 5 0 $ OF THE 45CA DOSE APPEARS IN THE EGG S H E L L , T H E R E F O R E , IF ONE ASSUMES E Q U I L I B R I U M BETWEEN THE E X T R A C E L L U L A R FLU IO ANO THE P L A S M A , IT FOLLOWS THAT 45CA MUST HAVE RETURNED TO THE BLOOD FROM THE E X T R A C E L L U L A R F L U I D . T H I S WOULO TEND TO LENGTHEN THE HALF L I F E OF THE EGG S H E L L C A L C I F I C A T I O N COMPONENT. THE OTHER FACTOR WHICH WOULD TEND TO PRODUCE A LONGER HALF L I F E IS THE PRESENCE OF OTHER E X P O N E N T I A L COMPONENTS WITH LONGER HALF L I V E S WHICH DO NOT APPEAR IN THE TWO HOUR BLOOD C U R V E . SUBTRACT ION OF THESE BY CURVE A N A L Y S I S WOULO DECREASE THE HALF L I V E S OF THE P R E C E E D I N G E X P O N E N T I A L S . HURWITZ ( 1 9 6 4 ) LOOKED AT THE 45CA D I S A P P E A R A N C E CURVE OVER A MUCH LONGER T IME INTERVAL THAN THE ONE USED H E R E . IF HIS CURVE IS S U B J E C T E D TO THE SAME TYPE OF K I N E T I C A N A L Y S I S PERFORMEO HERE ONE OBTAINS A MINIMUM OF THREE EXPONENTIAL COMPONENTS WITH HALF L I V E S OF 5,26 AND 111 HOURS. THIS- A N A L Y S I S WAS VERY C R U D E , S I N C E THE NUMBER OF POINTS WAS SO S M A L L , BUT THE GENERAL PATTERN CAN BE -73-o i S C E R N E D . S U B T R A C T I O N OF T H E S E E X P O N E N T I A L C O M P O N E N T S WOULD HAVE S H I F T E O T H E H A L F L I F E OF T H E S LOWEST C O M P O N E N T IN T H E TWO HOUR C U R V E TO A S M A L L E R V A L U E AND H E N C E C L O S E R TO T H E E X P E C T E D 1 2 0 M I N U T E S OF T H E EGG S H E L L C A L C I F I C A T I O N P R O C E S S . IT T H E R E -FORE A P P E A R S T H A T T H E S L O W E S T C O M P O N E N T OF T H E TWO HOUR BLOOD C U R V E WAS OUE TO EGG S H E L L C A L C I F I C A T I O N . WHEN T H E S T R E S S OF E G G S H E L L C A L C I F I C A T I O N WAS REMOVED T H I S E X P O N E N T I A L HAD A MUCH L O N G E R H A L F L I F E , BUT T H E I N D I V I D U A L V A R I A T I O N B E T W E E N T H E B I R D S WAS MUCH G R E A T E R THAN WHEN AN E G G WAS B E I N G C A L C I F I E D ( T A B L E X I I ) . T H I S COULD HAVE B E E N B E C A U S E O T H E R U N D E R L Y I N G K I N E T I C C O M P O N E N T S , S U C H AS BONE R E M O D E L L I N G , WERE P R E S E N T , BUT WHEN EGG S H E L L C A L C I F I C A T I O N C A U S E D A MAJOR D R A I N ON C A L C I U M M E T A B O L I S M T H E S E N O N - E S S E N T I A L P R O C E S S E S WOULO P R O B A B L Y HAVE V I R T U A L L Y C E A S E D . G E N E R A L L Y IN T H E B I R D S ON A P A U S E DAY T H E T H I R D E X P O N E N T I A L H A 0 A F A S T E R H A L F L I F E THAN IN B I R D S OUT OF L A Y . T H I S IS NOT S U R P R I S I N G S I N C E ONE WOULD E X P E C T L A Y I N G B I R D S TO HAVE A F A S T E R C A L C I U M M E T A B O L I S M THAN B I R O S WITH I N A C T I V E O V A R I E S . HOWEVER ONE B IRD ON A P A U S E DAY HAD AN E V E N SLOWER H A L F L I F E THAN T H E B I R O S OUT OF L A Y , SO T H A T I N D I V I D U A L V A R I A T I O N S WERE SO G R E A T T H A T NO C L E A R C U T C O N C L U S I O N S C O U L D BE DRAWN. THE F A S T E S T E X P O N E N T I A L WAS P R O B A B L Y M A I N L Y DUE TO E Q U I L I B -R A T I O N WITH T H E E X T R A C E L L U L A R F L U I 0 S ANO S O F T T I S S U E S WHILE T H E S L O W E S T E X P O N E N T I A L WAS OUE TO EGG S H E L L C A L C I F I C A T I O N . T H I S L E A V E S T H E S E C O N O E X P O N E N T I A L WHICH C O U L D BE DUE TO C A L C I U M A B S O R P T I O N , E X C H A N G E WITH B O N E , U R I N E F O R M A T I O N , OR A C O M -B I N A T I O N OF ANY OR A L L OF T H E S E . - 7 4 -T H E H A L F L I F E OF T H E S E C O N D E X P O N E N T I A L D ID NOT C H A N G E MUCH WITH E G G S H E L L C A L C I F I C A T I O N . IT WAS MUCH MORE V A R I A B L E WHEN THE B I R D S WERE ON A P A U S E DAY AND MAY P O S S I B L Y HAVE B E E N S L I G H T L Y SLOWER WHEN T H E B I R D S WERE OUT OF L A Y ( T A B L E X I L ) . M U E L L E R E_T A J L ( 1 9 6 4 ) SHOWED THAT T H E D I F F E R E N C E B E T W E E N BONE A C C R E T I O N ANO BONE R E S O R P T I O N WAS - 0 . 4 4 G / H E N / D A Y ON L A Y I N G DAYS AND 1.48 G / H E N / D A Y ON N O N - L A Y I N G D A Y S . T H E R E F O R E ONE WOULO E X P E C T A F A S T E R D I S A P P E A R A N C E OF L A B E L FROM T H E BLOOO DUE TO BONE D E P O S I T I O N ON N O N - L A Y I N G DAYS THAN ON L A Y I N G D A Y S . HURWITZ & 3AR ( 1 9 6 9 ) SHOWED THAT T H E A B S O R P T I O N OF C A L C I U M I N C R E A S E D FROM 4 0 $ TO 7 0 $ WHEN EGG S H E L L C A L C I F I C A T I O N WAS T A K I N G P L A C E . T H E R E F O R E HENS ON A P A U S E DAY WOULO BE A B S O R B I N G L E S S ANO T H I S WOULD T E N D TO SLOW THE D I S A P P E A R A N C E C U R V E . CA L C I U M E X C R E T E D IN T H E U R I N E I N C R E A S E S FROM 7.8 M E Q / O A Y ON L A Y I N G DAYS TO 13.9 M E Q / D A Y ON N O N - L A Y I N G D A Y S , T A Y L O R & K I R K L E Y ( 1 9 6 7 ) . THUS T H E E F F E C T O F U R I N E F O R M A T I O N ON THE 45CA D I S A P P E A R A N C E C U R V E WOULD BE TO D E C R E A S E THE H A L F L I F E OF T H E S E C O N D E X P O N E N T I A L IN HENS ON A P A U S E D A Y . | F ONLY ONE OF T H E S E T H R E E F A C T O R S WERE I N V O L V E D ONE WOULD E X P E C T TO S E E A C H A N G E IN THE S E C O N D E X P O N E N T I A L B E T W E E N B I R D S A C T I V E L Y C A L C I F Y I N G AN EGG ANO T H O S E WHICH WERE N O T . HOWEVER A B S O R P T I O N A C T S TO I N C R E A S E T H E H A L F L I F E OF THE S E C O N D E X P O N E N T I A L , WHILE BONE F O R M A T I O N AND U R I N E F O R M A T I O N A C T IN T H E O P P O S I T E D I R E C T I O N IN HENS ON A P A U S E 0 A Y• T H E C A L C U L A -T I O N S P E R F O R M E O A B O V E SHOWED THAT THE C H A N G E S IN T O T A L BLOOD - 7 5 -CALCIUM OUE TO THESE THREE FACTORS WERE OF THE SAME ORDER OF MAGNITUDE . T H E R E F O R E , IF THE SECOND EXPONENT IAL WERE DUE TO A COMBINATION OF ALL T H R E E , THE CHANGES IN EACH CAUSED BY EGG S H E L L C A L C I F I C A T I O N COULD P O S S I B L Y CANCEL OUT AND NO NET E F F E C T ON THE SECOND EXPONENTIAL WOULD BE S E E N . THE S L I G H T SLOWING OF THE SECOND EXPONENT IAL COMPONENT IN HENS OUT OF LAY COMPARED WITH THOSE IN A C T I V E LAY WAS TO BE E X P E C T E D B E C A U S E , P R E S U M E A B L Y , THESE BIRDS LACKED MEDULLARY BONE AND THEREFORE HAD L E S S BONE A C C R E T I O N . HURWITZ ( 1 9 6 5 ) STUDIED CALCIUM EXCHANGE IN VARIOUS SEGMENTS OF BONE AND FOUNO THAT THE TURNOVER RATE OF CALCIUM IN THE MEDULLARY SEGMENT WAS AT L E A S T 1 0 - 1 5 T IMES LARGER THAN THAT OF CORTICAL S E G M E N T S . HOWEVER THERE WAS MUCH MORE CALCIUM MASS IN THE CORT ICAL BONE AND THE ACCRET ION RATE WAS 1 . 5 0 , 0 . 2 3 AND 0 . 8 3 MG/HR IN THE FEMUR E N D S , CORTEX AND MEDULLARY SEGMENT R E S P E C T I V E L Y . T H E R E -FORE ALTHOUGH THE TURNOVER RATE IN THE MEDULLARY BONE MAY BE G R E A T E R , THE GREATER MASS OF THE OTHER SEGMENTS TENDS TO OVERIOE THE E F F E C T AS IT WOULD APPEAR IN A K I N E T I C A N A L Y S I S ; THUS ONLY A VERY MINOR E F F E C T WAS S E E N . ONE OF THE FUNDAMENTAL ASSUMPTIONS IN TRACER K I N E T I C S IS THAT THE SUBSTANCE BE ING STUDIEO IS UNIFORMLY D I S T R I B U T E D AT ALL T I M E S . T H I S I M P L I E S INSTANTANEOUS AND HOMOGENEOUS MIXING WITHIN A COMPARTMENT. 45CA WAS I N J E C T E D INTRAVENOUSLY AND A R T E R I A L SAMPLING BEGAN TWO MINUTES L A T E R . SlNCE THE C I R C U -L A T I O N TIME IN A HEN IS 2 . 8 S E C O N D S , S T U R K I E ( 1 9 6 5 ) , MIXING IN THE PLASMA WOULD OCCUR RAPIDLY SO THAT THIS ASSUMPTION APPEARED V A L I D . HOWEVER HURWITZ ( 1 9 6 8 ) HAS DEMONSTRATED THE T A B L E X I I I THE E F F E C T OF BLOCKADE OF VARIOUS BRANCHES OF THE AUTONOMIC NERVOUS SYSTEM ON THE INCORPORATION OF 4 5CA INTO THE EGG S H E L L TIME ( M I N . ) DRUG TYPE OF BLOCKADE io COUNTS I N EGG S H E L L NUMBER OF HENS 3 0 3 2 . 2 6 ± 1 . 9 4 * 3 ATROP1NE PARASYMPATHETI c 3 1 . 0 1 1 Dl BENZYL 1NE A L P H A - S Y M P A T H E T I c 3 1 . 1 5 1 PROPRANALOL B E T A - S Y M P A T H E T 1 c 3 3 . 1 2 1 1 2 0 5 1 . 3 6 ± 3 . 3 4 * 6 ATROPINE P A R A S Y M P A T H E Tic 4 7 . 3 7 1 Dl BENZYL 1NE A L P H A - S Y M P A T H E T I C 5 4 . 0 6 ± 8 . 8 7 * 2 PROPRANALOL B E T A - S Y M P A T H E T I C 5 4 . 6 0 1 3 . 9 1 * 2 * ± S . E . OF THE MEAN. - 7 6 -P R E S E N C E OF A S L 0 W LY— E X C H A N G I N G PROTE IN —80UND C A L C I U M F R A C T I O N IN T H E P L A S M A OF T H E L A Y I N G HEN WHICH C O M P R I S E S A B O U T 2 2 $ OF T H E T O T A L P L A S M A C A L C I U M ANO E X C H A N G E S AT A R A T E OF 0.11 M G / M I N / 1 0 0 M L . THE P R E S E N C E OF T H I S S L O W L Y - E X C H A N G I N G F R A C T I O N WOULD P R E V E N T I N S T A N T A N E O U S HOMOGENEOUS M I X I N G IN T H E P L A S M A AND H E N C E I N V A L I D A T E ONE OF T H E B A S I C A S S U M P T I O N S U S E D IN T H E K I N E T I C A N A L Y S I S . C O M P A R T M E N T A L A N A L Y S I S OF T H E 45CA D I S A P P E A R A N C E CURVE P R E S U P P O S E S S T E A O Y S T A T E C O N D I T I O N S . D U R I N G E G G S H E L L C A L -C I F I C A T I O N T H I S C O N D I T I O N IS NOT S T R I C T L Y T R U E . T A Y L O R & H E R T E L E N D Y ( 1 9 6 1 ) SHOWED T H A T T H E D I F F U S I B L E C A L C I U M F E L L 1 M G / 1 0 0 M L , A P P R O X I M A T E L Y A 4 $ F A L L IN T O T A L P L A S M A C A L C I U M . V/LNGET EJ_ AL. ( 1 9 5 8 ) SHOWED THAT T H E S H E L L G L A N D V E N O U S BLOOD HAD 2 0 $ L E S S T O T A L C A L C I U M THAN T H E A R T E R I A L B L O O D . T H E R E -FORE S T R I C T S T E A D Y S T A T E C O N D I T I O N S ARE NOT P R E S E N T . T H E M A T H E M A T I C A L T R E A T M E N T FOR N O N - S T E A D Y S T A T E S Y S T E M S IS VERY MUCH MORE C O M P L E X ANO HAS B E E N IGNORED IN F A V O U R OF T H E L E S S R I G O R O U S S T E A D Y S T A T E T R E A T M E N T . TH U S , WHILE T H E K I N E T I C A N A L Y S I S IS AN A P P R O X I M A T I O N I T DOES G I V E SOME IDEA OF T H E R E L A T I V E R A T E S OF E X C H A N G E B E T W E E N C O M P A R T M E N T S AND T H E F A T E OF C A L C I U M IN T H E L A Y I N G H E N . IN C O N C L U S I O N IT A P P E A R S T H A T THE T H R E E E X P O N E N T I A L S 0 8 T A I N E 0 IN T H E K I N E T I C A N A L Y S I S WERE NOT A L L S I M P L E F U N C T I O N S . THE F A S T E S T ONE WAS P R O B A B L Y A R E S U L T OF E Q U I L I B R A T I O N WITH T H E E X T R A C E L L U L A R F L U I D AND S O F T T I S S U E S AND THE SLOWEST WAS OUE M A I N L Y TO EGG S H E L L C A L C I F I C A T I O N . T H E I N T E R M E D I A T E E X P O N E N T I A L WAS P R O B A B L Y DUE TO A C O M B I N A T I O N OF A B S O R P T I O N , TABLE X IV THE E F F E C T OF VARIOUS NEURAL BLOCKING DRUGS ON THE HALF L I V E S OF THE EXPONENTIAL COMPONENTS C A L C U L A T E D FROM THE ARTERIAL PLASMA 4 5CA DISAPPEARANCE CURVE IN HENS A C T I V E L Y C A L C I F Y I N G AN EGG DRUG TYPE OF BLOCKADE HALF L I F E IN MINUTES 1o CA 4 5 DOSE IN EGG SHELL AFTER 2HR FAST I NTERMED1 ATE ( K 2 ) SLOW ( K 3 ) 1 6 5 . 4 7 ± 2 0 . 7 2 * 5 3 . 0 7 1 6 1 . 7 9 ± 1 4 . 0 2 4 6 . 0 4 1 3 9 . 7 9 - 6 . 0 1 51 . 7 7 2 . 8 5 ± 0 . 1 9 1 7 . 7 9 = 0 . 4 1 1 3 3 . 7 8 ± 7 . 4 9 5 6 . 1 3 3 . 2 7 ± 0 . 0 6 1 8 . 9 7 ± 0 . 4 6 1 3 8 . 0 1 ± 6 . 3 2 4 8 . 1 9 3 . 6 5 ± 0 . 0 1 1 8 . 0 4 ± 0 . 3 4 1 5 7 . 8 4 ± 7 . 6 6 5 3 . 2 3 ATROPINE PARASYMPATHETI c 2 . 8 0 ± 0 . 0 6 1 8 . 1 0 ± 0 . 4 0 1 4 8 . 8 0 ± 5 . 4 6 — 2 . 8 7 = 0 . 0 2 1 4 . 6 6 ± 0 . 3 8 1 4 9 . 5 4 t 6 . 1 3 4 7 . 3 7 Dl B E N Z Y L 1 NE A L P H A -SYMPATHETI c 3 . 0 0 + 0 . 0 6 1 7 . 5 9 ± 0 . 8 3 1 3 4 . 6 3 ± 5 . 8 6 4 5 . 1 9 2 . 6 2 ± 0 . 0 1 1 5 . 7 9 ± 0 . 1 7 1 3 5 . 5 3 ± 4 . 5 8 6 2 . 9 3 PROPRANALOL B E T A -S Y M P A T H E T 1 c 2 . 7 1 ± 0 . 0 3 1 8 . 4 0 ± 0 . 4 1 1 6 4 . 4 4 ± 1 7 . 2 8 5 0 . 6 9 3 . 3 1 ± 0 . 0 1 1 7 . 7 8 ± 0 . 2 9 1 1 7 . 9 1 ± 5 . 6 4 5 8 . 5 1 * ± S . E . ON THE L E A S T MEAN SQUARES S L O P E . - 7 7 -EXCHANGE WITH BONE AND URINE FORMATION. THE E F F E C T OF THE VARIOUS NEURAL BLOCKING DRUGS ON 45CA INCORPORATION INTO THE EGG SHELL AT 3 0 MIN AND TWO HOURS IS SHOWN IN TABLE XIII. AFTER 3 0 MIN APPROXIMATELY 3 0 $ OF THE TRACER 00SE HAD BEEN INCORPORATED INTO THE EGG S H E L L , REGAROLESS OF WHETHER THE ORUGS WERE P R E S E N T OR NOT. AFTER 1 2 0 MIN APPROXIMATELY 5 0 $ OF THE TRACER DOSE APPEARED IN THE EGG SHELL AND THE ORUGS A P P E A R E 0 TO HAVE NO E F F E C T ON INCORPORATION AT THIS TIME PERIOO E I T H E R . THUS THE DATA I M P L I E S THAT NONE OF THE ORUGS HAD ANY MARKED E F F E C T ON E ITHER THE RATE OF INCORPORATION OR THE A 8 S 0 L U T E AMOUNT OF CALCIUM INCORPORATED INTO THE EGG S H E L L . THE K I N E T I C PARAMETERS OF THE TRACER D I S A P P E A R A N C E C U R V E , DURING A C T I V E SHELL C A L C I F I C A T I O N , IN THE PRESENCE AND ABSENCE OF THE NEURAL BLOCKING DRUGS ARE SHOWN IN TABLE XIV AND SUMMARIZED IN TABLE XV. THE HALF L I F E OF THE SLOWEST COMPONENT WAS APPROXIMATELY 1 5 0 M I N , THAT OF THE INTERMEDIATE COMPONENT APPROXIMATELY 1 8 M I N , ANO THAT OF THE F A S T E S T APPROXIMATELY 3 M I N . THE PRESENCE OF THE NEURAL BLOCKING DRUGS HAD NO S I G N I F I C A N T E F F E C T ON ANY OF THESE COMPONENTS. THUS THE RESULTS WERE UNABLE TO DEMONSTRATE ANY E F F E C T S OF THE NEURAL BLOCKADE ON ANY A S P E C T OF CALCIUM METABOLISM IN THE L A Y I N G H E N . THE LACK OF E F F E C T OF A NEURAL BLOCKER ON A SYSTEM IS NOT D E F I N A T I V E PROOF THAT THE BRANCH OF THE NERVOUS SYSTEM BLOCKED BY THAT PARTICULAR DRUG IS NOT INVOLVED. THE BLOCKING DOSES S E L E C T E D FOR THESE EXPERIMENTS GAVE 7 0 - 8 0 $ B L O C K A D E , S INCE THE LARGE OOSES REQUIRED TO GIVE 1 0 0 $ BLOCKADE WOULD PROBABLY T A B L E XV A SUMMARY OF THE MEAN HALF L I V E S OF THE THREE EXPONENTIAL COMPONENTS C A L C U L A T E D FROM THE ARTERIAL PLASMA 45CA DISAPPEARANCE CURVE IN HENS UNDER VARIOUS CONDITIONS EGG I N SHELL GLAND PHYS1OLOG1CAL STATE OF HEN DRUG TYPE OF BLOCKADE HALF L I F E IN MINUTES NUMBER OF HENS FAST I NTERMED1 ATE ( K 2 ) SLOW ( K 3 ) YES ACTIVE LAY 3 . 2 6 ± - 0 . 3 3 * 1 8 . 2 7 ± 0 . 5 1 1 4 9 . 4 5 ± 1 2 . 5 7 6 ATROP1NE P A R A -SYMPATHET I c 2 . 8 4 ± 0 . 0 3 1 6 . 3 8 ± 1 . 7 2 1 4 9 . 1 7 ± 0 . 3 7 2 D1 BENZYL 1NE A L P H A -SYMPATHETIc 2 . 8 1 ± 0 . 1 9 1 6 . 6 9 ± 0 . 9 0 1 3 5 . 0 8 ± 0 . 4 5 2 PROPRANALOL B E T A -SYMPATHET1c 3 . 0 1 ± 0 . 3 0 1 8 . 0 9 ± 0 . 3 1 1 4 1 . 1 7 ± 2 3 . 2 6 2 No' ACTIVE LAY 2 . 6 1 ± 0 . 8 4 1 7 . 7 2 ± 2 . 5 4 2 5 8 . 9 7 ± 4 5 . 8 7 4 No OUT OF LAY 3 . 8 4 ± 0 . 3 6 2 2 . 5 5 ± 1 . 8 9 3 0 3 . 0 7 • ± 0 . 9 3 2 * MEAN ± S . E . ON THE MEAN -78-HAVE P R O D U C E D N O N - S P E C I F I C E F F E C T S . T H E 8 L 0 C K A 0 E WAS C A L -C U L A T E D U S I N G THE R E S P O N S E S OF THE C A R D I O V A S C U L A R S Y S T E M TO E X O G E N O U S D O S E S OF A G O N I S T S . HOWEVER IN A L L C A S E S IT A P P E A R S T H A T THE R E S P O N S E S OF THE C A R D I O V A S C U L A R S Y S T E M TO E X O G E N O U S D O S E S OF A G O N I S T S ARE B L O C K E D MORE E F F E C T I V E L Y THAN T H O S E TO M E D I A T O R R E L E A S E D L O C A L L Y AT THE N E R V E E N D I N G S , GOOOMAN & G l L M A N ( 1 9 6 5 ) . IN T H E C A S E OF A T R O P I N E T H I S HAS B E E N A T T R I B U T E D TO T H E R E L E A S E OF A C E T Y L C H O L I N E AT THE N E R V E E N O I N G S IN S U C H C L O S E P R O X I M I T Y TO THE R E C E P T I V E M E C H A N I S M THAT T H E A T R O P I N E IS U N A B L E TO G A I N A C C E S S TO THE R E C E P T O R S I T E S , T H E S O - C A L L E D " P R O X I M I T Y T H E O R Y " , A M B A C H E ( 1 9 5 5 ) . TH I S COULD A L S O A P P L Y TO T H E A D R E N E R G I C B L O C K E R S USED H E R E . A L T H O U G H O I B E N Z Y L I N E IS AN I R R E V E R S I B L E B L O C K E R BOTH A T R O P I N E AND P R O P R A N A L O L ARE C O M P E T I T I V E B L O C K E R S , C O M -P E T I T I V E B L O C K A D E MAKES D E F I N I T I O N OF 8 L O C K I N G OOSES D I F F I -C U L T , B E C A U S E AN I N C R E A S E IN THE A G O N I S T C O N C E N T R A T I O N WILL D I S P L A C E T H E B L O C K E R FROM R E C E P T O R S I T E S AND A R E S P O N S E CAN E N S U E . D O L L E R Y E_T AJ. ( 1 9 6 9 ) C I T E S E V E R A L S T U D I E S WITH P R O P R A N A L O L WHICH S U P P O R T T H I S C O N C E P T . S I N C E THE A B S O L U T E C O N C E N T R A T I O N S OF A G O N I S T NEAR T H E R E C E P T O R S I T E S ARE UNKNOWN, AND MAY L O C A L L Y BE VERY H I G H , T H E P O S S I B I L I T Y OF D I S P L A C E M E N T OF C O M P E T I T I V E B L O C K E R S C A N N O T BE R U L E D O U T . T H U S , WHILE T H E P H A R M A C O L O G I C A L S T U D I E S COULD HAVE D E M O N S T R A T E D A N E U R A L E F F E C T I F ONE HA 0 B E E N P R E S E N T , THE L A C K OF R E S P O N S E DOES NOT N E C E S S A R I L Y PROVE T H A T T H E N E R V O U S S Y S T E M IS NOT I N V O L V E O . B I B L I O G R A P H Y . A M B A C H E , N . ( 1 9 5 5 ) T HE USE AND L I M I T A T I O N S OF A T R O P I N E FOR P H A R M A C O L O G I C A L S T U D I E S ON A U T O N O M I C E F F E C T O R S . P H A R M A C O L . R E v 1 E w s 7 : 4 6 7 . A N D E R S O N , R . S . ( 1 9 6 7 ) A C I D - S A S E C H A N G E S IN T H E E X C R E T A OF T H E L A Y I N G H E N . V E T . R E C . 8 0 : 3 1 4 . A H L Q U I S T , R . P . ( 1 9 4 8 ) A S T U D Y OF THE A D E N O T R O P I C R E C E P T O R S . A M . J . P H Y S I O L . 1 5 3 : 5 8 6 . A H L Q U I S T , R . P . & L E V Y , B. ( 1 9 5 9 ) A D R E N E R G I C R E C E P T I V E M E C H A N I S M OF C A N I N E I L E U M . J . P H A R M A C O L . 1 2 7 : 1 4 6 . B AR , A . & H U R W I T Z , S . ( 1 9 6 9 ) T HE A C C U M U L A T I O N OF C A L C I U M IN L A Y I N G FOWL I N T E S T I N E IN VI T R O . B i O C H I M . B I O P H Y S . A C T A . 1 8 3 : 5 9 1 . B A R N E S , C D . & E L T H E R INGTON , L . G . ( 1 9 6 4 ) DRUG D O S A G E IN L A B O R A T O R Y A N I M A L S : A H A N D B O O K . U N I V E R S I T Y OF C A L -I F O R N I A P R E S S . B E L A N G E R , L . F . & T A Y L O R , T . G . ( 1 9 6 7 ) T HE M E C H A N I S M O F BONE R E S O R P T I O N IN L A Y I N G H E N S . A N A T . R E C . 1 5 7 : 2 1 1 . B L O O M , W . , B L O O M , M . A . & M C L E A N , F . C . ( 1 9 4 1 ) C A L C I F I C A T I O N AND O S S I F I C A T I O N . M E D U L L A R Y BONE C H A N G E S IN T H E R E -P R O D U C T I V E C Y C L E OF F E M A L E P I G E O N S . A N A T . R E C 8 1 ; 4 4 3 . B L O O M , M . A . , DOMM, L . U . , N A L B A N D O V , A . V . & B L O O M , W. ( 1 9 5 8 ) M E D U L L A R Y BONE OF L A Y I N G C H I C K E N S . A M . J . A N A T . 1 0 2 : 4 1 1 . 3 O L T O N , T.B. & BOWMAN, T . C . ( 1 9 6 9 ) A O E N O R E C E P T O R S IN T H E C A R D I O V A S C U L A R S Y S T E M OF T H E D O M E S T I C F O W L . E U R . J . P H A R -M A C O L . 5 : 1 2 1 . - 7 9 -- 8 0 -B R A D F I E L D , J . R . G . ( 1 9 5 1 ) R A D I O G R A P H I C S T U D I E S ON T H E F O R M A T I O N OF T H E H E N * S EGG S H E L L . J . E x P T L . B I O L . 2 8 : 9 . B R O N S C H , K . , L B R C H E R , K. & V I L L W O C K , J . ( 1 9 7 0 ) C A L C I U M R E T E N T I O N IN YOUNG P U L L E T S AND L A Y I N G HENS F E D A 4 7 C A -L A 8 E L L E D D I E T OF 0 I F F E R E N T C A L C I U M L E V E L S . A N N.BlOL. AN I M . 3 i O C H . 3i O P H Y S . T O , N ° H O R S - S E ' R I E 2 , 5 9 - 6 7 . B U N A G , R . D . & W A L A S Z E K , E . J . ( 1 9 6 2 ) C A R D I O V A S C U L A R P H A R -M A C O L O G Y OF THE D O M E S T I C F O W L . J A P A N . J . P A R M A C O L . H : 1 7 1 . B U R M E S T E R, B . R . ( 1 9 4 0 ) A S T U D Y OF T H E P H Y S I C A L AND C H E M -I C A L C H A N G E S OF T H E E G G D U R I N G I T S P A S S A G E THROUGH T H E I S T H M U S AND U T E R U S OF T H E H E N * S O V I D U C T . J . E X P . Z O O L . 8 4 : 4 4 5 . B U R M E S T E R, B . R . , S C O T T , H . M . & C A R D , L . E . ( 1 9 3 9 ) R ATE OF EGG S H E L L F O R M A T I O N IN T H E H E N . P R O C . 7 T H W O R L D ' S P O U L T R Y C O N G R . , P. 9 9 . C A M P O S , H . A . & U R Q U I L L A , P . R . ( 1 9 6 9 ) A C T I O N OF C O C A I N E AND C H R O N I C S Y M P A T H E T I C D E N E R V A T I O N ON V A G A L E S C A P E . J . P H Y S I O L . 2 0 0 : 3 1 1 . C A N O L I S H , J . K . & T A Y L O R , T . G . ( 1 9 7 0 ) T HE R E S P O N S E T I M E TO T H E P A R A T H Y R O I D HORMONE IN T H E L A Y I N G F O W L . J . E N D O C R . 4 8 : 1 4 3 . C H A S E , L . R . & A U R B A C H , G . D . ( 1 9 6 8 ) R ENAL A O E N Y L C Y C L A S E : A N A T O M I C A L L Y S E P A R A T E S I T E S FOR P A R A T H Y R O I O HORMONE AND V A S O P R E S S I N . S C I E N C E 1 5 9 : 5 4 5 . C H A S E , L . R . ?z A U R B A C H , G . D . ( 1 9 6 9 ) E F F E C T OF P A R A T H Y R O I D - 8 1 -HORMONE ON T H E C O N C E N T R A T I O N OF 3 ' 5 ' - A M P IN B O N E . C L I N . R E S . 1 7 : 3 8 0 . C H A S E , L . R . , F E O A K , S . A . & A U R B A C H , G.D. ( 1 9 6 9 ) A C -T I V A T I O N OF S K E L E T A L A D E N Y L C Y C L A S E BY P A R A T H Y R O I D HORMONE IN V I T R O . E N D O C R I N O L O G Y 8 4 : 7 6 1 . C H E N O W E T H , M . B . & V A N D Y K E , R . A . ( 1 9 6 9 ) A N A E S T H E S I A IN B I O M E D I C A L R E S E A R C H . F E O . P R O C . 2 8 : 1 3 8 3 . COMMON, R . H . ( 1 9 4 1 ) T H E C A R B O N I C A N H Y D R A S E A C T I V I T Y OF T H E H E N ' S O V I D U C T . J . A G R I C . S C I . C A M S . 3 1 _ : 4 1 2 . C O R R A D I N O , R . A . & W A S S E R M A N , R . H . ( 1 9 6 8 ) A C T I N O M Y C I N D I N H I B I T I O N OF V I T A M I N D3 — INDUCED C A L C I U M - B I N D I N G P R O T E I N ( C A B P ) F O R M A T I O N IN C H I C K DUODENAL M U C O S A . A R C H . B I O C H E M . B I O P H Y S . 1 2 6 : 9 5 7 . C O R R A D I N O , R . A . & W A S S E R M A N , R . H . ( 1 9 7 1 ) V I T A M I N D 3 : I N D U C T I O N OF C A L C I U M B I N D I N G P R O T E I N IN E M B R Y O N I C C H I C K I N T E S T I N E IN V I T R O . S C I E N C E 1 7 2 : 7 3 1 . D A L E , H .H . ( 1 9 0 6 ) ON SOME P H Y S I O L O G I C A L A C T I O N S OF E R G O T . J . P H Y S I O L . 3 4 : 1 6 3 . D E L U C A , H . F . ( 1 9 7 1 ) T H E R O L E OF V I T A M I N D AND I TS R E L A -T I O N S H I P TO PTH AND CT. R E C . P R O G . H O R M . R E S . 2 7 : 4 7 9 . D 1 A M A N T S T E i N , T. ( 1 9 6 6 ) USER D I E L O K A L E R O L L E OER CAR-B O A N H Y D R A T A S E IN H I N B L I C K A U F D I E E I S C H A L E N N E R K A L K U N G . A R C H I V . G E F L U G E L K . 3 0 : 3 1 0 . D l A M A N T S T E I N , T . & S C H L U N S , J . ( 1 9 6 4 ) L O K A L I S A T I ON UNO B E O E U T U N G OER K A R 8 0 A N H Y D R A S E IM U T E R U S VON L E G E H E N N E N . A CTA H I S T O C H E M . 3 D . 1 9 : 2 9 6 . D O L L E R Y , C . T . , P A T E R S O N , J . W . & C O N O L L Y , M . E . ( 1 9 6 9 ) C L I N I C A L P H A R M A C O L O G Y OF B E T A - R E C E P T O R - B L O C K I N G D R U G S . - 8 2 -C L i N . P H A R M A C O L . T H E R . 1 0 : 7 6 5 . D O W D L E , E . 8 . , S C H A C H T E R , D . & S C H E N K E R , H. ( 1960 ) R E Q U I R E -MENT FOR V I T A M I N 0 FOR A C T I V E T R A N S P O R T OF C A L C I U M BY T H E I N T E S T I N E . A M . J . P H Y S I O L . 198 :269 . D U N L O P , D . & S H A N K S , R.G. (1968) S E L E C T I V E B L O C K A D E OF A D R E N O C E P T I V E B E T A R E C E P T O R S IN T H E H E A R T . B R I T . J . P H A R M A C O L . 3 2 : 2 0 1 . E H R E N S P E C K , G . , S C H R A E R , H. & S C H R A E R , R . ( 1 9 6 7 ) SOME M E T A B O L I C A S P E C T S OF C A L C I U M MOVEMENT A C R O S S THE I S O -L A T E D A V I A N S H E L L G L A N D . P R 0 C . S O C . E X P T L . B I 0 L . M E 0 . 1 2 6 : 5 9 2 . E H R E N S P E C K , G . , S C H R A E R , H . & S C H R A E R , R . ( 1971 ) C A L C I U M T R A N S F E R A C R O S S I S O L A T E D A V I A N S H E L L G L A N D . A M . J . P H Y S I O L . 2 2 0 : 9 6 7 . E L J A C K , M .H . & L A K E , P . L . ( 1967 ) T H E C O N T E N T OF T H E P R I N C I P A L I N O R G A N I C IONS AND CARBON D I O X I D E IN U T E R I N E F L U I D S OF T H E D O M E S T I C F O W L . J . R E P R 0 D . F E R T . 15:127» F X N G E, R. , S C H M I D T - N l E L S O N , K. & R O B I N S O N , M. (1958 ) C O N T R O L OF S E C R E T I O N FROM T H E A V I A N S A L T G L A N D . A M . J . P H Y S I O L . 1 9 5 : 5 2 1 . F I T Z G E R A L D , J . D . ( 1969 ) P E R S P E C T I V E IN A D R E N E R G I C B E T A R E C E P T O R B L O C K A D E . C L I N . P H A R M A C O L . T H E R A P . 1 0 : 2 9 2 . F R A N K , F.R. & B U R G E R , R . E . ( 1 9 6 5 ) T H E E F F E C T OF CARBON D I O X I D E I N H A L A T I O N AND SOD IUM B I C A R B O N A T E I N G E S T I O N ON E G G S H E L L D E P O S I T I O N . P O U L T R Y S C I . 4 4 : 1 6 0 4 . F R A P S , R . M . (1955) EGG P R O D U C T I O N AND F E R T I L I T Y IN P O U L T R Y , IN P R O G R E S S IN THE P H Y S I O L O G Y OF FARM A N I M A L S - 8 3 -( E O . HAMMOND, J . ) B U T T E R W O R T H S S C I E N T I F I C P U B L I C A -T I O N S , LONDON , V O L . 2, P. 6 6 1 . F R A P S , R . M . (1970) P H 0 T 0 R E G U L A T I ON IN T H E O V U L A T I O N C Y C L E OF T H E D O M E S T I C FOWL . IN LA P H O T O R E G U L A T I ON DE LA  R E P R O D U C T I O N C H E Z L E S O I S E A U X E T L E S MAMMI F E R E S , C O L L O Q U E I N T E R N A T I O N A U X OU C . N . R . S . N O . 172 ( M O N T P E L L I E R , F R A N C E 1 9 6 7 ) . F R E E D M A N , S . L . & S T U R K I E , P . D . ( 1 9 6 3 A ) E X T R I N S I C N E R V E S OF T H E C H I C K E N ' S U T E R U S ( S H E L L G L A N D ) . A N A T . R E C . 1 4 7 : 4 3 1 . F R E E D M A N , S . L . & S T U R K I E , P . D . ( 1 9 6 3 B ) BLOOD V E S S E L S OF T H E C H I C K E N ' S U T E R U S ( S H E L L G L A N D ) . A M E R . J . A N A T . 1 1 3 ' 1 » G l a s s , O .S . (1926) T HE E F F E C T S OF A T R O P I N E , P H Y S O S T I G M I N E AND P I L O C A R P I N E ON T H E C A R D I A C V A G U S OF T H E F O W L . J . P A R M A C O L . 2 7 : 3 1 9 . G I L B E R T , A . B . (1967) F O R M A T I O N OF T H E E G G IN T H E D O M E S T I C C H . I C K E N . IN A O V A N C E S IN R E P R O D U C T I V E P H Y S I O L O G Y , VOL . 2 ( E D . A . M C L A R E N ) L OGOS P R E S S L O N D O N . G I L B E R T , A . B . & L A K E , P . E . (1963) T E R M I N A L I N N E R V A T I O N O F T H E U T E R U S AND V A G I N A OF T H E D O M E S T I C H E N . J . R E P R O D . F E R T . jj : 4 1 . G OODMAN, L . S . & G I L M A N , A . ( 1 9 6 5 ) T HE P H A R M A C O L O G I C A L 3 A S I S OF T H E R A P E U T I C S . M A C M I L L A N N . Y . G U T O W S K I , M . S . <?: M I T C H E L L , C . A . ( 1 9 4 5 ) C A R B O N I C A N H Y D R A S E IN THE C A L C I F I C A T I O N OF T H E E G G S H E L L . P O U L T R Y S c I . 2 4 : 1 5 9 . H A L L , K . N . & H E L B A C K A , N . V . ( 1 9 5 9 ) I MPROV ING A L B U M E N Q U A L I T Y . P O U L T R Y S c I . 3 8 : 1 1 1 . -84-H A R V E Y , 3 . C . , C O P E N , E . G . , E S K E L S O N , D . W . , G R A F F , S . R . , P O U L S E N , L . O . <fc R A S M U S S E N , D . L . (1954) AU T O N O M I C P H A R M A C O L O G Y OF T H E C H I C K E N WITH P A R T I C U L A R R E F E R E N C E TO A D R E N E R G I C B L O C K A D E . J . P H A R M . E X P . T H E R A . I J 2 : 8 . H U R W I T Z , S . (1964) BONE C O M P O S I T I O N AND 45CA R E T E N T I O N IN FOWL AS I N F L U E N C E D BY EGG F O R M A T I O N . A M . J . P H Y S I O L . 2 0 6 : 1 9 8 . H U R W I T Z , S . ( 1 9 6 5 ) C A L C I U M T U R N O V E R IN D I F F E R E N T BONE S E G M E N T S OF L A Y I N G FOWL . A M . J . P H Y S I O L . 2 0 8 : 2 0 5 » H U R W I T Z , S . (1968) C A L C I U M E X C H A N G E IN P L A S M A OF T H E FOWL . BiO C HiM .BiO P H Y S .AC T A . 156:589. H U R W I T Z , S . ( 1 9 7 0 ) TH E R O L E OF THE I N T E S T I N E IN C A L C I U M H O M E O S T A S I S IN T H E L A Y I N G H E N . A N N . B I O L . A N 1 M . 3 1 O C H . Bi O P H Y S . K ) , N ° H O R S - S E R I E 2, 69. H U R W I T Z , S . & B AR, A. (1968) A C T I V I T Y , C O N C E N T R A T I O N , AND L U M E N - B L O O D E L E C T R O C H E M I C A L P O T E N T I A L D I F F E R E N C E OF C A L C I U M IN T H E I N T E S T I N E OF T H E L A Y I N G H E N . J . N U T R . 95:647. H U R W I T Z , S . & B AR , A. (1969) IN T E S T I N A L C A L C I U M A B S O R P T I O N IN L A Y I N G FOWL ANO ITS I M P O R T A N C E IN C A L C I U M H O M E O S T A S I S . A M E R . J . C L I N . N U T R . 22:391. H U R W I T Z , S . d- G R I M I N G E R , P . ( 1 9 6 1 ) P A R T I T I O N OF C A L C I U M ANO P H O S P H O R O U S E X C R E T I O N IN T H E L A Y I N G H E N . NATURE 189:759. K R A I N T Z , L . & IN T S C H E R , K . (1969) E F F E C T OF C A L C I T O N I N ON T H E D O M E S T I C FOWL . C A N A D . J . PH Y S I 0 L . PH A RM A C OL . 47.8 51 3» L I F S H I T Z , F . , H A R R I S O N , H . C . & H A R R I S O N , H . E . (1969) IN F L U E N C E - 8 5 -OF P A R A T H Y R O I D F U N C T I O N UPON T H E IN V I T R O T R A N S P O R T OF C A L C I U M AND P H O S P H A T E BY THE RAT I N T E S T I N E . E N D O C R I N O L O G Y 8 4 : 9 1 2 . L L O Y D , S. & P I C K F O R O , M . ( 1 9 6 1 ) T HE P E R S I S T A N C E OF A D E P R E S S O R R E S P O N S E TO O X Y T O C I N IN T H E FOWL A F T E R D E N E R V A T I O N ANO B L O C K I N G A G E N T S . B R I T . J . P H A R M A C O L . 1 _ 6 : 1 2 9 . L U R C H E R , K. & H O D G E S , R . D . ( 1 9 6 9 ) SOME P O S S I B L E M E C H A N I S M S OF F O R M A T I O N OF T H E C A R B O N A T E F R A C T I O N OF E G G S H E L L C A L C I U M C A R B O N A T E . C O M P . B I 0 C H E M . P H Y S I O L . 2 8 : 1 1 9 . M O N G I N , P . & L A C A S S A G N E , L . ( 1 9 6 6 A ) R HYTHME R E S P I R A T O I R E E T P H Y S I O L O G I E DE LA F O R M A T I O N DE LA C O Q U I L L E DE L ' O E U F . A N N . BI O L . A N I M. BI O C H . BI O P H Y S . J S : 1 0 1 . M O N G I N , P . & L A C A S S A G N E , L . ( 1 9 6 6 B ) E Q U I L I B R E AC I D O - B A S I Q U E DU S A N G E T F O R M A T I O N DE LA C O Q U I L L E DE L ' O E U F . A N N . BI O L . AN I M . 3I O C H . BI O P H Y S . , 6 : 9 3 . M U E L L E R , C D . & S C O T T , H . M . ( 1 9 4 0 ) T H E P O R O S I T Y OF T H E E G G S H E L L IN R E L A T I O N TO H A T C H A B I L I T Y . P O U L T R Y S c I . 1 9 : 1 6 3 . M U E L L E R , W . J . ( 1 9 6 6 ) E F F E C T OF R A P I D T E M P E R A T U R E C H A N G E S ON A C I D - B A S E B A L A N C E IN S H E L L Q U A L I T Y . P O U L T R Y S C I . 4 5 : 1 1 0 9 . M U E L L E R , W . J . , B R U B A K E R , R . L . & C A P L A N , M . D . ( 1 9 6 9 ) E GG S H E L L F O R M A T I O N AND BONE R E S O R P T I O N IN L A Y I N G H E N S . F E D . P R O C . 2 8 : 1 8 5 1 . M U E L L E R , W . J . , S C H R A E R , R. & S C H R A E R , H. ( 1 9 6 4 ) C A L C I U M M E T A B O L I S M ANO S K E L E T A L O Y N A M I C S OF L A Y I N G P U L L E T S . J . N U T R . 8 4 : 2 0 . - 8 6 -MU R A D , F., BREWER , H . 3 . , J R . & VA U G H A N , M. ( 1 9 7 0 ) E F F E C T OF TCT ON 3 I 5 ' - C Y C I _ I C AMP F O R M A T I O N BY RAT K I D N E Y AND B O N E . P R O C . N A T . A C A D . S c i . 6 5 : 4 4 6 . N I C K E R S O N , M. & HO L L E N B E R G , N.K. ( 1 9 6 7 ) B L O C K A D E OF A L P H A -A D R E N E R G I C R E C E P T O R S . IN PH Y S I O L O G Y AND PH A R M A C O L O G Y , V O L . 4 , T H E N E R V O U S S Y S T E M PA R T D. ( E D S . RO O T , W.S. & HO F M A N N,F.G.) A C A D . P R E S S I N C . , N . Y . , P . 2 4 3 . NO R M A N , A.W., HA U S S L E R , M.R., AD A M S , T.H., M Y R T L E , J . F . , RO B E R T S , P. & H I B B E R D , K.A. ( 1 9 6 9 ) BA S I C S T U D I E S ON THE M E C H A N I S M OF A C T I O N OF V I T A M I N D . A M . J . C L I N . N u T R . 2 2 : 3 9 6 . NO R M A N , A.W., M Y R T L E , J . F . , M I D G E T T , R . J . & NO W I C K I , H.G. ( 1 9 7 1 ) 1 , 2 5 - D I H Y D R O X Y C H O L E C A L C I F E R O L : I D E N T I F I C A T I O N OF T H E P R O P O S E D A C T I V E FORM OF V I T A M I N D3 IN T H E I N T E S T I N E . S C I E N C E 1 7 5 : 5 1 . O ' DO R , R.K., P A R K E S , C O . & C O P P , D.H. ( 1 9 6 9 ) AMINO A C I D C O M P O S I T I O N OF SALMON C A L C I T O N I N . C A N . J . B I 0 C H E M . 4 7 : 8 2 5 . P O L I N , D. & S T U R K I E , P.D. ( 1 9 5 7 ) T H E I N F L U E N C E OF T H E P A R A T H Y R O I D S ON BLOOD C A L C I U M L E V E L S AND S H E L L D E P O S I T I O N IN L A Y I N G H E N S . E N D O C R I N O L O G Y 6 0 : 7 7 8 . P O S T E R N A K , T . H . , S U T H E R L A N D , E . W. & H E N I O N , W.F. ( 1 9 6 2 ) D E R I V A T I V E S OF C Y C L I C 5 ' 5 ' - A D E N O S I N E M O N O P H O S P H A T E . 3 I O C H I M . 3 I O P H Y S . A C T A . 6 5 : 5 5 8 . P O T T S , J . T . J R . , A U R B A C H , G.D. & SHERWOOD, L.M. ( 1 9 6 6 ) PA R A T H Y R O I D H O R M O N E : C H E M I C A L P R O P E R T I E S AND S T R U C T U R A L R E Q U I R E M E N T S FOR B I O L O G I C A L AND I M M U N O L O G I C A L A C T I V I T Y . R E C . P R O G . H O R M . R E S . 2 2 : 1 0 1 . - 8 7 -P O W E L L , C . E . & S L A T E R , I . H . ( 1 9 5 8 ) B L O C K I N G OF I N H I B I T O R Y R E C E P T O R S BY A D I C H L O R O A N A L O G OF I S O P R O T E R E N O L . J . P H A R -M A C O L . & E X P E R•THE R A P . 1 2 2 1 4 8 0 . R A S M U S S E N , H . & T E N N E N H O U S E , A . ( 1 9 6 8 ) C Y C L I C A D E N O S I N E M O N O P H O S P H A T E , C A + + , AND M E M B R A N E S . P R O C . N A T . A C A D . S C I . 5 9 : 1 3 6 4 . R O B E R T S O N , J . S . ( 1 9 5 7 ) T HE THEORY AND USE OF T R A C E R S IN D E T E R M I N I N G T R A N S F E R R A T E S IN B I O L O G I C A L S Y S T E M S . P H Y S I O L . R E V . 3 7 * 1 3 3 . R O B I S O N , G . A . , BU T C H E R , R . W . & S U T H E R L A N D , E . W . ( 1 9 6 7 ) A DENYL C Y C L A S E AS AN A D R E N E R G I C R E C E P T O R . A N N . N . Y . A C A D . S C I . 1 3 9 : 7 0 5 . R O B I S O N , G . A . , BU T C H E R , R . W . & S U T H E R L A N D , E . W . ( 1968 ) C Y C L I C AMP. A N N . R E V . 3 I O C H E M . 3 7 : 1 4 9 . R O B I S O N , G . A . , B U T C H E R , R . W . & S U T H E R L A N D , E . W . ( 1 9 7 1 ) C Y C L I C AMP. A C A D . P RESS , N . Y . S C H A C H T E R , D . , K I M B E R G , D . V . & S C H E N K E R , H. ( 1 9 6 1 ) A C T I V E T R A N S P O R T OF C A L C I U M BY I N T E S T I N E : A C T I O N AND B I O -A S S A Y OF V I T A M I N D . A M . J . P H Y S 1 O L . 2 0 0 : 1 2 6 3 . S C H M 1 D T - N I E L S O N , K. ( i 9 6 0 ) T H E S A L T S E C R E T I N G G L A N O OF M A R I N E B I R D S . C I R C U L A T I O N 2 1 : 9 5 5 » S I M K I S S , K. ( 1 9 6 1 ) C A L C I U M M E T A B O L I S M AND A V I A N R E P R O D U C T I O N . B I O L . R E V . 3 6 : 3 2 1 . S I M K I S S , K. ( 1 9 6 8 ) T H E S T R U C T U R E AND F O R M A T I O N OF T H E S H E L L AND S H E L L M E M B R A N E S . IN E G G Q U A L I T Y ( E D . C A R T E R , T . C ) O L I V E R & BO Y D , E D I N B U R G H , P. 3 . S P E E R S , G . M . , P E R E Y , D . Y . E . & BROWN, D . M . ( 1 9 7 0 ) E F F E C T OF - 8 8 -U L T I M O B R A N C H I A L E C T O M Y IN T H E L A Y I N G H E N . E N D O C R I N O L -OGY 8 7 : 1 2 9 2 . S T E E L , R . G . D . & T O R R I E , J . H . ( 1 9 6 0 ) P R I N C I P L E S AND P R O -C E D U R E S OF S T A T I S T I C S . MCGRAW-H I L L , N . Y . S T U R K I E , P . D . ( 1 9 6 5 ) A V I A N P H Y S I O L O G Y . C O R N E L L U N I V E R S I T Y P RESS , N . Y . T A Y L O R , A . N . & W A S S E R M A N , R . H . ( 1 9 6 7 ) V I T A M I N D ^ - I N D U C E D C A L C I U M - B I N D I N G P R O T E I N : P A R T I A L P U R I F I C A T I O N , E L E C -TR0PH0RE T I C V I S U A L I Z A T I O N , AND T I S S U E D I S T R I B U T I O N . A R C H . S i O C H E M . 3 I O P H Y S . 1 1 9 : 5 3 6 . T A Y L O R , T . G . ( 1 9 6 1 ) C A L C I U M A B S O R P T I O N AND M E T A B O L I S M IN T H E L A Y I N G H E N . IN N U T R I T I O N OF PlGS AND P O U L T R Y ( E D S . M O R G A N , J . T . & L E W I S , D . ) B U T T E R W O R T H S , L O N O O N , P. 1 4 8 - 1 5 7 . T A Y L O R , T . G . ( 1 9 6 5 ) C A L C I U M - E N D O C R I N E R E L A T I O N S H I P S IN T H E L A Y I N G H E N . P R O C . N U T R . S O C . 2 4 : 4 9 » T A Y L O R , T . G . ( 1 9 7 0 A ) HOW AN E G G S H E L L IS M A D E . S C I . A M . 2 2 2 : 8 8 . T A Y L O R , T . G . ( 1 9 7 0 s ) T HE R O L E OF THE S K E L E T O N IN EGG S H E L L F O R M A T I O N . AN N . B I OL . A N I M . 8 I 0 C H . B I 0 P H Y S . J_0, N ° HORS-S E R I E 2 , 8 3 - 9 1 . T A Y L O R , T . G . & B E L A N G E R , L . F . ( 1 9 6 9 ) T HE M E C H A N I S M OF BONE R E S O R P T I O N IN L A Y I N G H E N S . C A L C . T I S S . R E S . 4 : 1 6 2 . T A Y L O R , T . G . & H E R T E L E N D Y , F . ( 1 9 6 1 ) C H A N G E S IN BLOOD C A L C I U M A S S O C I A T E D WITH E G G S H E L L C A L C I F I C A T I O N IN T H E D O M E S T I C F O W L . I I . C H A N G E S IN T H E D I F F U S I B L E C A L C I U M . P O U L T R Y S C I . 4 0 : 1 1 5 . - 8 9 -T A Y L O R , T . G . & K I R K L E Y , J . ( 1 9 6 7 ) T H E A B S O R P T I O N AND E X C R E T I O N OF M I N E R A L S BY L A Y I N G HENS IN R E L A T I O N TO EGG S H E L L F O R M A T I O N . B R I T . P O U L T R Y S c I . 8 : 2 8 9 . T U R K , J . L . & M C G I N N I S , J . ( 1 9 6 4 ) I N F L U E N C E OF V I T A M I N D ON V A R I O U S A S P E C T S OF T H E R E P R O D U C T I V E P R O C E S S IN M A T U R E H E N S . P O U L T R Y S C I . 4 3 : 5 5 9 . U R I S T , M . R . ( 1 9 5 9 ) THE E F F E C T OF C A L C I U M D E P R I V A T I O N UPON T H E B L O O D , A D R E N A L C O R T E X , OVARY AND S K E L E T O N IN D O M E S T I C F O W L . R E C . P R O G . H O R M . R E S . 1 5 : 4 5 5 . U R I S T , M . R . ( 1 9 6 7 ) A V I A N P A R A T H Y R O I D P H Y S I O L O G Y : I N C L U D I N G A S P E C I A L COMMENT ON C A L C I T O N I N . A M E R . Z O O L . 7 : 8 8 3 . U R I S T , M . R . , D E U T S C H , N . M . , P O M E R A N T Z , G . & M C L E A N , F . C . ( 1 9 6 0 ) I N T E R R E L A T I O N S BETWEEN A C T I O N S OF P A R A T H Y R O I D HORMONE AND E S T R O G E N S ON BLOOD AND BONE IN A V I A N S P E C I E S . A M . J . P H Y S I O L . 1 9 9 : 8 5 1 . W A R R E N , D . C . & S C O T T , H . M . ( 1 9 3 5 ) THE BONE F A C T O R IN E G G P R O D U C T I O N . P O U L T R Y S C I . 1 4 : 1 9 5 . W A R R E N , D . C . & S C O T T , H . M . ( 1 9 3 6 ) I N F L U E N C E OF L I G H T ON O V U L A T I O N IN T H E FOWL . J . E X P . Z O O L . 7 4 : 1 3 7 . W A S S E R M A N , R . H . & K A L L F E L Z , F . A . ( 1 9 6 2 ) V I T A M I N D3 AND U N I D I R E C T I O N A L C A L C I U M F L U X E S A C R O S S T H E R A C H I T I C C H I C K D U O D E N U M . A M . J . P H Y S I O L . 2 0 3 : 2 2 1 - 2 2 4 . W A S S E R M A N , R . H . & T A Y L O R , A . N . ( 1 9 6 6 ) V I T A M I N D3-IN0UCEO C A L C I U M — B I N D I N G P R O T E I N IN C H I C K I N T E S T I N A L M U C O S A . S C I E N C E 1 5 2 : 7 9 1 . W A S S E R M A N , R . H . 6: T A Y L O R , A . N . ( 1 9 6 8 ) V I T A M I N D - D E P E N D E N T C A L C I U M B I N D I N G P R O T E I N R E S P O N S E TO SOME P H Y S I O L O G I C A L -90-AND N U T R I T I O N A L V A R I A B L E S . J . 3 I 0 L . C H E M . 2 4 3 ' 3 9 8 7 . W A S S E R M A N , R . H . « T A Y L O R , A . N . ( 1 9 6 9 ) SOME A S P E C T S OF T H E I N T E S T I N A L A B S O R P T I O N OF C A L C I U M WITH S P E C I A L R E F E R E N C E TO V I T A M I N D . IN M I N E R A L M E T A B O L I S M , AN  A D V A N C E D T R E A T I S E , V O L . 3 ( E D S . C O M A R , C L . & 3 R 0 N N E R , F . ) A C A D . P R E S S , N . Y . , p . 3 2 1 . W A S S E R M A N , R . H . , T A Y L O R , A . N . & K A L L F E L Z , F . A . ( 1 9 6 6 ) V I T A M I N D AND T R A N S F E R OF P L A S M A C A L C I U M TO I N T E S T I N A L LUMEN IN C H I C K S ANO R A T S . A M . J . P H Y S I 0 L . 211 : 4 1 9 . W I N G E T , C M . , S M I T H , A . H . <?: H O O V E R , G . N . ( 1 9 5 8 ) A R T E R I O -V E N O U S D I F F E R E N C E S IN P L A S M A C A L C I U M C O N C E N T R A T I O N IN T H E S H E L L G L A N O OF T H E L A Y I N G HEN O U R I N G S H E L L F O R M A T I O N . P O U L T R Y S C I . 3 7 : 1 3 2 5 . W I T T E R M A N , E . R . , C H E R I A N , A . G . & R A D D E , I . C ( 1 9 6 9 ) C A L -C I T O N I N C O N T E N T OF U L T I M 0 B R A N C H I AL BODY T I S S U E IN C H I C K S , P U L L E T S AND L A Y I N G H E N S . C A N . J . P H Y S I O L . P H A R M A C O L . 4 7 : 1 7 5 . A P P E N D I X » A D E S C R I P T I O N OF HOW A UNIFORM P O P U L A T I O N OF HENS WAS OBTAINED WHOSE EGGS WERE IN THE L I N E A R PORTION OF EGG S H E L L C A L C I F I C A T I O N S T U O I E S ON E G G S H E L L C A L C I F I C A T I O N R E Q U I R E O A UN IFORM P O P U L A T I O N OF B I R O S IN THE SAME S T A G E OF T H E C A L C I F I C A T I O N P R O C E S S . B U R M E S T E R EJ_ AJ. ( 1 9 3 9 ) ANO B R A O F I E L O ( 1 9 5 1 ) HAVE SHOWN THAT E G G S H E L L C A L C I F I C A T I O N S T A R T S A P P R O X I M A T E L Y F I V E HOURS A F T E R T H E EGG E N T E R S T H E S H E L L G L A N D AND P R O C E E D S AT A C O N S T A N T R A T E U N T I L J U S T B E F O R E T H E EGG IS L A I D SOME 1 5 HOURS L A T E R . T H E R E F O R E B I R D S T A K E N SOMEWHERE IN T H E M I D D L E OF T H I S 1 5 HOUR P E R I O D WOULD C O M P R I S E A GOOD E X P E R I M E N T A L P O P U L A T I O N . UNDER ORD INARY P H O T O P E R I O D S , I . E . L I G H T S 12 OR 14 HOURS OUT OF T H E 2 4 , T H E D O M E S T I C HEN L A Y S HER E G G S ON TWO OR MORE S U C C E S S I V E D A Y S , F A I L S TO LAY ON ONE D A Y , AND THEN A P P R O X I M A T E L Y R E P E A T S T H E P A T T E R N . T H E E G G S L A I D ON C O N S E C U T I V E DAYS C O N -S T I T U T E A S E Q U E N C E OR C L U T C H . T H E F I R S T E G G OF THE S E Q U E N C E IS L A I D D U R I N G THE E A R L Y OR M IDMORN ING H O U R S , S U B S E Q U E N T E G G S AT L A T E R HOURS ON S U C C E S S I V E DAYS U N T I L A S E Q U E N C E IS C O M -P L E T E D WITH LAY OF T H E T E R M I N A L EGG D U R I N G E A R L Y TO L A T E A F T E R N O O N H O U R S , F R A P S ( 1 9 5 5 ) . S L N C E EGG S H E L L C A L C I F I C A T I O N T A K E S P L A C E IN T H E L A S T 1 5 HOURS B E F O R E L A Y , OR O V I P O S I T I O N , MOST OF T H I S OCCURS AT N I G H T . IT A P P E A R E D A D V A N T A G E O U S TO C H A N G E T H E L A Y I N G P A T T E R N OF T H E E X P E R I M E N T A L B I R O S SO THAT E X P E R I M E N T S COULO BE C O N D U C T E D D U R I N G A NORMAL WORKING D A Y . NUMEROUS WORKERS HAVE SHOWN T H A T L I G H T P L A Y S A L A R G E R O L E IN THE C O N T R O L OF O V U L A T I O N IN H E N S , S E E F R A P S ( 1 9 7 0 ) FOR R E F E R E N C E S . WARREN & S C O T T ( 1 9 3 6 ) - 9 1 -F I G U R E 1 8 . D IAGRAM OF T H E C I R C U I T U S E D TO MON ITOR T H E T I M E OF O V I P O S I T I O N IN T H E E X P E R I M E N T A L H E N S . ONCE T H E EGG WAS L A I D IT R O L L E D INTO T H E TROUGH O U T S I O E T H E C A G E AND S T O P P E D L I G H T FROM T H E L A M P FROM H I T T I N G T H E LDR, T H U S S H U T T I N G O F F T H E C L O C K AT T H E T I M E OF L A Y . E X P L A N A T I O N OF S Y M B O L S . L AMP - No. 2 2 2 P R E F O C U S S E D L A M P . LDR - P H I L L I P S 3 8 7 3 / 0 3 0 2 L I G H T D E P E N D E N T R E S I S T O R . O P . A M P . - 1741 M OTOROLA I N T E G R A T E D C I R C U I T M O D U L E . E . F . - E M I T T O R F OLLOWER NPN T R A N S I S T O R MPS 8 3 4 . T R I A C - G ENERAL E L E C T R I C S C 4 0 8 (A/C S W I T C H ) . C LOCK - INGRAHAM 1 1 5 V , 2W, 6 0 C WALL E L E C T R I C C L O C K . T1 - HAMMONO T R A N S F O R M E R 2 6 2 E 6 . T2 - HAMMOND T R A N S F O R M E R 1 6 6 G 2 5 . D 1 - D 4 - MOTOROLA R E C T I F I E R D I O D E S I N 4 0 0 1 . Z 1 - Z 2 - Z ENER D I O D E S M OTOROLA MZ 1 0 0 0 - 1 7 . - 9 2 -S U C C E E D E O IN C O M P L E T E L Y R E V E R S I N G T H E L A Y I N G C Y C L E O F A GROUP OF HENS BY R E V E R S I N G T H E I R L I G H T S C H E O U L E . T H E B I R O S TOOK A B O U T 60 HOURS TO A D A P T TO A S U D D E N C H A N G E IN T H E I R L I G H T I N G R E G I M E , FROM L I G H T D U R I N G T H E DAY TO L I G H T D U R I N G T H E N I G H T . A F T E R T H I S T H E B I R D S ONLY L A I D D U R I N G T H E I R NEW L I G H T H O U R S , I . E . AT N I G H T . T H E C L U T C H E S OF E G G S L A I D SHOWEO T H E SAME T I M I N G P A T T E R N S AS T H E Y HAD D U R I N G NORMAL D A Y L I G H T H O U R S . HENS USED IN T H E E X P E R I M E N T S R E P O R T E D HERE HAD T H E I R L A Y I N G C Y C L E S R E V E R S E D IN A S I M I L A R M A N N E R . T H E Y WERE S U B -J E C T E D TO TWO S U C C E S S I V E 1 2 HOUR DARK P E R I O D S S E P A R A T E D BY A 30 M I N U T E L I G H T P E R I O O TO ALLOW F E E D I N G . UNDER T H E I R NEW L I G H T S C H E D U L E T H E L I G H T S WERE ON FROM 7 : 3 0 P M TO 9 * 3 0 AM. T H E H E N S ' OVI POSI TI ON P A T T E R N S WERE M O N I T O R E D BY U S I N G A L I G H T D E P E N D E N T R E S I S T O R (LDR ) TO G A T E A C L O C K AS SHOWN IN F I G U R E 1 8 . A L A M P WAS MOUNTED AT ONE END OF THE TROUGH INTO WHICH T H E E G G R O L L E D ONCE IT WAS L A I D , AND THE L I G H T D E P E N D E N T R E S I S T O R WAS AT T H E OTHER ENO OF T H E T R O U G H . T H I S M E A N T THAT T H E P R E S E N C E OF AN E G G IN T H E TROUGH S T O P P E D THE C L O C K . A SUMMARY OF T H E H E N S 1 L A Y I N G P A T T E R N S IS SHOWN IN T A B L E X V I . HENS WITH C L U T C H L E N G T H S FROM 2 - 5 E G G S WERE U S E D IN T H E C A L C I F I C A T I O N E X P E R I M E N T S . T H E Y WERE A N E S T H A T I ZED AT 1 1 : 0 0 A M -1 2 : O ONOON ON T H E DAY F O L L O W I N G T H E O V I P O S I T I O N OF T H E F I R S T E G G IN T H E C L U T C H AND T H E 45CA WAS I N J E C T E D B E T W E E N 1 - 2 P M . T H E R E F O R E T H E E G G S H E L L C A L C I F I C A T I O N S T U D I E S WERE C O N D U C T E D ON T H E S E C O N D E G G IN T H E C L U T C H 7 - 1 2 HOURS B E F O R E IT WOULD HAVE B E E N S P O N T A N E O U S L Y L A I D , I . E . IN THE M I D D L E OF T H E TABLE XVI SUMMARY OF THE T I M I N G OF OV I P O S I T I O N BY HENS ON A R E V E R S E D L I G H T SC H E D U L E ( L I G H T FROM 7*30PM TO 9*30A M ) NUMBER OF EGGS IN CY C L E EGG NUMBER ( T I M E OF L A Y ) TO T A L NUMBER OF CY C L E S NUMBER OF HENS 1 2 3 4 5 1 10PM- 4AM 1 5 5 2 9PM- 1AM 2AM-5AM 12 11 3 9PM-11PM 11PM-3AM 3AM - 6AM 1 8 1 3 4 7PM-1OPM 1OPM-1AM 12AM-3AM 3AM-7AM 7 5 5 7PM- 9PM 9PM-1AM 1OPM-1AM 12AM-3AM 4AM-7AM 5 2 -93-L I N E AR C A L C I F I C A T I O N P E R I O D . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0101928/manifest

Comment

Related Items