STUDIES OH THE CAUSE AHD INCIDENCE OF BLOOD SPOTS IH CHICKEN ECUS By PH IL IP GLENN STILES AN ABSTRACT S u b m itte d t o t h e S c h o o l f o r A d v an ced G ra d u a te S t u d i e s o f M ic h ig a n S t a t e U n i v e r s i t y o f A g r i c u l t u r e an d A p p lie d S c ie n c e i n p a r t i a l f u l f i l l m e n t o f th e re q u ire m e n ts f o r th e d e g re e o f DOCTOR OF PHILOSOPHY D e p a rtm e n t o f P o u l t r y S c ie n c e 1958 X ?■s PH ILIP GLENN STILES ABSTRACT The p r e s e n c e o f a "blood e g g to o r m e at s p o t i n an e g g c a u s e s t h a t be c l a s s e d a s C q u a l i t y i f i t i s l e s s t h a n o n e - e i g h t h o f an i n c h i n d ia m e te r a n d a s i n e d i b l e i f i t i s o v e r o n e - e i g h t h o f an in c h i n d ia m e te r and i s , th e re fo re , an eco n o m ic l o s s . T h is s t u d y a p p r o a c h e d t h e p ro b le m fro m a p h y s i o l o g i c a l a n d e n v i r o n ­ m e n ta l v ie w p o in t by a t t e m p t i n g to a d d e v id e n c e a s to t h e c a u s e a n d i n f l u e n c i n g f a c t o r s i n t h e i n c i d e n c e o f b lo o d s p o t s . A te c h n i q u e w as d e v e lo p e d w h ereb y t h e r e d b lo o d c e l l s o f a l a y i n g h e n w e re l a b e l e d w i th r a d i o a c t i v e p h o s p h o ru s (p 3 2 ) c o u l d be t r a c e d i n t o a b lo o d s p o t . B lo o d s p o t s fo rm e d a f t e r t h e P * ^ l a b e l e d c e l l s w e re i n j e c t e d h a d d e t e c t a b l e r a d i o a c t i v i t y when t h e b lo o d s p o t s c o n t a i n e d m ore th a n 0 .0 2 4 m i l l i l i t e r o f b l o o d . T h i s t e c h n i q u e may be u s e d to d e te r m in e t h e t o t a l b lo o d volum e in a h e n a n d i n a b lo o d s p o t by c o m p a rin g t h e i r c o u n t s p e r m in u te p e r u n i t o f volum e w ith t h e c o u n t s v o lu m e i n j e c t e d . More i m p o r t a n t , h o w e v e r, p e r m in u te o f t h e t h e m e th o d c a n be u s e d to d e te r m in e t h e tim e a b lo o d s p o t fo rm s i n h e n s w ith know n o v u l a t o r y c y c l e s . A h e n i n j e c t e d 9 h o u r s p r i o r to o v u la r- t i o n h a d a r a d i o a c t i v e b lo o d s p o t , w h ile a n o t h e r h e n i n j e c t e d 3 h o u r s a f t e r o v u l a t i o n h a d a n o n - a c t i v e b lo o d s p o t . I n c o m p a r in g n o rm a l a n d b lo o d s p o t e g g s o f a s i m i l a r c l u t c h p o s itio n , it was f o u n d t h a t t h e m ean o v i p o s i t i o n tim e o f e g g s c o n t a i n i n g b lo o d s p o t s o c c u r r e d a p p r o x im a te ly 3 0 to 50 m in u te s PH ILIP GLENN STILES s o o n e r th a n n o rm a l e g g s . A h i g h e r i n c i d e n c e o f b lo o d s p o t s w as f o u n d i n t h e f i r s t e g g o f a c l u t c h t h a n i n e g g s o f s u c c e e d in g c lu tc h p o s itio n s . No c o r r e l a t i o n w as fo u n d b e tw e e n t h e b a r o ­ m e tric p r e s s u r e o r n e t ch an g e s i n p r e s s u r e fo r th e a r e a and th e i n c i d e n c e o f b lo o d s p o t s i n e g g s l a i d 2 4 o r 48 h o u r s l a t e r . The m ean w e ig h t o f e g g s c o n t a i n i n g b lo o d s p o t s w as f o u n d to b e s i g ­ n i f i c a n t l y m ore t h a n t h e m ean w e ig h t o f n o rm a l e g g s i n s i m i l a r c lu tc h p o s itio n s . The a v e r a g e w e ig h t o f t h e b lo o d s p o t e g g s r a n g e d fro m 0 * 7 7 to 1*93 g ra m s h e a v i e r t h a n n o rm a l e g g s . P h y s ic a l d is tu rb a n c e tre a tm e n ts d id n o t s u b s t a n t i a l l y a f f e c t e g g p r o d u c t i o n , b lo o d s p o t i n c i d e n c e o r o t h e r i n t e r i o r q u a lity fa c to rs . S c a r in g b i r d s 3 t im e s a d ay d e c r e a s e d s h e l l t h i c k n e s s by 0 .0 0 0 3 1 o f a n i n c h . tr e a tm e n ts a ls o h a d l i t t l e in c id e n c e . H ig h a n d lo w f r e q u e n c y so u n d e f f e c t on e g g q u a l i t y o r b lo o d s p o t C o n tin u o u s l i g h t a n d s h o r t i n t e r m i t t e n t l i g h t p e r i o d s i n c r e a s e d b lo o d s p o t i n c i d e n c e , alb u m en h e i g h t , e g g w e ig h t a n d H augh s c o r e o v e r t h e n o rm a l 1 4 h o u r s o f l i g h t f o ll o w e d by 10 h o u rs o f d a rk n e ss. STUDIES ON THE CAUSE AND INCIDENCE OP BIOOD SPOTS IN CHICKEN EGGS 37 P H IL IP GLENN STILES A THESIS S u b m i tte d t o t h e S c h o o l f o r A d v an ced G ra d u a te S t u d i e s M ic h ig a n S t a t e U n i v e r s i t y o f A g r i c u l t u r e a n d A p p lie d S c ie n c e i n p a r t i a l f u l f i l l m e n t o f t h e r e q u i r e m e n t s f o r t h e d e g re e o f DOCTOR OP PHILOSOPHY D e p a rtm e n t o f P o u l t r y S c ie n c e ProQuest Number: 10008542 All rights reserved INFO RM ATIO N TO ALL USERS The quality o f this reproduction is dependent upon the quality o f the copy subm itted. In the unlikely event that the author did not send a com plete m anuscript and there are m issing pages, these will be noted. Also, if m aterial had to be removed, a note will indicate the deletion. uest ProQ uest 10008542 Published by ProQ uest LLC (2016). C opyright of the Dissertation is held by the Author. All rights reserved. This w o rk is protected against unauthorized copying under Title 17, United States Code M icroform Edition © ProQ uest LLC. ProQ uest LLC. 789 East Eisenhow er Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 ACKNOWIEDGEMENT The a u t h o r w is h e s t o e x p r e s s h i s s i n c e r e a p p r e c i a t i o n t o Dr* L. E. Dawson f o r h i s a s s i s t a n c e , a d v i c e , e n c o u r a g e m e n t, an d f o r h i s c r i t i c a l r e a d in g o f th e m a n u s c rip t. S i n c e r e t h a n k s a n d a p p r e c i a t i o n a r e a l s o e x te n d e d to D r. L* E. f f o l t e r i n k a n d D r. R. K. R in g e r f o r t h e i r s u g g e s t i o n s a n d a s s i s t a n c e in s e t t i n g up th e p r o j e c t , a n d t o Mr. E dw ard H. F a rm e r, Mr. J o s e p h M acN eil a n d M rs. B e t t y S m ith f o r t h e i r a s s i s t a n c e i n b re a k in g eg g s and re c o rd in g d a ta . I n a d d i t i o n h e w is h e s to t h a n k h i s w if e f o r t h e e n c o u r a g e ­ m e n t, a s s i s t a n c e , s u s t e n a n c e a n d u n d e r s t a n d i n g s h e h a s g iv e n ; to M rs. M a u ric e R i t c h e y f o r h e r t y p i n g o f t h e p u b l i c a t i o n s ; to a ll t h e m em bers o f t h e P o u l t r y S c ie n c e D e p a rtm e n t f o r t h e i r c o n s t a n t p e r s o n a l i n t e r e s t and h e lp ; a n d t o t h e A m e ric a n P o u l t r y a n d H a tc h e r y F e d e r a t i o n , w hose f u n d s h e l p e d make t h i s r e s e a r c h p o s s ib le . TABLE OP CONTENTS P ag e INTRODUCTION............................................................... 1 REVIEW OP LITERATURE........................................................................................ 3 I n c i d e n c e a n d E conom ic l o s s ................................................................ 3 O r i g i n ................................................................................................................... 5 N u t r i t i o n a l E f f e c t s ....................... . ......................................................... 7 G e n e tic E f f e c t s ............................................................................................. 9 P h y s i o l o g i c a l I n f l u e n c e ......................................................................... 11 E n v ir o n m e n ta l I n f l u e n c e .......................................................................... 15 D e t e c t i o n o f B lo o d S p o ts ....................................................................... 18 R a d i o a c t i v e P h o s p h o r u s ................................... . ...................................... 19 F e e d in g ........................ . 19 S u b c u ta n e o u s a n d I n t r a m u s c u l a r I n j e c t i o n s ......................... U p ta k e o f 19 i n t h e E g g ................................................................ 20 E f f e c t o f R a d i a t i o n on B lo o d ................................................. . . T e c h n iq u e s o f L a b e lin g HumanB lo o d C e l l s w ith P EXPERIMENT I . LABELING BIOOD SPOTS IN CHICKEN EGGS WITH RADIOACTIVE PHOSPHORUS.......................... E x p e r im e n ta l P r o c e d u r e ......................................... 22 32 ..22 24 24 R e s u l t s a n d D i s c u s s i o n ............................................................................ 26 EXPERIMENT I I . THE RELATIONSHIP OF OVLPOSITION TIME, CLUTCH POSITION, BAROMETRIC PRESSURE, AND EGG WEIGHT TO THE INCIDENCE OP BIOOD SPOTS IN EGGS........................................... 35 E x p e r im e n ta l P r o c e d u r e ............................................................................ 36 R e s u l t s a n d D i s c u s s i o n ........................... ................................................. 37 TABLE OP CONTENTS CONTINUED P age O v i p o s i t i o n T im e........................................................................................ 37 C lu tc h P o s i t i o n . ........................................................................................ 41 B a r o m e tr ic P r e s s u r e ................................................................................. 4 4 Egg W eight V a r i a t i o n s ............................................................................ 45 EXPERIMENT I I I . THE EFFECT OP PHYSICAL DISTURBANCE, SOUND AND LIGHT ON THE INCIDENCE OP BIOOD AND MEAT SPOTS AND OTHER EGG QUALITY FACTORS....................................................................... 48 E x p e r im e n ta l P r o c e d u r e ................................................................................. 49 R e s u l t s and D i s c u s s i o n ................................................................................. 53 Egg P r o d u c t i o n .................................................................... 53 B lo o d a n d M eat S p o t s .............................................................................. 57 Egg Q q a l i t y ..................... 65 DISCUSSION..................................................................................................................... 77 SUMMARY AND CONCLUSIONS..................................................................................... 81 LITERATURE CITED....................................................................................................... 87 APPENDIX.......................................................................................................................... 93 LIST OF TABLES T ab le P age 1 RADIOACTIVE PHOSPHORUS LOSS RATE IN THE BIOOD OF HEN 2 2 8 ................................................................................................................... 32 2 RADIOACTIVE PHOSPHORUS LOSS RATE IN HEN FECES; FECAL COUNTS FROM HEN 2 2 8 .................................... 33 3 THE AVERAGE TIME BETWEEN EGGS OF A CLUTCH AND BETWEEN CLUTCHES BASED ON MONTHLY AVERAGES................................................. 38 4 THE AVERAGE TIME BETWEEN EGGS OF A CLUTCH AND BETWEEN CLUTCHES BASED ON INDIVIDUAL HEN AVERAGES................................ 4 0 5 THE PERCENTAGE OF BLOOD SPOT EGGS FOUND IN THE DIFFERENT CLUTCH POSITIONS..................................................................... 4 2 6 NORMAL EGGS AND BIOOD SPOT EGGS LAID BEFORE AND AFTER 8 :0 0 A.M.................................................................................................. 43 7 CORRELATION BETWEEN BLOOD SPOTS AND BAROMETRIC PRESSURE................................................................................................................. 45 8 THE EFFECT OF INCREASING AND DECREASING BAROMETRIC PRESSURE ON THE INCIDENCE OF BIOOD SPOTS.................................. 46 A COMPARISON OF EGG WEIGHTS OF NORMAL EGGS AND BLOOD SPOT EGGS............................................................. 46 9 10 THE DISTRIBUTION OF TREATMENTS BY TIME PERIODS........................ 50 11 EGG PRODUCTION PRIOR TO, DURING, AND FOLLOWING THE PHYSICAL DISTURBANCE PHASE..................................................................... 54 12 EGG PRODUCTION DURING AND FOLLOWING THE SOUND PHASE 54 13 EGG PRODUCTION DURING AND FOLIO WING THE LIGHT PHASE 55 14 THE INCIDENCE OF BIOOD AND MEAT SPOTS PRIOR TO, DURING AND FOLLOWING THE PHYSICAL DISTURBANCE PHASE......................... 59 15 ANALYSIS OF VARIANCE FDR PERCENTAGE BLOOD SPOTS DURING TIME PERIOD 1 . ............................................................................................... 60 16 ANALYSIS OF VARIANCE FOR PERCENTAGE BLOOD SPOTS DURING TIME PERIOD 3 .................................................................................................... 60 LIST OF TABLES CONTINUED T a b le P ag e 17 THE INCIDENCE OF BIOOD AND MEAT SPOTS DURING AND FOLLOWING THE SOUND PHASE...................................................................... 62 18 ANALYSIS OF VARIANCE FOR PERCENTAGE BIOOD SPOTS DOPING TIME PERIOD 4 ..................................................................................................... 63 19 THE INCIDENCE OF BIOOD AND MEAT SPOTS DURING AND FOLLOWING THE LIGHT..PHASE...................................................................... 66 20 ANALYSIS OF VARIANCE FOR PERCENTAGE BIOOD SPOTS DURING TIME PERIOD 6 ..................................................................................................... 67 21 AVERAGE EGG QUALITY FACTORS PRIOR TO, DURING AND FOLLOWING THE PHYSICAL DISTURBANCE PHASE................................... 70 22 ANALYSIS OF VARIANCE FOR SHELL THICKNESS DURING TIME PERIOD 2 ................................................................................................................. 71 23 AVERAGE EGG QUALITY FACTORS DURING AND FOLLOWING THE SOUND PHASE......................................................................................................... 72 24 ANALYSIS OF VARIANCE FOR ALBUMEN HEIGHT DURING TIME PERIOD 4 ................................................................................................................. 72 25 ANALYSIS OF VARIANCE FOR ALBUMENHEIGHT DURING TIME PERIOD 5 FOLLOWING SOUND PHASE............................................................ 73 26 ANALYSIS OF VARIANCE FOR HAUGHSCORE DURING TIME PERIOD 5 FOLLOWING SOUND PHASE............................................................ 73 27 AVERAGE EGG QUALITY FACTORS DURING AND FOLLOWING THE LIGHT PHASE......................................................................................................... 75 28 ANALYSIS OF VARIANCE FOR ALBUMEN HEIGHT DURING TIME PERIOD 6 ................................................................................................................. 75 29 ANALYSIS OF VARIANCE FOR EGG WEIGHT DURING TIME PERIOD 6 .................................................................................................................................. 76 30 ANALYSIS OF VARIANCE FOR HAUGH SCORE DURING TIME PERIOD 6 .................................................................................................................................. 76 LIST OF FIGURES F ig u re 1. L o ss o f R a d i o a c t i v e P h o s p h o r u s fro m t h e B lo o d o f a H en..................................................... P ag e 31 2. R a d i o a c t i v e P h o s p h o ru s L o ss R a te i n Hen F e c e s .................. 34 3. Egg P r o d u c t i o n L u r in g E x p e rim e n t I I I ......................................... 56 4. P e r c e n t a g e B lo o d S p o ts D u rin g E x p e rim e n t I I I .........................68 L IST OF APPENDIX TABLES T a b le I II III IV V VI V II V III IX Page BIOOD SPOT - NORMAL EGO COMPARISONS FOR TIME OP LAY; ONE DAY SKIP BETWEEN CLUTCHES............................................ 93 BLOOD SPOT - NORMAL EGG COMPARISONS FOR TIME OP LAY; TWO DAY SKIP BETWEEN CLUTCHES............................................ 94 BIOOD SPOT - NORMAL EGG COMPARISONS FOR TIME OP LAY; TIME BETWEEN THE FIRST AND SECOND EGG OP A CLUTCH................................................................................................................ 95 BIOOD SPOT - NORMAL EGG COMPARISONS FOR TIME OP LAY; TIME BETWEEN THE SECOND AND THIRD EGG OP A CLUTCH................................................................................................................. 96 BIOOD SPOT - NORMAL EGG COMPARISONS FOR TIME OP LAY; TIME BETWEEN THIRD AND FOURTH EGG OPA CLUTCH.. . 97 BIOOD SPOT - NORMAL EGG COMPARISONS FOR TIME OP LAY; TIME BETWEEN ALL OP EGGS WITHIN ACLUTCH................. 98 PRODUCTION OP TOTAL EGGS AND BIOOD SPOT EGGS DURING THE 7 TIME PERIODS OP EXPERIMENT I I I ....................................... 99 ANALYSES OP VARIANCE FDR TOTAL BIOOD SPOTS......................... 100 ANALYSES OP VARIANCE FDR PERCENTAGE BIOOD SPOTS 101 X ANALYSES OP VARIANCE FOR SHELL THICKNESS.............................. 102 XI ANALYSES OP VARIANCE FOR EGG WEIGHT.......................................... 103 X II ANALYSES OP VARIANCE FOR HAUGH SCORE....................................... 1 0 4 INTRO BCJCTION T he p r o b le m o f b lo o d s p o t s i n c h i c k e n e g g s h a s e x i s t e d s i n c e t h e b e g i n n i n g o f c o m m e rc ia l eg g p r o d u c t i o n a n d c u r r e n t l y c a u s e s fro m 2 to 4 p e r c e n t o f t h e e g g s p r o d u c e d to b e c l a s s e d as lo s s . T h is p r o b le m h a s b e e n s t u d i e d fro m many d i f f e r e n t a s p e c t s a n d much i n f o r m a t i o n o n t h e s u b j e c t c a n b e fo u n d i n t h e lite ra tu re ; h o w e v e r, n o n e o f t h e r e s e a r c h w ork h a s b r o u g h t a b o u t t h e e l i m i n a t i o n o f b lo o d s p o t s a n d o n l y a few o f t h e s t u d i e s h a v e shown t h a t t h e i n c i d e n c e c o u l d b e c o n t r o l l e d to a l i m i t e d e x te n t. B lo o d s p o t s i n e g g s a r e n o t a p u b l i c h e a l t h h a z a r d a n d a r e c l a s s i f i e d as in e d ib le o r lo s s p u re ly fo r p s y c h o lo g ic a l re a so n s. A b r i g h t r e d b lo o d s p o t sh o w in g o n a l u s t r o u s y e llo w y o l k d o e s n o t h a v e an a p p e t i z i n g a p p e a r a n c e ; t h e r e f o r e , h o u s e w if e r e f u s e s to e a t i t t h e m odem a n d c o n s e q u e n t l y may n o t make f u r t h e r e g g p u r c h a s e s fro m t h e same s o u r c e . The eco n o m ic l o s s e s due to n o t p u r c h a s i n g e g g s a f t e r f i n d i n g o n e o r m ore b lo o d s p o t s c a n n o t b e a c c u r a t e l y m e a s u re d , b u t a r e u n d o u b te d ly o f m a jo r im p o r ta n c e . I n t h i s s t u d y t h e b lo o d s p o t p r o b le m w as i n v e s t i g a t e d fro m t h e p h y s i o l o g i c a l v ie w p o in t by u t i l i z i n g r a d i o a c t i v e i s o t o p e t r a c e r s w ith o v u l a t o r y c y c l e a n a l y s i s an d a l s o p o in t o f th e in f lu e n c e o f s p e c if ic fro m t h e s t a n d ­ e n v ir o n m e n ta l e l e m e n ts . The f i r s t e x p e r im e n t c o n s i s t e d o f d e v e lo p i n g a p r o c e d u r e f o r l a b e l i n g t h e r e d b lo o d c e l l s i n a l a y i n g h e n i n s u c h a way t h a t b lo o d s p o t s - 1 - - 2 - i n a n e g g fro m t h i s h e n w o u ld show r a d i o a c t i v i t y a m o u n ts . in d e te c ta b le T h u s, by l a b e l i n g t h e b l o o d a t a s p e c i f i c tim e i n t h e o v u l a t o r y c y c l e o n e c o u l d d e te r m in e when a b lo o d s p o t was fo rm e d . A n o th e r o b j e c t i v e , to s t u d i e d i n t h e s e c o n d e x p e r im e n t , w as d e te r m in e i f o v u l a t i o n tim e was d i f f e r e n t f o r e g g s c o n t a i n i n g b l o o d s p o t s t h a n f o r n o rm a l e g g s . I n a d d i t i o n , t h i s e x p e r im e n t w as d e s i g n e d t o d i s c o v e r i f t h e r e w as a r e l a t i o n s h i p b e tw e e n t h e i n c i d e n c e o f b lo o d s p o t s a n d e g g c l u t c h p o s i t i o n , b a r o m e t r i c p re ssu re , e g g w e ig h t, a n d e g g alb u m en h e i g h t . A t h i r d o b j e c t i v e o f t h i s r e s e a r c h w as to i n v e s t i g a t e t h e in flu e n c e o f p h y s ic a l d is tu rb a n c e , sound, and l i g h t v a r i a t i o n s on t h e i n c i d e n c e o f b lo o d s p o t s a n d o t h e r eg g q u a l i t y c h a r a c t e r i s t i c s . E a c h o f t h e e n v ir o n m e n ta l c o n d i t i o n s w e re s t u d i e d s e p a r a t e l y to d e te r m in e t h e i r i n d i v i d u a l e f f e c t s . REVIEW OE LITERATURE I n c i d e n c e e n d Econom ic L o s s The o c c u r r e n c e o f h lo o d s p o t s i n c h i c k e n e g g s h a s b e e n known s in c e e a r ly h is to r y . A c c o rd in g to R o m an o ff a n d R o m a n o ff ( 1 9 4 9 ) , A r i s t o t l e b e l i e v e d b lo o d s p o t s r e s u l t e d fro m p r e m a tu r e y o l k e x ­ p u ls io n . I n 1600 E a b r ic u s th o u g h t t h e p r e s e n c e o f b lo o d s p o t s w as e v id e n c e t h a t e g g y o l k s a r e fo rm e d d i r e c t l y fro m b l o o d . Many c o n c e n t r a t e d s t u d i e s o f b lo o d a n d m eat s p o t s h a v e b e e n made s i n c e 1930. T h e re i s a c o n s i d e r a b l e eco n o m ic l o s s due to b l o o d s p o t s . D awson (1 9 5 3 a n d 1 9 5 5 ) r e p o r t e d a $ 7 0 0 ,0 0 0 a n n u a l l o s s t o M ic h ig a n p r o d u c e r s i n 1953 a n d o v e r o n e m i l l i o n d o l l a r s i n 1955 d u e to r e ­ m o v in g b lo o d s p o t e g g s a f t e r c a n d l i n g . The t o t a l eco n o m ic l o s s i s u n d o u b te d ly much g r e a t e r th a n t h e s e f i g u r e s s i n c e t h e s m a ll b lo o d s p o t s t h a t a r e n o t d e t e c t e d by t h e c a n d l e r s may c a u s e a lo w e r c o n su m e r dem and a n d c o n s e q u e n t l y fe w e r e g g s a r e p u r c h a s e d a n d c o n su m e d . One M ic h ig a n e g g b u y in g s t a t i o n h a d a c a n d l i n g l o s s o f l i p e r c e n t o f a l l i t s e g g s due to b lo o d and m e at s p o t s . e g g s b ro k e n o u t fro m 2 ,4 3 8 Hew H a m p sh ire h e n s , In Dawson a n d R ic h a r d ­ s o n (1 9 5 2 ) fo u n d 6 4 .8 p e r c e n t o f t h e e g g s c o n t a i n e d s p o t s . Of t h e s e 48 p e r c e n t w e re on t h e y o l k , 27 p e r c e n t w e re f r e e f l o a t i n g i n t h e a lb u m e n , a n d 25 p e r c e n t w e re i n t h e c h a l a z a . T w enty p e r ­ c e n t o f t h e e g g s w i t h s p o t s h a d th em i n m ore t h a n o n e p o s i t i o n . - 3 - - 4 - F iv e p e r c e n t o f t h e y o l k s p o t s w e re w h ite w h i le 50 p e r c e n t w e re r e d ; h o w e v e r, i n t h e alb u m en 50 p e r c e n t w e re w h i t e , 2 p e r c e n t w e re re d , in a n d t h e r e m a in d e r w e re a s h a d e o f h ro w n . t h e c h a l a z a w e re h ro w n . M ille r e t a l . M ost o f t h e s p o t s (1 9 5 7 ) r e p o r t e d e g g s s e l e c t e d fro m a g r a d i n g s t a t i o n p r i o r to c a n d l i n g c o n t a i n e d o v e r 3 p e r c e n t b lo o d s p o t s w ith 0 .9 6 p e r c e n t b e i n g o v e r o n e - e i g h t h o f an in c h i n d ia m e te r, lubst b lo o d s p o t s w e re o n e - t h i r t y - s e c o n d o f an i n c h o r l e s s i n d i a m e t e r . U a lb a n d o v a n d C a rd (1 9 4 7 ) r e p o r t e d t h e i n c i d e n c e o f b lo o d s p o t e g g s fro m 10 a d d w e s te r n L e g h o rn f l o c k s a v e r a g e d 1 2 .5 p e r c e n t . O f t h i s 7 . 7 p e r c e n t w e re -under o n e - e i g h t h o f an i n c h i n d i a m e t e r . M eat s p o t s w e re f o u n d i n 1 2 .8 p e r c e n t o f t h e e g g s w i t h 1 0 .2 p e r ­ c e n t b e i n g u n d e r o n e - e i g h t h o f an i n c h i n d i a m e t e r . The sam p le o f e g g s fro m h e a v y b r e e d s c o n t a i n e d 4 1 .7 p e r c e n t b lo o d a n d m eat s p o t s o f w h ic h 1 1 * 4 p e r c e n t w e re o v e r o n e - e i g h t h o f an i n c h i n d ia m e te r. M e r r i t t (1 9 5 0 ) r e p o r t e d 20 p e r c e n t o f t h e t u r k e y e g g s e x a m in e d c o n t a i n e d e i t h e r b lo o d o r m e a t s p o t s , m o s tly w h ite m eat s p o ts . “W hite m eat s p o t s w ere fo u n d i n 31 p e r c e n t o f t h e d u ck e g g s b r o k e n o u t a n d 19 p e r c e n t o f t h e p h e a s a n t e g g s c o n t a i n e d m e a t s p o ts * A n o th e r p o s s i b l e eco n o m ic l o s s may r e s u l t fro m t h e e g g b e i n g m ore s u s c e p t i b l e to b a c t e r i a l s p o i l a g e when i t c o n t a i n s a b lo o d s p o t due t o t h e m ore e a s i l y a s s i m i l a b l e n u t r i e n t m a t e r i a l a v a i l a b l e f o r b a c t e r i a (R o m a n o ff a n d R o m an o ff, 1 9 4 9 ). - 5 - O rig in N a lb a n d o v a n d C a rd (1 9 4 4 ) r e p o r t e d t h a t b l e e d i n g may o c c u r a t o v u l a t i o n a n d c a u s e b lo o d s p o t s , in s ta n c e s . b u t t h i s h a p p e n s o n ly i n r a r e More f r e q u e n t l y b lo o d s p o t s a r e c a u s e d by i n t r a - f o l l i c u l a r b l e e d i n g , w h ic h may h a v e o c c u r r e d s e v e r a l d a y s b e f o r e o v u la tio n . I n o b s e r v a t i o n s o f l a p a r o t o m i z e d h e n s , th e y fo u n d y o l k s r e l e a s e d w ith b o th l a r g e an d s m a l l b lo o d c l o t s a d h e r i n g t o th e m a lth o u g h no h e m o rrh a g e h a d ta k e n p l a c e a t o v u l a t i o n . H e m o rrh a g e s may t a k e p l a c e a n y w h e re i n th e f o l l i c l e . S h e e t-lik e o r n a r r o w , e l o n g a t e d m a s s e s o f b lo o d on t h e y o l k a r e c a u s e d byl a r g e m a s s e s o f b l o o d d i f f u s i n g b e tw e e n t h e y o l k a n d f o l l i c l e . A s e v e r e h e m o rrh a g e c a n c a u s e a w id e s h e e t o f b lo o d to c o v e r m o st o f t h e y o l k s u r f a c e , o r may a c c u m u la te i n t h e h o llo w f o l l i c l e s t a l k b e co m in g c o n i c a l o r g l o b u l a r i n s h a p e an d may e v e n t u a l l y b e fre e f l o a t i n g i n t h e a lb u m e n . S ub-m em branous c l o t s a r e m ost comnx>nly fo u n d a t t a c h e d to t h e v i t e l l i n e m em brane a n d re m a in t h e r e th r o u g h o u t o v u l a t i o n . I b r many y e a r s m eat s p o t s w e re r e p o r t e d a s b e in g d e g e n e r a te b l o o d s p o t s c a u s e d b y n o rm a l c h a n g e s i n pH o r te m p e r a t u r e o r b o th , a n d t h a t t h e p r e s e n c e o f a m eat s p o t i n d i c a t e d t h e b l e e d i n g to o k p l a c e s e v e r a l d a y s b e f o r e o v u l a t i o n , w h ile b lo o d s p o t s w e re c a u s e d b y b l e e d i n g im m e d ia te ly p r i o r to o v u l a t i o n (H a lb a n d o v an d C a rd , 1 9 4 7 ). N a lb a n d o v a n d C a rd , (1 9 4 4 ) fo u n d t h a t a r t i f i c i a l b lo o d c l o t s w e re c h a n g e d i n t o m e a t s p o t s a f t e r 10 d a y s a t 88 d e g r e e s - 6 - F a h r e n h e i t a n d a pH o f 8 .0 to 9 . 2 , E x p o s u re to a i r a n d u n s t e r i l e t e c h n i q u e h a s t e n e d t h e t r a n s f o r m a t i o n o f b lo o d c l o t s to m e at s p o t s . M e r r i t t (1 9 5 0 ) r e p o r t e d h i g h t e m p e r a t u r e s (4 0 d e g r e e s c e n t i ­ g r a d e ) c a u s e d b lo o d c l o t s to becom e l i m i t e r i n c o l o r , w h e re a s re frig e ra tio n t e m p e r a t u r e s (4 d e g r e e s c e n t i g r a d e ) d id n o t. H is w o rk i n d i c a t e d t h a t an y d e g e n e r a t i o n o f b lo o d s p o t s i n t o m e a t s p o t s a f t e r o v i p o s i t i o n w as i n s i g n i f i c a n t . M eat s p o t s w e re much m ore p r e v a l e n t i n e g g s fro m h e a v y b r e e d s t h a n i n e g g s fro m l i g h t b re e d s o f c h ic k e n s . M e r r i t t s t a t e d t h a t i t was c o n c e i v a b l e t h a t a l a r g e p e r c e n t a g e o f t h e w h i te m e at s p o t s r e s u l t e d fro m accum u­ l a t i o n s o f o v i d u c a l e x c r e t i o n s , w h ic h i s i n p a r t i a l a g re e m e n t w ith L u c a s (1 9 4 6 ) b u t i n d is a g r e e m e n t w i t h IT albandov a n d C a rd ( 1 9 4 4 ) , who s t a t e d w h i t e m e a t s p o t s r e s u l t e d fro m b lo o d c l o t d e g e n e r a t i o n . A tte m p ts to in d u c e i n c l u s i o n s i n e g g s th r o u g h o p e r a t i v e an d i n ­ s e r t i o n t e c h n i q u e s i n t h i s s tu d y w ere u n s u c c e s s f u l . B u rm e s te r a n d C a rd (1 9 3 8 ) made a m ic r o s c o p ic e x a m i n a tio n o f m e a t s p o t s w h ic h show ed t h a t d a r k r e d m eat s p o t s c o n s i s t e d o f l a r g e m a s s e s o f r e d b lo o d c o r p u s c l e s s u r r o u n d e d by a t h i n l a y e r o f p ro te in . V ery l i t t l e d e g e n e r a t i o n o f r e d b lo o d c e l l s to o k p l a c e i n t h e d a r k r e d m eat s p o t s . Some i n i t i a l d e g e n e r a t i o n c h a n g e s h a d t a k e n p l a c e i n t h e b ro w n is h c o l o r e d m e at s p o t s . T h e re w as a w id e v a r i a t i o n i n t h e m ic r o s c o p ic s t r u c t u r e o f m eat s p o t s r a n g i n g fro m t h o s e w ith a l a r g e am ount o f n o n - c e l l u l a r p r o t e i n s a n d v e r y few r e d c e l l s to t h o s e sh o w in g no e v id e n c e o f 7 - c e l l s i n any p h a s e o f d e g e n e r a t i o n . A ll h u t 3 p e r c e n t o f t h e m eat s p o t s e x a m in e d h a d e v id e n c e o f h a v in g c o n t a i n e d r e d "blood c e l l s . T h e re w e re n o n o rm a l o r a b n o rm a l c e l l u l a r t i s s u e s o t h e r t h a n b lo o d f o u n d i n an y m eat s p o t e x c e p t f o r a few b a c t e r i a fo u n d i n a s m a ll p e r c e n t a g e o f t h e m e at s p o t s . H e lb a c k a a n d Sw anson (1 9 5 8 ) fo rm e d t h e o p i n i o n t h a t c o l o r e d m e a t s p o t s fo u n d i n w h i t e s h e l l e d e g g s a r e n o r m a lly d e g e n e r a t e d b l o o d , b u t t h o s e f o u n d i n brow n s h e l l e d e g g s a p p e a r e d t o b e o f tw o k i n d s : (1 ) t h o s e h a v in g t h e i r o r i g i n i n d e g e n e r a t e d b lo o d a n d b e in g n o n - f l u o r e s c e n t u n d e r u l t r a - v i o l e t l i g h t ; o r ( 2 ) th o s e c o n t a i n i n g a c o n s i d e r a b l e < ? u a n tity o f c a l c i u m an d b e in g f l u o r e s ­ c e n t b e c a u se o f t h e i r p o rp h y rin c o n te n t. P ro to p o rp h y rin i s th e p ig m e n t i n c o l o r e d m e at s p o t s t h a t g i v e t h e f l u o r e s c e n t p r o p e r t y . T hey c o n c lu d e d t h a t t h e m a j o r i t y o f m eat s p o t s i n s h e l l e g g s do n o t h a v e t h e i r o r i g i n i n d e g e n e r a t e d b lo o d . N u tr itio n a l E ffe c ts T he i n c i d e n c e o f b lo o d s p o t s i s i n f l u e n c e d to some e x t e n t by n u t r i t i o n a l f a c t o r s . N a lb a n d o v a n d C a rd (1 9 4 7 ) r e p o r t e d a l a r g e d e c r e a s e i n t h e num b er o f b lo o d s p o t s i n t h e e g g s fro m h e n s a l l o w e d to r a n g e f o r 3 o r 4 w e e k s. They assu m e d t h a t f r e s h g r e e n g r a s s c o u ld p r e v e n t f o l l i c u l a r h e m o rrh a g e s . th is , I n d is a g r e e m e n t w it h D en to n (1 9 4 7 ) i n d i c a t e d t h a t t h e i n g e s t i o n o f g r e e n g r a s s a n d r a n g e h a d no a p p r e c i a b l e e f f e c t on t h e i n c i d e n c e o f b lo o d a n d - 8 - m eat s p o ts . He a l s o fo u n d t h a t v i t a m i n K h a d no e f f e c t o n t h e i r in c id e n c e . C a r v e r a n d H e n d e rs o n (1 9 4 8 ) r e p o r t e d t h a t t h e a d d i t i o n o f ru tin a n d a s c o r b i c a c i d to t h e r a t i o n d i d n o t r e d u c e t h e i n c i d e n c e o f h lo o d s p o t s . S a u te r, S ta d e lm a n a n d C a r v e r (1 9 5 2 ) fo u n d t h a t h e n s f e d a r a t i o n c o n t a i n i n g 10 p e r c e n t a l f a l f a c o n s i s t e n t l y p r o d u c e d a lo w e r i n c i d e n c e o f b o th l a r g e an d s m a ll b lo o d s p o t s t h a n d i d h e n s f e d a r a t i o n c o n t a i n i n g no a l f a l f a . ra tio n The L e g h o rn s o n t h e a l f a l f a a v e r a g e d 0 . 6 p e r c e n t lo w e r b lo o d s p o t i n c i d e n c e an d Hew Hamp s h i r e s a v e r a g e d 5 .9 p e r c e n t lo w e r i n c i d e n c e . B e a r s e , M cC la ry a n d S a x e n a (1 9 5 3 ) r e p o r t e d t h a t h e n s r e ­ c e i v i n g lo w l e v e l s o f v i t a m i n A (2 0 0 I n t e r n a t i o n a l u n i t s p e r 100 g ra m s f e e d ) show ed a s i g n i f i c a n t l y h i g h e r b lo o d s p o t i n c i d e n c e th a n h e n s f e d h i g h e r v i t a m i n A l e v e l s . B e a rs e (1 9 5 5 ) fo u n d t h a t a d e c r e a s e i n v i t a m i n A b e lo w 2000 I n t e r n a t i o n a l u n i t s p e r p o u n d c a u s e d a n i n c r e a s e i n t h e b lo o d s p o t i n c i d e n c e . L e v e ls o f v ita m in A a b o v e 1700 U n i t e d S t a t e s P h a r m a c o p e ia u n i t s c a u s e d t h e maximum b e n e f i t i n t h i s s tu d y . H ens f e d r a t i o n s c o n t a i n i n g o n ly 908 I n t e r n a t i o n a l u n i t s o f v ita m in A c o n s is t e n t ly l a i d eggs o f a h i g h e r b lo o d s p o t i n c i d e n c e th a n d i d s i m i l a r h e n s f e d 2000 I n t e r ­ n a t i o n a l u n i t s o f v i t a m i n A. V ita m in B^g s u p p le m e n ta t io n a p p e a r e d t o h a v e some e f f e c t i n l o w e r i n g t h e b lo o d s p o t i n c i d e n c e . B e a rs e (1 9 5 8 ) r e p o r t e d t h a t b i r d s w h ic h h a d r e c e i v e d - 9 - a d d i t i o n a l l e v e l s o f v i t a m i n s D, E a n d K s e p a r a t e l y show ed no d i f f e r e n c e i n h l o o d s p o t i n c i d e n c e ; h o w e v e r, t h e a d d i t i o n o f e x c e s s v i t a m i n s A* D, E a n d K t o g e t h e r d e c r e a s e d h lo o d s p o t s s lig h tly . B i r d s f e d 6000 I n t e r n a t i o n a l u n i t s o f v i ta m in A p e r p o n n d h a d fe w e r b lo o d s p o t s th a n d i d b i r d s f e d 3000 I n t e r n a t i o n a l u n i t s o f v i t a m i n A. I n c r e a s i n g t h e e n e r g y l e v e l fro m 1115 to 1450 C a l o r i e s p e r p o u n d r e s u l t e d i n a n i n c r e a s e i n b lo o d s p o t s . G e n e tic E f f e c t s More h a s b e e n a c c o m p lis h e d to w a r d r e d u c i n g t h e i n c i d e n c e o f b lo o d s p o t s by a s y s t e m a t i c g e n e t i c s e l e c t i o n o f b r e e d i n g s t o c k t h a n by a n y o t h e r m e an s. Q a in n a n d G o d fre y (1 9 4 0 ) r e p o r t e d t h e r e w e re s i g n i f i c a n t b r e e d a n d f a m il y d i f f e r e n c e s i n t h e b lo o d s p o t s o c c u r r i n g i n b o th t h e y o l k a n d t h e a lb u m e n . They fo u n d no s i g n i ­ f i c a n t c o r r e l a t i o n b e tw e e n p e r c e n t a g e o f b lo o d s p o t s a n d e g g p ro d u c tio n , e g g w e i g h t , o r b o d y w e ig h t. J e f f r e y (1 9 4 5 ) r e p o r t e d m a rk e d b r e e d d i f f e r e n c e s e x i s t i n g i n t h e p e r c e n t a g e o f e g g s w ith r e d m e at s p o t s . Brown e g g s p r o ­ d u c e d b y h e a v y b r e e d s c o n t a i n e d b e tw e e n 1 2 .6 a n d 2 0 .3 p e r c e n t r e d m e a t s p o t s , w h e re a s S in g l e Comb W h ite L e g h o rn e g g s h a d a s lo w a s z e ro p e r c e n t r e d m eat s p o ts . S i m i l a r b r e e d d i f f e r e n c e s w e re fo u n d f o r p a l e m eat s p o t s . L e r n e r an d T a y lo r (1 9 4 7 ) r e p o r t e d no s i g n i f i c a n t r e d u c t i o n i n b lo o d s p o t i n c i d e n c e when b lo o d s p o t e g g s w e re rem o v ed b e f o r e - 10 - t h e e g g s w e re s e t i n t h e i n c u b a t o r s d u r i n g t h e h a t c h i n g s e a s o n . By s e l e c t i n g f o r a h i g h an d lo w i n c i d e n c e o f b lo o d s p o t s i n a s t r a i n o f B hode I s l a n d B ed s w h ic h h a d an o r i g i n a l a v e r a g e o f 6 1 .9 p e r c e n t b lo o d s p o t s , Q ninn et_ a l . (1 9 4 8 ) d e v e lo p e d two s e p a r a t e s tr a in s a f te r s ix y e a rs o f s e le c tio n . One s t r a i n a v e r a g e d 80 p e r c e n t b lo o d s p o t s a n d t h e o t h e r 20 p e r c e n t . s e a s o n a p p e a re d to h av e l i t t l e E n v iro n m e n t an d i n f l u e n c e o n b lo o d s p o t i n c i d e n c e i n t h i s s tu d y . Q o inn a n d L e e (1 9 4 9 ) c r o s s e d E hode I s l a n d B ed s w ith W h ite L e g h o rn s a n d f o u n d no a p p a r e n t r e l a t i o n s h i p b e tw e e n s h e l l c o l o r a n d t h e p e r c e n t a g e o f b lo o d s p o t s i n e g g s fro m t h e 5^ g e n e r a t i o n o f th is c ro ss. J e f f r e y and W alk er (1 9 5 0 ) r e p o r t e d t h e r e i s an in d e p e n d e n c e i n t h e i n c i d e n c e o f b lo o d s p o t s a n d m e at s p o t s a n d t h a t s e p a r a t e g e n e s p r o b a b ly c o n t r o l t h e i r p r o d u c t i o n . T h e ir e v i d e n c e show ed t h a t d a r k o r m edium brow n e g g s c o n t a i n e d m ore c o l o r e d m eat s p o t s a n d fe w e r w h ite m eat s p o t s th a n l i g h t e r c o l o r e d eg g s. S ta d e lm a n e t a l . (1 9 5 2 ) r e p o r t e d t h e r e w as no r e l a t i o n s h i p b e tw e e n b lo o d s p o t i n c i d e n c e a n d s h e l l c o l o r o r b e tw e e n t h e i n c i ­ d e n c e o f b lo o d a n d m eat s p o t s . T h e re was a s i g n i f i c a n t r e l a t i o n ­ s h i p f o u n d b e tw e e n s h e l l c o l o r a n d t h e c o l o r o f m e a t s p o t s . L e r n e r , T a y lo r a n d Lowry ( 1 9 5 1 ) , by o r d i n a r y c o m m e rc ia l c a n d lin g , d e t e c t e d b lo o d s p o t s i n 0 . 5 to 1 .3 3 p e r c e n t o f t h e e g g s fro m a p r o d u c t i o n b r e d f l o c k o f S i n g l e Comb W h ite L e g h o rn s . T h is - 11 - p e r c e n t a g e i n c r e a s e d to 23 p e r c e n t by s e l e c t i o n f o r a h i g h i n c i ­ dence. Dawson a n d B ic h a r d s o n (1 9 5 2 ) r e p o r t e d L e g h o rn s h a d a lo w e r i n c i d e n c e o f "blood a n d m eat s p o t s t h a n d i d t h e h e a v y b r e e d s o n a b ro k e n o u t b a s i s . A s e a s o n a l v a r i a t i o n and a s t r a i n v a ria tio n w e re r e p o r t e d f o r b o th S in g l e Comb W h ite L e g h o rn s a n d h e a v y b r e e d s . Dawson a t a l . (1 9 5 4 ) fo u n d c o n s i d e r a b l e v a r i a t i o n b e tw e e n s t r a i n s a n d i n d i v i d u a l s f o r a lb u m e n f i r m n e s s , a n d t h e i n c i d e n c e o f b lo o d a n d m eat s p o t s . s h e ll th ic k n e s s , M cC lary a n d B e a r s e (1 9 5 4 ) s e l e c t e d f o r ab o v e a v e r a g e s h e l l t h i c k n e s s among s e v e r a l S i n g l e Comb W hite L e g h o rn s a n d fo u n d t h e y h a d a h i g h e r b lo o d s p o t i n c i d e n c e th a n s t r a i n s n o t s e l e c t e d f o r s h e l l t h i c k n e s s . A p o s i­ t i v e g e n e t i c c o r r e l a t i o n o f 0 . 4 4 w as c a l c u l a t e d f o r e g g s h e l l th ic k n e s s and p e r c e n ta g e o f b lo o d s p o ts . F a r n s w o r th a n d N o rd s k o g (1 9 5 5 ) e s t i m a t e d t h e h e r i t a b i l i t y o f b l o o d s p o t s to b e 0 .3 2 f o r a p p r o x im a te ly 15 e g g s c a n d l e d p e r hen. No s i g n i f i c a n t c o r r e l a t i o n s w e re fo u n d when b lo o d s p o t s w e re c o m p a re d w i t h e g g p r o d u c t i o n , s e x u a l m a t u r i t y , o r e g g w e ig h t. P h y s io lo g ic a l In flu e n c e L u c a s (1 9 4 6 ) made s m e a rs fro m b lo o d s p o t s fro m f r e s h e g g s a n d fo u n d g r e a t v a r i a b i l i t y i n t h e r e l a t i v e c e l l ty p e f r e q u e n c i e s . T he r a n g e o f ly a q o h o c y te s w as fro m 3 t o 60 p e r c e n t , e o s i n o p h i l s fro m 8 to 74 p e r c e n t , b a s o p h i l s fro m 0 to 19 p e r c e n t , p h a g e s fro m 4 to 85 p e r c e n t . a n d m acro­ D i f f e r e n t i a l b lo o d c o u n t s a v e r a g e d - 12 - 3 8 . 3 p e r c e n t ly m p h o c y te s a n d m o n o c y te s , 5 2 .3 p e r c e n t e o s i n o p h i l s ( a l l t y p e s ) a n d 1 0 .5 p e r c e n t b a s o p h i l s . The ly m p h o c y te -m o n o c y te g r o u p a p p e a r e d m o re s u s c e p t i b l e to dam age th a n t h e o t h e r tw o . e v id e n c e i n t h i s The s tu d y i n d i c a t e d t h a t t h e c o n n e c t i v e t i s s u e c e l l s a n d c e l l s o f e x t r a v a s c u l a r o r i g i n w ere c a r r i e d i n t o th e egg. H e lb a c k a a n d Sw anson (1 9 5 8 ) r e p o r t e d t h a t c o l o r e d m eat s p o t s h a v e a f l u o r e s c e n t p r o p e r t y s i m i l a r to t h a t o f e g g s h e l l p ig m e n ts w hen e x p o s e d to u l t r a v i o l e t l i g h t . N e i t h e r b lo o d s p o t s n o r d e ­ g e n e r a t e d b lo o d p o s s e s s t h i s q u a l i t y . S ig n ific a n t c o rre la tio n s w e re fo u n d b e tw e e n e g g s h e l l c o l o r a n d m eat s p o t c o l o r , m eat s p o t c o l o r a n d m eat s p o t f l u o r e s c e n c e , s p o t flu o re s c e n c e . tis s u e a n d e g g s h e l l c o l o r a n d m eat The o v i d u c t s o f a u t o p s i e d h e n s c o n t a i n e d s i m i l a r to m e at s p o t s i n e g g s . t r a t e d m a in ly i n t h e u t e r u s . T h e s e t i s s u e s w e re c o n c e n ­ H ens w i t h a h i g h i n c i d e n c e o f m eat s p o ts in t h e i r eg g s u s u a lly h a d a h ig h in c id e n c e o f a b n o rm a litie s o f th e o v id u c t. Age a p p e a r s to h a v e some e f f e c t o n b lo o d s p o t i n c i d e n c e . N a lb a n d o v a n d C a rd (1 9 4 4 ) r e p o r t e d 4 4 p e r c e n t o f a l l h e n s sa m p le d l a i d e g g s c o n t a i n i n g b lo o d s p o t s t h e i r f i r s t y e a r . The b lo o d s p o t i n c i d e n c e d e c r e a s e d i n t h e s e c o n d l a y i n g y e a r f o r 60 p e r c e n t o f t h e h e n s a n d i n c r e a s e d f o r 35 p e r c e n t . Je ffre y (1 9 4 5 ) r e p o r t e d t h e s i z e o f p a l e an d r e d m e at s p o t s t e n d e d to d e c r e a s e a s t h e la y in g y e a r p ro g re sse d . L e m e r (1 9 4 6 ) fo u n d t h a t b i r d s n o t s u r ­ v i v i n g t h e i r f i r s t l a y i n g y e a r h a d a lo w e r b lo o d s p o t i n c i d e n c e th a n th o s e t h a t li v e d in to t h e i r seco n d la y in g y e a r . The d i f f e r e n c e - 13 - w as n o t s i g n i f i c a n t . T h e re i s a g r a d u a l d e c r e a s e i n e g g w e ig h t w i t h e a c h s u c c e e d ­ in g egg in a c lu tc h , en d t h e l o n g e r t h e c l u t c h t h e g r e a t e r i s t h e d e c r e a s e fro m t h e f i r s t to t h e l& s t e g g , h u t t h e s m a l l e r i s th e d e c r e a s e o f e a c h e g g w i t h i n t h e c l u t c h (B e n n io n a n d W arren , 1 9 3 3 ). S in c e a l l o f t h e e g g s o f a c l u t c h a r e n o t e a u a l i n s i z e i t i s l o g i c a l to assum e t h a t t h i s c o u l d h a v e an i n f l u e n c e o n b lo o d o r m eat s p o ts . The r e l a t i o n s h i p b e tw e e n s i z e and c o l o r o f m eat s p o t s t o c l u t c h p o s i t i o n w as d e te r m i n e d by J e f f r e y ( 1 9 4 5 ) . T in y p a l e m e a t s p o t s r e a c h e d a p e a k o f 3 2 .1 p e r c e n t a t t h e num ber 5 c l u t c h p o s itio n , tio n . th e n d e c l i n e d to 2 0 .8 p e r c e n t a t t h e s i x t h c l u t c h p o s i ­ A n o th e r i n c r e a s e f o ll o w e d u n t i l a h i g h p e a k o f 4 1 .2 p e r c e n t w as r e a c h e d . The p e r c e n t a g e o f l a r g e r e d m e at s p o t s w as h i g h e s t i n e g g s o f c l u t c h p o s i t i o n 1 ( 1 4 .9 p e r c e n t ) f o l l o w e d by a s h a r p d e c l i n e u n t i l a s e c o n d p e a k w as r e a c h e d a t p o s i t i o n s 6 , 7 an d 8 . Ho l a r g e r e d m eat s p o t s w e re fo u n d i n e g g s l a i d a f t e r t h e 9 t h c l u t c h p o s itio n . T h e re w as no s i g n i f i c a n t d i f f e r e n c e i n t h e i n c i d e n c e o f m e a t s p o t s p r e v i o u s to a n d f o l l o w i n g t h e p a u s e i n e g g p r o d u c t i o n . T he f i r s t e g g l a i d f o l l o w i n g a p a u s e c o n s t i t u t e d a v e ry s i g n i f i c a n t c h a n g e i n b o th e g g s h e l l c o l o r a n d m e at s p o t c o l o r . (1 9 5 2 ) r e p o r t e d l i t t l e S a u te r e t a l . c o r r e l a t i o n b e tw e e n t h e num ber o f p a u s e s ( 1 4 d a y s o r m o re) a n d t h e o v e r a l l p e r c e n t a g e o f b lo o d s p o t s . te n s ity an d p e r s i s t e n c y o f l a y w e re r e p o r t e d t o h a v e l i t t l e In ­ e ffe c t o n b lo o d s p o t s . S ta d e lm a n a n d S a u te r (1 9 5 2 ) r e p o r t e d t h a t h e n s t h a t p ro d u c e - 14 - 1 2 c o n s e c u t i v e ‘b lo o d s p o t f r e e e g g s l a y l e s s t h a n 20 p e r c e n t b lo o d s p o t s w i t h 99 p e r c e n t r e l i a b i l i t y p e rc e n t r e lia b ility . a n d l e s s t h a n 15 p e r c e n t w it h 95 T h i r ty - s ix c o n s e c u tiv e c le a r eggs in d ic a te h e n s l a y l e s s t h a n 5 p e r c e n t b lo o d s p o t s w ith 99 p e r c e n t r e l i a b i l i t y . S t r a i n a n d J o h n s o n (1 9 5 7 ) fo u n d e g g s i n t h e f i r s t c l u t c h p o s i t i o n h a d t h e h i g h e s t i n c i d e n c e o f b lo o d s p o t s ( 1 2 .9 p e r c e n t ) and th e seco n d c lu tc h p o s i t i o n h a d th e seco n d h ig h e s t in c id e n c e ( 1 0 . 1 p e r c e n t ) o f b lo o d s p o t s . B a s t i a n a n d Z arro w (1 9 5 5 ) d e v e lo p e d a h y p o t h e s i s f o r th e a sy n c h ro n o u s o v u la to r y c y c le o f la y in g h e n s. They r e p o r t e d o v u ­ l a t i o n i s n o t d e p e n d e n t o n o r c a u s e d by o v i p o s i t i o n , b u t m ore by t h e r e l e a s e o f l u t e i n i z i n g h o rm o n e fro m t h e p i t u i t a r y 6 to 8 h o u r s b e fo re o v u la tio n . T h is b eco m es l a t e r w ith e a c h eg g i n t h e c l u t c h a n d t h u s c a u s e s e a c h s u c c e e d i n g eg g to be o v u l a t e d l a t e r . They b e l i e v e d t h a t 2 s e p a r a t e a n d in d e p e n d e n t c y c l e s i n t e r a c t i n s u c h a way a s to r e s u l t i n the- t y p i c a l o v u l a t o r y c y c l e o f t h e h e n . T hese 2 c y c le s a r e : (l) t h e 24 h o u r d a y - n i g h t rh y th m ; a n d ( 2 ) t h e r h y th m ic m a t u r a t i o n o f o v a r i a n f o l l i c l e s . The i m p l i c a t i o n s d ra w n fro m t h e s e two c y c l e s a r e t h a t e a c h s u c c e e d in g ovum i n a c l u t c h i s o v u l a t e d a t a p r o g r e s s i v e l y e a r l i e r s t a t e o f d e v e lo p m e n t and i s th e r e f o r e s m a lle r. l e s s m a tu r e , S u c c e e d in g o v a w i t h i n a c l u t c h , b e in g s h o u ld r e q u i r e p r o g r e s s i v e ly h ig h e r l e v e l s o f l u t e i n - i z in g h o rn o n e to o v u l a t e th e m a t e q u a l p e r i o d s p r i o r to t h e i r e x p e c te d o v u la tio n . - 15 - E n v ir o n m e n ta l I n f l u e n c e s The e n v ir o n m e n t w i t h i n w h ic h a h e n i s p l a c e d c e r t a i n l y h a s a m a jo r i n f l u e n c e o n "both e g g p r o d u c t i o n a n d eg g q u a l i t y . The i n f l u e n c e o f t h e e n v iro n m e n t o n t h e i n c i d e n c e o f b lo o d s p o t s h a s b een d iv id e d in to t h e e f f e c t s o f s e a s o n , l i g h t an d a c t i v i t y , sound, and i r r a d i a t i o n . S i g n i f i c a n t s e a s o n a l e f f e c t s o n b lo o d s p o t i n c i d e n c e h a v e b e e n n o t e d b y L e r n e r a n d S m ith ( 1 9 4 2 ) , J e f f r e y T a y lo r ( 1 9 4 7 ) , Jo h n so n (1 9 5 7 ). S a u t e r et_ *&. ( 1 9 5 2 ) , ( 1 9 4 5 ) , L e r n e r an d Dawson (1 9 5 5 ) an d S t r a i n an d S e a s o n a l i n f l u e n c e s w ere r e p o r t e d to b e n o n - s i g ­ n i f i c a n t by D en to n (1 9 4 7 ) a n d K in g a n d H a l l ( 1 9 5 5 ) . (1 9 4 5 ) Je ffre y fo u n d ( f r o m D ecem ber u n t i l t h e f o l l o w i n g A u g u st) a m ark ed s e a s o n a l d e c l i n e i n t h e i n c i d e n c e o f a l l s i z e s o f b lo o d s p o t s f o r a l l b r e e d s o f c h i c k e n s e x a m in e d e x c e p t t h e B a r r e d P ly m o u th R o ck s. B lo o d s p o t i n c i d e n c e i n e g g s fro m B a r r e d P ly m o u th R o c k s i n c r e a s e d u n t i l M arch a n d t h e n d e c l i n e d . L e r n e r a n d T a y lo r (1 9 4 7 ) fo u n d a s e a s o n a l i n c r e a s e i n b lo o d s p o t i n c i d e n c e b e g in n in g w ith p r o d u c ­ t io n i n th e p u l l e t y e a r and re a c h in g a peak in Ju n e. S a u te r e t a l . (1 9 5 2 ) r e p o r t e d t h a t t h e l o w e s t b lo o d s p o t i n c i d e n c e o c c u r r e d i n m id D ecem ber a n d h i g h p e a k s i n F e b r u a r y , M arch a n d J u l y . (1 9 5 5 ) Dawson fo u n d t h e h i g h e s t b lo o d s p o t i n c i d e n c e i n F e b r u a r y , M arch and A p ril. S t r a i n a n d J o h n s o n (1 9 5 7 ) fo u n d t h e lo w e s t i n c i d e n c e i n O c to b e r . Many w o r k e r s h a v e s t u d i e d t h e e f f e c t o f l i g h t o n eg g q u a l i t y - 16 - and p ro d u c tio n . F e n g u i t e a n d Thom pson (1 9 3 3 ) r e p o r t e d t h a t c o n ­ t i n u o u s l i g h t s d i d n o t s i g n i f i c a n t l y c h a n g e t h e t o t a l num ber o f e g g s l a i d b u t d i d c a u s e m ore e g g s to b e l a i d i n t h e w i n t e r . P a rk - h u r s t (1 9 3 3 ) fo u n d no s i g n i f i c a n t d i f f e r e n c e i n t h e e g g w e ig h t o f W h ite L e g h o rn p u l l e t s i n l i g h t e d p e n s a n d u n l i g h t e d p e n s . e t a l. M u e lle r (1 9 5 1 ) r e p o r t e d t h a t t h e e f f e c t s o f l i g h t o n e g g q u a l i t y a n d b lo o d s p o t i n c i d e n c e w e re i n c o n s i s t e n t . Egg q u a l i t y w as h i g h e s t u n d e r c o n t r o l l e d l i g h t and te m p e ra tu re c o n d itio n s . U ls o n and A b p la n a lp (1 9 5 6 ) r e p o r t e d t h a t i n t e r m i t t e n t l i g h t a t r e g u l a r c y c l e s o f 4 h o u r s g a v e h i g h e r e g g p r o d u c t i o n t h a n t h e same am ount o f c o n ­ tin u o u s lig h tin g . Egg p r o d u c t i o n o b t a i n e d u n d e r s h o r t p h o t o p e r i o d s w as n o t p r o p o r t i o n a t e to t h e am ount o f l i g h t g i v e n . The minimum am ount o f l i g h t n e e d e d f o r an a l l - o r - n o n e r e s p o n s e f o r m a i n t a i n i n g e g g p r o d u c t i o n i n p u l l e t s i s p r o b a b l y l e s s t h a n 6 e v e n ly s p a c e d o n e - m in q te p h o t o p e r i o d s i n 34 h o u r s . th a n p u l l e t s to l i g h t f l u c t u a t i o n s , H ens w ere m ore s u s c e p t i b l e a n d g o o d l a y e r s w e re m ore r e s i s t a n t to c h a n g e s t h a n p o o r l a y e r s . L an so n a n d S t u r k i e (1 9 5 8 ) r e p o r t e d t h e tim e o f o v i p o s i t i o n c o u l d be a l t e r e d a s much a s 6 h o u r s by t h e m an n er o f p la c e m e n t o f o n e - h a l f h o u r l i g h t p e r i o d s . When t h e am ount o f d a r k n e s s was r e d u c e d to 3 .5 h o u r s p e r d a y , e g g s w e re l a i d a n a v e r a g e o f 14 h o u r s a f t e r t h e b e g i n n in g o f t h e d a r k p e rio d . P u l l e t s r e c e i v i n g 10 h o u r s o f d a r k n e s s l a i d e g g s an a v e r a g e o f 16 h o u r s a f t e r t h e s t a r t o f th e d a r k p e r i o d . D a rk n e s s d i d n o t h a v e an a c c u m u la tiv e e f f e c t i n c o n t r o l l i n g t h e tim e o f - 17 - o v i p o s i t i o n w hen o n e - h a l f h o u r s o f d a r k n e s s w e re s e p a r a t e d by o n e h a l f h o u rs o f l i g h t . O stm an n a n d B i e l l i e r (1 9 5 8 ) fo u n d t h a t h e n s p u t o n r h y th m ic l i g h t - d a r k Md a y s ” o f l e s s t h a n 24 h o u r s i n l e n g t h d e c r e a s e d t h e i r a v e ra g e c lu tc h le n g th and h e n -d a y egg p ro d u c tio n . I n c r e a s i n g p e r i o d s o f d a y - l e n g t h p r o g r e s s ! v e l y a d v a n c e d t h e tim e o f o v i p o s i t i o n to a n e a r l i e r h o u r o f t h e d a y . J e f f r e y a n d P in o (1 9 4 3 ) f r i g h t e n e d l a y i n g h e n s w ith h a r k ­ i n g d o g s an d fo u n d t h a t t h i s t r e a t m e n t d i d n o t i n c r e a s e t h e i n c i ­ d e n c e o f b lo o d s p o t s i n e g g s . They a l s o r e p o r t e d t h a t e g g s fro m b i r d s k e p t i n c a g e s h a d a lo w e r b lo o d s p o t i n c i d e n c e t h a n d i d e g g s fro m b i r d s on t h e f l o o r . I n c o n t r a s t to t h i s McDowell (1 9 5 8 ) r e ­ p o r t e d t h a t e g g s fro m c a g e d b i r d s h a d m ore b lo o d s p o t s t h a n e g g s fro m f l o o r b i r d s . Van Went (1 9 5 4 ) t e s t e d t h e e f f e c t s o f u l t r a s o n i c s h o r t w av es o n h u m a n s. in and u l t r a s He fo u n d t h e human bod y p a r t s i n f l u e n c e d a m e c h a n ic a l way by u l t r a s o n i c w av es s u f f e r e d fro m t r e a t m e n t w i t h lo w f r e q u e n c i e s r a t h e r t h a n h i g h f r e q u e n c i e s . U ltra s o n ic w a v es c a u s e d h e m o ly s is an d u l t r a s h o r t w av es a p p l i e d d i r e c t l y to th e body c a u se d v a s o d ila tio n . S m ith et^ a l . (1 9 5 6 ) r e p o r t e d no i n c r e a s e i n b lo o d s p o t t i n g by o v i d u c t i r r a d i a t i o n . The o v a r y w as s h i e l d e d fro m t h e r a d i a t i o n a n d show ed no e v id e n c e o f r a d i a t i o n dam age. A fte r i r r a d i a t i o n th e a lb u m e n c o n t a i n e d a n o p a q u e m a t e r i a l g i v i n g t h e alb u m en a m ilk y a p p e a ra n c e . shapes. The e g g s h e l l s fro m t r e a t e d h e n s o f t e n h a d a b n o rm a l - 18 - D e t e c t i o n o f B lo o d S p o ts A t warm t e m p e r a t u r e s t h e alb u m en b eco m es m ore w a te r y w ith a g e a n d a llo w s t h e y o l k to come c l o s e r to t h e s u r f a c e e n a b l i n g t h e c a n d l e r to d e t e c t m ore b lo o d s p o t s . J e n s e n et_ a l . (1 9 5 2 ) r e p o r t e d t h a t a l a r g e r p e r c e n t a g e o f b lo o d s p o t s p r e s e n t c a n b e d e t e c t e d by c a n d l i n g e g g s a f t e r t h e y a r e s e v e r a l d a y s o l d t h a n when t h e y a r e f i r s t l a i d . A p p ro x im a te ly 40 p e r c e n t o f t h e b lo o d s p o t s d e g e n e r a t e d o r d i s a p p e a r e d when e g g s w ere s t o r e d s e v e r a l w eeks. B lo o d s p o t s l e s s t h a n o n e - e i g h t h in c h i n d ia m e te r d i s a p ­ p e a r e d i n l a r g e r p e r c e n t a g e s t h a n t h o s e o v e r o n e - e i g h t h o f an in c h . B r a n t e t, a l . (1 9 5 3 ) r e p o r t e d t h a t e g g s c o n t a i n i n g b lo o d s p o t s o v e r o n e - e i g h t h o f an i n c h i n s iz e c o n s is te n tly a b so rb ed l e n g t h s o f l i g h t n e a r 575 m i l l i m i c r o n s . An i n s t r u m e n t was d e­ v e l o p e d t h a t a u t o m a t i c a l l y s c a n n e d e g g s w ith l i g h t w av es b e tw e e n 5 8 5 an d 565 m i l l i m i c r o n s . E g g s c o n t a i n i n g no b lo o d c o u l d b e s e g ­ r e g a t e d w ith 9 9 .7 p e r c e n t a c c u r a c y . Eggs c o n t a i n i n g b lo o d s p o t s o v e r o n e - e i g h t h o f a n in c h i n d ia m e te r w e re s e g r e g a t e d w it h 9 8 .1 p e r c e n t a c c u r a c y , w h ile th o s e w i t h b lo o d s p o t s l e s s th a n o n e e i g h t h o f an i n c h i n d ia m e te r w ere i d e n t i f i e d w ith o n ly 2 8 .6 p e r ­ c e n t a cc u ra cy . Hood (1 9 5 6 ) r e p o r t e d t h a t an e l e c t r o n i c m a c h in e m a n u f a c tu r e d by I d b r a s c o p e , In c o rp o ra te d , c o u ld a u to m a tic a lly d e t e c t a n d s e g r e g a t e b lo o d s p o t e g g s a t t h e r a t e o f 7 ,2 0 0 e g g s p e r h o u r. - 19 - R a d i o a c t i v e P h o s p h o ru s T h e r e a r e many r e p o r t s i n t h e l i t e r a t u r e r e g a r d i n g t h e u s e o f r a d i o a c t i v e p h o s p h o r u s w ith c h i c k e n s . P ra c tic a lly a ll o f t h e s e r e p o r t s i n v o l v e d f e e d i n g F *^ o r i n j e c t i n g i t in tra r m u s c u la rly and th e n r e c o r d in g th e p h o sp h o ru s u p ta k e in th e v a r io u s t i s s u e s a n d c o m p o n e n ts o f t h e e g g . F e e d in g . — S h i r l e y a t a l . (1951) r e p o r t e d t h a t f o ll o w i n g a n o r a l a d m i n i s t r a t i o n o f F®®, t h e d o s e t h a t was e x c r e t e d i n th e e x c r e m e n t d u r in g t h e f i r s t , second, th ir d , an d f o u r t h d a y s was a p p ro x im a te ly 3 5 , 7 , 1 , and l e s s th a n 1 p e r c e n t r e s p e c t i v e l y . A f te r th e te n th day th e v a lu e s l e v e l e d o f f w ith in th e ra n g e o f 0 .0 0 0 1 to 0 .0 0 1 p e r c e n t . No F ^ t h e e x c r e m e n t a f t e r 65 d a y s . i s o t o p e c o u l d "be d e t e c t e d i n A f t e r 130 d a y s P0 *5 c o u l d "be de­ t e c t e d i n s e v e r a l o f t h e "bones, "but n o t i n t h e m u s c le , n e r v e tis s u e , "blood o r e g g s . C le g g et_ a l . to (1 9 5 4 ) f e d r a d i o a c t i v e p h o s p h o r u s to h e n s d e te r m in e t h e d i s t r i b u t i o n o f p h o s p h o r u s i n e a c h o f t h e e l e c t r o p h o r e t i c c o m p o n e n ts . They d e v e lo p e d an e x t r a c t i o n p r o ­ c e d u re f o r p re p a rin g a c l e a r s o lu tio n c o n ta in in g a l l o f th e y o lk p ro te in s . P a t r i c k a n d S c h w e itz e r (1 9 5 2 ) fo u n d t h a t v i t a m i n D i s r e q u i r e d f o r p h o s p h o r u s a b s o r p t i o n fi'om t h e d i g e s t i v e t r a c t . S u b c u ta n e o u s a n d I n t r a m u s c u l a r I n j e c t i o n s . — S h i r l e y e t a l . (1 9 5 2 ) u s i n g r a d i o a c t i v e p h o s p h o r u s a n d c a l c i u m fo u n d t h a t t h e - 20 - duodenum , s m a ll i n t e s t i n e , c e c a , and c lo a c a a re th e p r in c ip le s i t e s o f c a lc iu m and p h o sp h o ru s e x c r e tio n . e x c r e te d in th e la r g e i n t e s t i n e . V ery l i t t l e w as T h e se same w o r k e r s i n 1954 d e t e r m i n e d t h e r a t e o f d e p o s i t i o n an d t u r n o v e r o f t h e i s o t o p e s i n 18 p r i n c i p l e t i s s u e s a t i n t e r v a l s “b e tw e e n 0 .2 5 h o u r s a n d 21 d ay s a f t e r in tra m u s c u la r i n je c tio n s o f D u rin g th e sam e y e a r t h e s e w o r k e r s r e p o r t e d t h a t two h e n s r e c e i v i n g a p p r o x im a te ly 20 m illic u rie s o f P 32 i n t r a m u s c u l a r l y o v e r a p e r i o d o f 7 n u n th s d i e d w i t h i n 8 m o n th s o f t h e i n i t i a l a d m i n i s t r a t i o n . E ach h e n h a d l o s t a p p r o x im a te ly 200 g ra m s o f w e ig h t h u t show ed no r e d u c e d v i t a l i t y u n t i l d e a th . H ow ever, t h e y show ed g r e a t n u m b e rs o f p e t e c h i a l h e m o rrh a g e s th ro u g h o u t th e h e a r t , o v a r i e s , r i g h t lo b e o f th e l i v e r , in te s tin a l tra c t, o f th e P 32 an a b n o rm a l g r e e n s u b s ta n c e i n t h e s m a ll a n d b lo o d i n t h e c e c a . in je c te d in to a n d magnum, a. n e c r o t i c A p p ro x im a te ly 43 p e r c e n t t h e h e n s w as e x c r e t e d i n t h e e x c re m e n t w i t h i n 10 d a y s a f t e r a d m i n i s t r a t i o n . In g e n e ra l, th e h en i s v e ry r e s i s t a n t to r a d i a t i o n dam age. U p ta k e o f F ^ i n t h e E gg. — L o re n z , P e rlm a n , and C h a ik o ff (1 9 4 3 ) r e p o r t e d t h a t when P * ^ w as i n j e c t e d s u b c u t an e o u s l y , s h e l l s fro m e g g s l a i d w i t h i n o n e h o u r show ed r a d i o a c t i v i t y . A c tiv ity w as g r e a t e s t i n t h o s e s h e l l s t h a t w e re b e in g fo rm e d a t t h e tim e o f in je c tio n . No a p p r e c i a b l e a c t i v i t y was fo u n d i n t h e alb u m en o f eg g s fo r 24 h o u r s . R a d i o a c t i v i t y r e a c h e d a maximum i n 24 to 54 h o u rs and th e n s t e a d i l y d e c lin e d . Y o lk s show ed o n ly t r a c e s - 21 - o f F ^ 2 d u r in g t h e f i r s t 2 4 h o u r s f o l l o w i n g i n j e c t i o n . Maximum y o l k a c t i v i t y w as f o u n d i n t h e p e r i o d b e tw e e n 98 t o 1 4 6 .8 h o u r s a f te r in je c tio n . O 'N e i l (1 9 4 8 ) s u b s t a n t i a t e d t h i s e v id e n c e when h e f o u n d a maximum a c t i v i t y i n t h e s h e l l w i t h i n 24 h o u r s , 48 to 72 h o u rs fo r th e w h ite , a n d 144 h o u r s f o r t h e y o lk . R a n d le s (1 9 5 4 ) r e p o r t e d m o st o f t h e p h o s p h o r u s i n t h e f r e s h egg i s fo u n d i n t h e y o l k a s p h o s p h o l i p i d c o m p le x e s . I n t h e d e v e l­ o p i n g em bryo t h e s e l i p i d s becom e d i s s o c i a t e d a n d t h e f r e e p h o s ­ p h a t e i s m o b i l i z e d t o t h e l i v i n g o r g a n is m . Ifcr u s i n g an a l l a n t o i c i n j e c t i o n o f a p h o s p h a te b u f f e r l a b e l e d w ith p h o s p h o r u s , h e fo u n d v e ry l i t t l e o r none o f th e tr a c e r d if f u s e d in to th e y o lk s a c ; and c o n c l u d e d t h a t t h e r e i s o n ly a u n i d i r e c t i o n a l d i f f u s i o n o f p h o s ­ p h a te o u t o f th e y o lk s a c . S m ith , W in g e t, an d B la c k a r d (1 9 5 4 ) r e p o r t e d t h a t u p o n i n ­ je c tin g i n t r a m u s c u l a r l y l e s s th a n 2 p e r c e n t o f t h e d o s e was t r a n s f e r r e d to t h e s h e l l a n d l e s s th a n 1 p e r c e n t w as t r a n s f e r r e d to t h e w h ite i n 1 2 d a y s , w h ile 1 0 .8 p e r c e n t was t r a n s f e r r e d to t h e y o l k i n t h e same p e r i o d o f tim e . I n t h i s s tu d y h e u s e d t h e f o l l o w ­ i n g f o r m u la s . S p e c i f i c A c t i v i t y - me P ^ _________________ mgms. pho s p h o r u s O r g a n ic S p e c i f i c A c t. ^ T o t a l a c t i v i t y - i n o r g a n i c a c t i v i t y _______ T o t a l p h o s p h o ru s - i n o r g a n i c p h o s p h o ru s S ta n d a r d S p e c i f i c A c t. - me P5 2 / mgm p h o s p h o ru s _______ me i n j e c t e d / k g body w t. - 22 - E f f e c t o f R a d i a t i o n o n B lo o d . — D ixon (1 9 4 8 ) g a v e l e t h a l a n d s u b l e t h a l do s e e o f t o 17 d ay o l d c h i c k s a n d o b s e r v e d t h e r e d b l o o d c e l l s a n d v a r i o u s l e u c o c y t e s u n t i l d e a t h , o r a t a tim e o f s a c r i f i c e 17 d a y s l a t e r . a f t e r 7 days o f in te n s e P 32 He fo u n d v i r t u a l a g r a n u l o c y t o s i s ra d ia tio n s . c r e a s e o f t o t a l r e d an d w h ite c e l l s . et a l. T h e re was a m a rk e d de­ I n c o n t r a s t to t h i s S h i r l e y (1 9 5 4 ) r e p o r t e d F *^ h a d no o b s e r v e d e f f e c t o n t h e t o t a l r e d a n d w h ite c e l l c o u n t s , h o w e v e r, no m o n o c y te s o r n e u t r o p h i l e s w e re o b s e r v e d f o r t h r e e w eeks p r i o r t o d e a t h . T e c h n iq u e s o f L a b e lin g Human B lo o d C e l l s w ith p 5 2 . — B e i e r w a lte rs , Jo hnson, an d S o l a r i (1 9 5 7 ) r e p o r t e d a t e c h n i q u e f o r b lo o d v o lu m e d e t e r m i n a t i o n s i n hum ans u s i n g r a d i o a c t i v e p h o s p h o r u s . T h e ir t e c h n i q u e w as a s f o l l o w s : in to a h e p e rin iz e d s y rin g e . s te rile , Draw 15 m i l l i l i t e r s o f b lo o d F l a c e 5 m i l l i l i t e r s o f b lo o d i n t o r u b b e r - c a p p e d , c e n t r i f u g e tu b e , a an d i n c u b a t e a t 37 de­ g r e e s c e n t i g r a d e w i t h c o n s t a n t r o t a t i o n w ith 0 . 5 m i l l i c u r i e o f ra d io a c tiv e is o to n ic d ih y d r o g e n p h o s p h a te w ith pH o f 7 . 3 . At th e e n d o f 2 h o u r s t h e r e d c e l l s a r e w a sh e d 3 tim e s by a d d in g i s o t o n i c s a lin e s o lu tio n , c e n trifu g in g , a n d re m o v in g t h e s u p e r n a t a n t f l u i d . A f t e r t h e t h i r d w a s h in g w it h s a l i n e , some o f t h e p la s m a o b t a i n e d fr o m t h e r e m a in d e r o f t h e p a t i e n t ' s b lo o d a n d c o n t a i n i n g no F * ^ , i s a d d e d t o t h e c e l l s to r e c o n s t i t u t e w h o le b lo o d w ith a h e m a to ­ c r i t o f a p p r o x im a te ly 4 0 . in je c te d in to One m i l l i l i t e r o f t h i s b lo o d i s t h e n an a n t e c u b i t a l v e i n , a n d u s i n g t h e same n e e d l e an d - 23 - s y r i n g e 1 m i l l i l i t e r i s p l a c e d i n a 2000 m i l l i l i t e r w a te r i s a d d e d to t h e 2 l i t e r m ark ( S t a n d a r d ) . f l a s k and F i f t e e n m in u te s a f t e r i n j e c t i o n , 5 . 0 m i l l i l i t e r s o f b lo o d i s draw n fro m t h e a n t e c u b i t a l v e i n i n t h e o p p o s i t e arm a n d p l a c e d i n a d ry o x a l a t e tu b e . T h en 0 . 1 m i l l i l i t e r o f t h i s b lo o d s a m p le , a n d t h e s t a n d a r d , each p ip e tte d in to alum inu m p l a n c h e t s i n t r i p l i c a t e a re a n d c o u n te d i n a p i g u n d e r a G e ig e r - M u e lle r t u b e . T o t a l R ed C e l l V ol. r CFM / 0 . 1 m l. S t a n d a r d X 2 .0 0 0 X h e m a t o c r i t CPM / 0 . 1 i d . b lo o d B lo o d Volume r T o t a l r e d c e l l vo lu m e H e m a to c r it EXPERIMENT I . LABELING- BLOOD SPOTS IN CHICKEN EGOS WITH RADIOACTIVE PHOSPHORUS T he o b j e c t i v e o f t h i s e x p e r im e n t was to d e v e lo p a p r o c e d u r e f o r l a b e l i n g t h e r e d b lo o d c e l l s i n a l a y i n g h e n i n s u c h a way t h a t b l o o d s p o t s i n an e g g fro m t h i s h e n w o u ld show r a d i o a c t i v i t y i n d e t e c t a b l e a m o u n ts . R a d i o a c t i v e p h o s p h o r u s (P®^) d e c a y s i n t o e m is s io n o f a b e ta p a r t i c l e . s u lfu r ( S ^ ) by The b e t a p a r t i c l e i s a n e g a t i v e l y c h a r g e d e l e c t r o n a r i s i n g fro m a n e u t r o n i n t h e n u c l e u s o f t h e a to m . T h is r e a c t i o n c a n b e shown a s : ^,32 -------------- > 15 32 16S o - 1B •+" R a d io a c tiv e p h o sp h o ru s h a s a h a l f l i f e o f 1 4 .3 d a y s . S in c e r e d b l o o d c e l l s a b s o r b p h o s p h o r u s - 3 2 when i n c u b a t e d w ith r a d i o a c t i v e is o to n ic so d iu m d ih y d r o g e n p h o s p h a te , s u c h l a b e l e d c e l l s c a n be t r a c e d t h r o u g h o u t t h e body o f t h e h e n . I f th e la b e le d c e l l s a re i n j e c t e d i n t o a b i r d a t known t im e s o f t h e o v u l a t o r y c y c l e , t h e tim e o f su b se q fu e n t b l o o d s p o t f o r m a t i o n c a n b e c o r r e l a t e d to t h i s c y c le . B e irw a lte rs , J o h n s o n a n d S o l a r i (1 9 5 7 ) r e p o r t e d a t e c h ­ n i q u e f o r b lo o d vo lu m e d e t e r m i n a t i o n s i n hum ans u s i n g r a d i o a c t i v e p h o sp h o ru s. A m o d i f i c a t i o n o f t h e i r t e c h n i q u e was f o ll o w e d i n d e v e l o p i n g t h i s p r o c e d u r e f o r l a b e l i n g b lo o d s p o t s i n e g g s . E x p e r im e n ta l P r o c e d u r e I b u r S i n g l e Comb W h ite L e g h o rn h e n s w ith a known h i g h b lo o d - 24 - - 25 - s p o t i n c i d e n c e w e re k e p t i n s e p a r a t e c a g e s a n d c h e c k e d h o u r l y d u rin g th e day to to d e te r m in e t h e i r e x a c t o v u l a t o r y c y c l e . a la c k in s u f f ic ie n t q u a n titie s o f P a t a tim e i n d e v e lo p i n g t h i s t e c h n i q u e . r a d io a c tiv e is o to n ic 32 Cue o n ly o n e h e n was u s e d A know n v o lu m e o f so d iu m d ih y d r o g e n p h o s p h a te (U a Hg PO^) w as n e u t r a l i z e d t o a pH o f 7 . 3 "by t h e a d d i t i o n o f so d iu m h y ­ d ro x id e . A o n e la m b d a sam p le ( 0 .0 0 1 m i l l i l i t e r ) was ta k e n to d e t e r m i n e t h e am ount o f r a d i o a c t i v i t y e x p o s e d to t h e b lo o d c e l l s . P o u r a n d o n e - h a l f h o u r s b e f o r e t h e h e n w as due to o v u l a t e , a 5 m i l l i l i t e r b lo o d s a m p le w as w ith d ra w n fro m t h e b r a c h i a l v e i n i n a h e p a r in iz e d s y rin g e . The r a d i o a c t i v e i s o t o n i c d ih y d r o g e n p h o s p h a t e w as c a r e f u l l y a d d e d to t h e 5 m i l l i l i t e r s o f w h o le b l o o d a n d th o r o u g h l y m ix e d w i t h b u b b l i n g a i r . i m m e d ia te ly d i v i d e d i n t o The m i x tu r e w as f o u r e q u a l am o u n ts i n f o u r W arburg f l a s k s a n d p l a c e d i n a W arburg w a te r b a th to i n c u b a t e . The W arb u rg w a te r b a t h m a i n t a i n e d a c o n s t a n t t e m p e r a t u r e a t 39 d e g r e e s c e n t i g r a d e an d k e p t t h e f l a s k s i n a c o n t i n u o u s s h a k in g m ovem ent. h o u rs. The o p tim u m i n c u b a t i o n tim e was a p p r o x im a te ly t h r e e C u rin g t h i s p e r i o d t h e f l a s k s w ere a llo w e d p l e n t y o f f r e s h a i r to p r e v e n t t h e c e l l s fro m b e c o m in g d e o x y g e n a te d . A f t e r t h e m i x t u r e o f b lo o d a n d P * ^ h a d c o m p le te d t h e i n c u ­ b a t i o n p e r i o d , t h e c e l l s w e re s e p a r a t e d by c e n t r i f u g a t i o n a t 3 000 r e v o l u t i o n s p e r m in u te f o r 12 m in u te s . s u p e m a t e n t a n d b lo o d c e l l s w e re n o t e d . The v o lu m e s o f b o th T h re e 0 .0 1 m i l l i l i t e r s a m p le s o f s u p e r n a t a n t w e re t e s t e d f o r r a d i o a c t i v i t y . P h y s io ­ l o g i c a l s a l i n e w as a d d e d to t h e c e l l s to b r i n g t h e t o t a l volum e to 5 m i l l i l i t e r s . fu lly T h is m i x t u r e w as th o r o u g h l y b le n d e d by c a r e ­ b u b b lin g a i r th r o u g h i t , t h u s m in im iz in g t h e m e c h a n ic a l dam age to t h e c e l l s c a u s e d by u s i n g a s t i r r i n g r o d . A g la s s s t i r r i n g r o d w as u s e d to m ix t h e P 32 w ith t h e c e l l s i n t h e f i r s t tw o p r e l i m i n a r y t r i a l s , b u t b u b b lin g a i r was u s e d i n l a t e r t r i a l s . T he c e l l s w e re th e n w a sh e d by c e n t r i f u g i n g a t 3 000 r e v o l u t i o n s p e r m in u te f o r 10 m in u te s , a f t e r w h ic h t h e s u p e r n a t a n t was a g a in re m o v e d a n d t h r e e s a m p le s o f 0 . 2 m i l l i l i t e r s w e re t a k e n . T h is w a s h in g p r o c e s s was r e p e a t e d t h r e e tim e s to i n s u r e t h e re m o v a l o f a l l e x c e s s F 3 ^ fro m t h e s u r f a c e o f t h e b lo o d c e l l . The l a b e l e d c e l l s w ere r e s u s p e n d e d i n p h y s i o l o g i c a l s a l i n e i n t h e e x a c t h e m a t o c r i t p r o p o r t i o n a n d w e re i n j e c t e d i n t o t h e b r a c h i a l v e i n a t a known s t a g e o f t h e o v u l a t o r y c y c l e . fo r ra d io a c tiv ity S a m p le s a n d b lo o d v o lu m e m e asu re m e n t w ere ta k e n o f t h e b lo o d i n j e c t e d a n d a l s o o f t h e c i r c u l a t i n g b lo o d a t v a r i o u s t im e s a f te r in je c tio n . F e c a l s a m p le s w e re ta k e n h o u r l y f o r th e f i r s t fe w h o u r s a f t e r i n j e c t i o n and d a ily t h e r e a f t e r . A ll o f t h e s a m p le s w e re d r i e d f o r 24 h o u r s a t room t e m p e r a t u r e b e f o r e th e y w e re c o u n te d . R e s u l t s a n d D is c u s s i o n The w h o le b lo o d h a d s t r o n g r a d i o a c t i v i t y w i t h i n 10 m in u te s a f t e r i n j e c t i o n w h ile th e p la s m a r e g i s t e r e d no a c t i v i t y f o r 45 - 27 - h o u rs. T h is i n d i c a t e d t h a t a l l o f t h e P rzO w as c o n t a i n e d w i t h i n t h e b l o o d c e l l s a n d d i d n o t e s c a p e u n t i l n o rm a l m e ta b o li c t r a n s f e r s h i f t e d some o f i t in to t h e p la s m a a n d t i s s u e s . The d i s a p p e a r a n c e o f P 3 3 fro m t h e c e l l s w as r a p i d a s i l l u s t r a t e d i n F ig u r e 1 . s e q u e n tly , o n l y to Con­ t h i s p r o c e d u r e g i v e s r a d i o a c t i v i t y w h ic h i s c o n f i n e d t h e b lo o d c e l l s w i t h i n t h e b lo o d s p o t i f t h e c e l l s a r e i n j e c t e d w i t h i n a few h o u r s p r i o r to b lo o d s p o t f o r m a t i o n . If t h e tim e b e tw e e n i n j e c t i o n a n d b lo o d s p o t f o r m a ti o n i s i n c r e a s e d t o m ore t h a n 24 h o u r s , t h e y o l k a l s o b ecom es r a d i o a c t i v e . !E$y k n o w in g t h e e x a c t o v u l a t o r y c y c l e o f t h e h e n a n d by ti m i n g t h e P i n j e c t i o n to f i t in to t h i s c y c le , s p o t f o r m a ti o n c a n be d e te r m in e d . e g g s t h a t show s i z a b l e b lo o d s p o t s , t h e e x a c t tim e o f b lo o d Due to t h e lo w p e r c e n t a g e o f s e v e r a l h e n s s h o u ld be i n ­ j e c t e d a t o n e tim e to c a r r y o u t t h e e x p e r im e n t p r o p e r l y . Hen 228 w as i n j e c t e d a p p r o x im a te ly 3 .5 h o u r s a f t e r t h e p r e d i c t e d o v u l a t i o n tim e o f a n eg g c o n t a i n i n g a l a r g e b lo o d s p o t . T he b lo o d s p o t w as n o t r a d i o a c t i v e sh o w in g t h a t t h i s h e n , w hose n o rm a l tim e b e tw e e n o v u l a t i o n a n d o v i p o s i t i o n was 28 h o u r s , c o u l d n o t h a v e fo rm ed t h e b lo o d s p o t b e tw e e n t h e tim e o f i n j e c t i o n an d o v ip o s itio n . On a n o th e r d a t e H en 1 5 9 , w hose n o rm a l tim e b e tw e e n o v u l a t i o n a n d o v i p o s i t i o n w as a l s o 28 h o u r s , was i n j e c t e d w ith l a b e l e d c e l l s 9 h o u r s p r i o r to o v u l a t i o n . T h is o v u l a t i o n p r o ­ d u c e d an e g g c o n t a i n i n g a b lo o d s p o t w h ich d i d show d e t e c t a b l e ra d io a c tiv ity , t h u s sh o w in g t h a t t h e f o r m a t i o n o f t h e b lo o d s p o t - 28 - o c c u r r e d w i t h i n t h a t 9 h o u r p e r i o d o f tim e p r i o r to o v u l a t i o n . F u r t h e r e x p e r im e n ts u s i n g t h i s m e th o d s h o u ld show m ore a c c u r a t e l y t h e tim e o f b lo o d s p o t f o r m a t i o n . B lo o d s p o t s fo rm e d w i t h i n 48 h o u r s a f t e r t h e l a b e l e d c e l l s h a v e b e e n i n j e c t e d show d e t e c t ­ a b l e r a d i o a c t i v i t y i f t h e b lo o d s p o t s c o n t a i n m ore t h a n 0 .0 2 4 m i l l i l i t e r s o f b lo o d . B lo o d volum e o f t h e h e n c a n b e d e te r m in e d s i m u l t a n e o u s l y by d e te rm in in g a c c u r a te ly th e t o t a l r a d i o a c t i v i t y i n j e c t e d an d t h e n s a m p lin g t h e b lo o d a t 10 m in u te i n t e r v a l s a f t e r i n j e c t i o n . The b lo o d v o lu m e (V ^) c a n b e c a l c u l a t e d fro m t h e f o l l o w i n g f o r ­ m u la :Vt r (v o lu m e i n j e c t e d ) (CPM /ml. i n j e c t e d ) CFM / m l. sa m p le T he vo lu m e o f w h o le b lo o d i n a b lo o d s p o t (F b s ) c®11 b e c a l c u ­ l a t e d b y :V^e r CPM o f b lo o d s p o t CPM / m l. sam p le CPM m eans c o u n t s p e r m in u te . la s is a s fo llo w s : An i l l u s t r a t i o n u s i n g t h e s e fo rm u ­ I n j e c t a h e n w ith 2 m i l l i l i t e r s o f r a d i o a c t i v e b l o o d w h ic h show ed 1 6 8 ,0 0 0 c o u n t s p e r m in u te p e r m i l l i l i t e r a c tiv ity . I f t h e w ith d ra w n s a m p le s o f b lo o d a f t e r t h e i n j e c t i o n h a d 2800 c o u n t s p e r m in u te p e r m i l l i l i t e r th e n t h e t o t a l b lo o d v o lu m e (V ^) w o u ld b e : Vt - ( 2 x 1 6 8 .0 0 0 ) s 120 m l. 2800 I f a b lo o d s p o t h a d a t o t a l o f 132 c o u n t s p e r m in u te , i t s volum e - 29 - (V-bs ) w o u ld h e i ­ s t s - 132 2 800 • .0 4 7 a d . A l l o f t h e c o u n t s s h o u ld be made a t t h e same tim e to r u l e o u t t h e d ecay o f th e is o to p e . The r a t e o f d i s a p p e a r a n c e o f P ^ 2 fro m th e b lo o d a p p e a r e d to f o l l o w a sm ooth h y p e r b o l i c c u r v e a s shown i n F ig u r e 1 . The d i s a p p e a r a n c e w as m ost r a p i d d u r i n g t h e f i r s t few h o u r s o f t h e f i r s t day a f t e r i n j e c t i o n . T h i s was p r o b a b ly due to t h e h ig h e x c r e t i o n r a t e i n t o t h e f e c e s a n d e g g s a n d t h e r a p i d re m o v a l o f V3 2 by t h e r e t i c u l o e n d o t h e l i a l s y s te m . The d is a p p e a r a n c e r a t e l e v e l e d o f f c o n s i d e r a b l y by 5 d a y s a f t e r a d m i n i s t r a t i o n a n d b e­ cam e u l t i m a t e l y t a n g e n t to th e n o rm a l b a c k g ro u n d l e v e l o f b lo o d ra d io a c tiv ity . The d is a p p e a r a n c e o f r a d i o a c t i v i t y i n t h e f e c e s f o l l o w i n g P in je c tio n is show n i n F ig u r e 2 . A Lthough some a c t i v i t y ap ­ p e a r e d i n t h e f e c e s w i t h i n a few m in u te s a f t e r a d m i n i s t r a t i o n , t h e m axim im r a d i o a c t i v i t y a p p e a r e d i n 15 to 24 h o u r s . A fte r t h i s i n i t i a l i n c r e a s e d u r in g t h e f i r s t day* a s h a r p i r r e g u l a r d e c lin e in a c t i v i t y f o ll o w e d f o r t h e n e x t few d a y s . r a d i o a c t i v i t y c o n t i n u e d f o r s e v e r a l w e e k s. w ith S h i r l e y e t a l . S lig h t T h i s i s i n a g re e m e n t (1 9 5 1 ) who a d m i n i s t e r e d P3S o r a l l y . M o d i f i c a t i o n s o f th e i n i t i a l t e c h n i q u e w ere made d u r i n g la te r tria ls . S in c e o n ly a s m a ll p e r c e n t a g e o f t h e t o t a l P32 - 30 - w as a b s o r b e d by t h e r e d b lo o d c e l l s , s u p e r n a ta n t m ix tu re . much was l e f t o v e r i n t h e A p o r t i o n o f t h i s c o u l d be s a l v a g e d f o r s i m i l a r e x p e r im e n t s by a d d in g i t to o t h e r b lo o d c e l l s p e a ti n g th e in c u b a tio n p r o c e s s . E ggs w ith b lo o d s p o t s w e re b ro k e n o u t i n t o F a h re n h e it. an d r e ­ a p e t r i d i s h a n d f r o z e n a t m in u s 4 0 d e g r e e s T h is a i d e d i n a c l e a n re m o v a l o f t h e b lo o d s p o t fro m t h e y o l k a n d p r e v e n t e d m i g r a t i o n o f t h e t r a c e r . - 31 - 2000 (Z UJ GL 1500 UJ h3 Z 1000 ” cz UJ CL CO H Z 500 -- D o o 0 1 2 3 4 5 6 DAYS A F T E R I N J E C T I O N F ig u re 1. L o ss o f R a d i o a c t i v e P h o s p h o ru s fro m t h e B lo o d o f a Hen. - 32 - TABLE 1 RADIOACTIVE PHOSPHORUS LOSS RATE IN THE BIOOD OF HEN 228 S am ple Amount ml O rig in a l O rig in a l p 0 .0 0 1 0 .0 0 1 B lo o d i n j e c t e d B lo o d i n j e c t e d B lo o d i n j e c t e d B lo o d 10 m in. a f t e r i n j . B lo o d 1 0 ndn. a f t e r i n j . B lo o d 10 ndn. a f t e r i n j . B lo o d 20 min. a f t e r i n j . B lo o d 20 m in. a f t e r i n j . B lo o d 20 min* a f t e r i n j . B lo o d 30 min. a f t e r i n j . B lo o d 3 0 min. a f t e r i n j . B lo o d 30 m in. a f t e r i n j . B lo o d 24 h r s. a f t e r i n j . B lo o d 24 h r s. a f t e r i n j . B lo o d 24 h r s . a f t e r i n j . B lo o d 120 h r s. a f t e r i n j . B lo o d 120 h r s. a f t e r i n j . B lo o d 120 h r s. a f t e r i n j . I. 0 .1 0 .1 0 .1 0 .4 0 .4 0 .4 0 .4 0 .4 0 .4 0 .4 0 .4 0 .4 0 .2 0 .2 0 .2 0 .5 0 .5 0 .5 T o ta l1 count C o u n t p e r m inute^ p e r ml 8929 9441 Ave. 8 ,9 2 9 ,0 0 0 9 .4 4 1 .0 0 0 9 .1 8 5 .0 0 0 Ave. 1 9 6 ,8 1 0 1 8 6 ,5 7 0 1 9 6 ,8 1 0 1 9 3 ,3 9 7 A ve. 1 ,9 2 2 2 ,0 8 2 2 .0 4 2 2 ,0 1 5 Ave. 1 ,8 4 2 2 ,1 2 2 1 .9 6 2 1 ,9 7 5 Ave. 1 ,8 8 2 1 ,8 8 2 1 .9 6 2 1 ,9 0 9 Ave. 855 960 825 880 Ave. 32 26 16 25 19681 18657 19681 769 833 817 737 849 785 753 753 785 171 192 165 16 13 8 C o r r e c t e d f o r a b a c k g ro u n d o f 31 c o u n ts p e r m in u te . - 33 - TABLE 2 RAEIO ACTIVE PHOSPHORUS LOSS RATE IN HEN FECES; FECAL COUNTS FROM HEN 228 S am p le nu m b er S am ple w e ig h t g ra m s H o u rs a f t e r in je c tio n T o ta l c o u n ts p e r m in u te p e r gram-*- 1 0 .5 2 337 2 1 .0 15 808 3 1 .0 39 475 4 1 .0 63 501 5 1 .0 87 65 6 1 .0 111 71 1. C o r r e c t e d f o r a "background c o u n t o f 37 c o u n t s p e r m in u te . 700 ■ 600 - MINUTE 400 PER 300 COUNTS PER GRAM - 34 - 200 - " - I00-- 10 20 30 40 50 60 70 80 90 100 110 HOURS AFTER IN J E C T IO N ]H.gure 2 . B a d io a o tiv e P h o sp h o ru s L oss B a te i n Hen tfeces. EXPERIMENT I I . THE RELATIONSHIP OT OVIPOSITION TIME, CUJTCH PO SITIO N, Bj&OMETRIC PRESSURE, M B EGG HEIGHT TO THE INCIDENCE OE BLOOD SPOTS IN EGGS T h i s e x p e r im e n t was d e s ig n e d to d e te r m in e i f t h e tim e o f o v u l a t i o n ( b a s e d on o v i p o s i t i o n tim e ) w as d i f f e r e n t f o r e g g s c o n t a i n i n g b lo o d s p o t s t h a n f o r n o rm a l e g g s . In a d d itio n , th e e x p e r im e n t w as d e s i g n e d to d i s c o v e r i f t h e r e w as an i n f l u e n c e o n t h e i n c i d e n c e o f b lo o d s p o t s due t o e g g c l u t c h p o s i t i o n , b a r o m e t r i c p r e s s u r e , e g g w e ig h t a n d e g g alb u m en h e i g h t . la tte r T h ese f o u r c o n d i t i o n s w e re s t u d i e d b e c a u s e t h e i r e f f e c t ( i f a n y ) c o u l d be i n v e s t i g a t e d w i th o u t a l t e r i n g t h e e x p e r i m e n t ’ s m ain o b je c tiv e , tio n w h ic h d e a l t w ith t h e r e l a t i o n s h i p o f tim e o f o v u l a ­ a n d o v i p o s i t i o n to b lo o d s p o t i n c i d e n c e . Some s t u d i e s o f t h i s n a t u r e h a v e b e e n r e p o r t e d i n t h e l i t e r a t u r e , h o w e v e r, n o n e w e re fo u n d to d e a l w ith t h e i n f l u e n c e o f o v u l a t i o n tim e o n b lo o d s p o t s . Q u in n a n d G o d fre y (1 9 4 0 ) fo u n d n o s i g n i f i c a n t c o r r e l a t i o n b e tw e e n t h e p e r c e n t a g e o f b lo o d s p o t s a n d e g g p r o d u c t i o n , e g g w e ig h t, o r body w e ig h t. Je ffre y (1 9 4 5 ) s t u d i e d th e r e l a t i o n s h i p b e tw e e n t h e s i z e a n d c o l o r o f m e at s p o t s an d t h e p o s i t i o n o f a b n o rm a l e g g s i n t h e c l u t c h . He fo u n d t h a t t h e p e r c e n t a g e o f l a r g e r e d m eat s p o t s was h i g h e s t i n th e e g g s i n c l u t c h p o s i t i o n 1 f o ll o w e d by a s h a r p d e c l i n e u n t i l a s e c o n d p e a k w as r e a c h e d a t p o s i t i o n s 6 , 7 a n d 8 . - 35 - S m a ll m e at s p o t s w e re - 36 - m ost p r e v a le n t a t th e f i f t h c l u t c h p o s i t i o n . S tr a in and Joh n so n (1 9 5 7 ) r e p o r t e d t h a t e g g s i n t h e f i r s t c l u t c h p o s i t i o n h a d t h e h i g h e s t i n c i d e n c e o f b lo o d s p o t s a n d t h e s e c o n d c l u t c h p o s i t i o n h a d t h e s e c o n d h i g h e s t i n c i d e n c e o f b lo o d s p o t s . They fo u n d no s i g n i f i c a n t i n f l u e n c e o f c l u t c h p o s i t i o n o n m e at s p o t s . E x p e r im e n t a l P r o c e d u r e E ggs w ere c o l l e c t e d h o u r l y e v e r y d ay f o r 7 m o n th s fro m 61 y e a r l i n g W hite L e g h o rn h e n s h e l d i n c a g e s . frig e ra te d T h e s e e g g s w e re r e ­ (6 0 d e g r e e s F a h r e n h e i t ) u n t i l b ro k e n o n a g l a s s p l a t e w i t h m i r r o r s b o th u n d e r n e a t h a n d b e h in d to a ll o w f u l l o b s e r v a t i o n o f th e eggs f o r b lo o d s p o ts . a f t e r t h e y w e re l a i d , A l l e g g s w e re b ro k e n w i t h i n 24 h o u r s an d o v i p o s i t i o n tim e , w e ig h t, U n ite d S t a t e s D e p a rtm e n t o f A g r i c u l t u r e s c o r e d , b lo o d s p o t s a n d o t h e r a b n o rm a l­ i t i e s w e re r e c o r d e d d a i l y . Album en h e i g h t , Hangh s c o r e and s h e l l t h i c k n e s s w e re r e c o r d e d 4 d a y s a w eek. The a c c u r a c y o f tim e t h e e g g s w e re l a i d was w i t h i n o n e h o u r , t h e egg w e ig h t was w i t h i n o n e g ra m , t h e a lb u m e n h e i g h t w as w i t h i n 0 . 1 m i l l i l i t e r , t h i c k n e s s w as w i t h i n 0 .0 0 0 1 i n c h . and th e s h e l l The s i z e o f e a c h b lo o d s p o t was d i f f e r e n t i a t e d a s b e in g l a r g e - o v e r o n e - e i g h t h o f an i n c h i n d i a m e t e r , m edium - b e tw e e n o n e - s i x t e e n t h a n d o n e - e i g h t h o f an i n c h i n d i a m e t e r , a n d s m a ll - l e s s th a n o n e - s i x t e e n t h o f an in c h i n d ia m e te r. 1. An e x p l a n a t i o n o f t h e U n i t e d S t a t e s D e p a rtm e n t o f A g r i c u l t u r e s c o r e a n d H angh s c o r e c a n be o b t a i n e d fro m a p u b l i c a t i o n p r e ­ p a r e d by L e s t e r K i l p a t r i c k , A. W. B r a n t a n d H. L. S h r a d e r , 1958. E q u ip m en t a n d m e th o d s f o r m e a s u r in g eg g q u a l i t y . AMS Number 24-6, U. S .D .A ., W a s h in g to n , D. C. - 37 - The b a r o m e t r i c p r e s s u r e d a t a w e re o b t a i n e d fro m t h e U n i t e d S t a t e s W e a th e r B u re a u i n E a s t L a n s in g . T h e s e d a t a w ere a c q u i r e d a p p r o x i m a t e l y o n e m ile fro m w h e re t h e c h i c k e n s w e re h o u s e d . At t h i s s t a t i o n t h e p r e s s u r e was r e c o r d e d a t 7 :3 0 a . m. a n d 1 :3 0 p .m . C o r r e l a t i o n s w ere made b e tw e e n t h e p r e s s u r e a t t h e s e tim e s w ith b l o o d s p o t s i n e g g s o v u l a t e d d u r i n g t h e sam e tim e p e r i o d an d a l s o i n t h o s e o v u l a t e d a t d i f f e r e n t tim e i n t e r v a l s u n t i l 2 d a y s la te r. The d a t a f o r t h i s s tu d y i n c l u d e d a l l o f t h e p r e s s u r e p e r i o d s a n d a l l o f t h e e g g s fro m t h e h e n s on t h e e x p e r im e n t e v e r y day b e tw e e n D ecem ber 1 2 , 1956 th r o u g h May 2 , 1 9 5 7 . R e s u l t s a n d D is c u s s i o n O v i p o s i t i o n T im e : — The tim e b e tw e e n t h e l a y i n g o f e g g s w i t h i n a c l u t c h a n d b e tw e e n c l u t c h e s was c o m p a re d f o r n o rm a l e g g s a n d e g g s c o n t a i n i n g b lo o d s p o t s . T a b le 3 show s t h e r e s u l t s when a l l o f t h e e g g s w e re g ro u p e d i n m o n th ly u n i t s a n d t h e s e u n i t s a n a ly z e d s t a t i s t i c a l l y . * 1. A l l t h e s t a t i s t i c a l p r o c e d u r e s e m p lo y e d i n t h i s t h e s i s w e re th o s e o f W alk er a n d Lev ( 1 9 5 3 ) . - 38 - TABLE 3 THE AVERAGE TIME BETWEEN EGGS OE A CLUTCH AND BETWEEN CLUTCHES BASED ON MONTHLY AVERAGES N orm al e g g s A v e ra g e tim e b e­ T o t a l tw e e n e g g s eggs (h o u rs) B lo o d s n o t e g g s A v erag e tim e be­ T o t a l tw e e n e g g s (h o u rs) eggs D iff­ e re n c e (m in .) S ta n d a rd e rro r (m in . ) B e tw e e n c l u t c h e s 1 day s k ip 1163 4 3 .2 3 140 4 2 .6 7 3 3 .6 * 1 8 .0 2 day s k ip 88 6 9 .4 5 9 6 8 .6 7 4 9 .3 3 4 .8 B e tw e e n e g g s o f a c l u t c h 2nd1 1109 2 6 .5 7 91 2 6 .0 2 3 3 .0 * 1 4 .8 3 rd 1 713 2 6 .7 2 42 2 6 .0 8 38 • 4 2 5 .9 4 th 1 375 2 6 .0 8 7 2 5 .9 3 9 .0 4 3 .4 A ll2 2 635 2 6 .3 4 172 2 5 .7 9 3 3 .0 * * 1 2 .1 * S ig n if ic a n t a t th e 5 p e rc e n t le v e l ** S i g n i f i c a n t a t t h e 1 p e r c e n t l e v e l . 1 . B e f e r s to t h e tim e b e tw e e n t h i s e g g an d t h e o n e im m e d ia te ly p re c e d in g i t . 2 . B e f e r s to t h e a v e r a g e tim e b e tw e e n a l l o f th e e g g s w i t h i n t h e c lu tc h re g a rd le s s o f c lu tc h p o s itio n . - 39 - The d a t a i n T a b le 3 r e v e a l t h a t t h e tim e i n t e r v a l b e tw e e n s u c c e s s i v e e g g s i n a c l u t c h w as l e s s f o r e g g s c o n t a i n i n g b lo o d s p o t s t h a n f o r n o rm a l e g g s . W here t h e r e w ere e n o u g h e g g s f o r t h e d a t a to b e s i g n i f i c a n t , t h e d i f f e r e n c e i n t h e mean l a y i n g t i m e s a v e r a g e d 33 m in u te s l e s s to l a y an e g g c o n t a i n i n g a b lo o d s p o t t h a n to l a y a n o rm a l e g g . The s t a n d a r d e r r o r o f t h e mean f o r t h e tim e i n t h e o n e d a y s k i p b e tw e e n c l u t c h e s , f i r s t and seco n d egg in a c lu tc h , b e tw e e n th e a n d b e tw e e n a l l o f t h e e g g s w i t h i n a c l u t c h r a n g e d fro m 12 to 18 m in u te s . When t h e d a t a w e re a n a l y z e d o n a n i n d i v i d u a l h e n b a s i s , t h e r e s u l t s w e re s i m i l a r to t h o s e fo u n d when m o n th ly a v e r a g e s w e re c o m p a re d . T h e s e a v e r a g e i n d i v i d u a l h e n c o m p a r is o n s a r e show n i n T a b le 4 . O n ly t h e tim e c o m p a r is o n s f o r a o n e day s k i p b e tw e e n c lu tc h e s , b e tw e e n t h e f i r s t a n d s e c o n d eg g o f a c l u t c h , an d b e tw e e n a l l o f t h e e g g s w i t h i n a c l u t c h w e re a n a ly z e d o n an in d iv id u a l hen b a s is . The a v e r a g e f i g u r e f o r e a c h h e n was t r e a t e d a s a u n i t a n d t h e s e u n i t s w e re a n a ly z e d s t a t i s t i c a l l y . A h i g h l y s i g n i f i c a n t d i f f e r e n c e i n o v i p o s i t i o n tim e was fo u n d b e tw e e n b lo o d s p o t e g g s an d n o rm a l e g g s when t h e a v e r a g e tim e s b e tw e e n e g g s l a i d a f t e r a o n e d a y s k i p w ere c o m p a re d an d a l s o w hen a l l e g g s w i t h i n a c l u t c h d i s r e g a r d i n g c l u t c h p o s i t i o n w e re c o m p a re d . T h e se d i f f e r e n c e s w e re 5 5 .8 m in u te s f o r t h e o n e day s k i p a n d 4 8 .0 m in u te s when a l l o f t h e e g g s w ere c o m p a re d w ith - 40 - TABLE 4 THE AVERAGE TIME BETWEEN EGGS 0 E A CLUTCH AND BETWEEN CLUTCHES BASED ON INDIVIDUAL HEN AVERAGES N orm al e g g s • A v e rag e tim e b e­ tw e e n e g g s T o ta l eggs (h o u rs) B lo o d s p o t e g g s A v erag e tim e b e­ T o t a l tw e e n e g g s (h o u rs) eggs D iff­ e re n ce ( m in .) S tan d ar< e rro r (m in . ) B etw e e n c l u t c h e s 1 day s k i p 937 4 3 .0 1 109 4 2 .0 8 5 5 .8 * * 1 5 .8 B e tw e e n e g g s o f a c l u t c h 2nd1 A ll * 931 2 6 .3 1 74 2 5 .5 7 4 4 .4 * 2 4 .4 2317 2 6 .4 8 147 2 5 .6 8 4 8 .0 * * 1 9 .8 S ig n if ic a n t a t th e 5 p e rc e n t le v e l. ** S i g n i f i c a n t a t t h e 1 p e r c e n t l e v e l . 1 . Tim e b e tw e e n t h i s egg an d t h e o n e p r e c e d i n g i t . - 41 - t h e b lo o d s p o t e g g s r e o u i r i n g l e s s tim e to b e l a i d i n b o th c a s e s . When t h e o v i p o s i t i o n tim e s o f e g g s i n t h e s e c o n d c l u t c h p o s i t i o n w e re c o m p a re d , t h e d i f f e r e n c e a v e r a g e d 4 4 . 4 m in u te s l e s s f o r b lo o d s p o t e g g s a n d was s i g n i f i c a n t a t t h e 5 p e r c e n t l e v e l . As r e p o r t e d i n T a b le 4 t h e s t a n d a r d e r r o r w as 1 5 .8 m in u te s f o r t h e o n e day s k i p b e tw e e n c l u t c h e s , 2 4 .4 m in u te s f o r t h e tim e b e tw e e n t h e f i r s t a n d s e c o n d eg g o f a c l u t c h , a n d 1 9 .8 m in u te s f o r t h e tim e b e tw e e n a l l o f t h e e g g s w i t h i n a c l u t c h . C l u tc h P o s i t i o n : — S in c e t h e b i r d s w ere n o t t r a p p e d u n t i l 8 :0 0 a .m . a n d l i g h t s w e re t u r n e d on a t 4 : 3 0 p .m ., i t w as s u s p e c t e d t h a t t h e d a t a m ig h t b e s l i g h t l y b i a s e d a s t h e f i r s t e g g o f a c l u t c h h a d a g r e a t e r p r o b a b i l i t y o f b e in g l a i d b e f o r e 8 :0 0 a.m . a n d h a d a g r e a t e r p r o b a b i l i t y o f c o n t a i n i n g a b lo o d s p o t th a n d i d t h e e g g s o f s u c c e e d in g c l u t c h p o s i t i o n s . T h is was t r u e i n t h e s e d a t a a n d a g r e e s w ith t h e f i n d i n g s o f J e f f r e y S tra in an d J o h n s o n ( 1 9 5 7 ) . (1 9 4 5 ) an d The p e r c e n t a g e s o f b lo o d s p o t s fo u n d i n e g g s o f t h e v a r i o u s c l u t c h p o s i t i o n s a r e r e p o r t e d i n T a b le 5 . The C hi S q u a re v a l u e o f 2 5 .6 4 r e p o r t e d i n T a b le 5 was s i g n i f i c a n t a t t h e 1 p e r c e n t l e v e l sh o w in g t h a t t h e v a r i a t i o n b e tw e e n t h e o b s e r v e d a n d t h e e x p e c t e d v a l u e s i n T a b le 5 w e re s ig n ific a n tly d iffe re n t, t h a t i s , m ore b lo o d s p o t e g g s w e re f o u n d i n t h e f i r s t c l u t c h p o s i t i o n a n d fe w e r i n t h e t h i r d an d f o u r t h p o s i t i o n s th a n c o u l d b e a c c o u n te d f o r o n t h e b a s i s o f c h a n c e a lo n e . - 42 - TABLE 5 THE PERCENTAGE OP BLOOD SPOT EGGS FOUND IN THE DIFFERENT CIIJTCH POSITIONS C lu tc h p o s itio n N orm al e g g s O b s e rv e d E x p e c te d B lo o d s p o t e g g s O b s e rv e d E x p e c te d P e rc e n ta g e b lo o d s p o t eggs 1 111 8 1 1 4 7 .5 120 9 0 .5 9 .6 9 2 966 9 6 5 .8 76 7 6 .2 7 .2 8 3 651 6 3 9 .6 39 5 0 .4 5 .6 5 4 353 3 3 5 .5 9 2 6 .5 2 .5 0 5 158 1 5 7 .6 12 1 2 .4 7 .0 6 X2 r 2 5 .6 4 * * ** S i g n i f i c a n t a t t h e 1 p e r c e n t l e v e l . - 43 - A C hi S q u a re f r e q u e n c y t e s t w as p e r f o r m e d on t h e d a t a to d e t e r m i n e i f t h e r e w as a s i g n i f i c a n t d i f f e r e n c e b e tw e e n n o rm a l e g g s a n d b lo o d s p o t e g g s l a i d a t o r b e f o r e 8 :0 0 a .m . T h e se d a t a a r e shown i n T a b le 6 . TABLE 6 NORMAL EGGS AND BLOOD SPOT EGGS LAID BE30RE AND AETER 8 :0 0 am. B e f o r e 8 :0 0 a.m . A f t e r 8 :0 0 a.m . N o rm al e g g s O b s e rv e d 730 277 4 E x p e c te d 7 4 7 .0 1 2 7 5 6 .9 9 X2 - 6 .8 3 + B lo o d s p o t e g g s O b s e rv e d 75 197 E x p e c te d 5 7 .9 9 2 1 4 .0 1 * S ig n ific a n t a t th e 5 p e rc e n t le v e l. The C hi S q u a re v a lu e show ed t h a t some b i a s e x i s t e d a s s i g n i f i c a n t l y m ore e g g s c o n t a i n i n g b lo o d s p o t s w e re l a i d b e f o r e 8 :0 0 a .m . th a n o n e w o u ld n o r m a l ly e x p e c t . th is , I t i s c o n c lu d e d fro m t h a t t h e d i f f e r e n c e i n t h e a v e r a g e tim e t h a t b lo o d s p o t a n d n o rm a l e g g s a r e l a i d i s a s l i g h t u n d e r e s t i m a t e , in o th e r w o rd s , t h e d i f f e r e n c e o f 33 m in u te s shown i n T a b le 3 i s s lig h tly - 44 - u n d e r th e p ro b a b le t r u e d if f e r e n c e . The d i f f e r e n c e s r a n g i n g f r o m 4 4 .4 t o 5 5 .8 m in u te s i n T a b le 4 a r e p r o b a b ly m ore a c c u r a t e in a c c o r d a n c e to t h e t r u e d i f f e r e n c e b e c a u s e t h e s y s te m o f a n a l y z i n g t h e s e d a t a p u t l e s s e m p h a s is on e g g s l a i d p r i o r to 8 : 0 0 a . m. B a r o m e tr ic P r e s s u r e ; — S in c e c h a n g e s i n b a r o m e t r ic p r e s s u r e c a n h a v e c e r t a i n p h y s i o l o g i c a l e f f e c t s on a n im a ls , th is a n a l y s i s was c o n d u c te d to d e te r m in e i f t h e r e was a c o r r e ­ l a t i o n b e tw e e n b a r o m e t r i c p r e s s u r e a n d t h e i n c i d e n c e o f b lo o d s p o t s i n e g g s l a i d a t v a r i o u s tim e i n t e r v a l s a f t e r t h e p r e s s u r e w as r e c o r d e d . The tim e i n t e r v a l s u s e d i n t h e c o m p a r is o n s w ere f o r t h o s e e g g s l a i d 24 to 2 8 . 5 , 29 to 3 3 , an d 48 to 52 h o u r s la te r. T he r e s u l t s , shown i n T a b le 7 , i n d i c a t e d t h a t t h e r e was n o s i g n i f i c a n t c o r r e l a t i o n b e tw e e n h i g h o r lo w b a r o m e tr ic p r e s s u r e s a n d t h e i n c i d e n c e o f b lo o d s p o t s . To d e te r m in e i f a d i f f e r e n c e o c c u r r e d i n t h e f r e q u e n c y o f b lo o d s p o t s w hen t h e b a r o m e t r i c p r e s s u r e was i n c r e a s i n g a s c o m p a re d t o when i t a p p lie d . was d e c r e a s i n g , a Chi S q u a re a n a l y s i s was The b a r o m e t r i c p r e s s u r e c h a n g e was d e te r m in e d by t h e d i f f e r e n c e i n p r e s s u r e fro m 7 :3 0 a .m . to 1 :2 0 p .m . w ith t h i s t im e r a n g e b e in g t h e o n ly o n e c o n s i d e r e d . - 45 - TABLE 7 CORRELATION BETWEEN BLOOD SPOTS AND BAROMETRIC PRESSURE Tim e b e tw e e n t h e p r e s s u r e p e r i o d an d when t h e e g g s w e re l a i d ( h o u r s ) C o rre la tio n c o e ffic ie n t t v a lu e ^ .0 1 5 6 .2 6 1 .0 1 8 3 .3 0 7 .0 1 3 .2 1 8 2 4 - 2 8 .5 29 - 33 48 - 52 1. - A t v a lu e o f g r e a t e r t h a n 1 .9 6 i s r e q u i r e d t o be s i g n i f i c a n t a t th e 5 p e r c e n t l e v e l . The r e s u l t s o f t h e d a t a shown i n T a b le 8 i n d i c a t e d t h a t i n c r e a s e s o r d e c r e a s e s i n b a r o m e tr ic p r e s s u r e h a d no e f f e c t on t h e f r e q u e n c y o f b lo o d s p o t s a s n o n e o f t h e C hi S q u a re v a l u e s re a c h e d th e s i g n i f i c a n t le v e l . Egg W eight V a r i a t i o n s : — A c o m p a r is o n was made b e tw e e n t h e a v e r a g e w e ig h t o f n o rm a l e g g s a n d e g g s c o n t a i n i n g b lo o d s p o t s d u r i n g t h e m onth o f May, 1 9 5 7 . O nly t h e e g g s l a i d i n s i m i l a r c l u t c h p o s i t i o n s w e re c o m p a re d . T a b le 9 show s t h e r e s u l t s o f t h i s c o m p a r is o n . The r e s u l t s a s shown i n T a b le 9 i n d i c a t e d t h a t n o rm a l e g g s w e ig h e d s i g n i f i c a n t l y l e s s t h a n d i d b lo o d s p o t e g g s when o n l y e g g s i n t h e f i r s t c l u t c h p o s i t i o n w e re c o m p a re d , an d when a l l o f t h e e g g s d i s r e g a r d i n g c l u t c h p o s i t i o n w e re c o m p a re d . T h e r e was no s i g n i f i c a n t d i f f e r e n c e fo u n d i n t h e eg g w e ig h ts - 46 - TABLE 8 THE EFFECT OF INCREASING AND DECREASING BAROMETRIC PRESSURE ON THE INCIDENCE OF BLOOD SPOTS B a r o m e t r ic p re ssu re change Num ber o f e g g s w i t h B lo o d s p o t s 2 0 1 2 3 4 In c re a s in g D e c r e a s in g 2 4 t o 2 8 .5 h o u r s a f t e r p r e s s u r e c h a n g e (d a y s) 16 16 11 9 6 21 32 20 7 2 In c re a s in g D e c r e a s in g 29 to 33 h o u r s a f t e r p r e s s u r e c h a n g e (d a y s) 35 2 0 15 6 3 1 45 24 9 In c re a s in g D e c r e a s in g 48 to 52 h o u r s ( 18 14 20 26 1. C hi S q u a re v alu e^ - a f t e r p r e s s u r e change d ay s) 12 8 2 8 7 16 6 .9 6 2 1 .0 4 4 1 .8 7 9 A C hi S q u a re v a l u e o f 11*1 o r g r e a t e r i s r e q u i r e d to h e s i g n i f i c a n t a t th e 5 p e r c e n t l e v e l . 2 . The n u m b e rs i n t h e body o f t h e t a b l e a r e t h e f r e q u e n c y t h a t th e nu m b er o f b lo o d s p o t e g g s w e re o b s e r v e d d u r i n g e a c h tim e p e r i o d . TABLE 9 A COMPARISON OF EGG WEIGHTS OF NORMAL EGGS AND BIOOD SPOT EGGS C lu tc h N orm al e g g s p o s i t i o n Number A ve. w t. (g ra m s ) 1 2 A ll 203 169 630 6 1 .0 1 5 6 1 .0 5 9 6 0 .5 5 6 B lo o d s p o t e g g s Number Ave. w t. (g ra m s ) 55 22 98 6 2 .9 4 5 6 0 .2 7 3 6 2 .0 9 2 ** S i g n i f i c a n t a t t h e 1 p e r c e n t l e v e l . Wei g h t S ta n d a r d d i f f e re n c e e r r o r (g ra m s ) (g ra m s ) 1 .9 3 * * .7 9 1 .5 4 * * 0 .3 1 6 1 .0 2 6 0 .3 4 5 - 47 - w hen o n l y t h e e g g s o f t h e s e c o n d c l u t c h p o s i t i o n w e re c o m p a re d . T h i s a n a l y s i s w as d o n e hy u s i n g t h e m o d if i e d t t e s t c o m p a rin g m eans. The s t a n d a r d e r r o r s w ere 0 .3 1 6 g ram s f o r t h e f i r s t c l u t c h p o s i t i o n , 1 .0 2 6 g ra m s f o r t h e s e c o n d c l u t c h p o s i t i o n , an d 0 .3 4 5 g ra m s f o r a l l c l u t c h p o s i t i o n s . S in c e e g g s c o n t a i n i n g b lo o d s p o t s w ere o v u l a t e d s o o n e r a n d w e ig h e d m ore th a n n o rm a l e g g s , i t was p r o b a b l e t h a t t h e b lo o d s p o t e g g s w e re a b n o rm a l a t o r p r i o r to o v u l a t i o n o r t h a t a m al­ f u n c t i o n o c c u r r e d i n t h e r e p r o d u c t i v e o r g a n s w h ich c a u s e d a h e m o rrh a g e o f t h e c a p i l l a r i e B a n d t h u s , b lo o d s p o t s w e re fo rm e d . T h e r e a r e p r o b a b ly o t h e r p h y s i c a l o r b io c h e m ic a l a b n o r m a l i t i e s a s s o c i a t e d w ith t h i s c a p i l l a r y fra g ility . As m ore o f t h e s e f a c t o r s a n d t h e i r a s s o c i a t i o n s becom e know n, i t w i l l be e a s i e r to d e te r m in e why i n t r a f o l l i c u l a r b l e e d i n g fo rm s b lo o d s p o t s . The same e g g s w h ic h w e re u s e d f o r w e ig h t a n a l y s i s w e re a l s o m e a s u re d f o r alb u m en h e i g h t o n a f o u r day a w eek sam p le b a s is . No d i f f e r e n c e s w e re o b s e r v e d i n th e alb u m en h e i g h t o f b l o o d s p o t e g g s a n d n o rm a l e g g s . EXPERIMENT I I I . THE EFFECT OF PHYSICAL DISTURBANCE, SOUND AND LIGHT ON THE INCIDENCE OF BLOOD AND MEAT SPOTS AND OTHER EGG QUALTTY FACTORS R e s e a r c h w o r k e r s h a v e b e e n t r y i n g f o r many y e a r s to a s s o ­ c ia te t h e i n c i d e n c e o f b lo o d a n d m eat s p o t s w ith v a r i o u s e n v ir o n ­ m e n ta l f a c t o r s t h a t w o u ld i n f l u e n c e a l a y i n g h e n 's e m o tio n s o r i n a n y o t h e r way r e s u l t i n t h e a b n o rm a l p r o d u c t i o n o f e g g s . a n d P in o Je ffre y (1 9 4 3 ) f r i g h t e n e d b i r d s w ith d o g s a n d d i d n o t g e t a n i n c r e a s e i n t h e i n c i d e n c e o f b lo o d s p o t s . J e f f r e y (1 9 4 5 ) fo u n d b i r d s h e l d i n c a g e s h a d a s l i g h t l y h i g h e r b lo o d s p o t i n c i d e n c e t h a n d i d s i m i l a r b i r d s on t h e f l o o r . The e n v ir o n m e n ta l f a c t o r s o f p h y s i c a l d i s t u r b a n c e , so u n d a n d l i g h t w e re s t u d i e d i n t h r e e s e p a r a t e p h a s e s i n t h i s e x p e r i ­ m e n t. I t w as th o u g h t t h a t by k e e p in g e a c h o f t h e s e p h a s e s s e p a r a t e and c o m p a r in g d i f f e r e n t ty p e s o f p h y s i c a l d i s t u r b a n c e , s o u n d an d l i g h t , c o u ld be s tu d ie d . th e e x a c t n a tu r e o f t h e i r in f lu e n c e , i f an y , A lth o u g h t h e r e a r e many o t h e r e n v ir o n m e n ta l f a c t o r s s u c h a s t e m p e r a t u r e , h u m i d i ty , o x y g e n an d c a r b o n d io x id e l e v e l s t h a t c o u l d b e s t u d i e d , i t w as f e l t t h a t t h e t h r e e c h o s e n w e re t h e m o st i m p o r t a n t . P h y s i c a l d i s t u r b a n c e c a u s e s b o th a m u s c u la r a n d e m o tio n a l s t r e s s o n t h e b i r d s t h a t m ig h t i n c r e a s e t h e b lo o d p r e s s u r e , h e a r t r a t e , o r c e r t a i n horm one r e l e a s e s to th e p o in t t h a t f r a g i l e c a p i l l a r i e s in th e o v a r ie s a re ru p tu re d . S o u n d a l s o c a u s e s an e m o tio n a l s t r e s s g r e a t e r th a n n o rm a l when - 48 - - 49 - t h e s o u n d i s c o n t i n u o u s o r o f an e x tre m e p i t c h o r i n t e n s i t y . L a y e r s w ere s u b j e c t e d to i n t e r m i t t e n t h i g h p i t c h so u n d w a v es, lo w p i t c h s o u n d w a v e s , m e n t 's s o u n d p h a s e . an d c o n t i n u o u s so u n d s d u r i n g t h e e x p e r i ­ L ig h t i n c o m b in a tio n w ith a c t i v i t y a f f e c t s t h e s e c r e t i o n o f l u t e i n i z i n g h o rm o n e w h ic h i n t u r n a f f e c t s t h e o v u la tio n c y c le . S in c e t h e p r e v i o u s e x p e r im e n t i n d i c a t e d t h a t b o t h t h e tim e o f l a y and th e p o s i t i o n o f th e eg g w i t h i n a c l u t c h i n f l u e n c e b lo o d s p o t i n c i d e n c e , i t i s c o n c e i v a b l e t h a t l i g h t , e i t h e r i n t e r m i t t e n t l y o r c o n tin u o u s ly , c o u ld d i r e c t l y a f f e c t i n t r a f o l l i c u l a r b le e d in g . E x p e r im e n ta l P r o c e d u r e F iv e h u n d r e d y e a r l i n g D e k a lb h y b r i d h e n s w e re ra n d o m ly d i v i d e d i n t o 4 p e n s o f 125 b i r d s . A s c h e d u le was s e t u p t o e v a l u a t e egg p r o d u c t i o n a n d q u a l i t y b e g in n in g w i th a no t r e a t ­ m en t c o n t r o l p e r i o d , a f t e r w h ic h t h e v a r i o u s p h a s e s o f t h e ex ­ p e r i m e n t w e re c a r r i e d o u t i n s u c c e s s i o n a s f o l l o w s : d i s t u r b a n c e p h a s e , no t r e a t m e n t c o n t r o l p e r i o d , no t r e a t m e n t c o n t r o l p e r i o d , c o n tr o l p e rio d . lig h t ph ase, T a b le 10 i l l u s t r a t e s p h y s ic a l so u n d p h a s e , an d a no t r e a t m e n t th e o v e r a l l p la n o f th e e x p e r im e n t . A l l o f t h e b i r d s w e re t r a p n e s t e d e x c e p t i n p e n C o f tim e p e r i o d 2 , w h ic h was t h e c o n t r o l p e n f o r t h e p h y s i c a l d i s t u r b a n c e p h ase. A ll o f t h e e g g s w ere b ro k e n a n d i n s p e c t e d f o r s p o t s e a c h - 50 - TABLE 10 THE DISTRIBUTION OE TREATMENTS BY TIME PERIODS Tim e p e rio d Pen D a te x A 1 8 /1 3 - 9 /1 ? 2 9 / 1 8 - 9 /2 8 3 9 /3 0 - 1 0 /1 4 4 1 0 /1 5 - 1 0 /3 4 H ig h w h is tle 5 1 0 /2 5 - 1 1 /1 6 6 1 1 /1 9 - 1 2 /2 0 15 m in . lig h t p e rio d s in te r­ m itte n t 7 1 3 /2 2 - 1 /8 B C D C o n tr o l p e r i o d S hake "b ird s S c a re "b ird s C o n tr o l N orm al t r a p p i n g C o n tro l p e r io d R a d io Low w h is tle C o n tr o l C o n tro l p e r io d 24 h o u r lig h t C o n tr o l 1 .7 5 h o u r s l i g h t 1 .2 5 h o u r s d a r k in te rm itte n t C o n tro l p e r io d 1 . The p e r i o d f o r t h e e n t i r e e x p e r im e n t was fro m A u g u st 1 2 , 1 9 5 7 , to Ja n u a ry 8, 1958. - 51 - d a y th r o u g h o u t t h e t r e a t m e n t p e r i o d . D u rin g t h e p e r i o d s b e tw e e n t r e a t m e n t s e g g s w e re b r o k e n a n d i n s p e c t e d o n ly e v e r y s e c o n d d a y . A ra n d o m sa m p le o f 15 e g g s p e r p e n was m e a s u re d o n e a c h day o f b r e a k i n g f o r a lb u m e n q u a l i t y . A ll o f t h e e g g s w e re b ro k e n w i t h i n 2 4 h o u r s a f t e r l a y i n g a n d w e re e x a m in e d f o r t h e p r e s e n c e o f b lo o d and m eat s p o ts . T he b r e a k i n g w as d o n e on a g l a s s p l a t e w ith m ir ­ r o r s b o t h u n d e r n e a t h a n d b e h in d t h e g l a s s p l a t e to a llo w f u l l v i s i o n o f t h e b ro k e n o u t e g g . The e g g w e ig h t was m e a s u re d w i t h i n 1 g ram , t h e U n ite d S t a t e s D e p a rtm e n t o f A g r i c u l t u r e s c o r e was b a s e d o n t h e s t a n d a r d U n ite d S t a t e s D e p a rtm e n t o f A g r i c u l t u r e egg q u a lity c h a rt, t h e alb u m en h e i g h t was m e a s u re d w ith a m ic ro ­ m e te r w i t h i n 0 . 1 m i l l i m e t e r a n d t h e s h e l l t h i c k n e s s was m e a s u re d w i t h a n o th e r m ic r o m e te r w i t h i n 0 .0 0 0 1 i n c h e s . Time p e r i o d 2 was t h e p h y s i c a l d i s t u r b a n c e p h a s e o f t h e e x p e r im e n t . I n p e n A t h e h e n s w e re s h a k e n m i l d l y f o r a b o u t 20 t o 3 0 s e c o n d s a s th e y w e re re m o v e d fro m t h e t r a p n e s t s . c a u s e d them to r u f f l e t h e i r f e a t h e r s w ith e a c h s h a k in g . T h is The b i r d s i n p e n B w e re c h a s e d a ro u n d t h e p e n t h r e e tim e s a d ay by v i o l e n t l y w a v in g two l a r g e p i e c e s o f w h ite c l o t h , Uo so u n d s w e re u s e d a n d t h e b i r d s w e re n o t h a n d l e d o t h e r t h a n d u r in g n o rm a l t r a p n e s t i n g . m in u te s i n d u r a t i o n . E ach t r e a t m e n t p e r i o d was fro m 3 to 5 At t h e e n d o f t h i s p e r i o d t h e h e n s ap­ p e a r e d t i r e d a n d a c c u s to m e d to t h e c h a s i n g to t h e p o i n t t h a t t h e y w o u ld no l o n g e r f l y u p , b u t w o u ld s t i l l r u n fro m t h e c h a s e r . - 52 - By t h e e n d o f t h e 1 0 t h day o f t r e a t m e n t t h e b i r d s w o u ld n o t s c a r e e a s ily a n d w o u ld n o t f l y u p r e a d i l y , chased. b u t w o u ld s t i l l r u n when P e n C w as t h e n o n - t r a p n e s t e d c o n t r o l p e n . w e re n o t a b n o r m a lly d i s t u r b e d i n any w ay. n e s te d c o n tro l pen. c o n d itio n s , T h e se b i r d s P e n D was t h e t r a p - T h e se b i r d s w e re t r a p n e s t e d u n d e r n o rm a l a n d w e re n o t o t h e r w i s e a b n o r m a lly d i s t u r b e d . D u rin g t h i s p e r i o d a l l o f t h e e g g s w ere s a v e d an d w ere b ro k e n f o r q u a l i t y m e a s u re m e n ts a n d t h e p r e s e n c e o f b lo o d s p o t s . A f t e r a two w eek no t r e a t m e n t c o n t r o l p e r i o d t h e s o u n d p o r t i o n o f t h e e x p e r im e n t w as i n i t i a t e d . s ile n t I n p e n A, a common ty p e dog w h i s t l e w ith a f r e o u e n c y a b o v e t h e human a u d i b l e r a n g e w as b lo w n f o r a o n e m in u te d u r a t i o n o n c e e a c h h o u r fro m 7 :3 0 a.m . u n t i l 4 :3 0 p .m . e a c h d a y . T h e re w as a s l i g h t so u n d le a k a g e to p e n B b u t n o t to t h e o t h e r p e n s . I n p e n 3 a r a d i o was p la y e d m edium lo u d ( s t a t i o n TSILS) f o r 24 h o u r s p e r d a y . The t r e a t m e n t i n p e n C c o n s i s t e d o f a lo w f r e q u e n c y w h i s t l e blow n f o r a d u ra ­ t i o n o f o n e m in u te o n c e an h o u r b e tw e e n 7 :3 0 a .m . a n d 4 :3 0 p .m . each day. P e n D w as t h e c o n t r o l p e n a n d h a d no a b n o rm a l so u n d a d m in is te re d . T h e re was a l a r g e f e e d room b e tw e e n t h i s p e n a n d th e o t h e r p e n s , th u s p r e v e n tin g a sound le a k a g e . A t h r e e week no t r e a t m e n t c o n t r o l p e r i o d f o ll o w e d t h i s p h a s e o f t h e e x p e r im e n t . The l i g h t p h a s e o f t h e e x p e r im e n t f o llo w e d t h e t h r e e week c o n tr o l p e rio d . P a p e r s a c k s w e re n a i l e d o v e r t h e w indow s an d a s m a l l v e n t i l a t i o n o p e n in g 6 i n c h e s w id e was c o v e r e d w ith b u r l a p - 53 - to a llo w a i r c i r c u l a t i o n . The p e n s w e re a l l u n if o r m i n t h e i r w indow c o v e r i n g a n d v e n t i l a t i o n . Two new 100 w a tt b u lb s w e re p la c e d in each o f th e fo u r p e n s. The d i f f e r e n t l i g h t p e r i o d s w e re s w i tc h e d o n and o f f w i t h e l e c t r i c a l tim e s w i t c h e s . A 56 l i g h t p e r i o d s a n d 4 0 d a r k p e r i o d s w ere a d m i n i s t e r e d . I n pen Each p e r i o d w as 15 m in u te s i n d u r a t i o n f o r a t o t a l o f 14 h o u r s l i g h t a n d 10 h o u r s o f d a r k n e s s . At c e r t a i n i n t e r v a l s i t was n e c e s s a r y t o h a v e 2 c o n s e c u t i v e l i g h t p e r i o d s due to i n t h e tim e c l o c k . a la c k o f f l e x i b i l i t y No l i g h t p e r i o d was l o n g e r th a n 30 m in u te s w i t h o u t an i n t e r v e n i n g d a r k p e r i o d o f 15 m in u te s . on 24 h o u r s a d a y i n p e n B. L i g h t s w e re P e n C was t h e c o n t r o l p e n an d h a d 1 4 c o n t i n u o u s h o u r s o f l i g h t a n d 10 c o n ti n u o u s h o u r s o f d a r k n e s s d u rin g e ac h 24 h o u r p e r io d . Pen D h ad a lte r n a t in g p e rio d s o f 1 .7 5 h o u r s l i g h t w ith 1 .2 5 h o u r s o f d a r k n e s s to g i v e a t o t a l o f 1 4 h o u r s o f l i g h t a n d 10 h o u r s o f d a r k n e s s d u r in g e a c h 24 h o u r p e rio d . A two w eek n o n t r e a t m e n t c o n t r o l p e r i o d f o llo w e d t h e l i g h t p h a s e o f t h e e x p e r im e n t . R e s u l t s an d D is c u s s i o n Egg P r o d u c t i o n : — The v a r i a t i o n s in eg g p r o d u c t i o n d u r in g t h i s e x p e r im e n t a r e shown i n T a b le s 1 1 , 12 a n d 1 3 . - 54 - TABLE 11 EGG PRODUCTION PRIOR TO, BORING, AND FOLLOWING THE PHYSICAL DISTURBANCE PHASE Tim e p e rio d Days Pen T re a tm e n t 1 1 1 1 22 22 22 22 A B C D None None None None 2 2 2 2 10 10 10 10 A B C D Shake "b ird s S c a r e “b i r d s None N o rm al t r a p p i n g 3 3 3 3 8 8 8 8 A B C D None N one None None iT o ta l 1427 1286 1 489 1332 E sg s P r o d u c e d P e rc e n t A v e ra g e n o . p e r day 6 4 .8 6 5 8 .4 5 6 7 .6 8 6 0 .5 4 5 5 .6 8 5 0 .1 8 5 7 .8 5 5 1 .0 9 559 527 629 555 5 5 .9 5 2 .7 6 2 .9 5 5 .5 4 9 .4 7 4 6 .2 3 5 4 .7 0 4 7 .0 3 434 422 467 401 5 4 .2 5 5 2 .7 5 5 8 .3 8 5 0 .1 2 4 8 .0 1 4 6 .2 7 5 0 .7 7 4 2 .4 8 TABLE 12 EGG PRODUCTION DURING AND FOLLOWING THE SOUND PHASE Tim e p e rio d Days Pen T re a tm e n t T o ta l Eggs P ro d u c e d A v e rag e n o . P e r c e n t p e r day 4 4 4 4 10 10 8 10 A B C D High, w h i s t l e 24 h o u r r a d i o Low w h i s t l e None 509 462 417 468 5 0 .9 4 6 .2 5 2 .1 2 4 6 .8 4 5 .4 5 3 7 .0 2 4 6 .1 3 4 0 .3 4 5 5 5 5 11 11 11 11 A B C D None None None N one 4 79 476 465 439 4 3 .5 4 4 3 .2 7 4 2 .2 7 3 9 .9 1 3 8 .8 8 3 7 .9 6 3 7 .7 4 3 4 .4 0 - 55 - TABLE 13 EGG FRODUCTIOH DUHIHG AHD HOLLOT O G THE LIGHT PHASE Tim e p e rio d 6 6 6 6 7 7 7 7 Days Pen T r e a tm e n t T o ta l 18 18 18 18 8 8 8 8 A B C D A B C D E gss P ro d u ce d A v e rag e n o . p e r dav P e rc e n t 15 m in. I t . p e r . 24 h o u r s l i g h t C o n tro l 1 .7 5 h r s . I t . a n d 1 .2 5 h r s . d a r k p e r 3 h o u rs 745 887 888 4 1 .3 9 4 9 .2 8 4 9 .3 3 4 6 .7 2 4 3 .6 1 4 4 .9 4 762 4 2 .3 3 3 7 .4 6 Hone Hone H one H one 330 402 423 348 4 1 .2 5 5 0 .2 5 5 2 .8 8 4 3 .5 0 3 6 .8 3 4 6 .1 0 4 7 .2 1 3 8 .8 4 At t h e b e g in n in g o f t h e e x p e r im e n t t h e b i r d s i n a l l 4 p e n s p r o d u c e d e g g s a t a r a t e o f o v e r 50 p e r c e n t w i t h t h e b i r d s i n p e n C h a v in g t h e h i g h e s t p r o d u c t i o n ( 5 7 .8 5 p e r c e n t ) B t h e lo w e s t ( 5 0 .1 8 p e r c e n t ) . an d th o s e i n p e n D u rin g t h e p h y s i c a l d i s t u r b a n c e p h a s e t h e b i r d s i n a l l t h e p e n s d ro p p e d fro m 3 to 6 p e r c e n t i n p r o d u c t i o n w ith t h e t r e a t m e n t o f s h a k in g t h e b i r d s a s t h e y w e re t r a p p e d sh o w in g t h e l a r g e s t d ro p i n p r o d u c t i o n . The a v e r a g e p e r c e n t a g e p r o d u c t i o n fro m e a c h p e n d u r in g t h e d i f f e r e n t tim e p e r i o d s i s r e p o r t e d i n E ig u r e 3 . T h is g r a p h i l l u s t r a t e s th e g e n e r a l d e c l i n e i n e g g p r o d u c t i o n fro m t h e b e g in n in g o f t h e e x p e r im e n t u n t i l tim e p e r i o d 5 ; a f t e r w hich, t h e r e w as a s l i g h t i n c r e a s e i n eg g p r o d u c t i o n d u r i n g tim e p e r i o d s 6 a n d 7 e x c e p t f o r t h e b i r d s i n p e n A* w h ich show ed a d e c l i n e d u r i n g tim e p e r i o d 7 . 6 0 r Figure 3 . NOlJLDDQOUd III O to CO oJ ■i 1N3 D U 3 d sA-J During Experiment PERIOD (J CD Egg Production TIME - 56 - < UJ LLi R TIME OP LAY; ONE DAY SKIP BETWEEN CLUTCHES M onth T o ta l tim e (h o u rs) N o rm al E g g s T o t a l 3no. Ave. tim e /e g g n o rm a l eggs (h o u rs) B lo o d S p o t Eggs T o ta l T o ta l no. tim e b lo o d (h o u r s ) sp o t eggs D ec. 5 4 2 7 .5 125 4 3 .4 2 381 Jan. 8 2 2 4 .5 191 4 3 .0 6 E eb. 9531 222 M ar. 9 6 0 8 .5 A p r. Ave. tim e / eg^ (h o u rs) 9 4 2 .3 3 7 1 0 .5 17 4 1 .7 9 4 2 .9 3 7 2 2 .5 17 4 2 .5 0 223 4 3 .0 9 1 2 2 2 .5 29 4 2 .1 6 8222 189 4 3 .5 0 953 22 4 3 .3 2 May 6 2 0 7 .5 144 4 3 .1 2 1 5 8 6 .5 37 4 2 .8 8 J-une 3001 69 4 3 .4 9 3 9 3 .5 9 4 3 .7 2 5 0 232 1163 3 0 2 .6 1 5 9 6 9 .5 140 2 8 9 .7 0 A v e ra g e 7176 166 4 3 .2 3 8 5 2 .8 20 4 2 .6 7 T o ta l - 94 - .APPENDIX TABLE I I BIOOD SPOT - NORMAL EGG COMPARISONS K)R TIME OF LAT; TO) DAT SKIP BETWEEN CLUTCHES M onth T o ta l tim e (h o u rs) N orm al S e e s T o ta l no. Ave. t im e / e g g n o rm a l eggs (h o u rs) B lo o d S p o t Eggs T o ta l T o ta l no. tim e b lo o d (h o u r s ) s p o t eggs Ave. tim e /e , (h o u rs D ec. 1 2 4 7 .5 18 6 9 .3 1 - Jan. 6 8 4 .5 10 6 8 .4 5 136 2 6 8 .0 0 Feb. 4 2 1 .0 6 7 0 .1 7 68 1 6 8 .0 0 M ar. 1 3 3 1 .5 19 7 0 .0 8 144 2 7 2 .0 0 A p r. 1 1 0 7 .0 16 6 9 .1 9 2 0 9 .5 3 6 9 .8 3 May 1 1 0 0 .5 16 6 8 .7 8 6 5 .5 1 6 5 .5 June 2 1 0 .5 3 7 0 .1 7 6 1 0 2 .5 88 T o ta l A v e ra g e 8 7 1 .8 1 2 .6 4 8 6 1 .5 6 9 .4 5 - _ _ 6 2 3 .0 9 1 2 4 .6 1 .8 - — 3 4 3 .3 3 6 8 .6 7 - 95 - APPENDIX TABLE I I I BIOOD SPOT - FORMAL EGG COMPARISONS TOR TIME OP LAI; TIME BETWEEN THE PIRST AND SECOND EGG OP A CLUTCH M onth T o ta l tim e (h o u rs) N orm al E ggs T o ta l no. Ave. tim e /e g g n o rm a l eggs (h o u rs) B lo o d S p o t E ggs T o ta l no. T o ta l b lo o d tim e (h o u rs) sp o t eggs Ave. ti m e / e^ (h o u rs' D ec. 2890 111 2 6 .0 4 1 2 6 .5 5 2 5 .3 0 J an. 5 003 187 2 6 .7 5 3 0 7 .5 12 2 5 .6 2 Peb. 5 4 3 4 .5 200 2 7 .1 7 3 6 3 .5 14 2 5 .9 6 M ar. 5924 222 2 6 .6 8 4 1 9 .5 16 2 6 .2 2 A p r. 4690 178 2 6 .3 5 4 1 2 .0 16 2 5 .7 5 May 3 963 150 2 6 .4 2 5 0 0 .0 19 2 6 .3 2 June 1566 61 2 5 .6 7 2 3 9 .0 9 2 6 .5 6 T o ta l 2 9 4 7 0 .5 1109 1 8 5 .0 8 2 3 6 8 .0 91 1 8 1 .7 3 2 6 .4 4 3 3 8 .3 13 2 5 .9 6 A v e ra g e 4 2 1 0 .1 1 5 8 .4 - 96 - APPENDIX TABLE IV BIOOD SPOT - NORMAL EGG COMPARISONS ROD TIME OF LAY; TIME BETWEEN THE SECOND AND THIRD EGG- OF A CLOTCH M o n t h _________ N orm al E g g s_________ T o ta l Ave. T o ta l no. tim e tim e / e g g n o rm a l (h o u rs) eggs (h o u rs) D ec. 2113 J ©n. B lo o d S p o t Eggs T o ta l no. T o ta l tim e b lo o d sp o t eggs (h o u rs) Ave. tim e / e,| (h o u rs' 79 2 6 .7 5 7 3 .0 3 2 4 .3 3 3 1 7 3 .5 117 2 7 .1 2 7 6 .5 3 2 5 .5 0 Feb. 2804 103 2 7 .2 2 1 5 8 .5 6 2 6 .4 2 M ar. 3910 147 2 6 .6 0 1 3 9 .5 5 2 7 .9 0 A p r. 3002 115 2 6 .1 0 3 1 4 .0 12 2 6 .1 7 May 2 8 1 9 .5 106 2 6 .6 0 2 3 1 .5 9 2 5 .7 2 June 1 2 2 7 .5 46 2 6 .6 8 1 0 2 .5 4 2 5 .6 2 1 9 0 4 9 .5 713 1 8 7 .0 7 1 0 9 5 .5 42 1 8 1 .6 6 2 6 .7 1 5 6 .5 6 2 5 . 96 T o ta l A v e ra g e 2 7 2 1 .4 1 0 1 .9 - 97 - APPENDIX TABLE V BLOOD SPOT - NORMAL EGG COMPARISONS FOR TIME OF LAX; TIME BETWEEN THIRD AND FOURTH EGG OF A CLUTCH M onth T o ta l tim e (h o u rs) N orm al S e e s Ave. T o ta l no. tim e /e g g n o rm a l eggs (h o u rs) B lo o d T o ta l tim e (h o u rs) S p o t B ess T o ta l no. "blood sp o t eggs Ave. tim e /e g ^ (h o u rs) D ec. 1 0 0 9 .5 39 2 5 .8 8 - - - J an. 1 2 6 6 .0 47 2 6 .9 4 24 1 2 4 .0 0 Feb. 1 2 2 3 .0 47 2 6 .0 2 28 1 2 8 .0 0 M ar. 1 8 8 3 .0 71 2 6 .5 2 52 2 2 6 .0 0 A p r. 2 0 4 3 .5 79 2 5 .8 7 28 1 2 8 .0 0 May 1 572*0 61 2 5 .7 7 - - - June 7 8 3 .0 31 2 5 .2 6 4 9 .5 2 2 4 .7 5 T o ta l 9 7 .8 0 .5 375 1 8 2 .2 6 1 8 1 .5 7 1 3 0 .7 5 A v e ra g e 1 3 9 7 .2 2 6 .0 4 3 6 .3 5 3 .6 1 .4 2 6 .1 5 - 98 - APPENDIX TABLE VI BLOOD SPOT - NORMAL EGG COMPARISONS TOR TIME OE LAY; TIME BETWEEN ALL OE THE EGGS WITHIN A CLUTCH N orm al Ite e s Ave. T o ta l no. ti m e / e g g n o rm a l eggs (h o u rs) B lo o d T o ta l T o ta l no. tim e b lo o d (h o u rs) sp o t eggs (0 T o ta l tim e (h o u rs) a p so M onth Ave. tim e / e g t (h o u rs) D ec. 8346 320 2 6 .0 8 2 5 2 .5 10 2 5 .2 5 Jan . 1 0 6 1 3 .5 396 2 6 .8 0 408 16 2 5 .5 0 T eh. 1 0 2 9 2 .5 382 2 6 .9 4 6 2 4 .5 24 2 6 .0 2 MaT. 13266 501 2 6 .4 8 736 28 2 6 .2 9 A p r. 1 1 3 8 3 .5 437 2 6 .0 5 985 38 2 5 .9 2 May 11125 425 2 6 .1 8 1 0 1 2 .5 39 2 5 .7 0 4 4 9 5 .5 174 2 5 .8 4 439 17 2 5 .8 2 6 9 5 2 2 .5 2635 1 8 4 .3 7 4 4 5 7 .5 172 1 8 0 .5 0 9 9 3 1 .8 376 2 6 .3 4 6 3 6 .8 June T o ta l A v e ra g e 2 4 .6 2 5 .7 9 - 99 - APPENDIX TABLE 711 PRODUCTION OP TOTAL EGOS AND BLOOD SPOT EGGS DURING THE 7 TIME PERIODS OP EXPERIMENT I I I P e rio d Pen 1 A B C D None None None None 22 22 22 22 1427 1286 1489 1332 5 5 .6 8 5 0 .1 8 5 7 .8 5 5 1 .0 9 101 76 82 52 7 .0 8 5 .9 1 5 .5 1 3 .9 0 40 39 38 27 2 A B. C D Shake "b ird s S c a r e "b ird s No t r a p p i n g N orm al t r a p 10 10 10 10 559 527 629 555 4 9 .4 7 4 6 .2 3 5 4 .7 0 4 7 .0 3 43 37 37 29 7 .6 9 7 .0 2 5 .8 8 5 .2 2 29 22 23 3 A B C D N one None None None 8 8 8 8 434 422 467 401 4 8 .0 1 4 6 .2 7 5 0 .7 7 4 2 .4 8 30 33 21 12 6 .9 1 7 .8 2 4 .5 0 2 .9 9 19 21 16 11 4 A 10 10 509 462 4 5 .4 5 3 7 .0 2 43 45 8 .4 5 9 .7 4 26 23 D H ig h f r e q . whi s t l e 24 h r . r a d i o low f r e q . w h is tle No s o u n d 8 10 417 468 4 6 .1 3 4 0 .3 4 38 22 9 .1 1 4 .7 0 30 19 5 A B C D None None None None 11 11 11 11 479 476 465 439 3 8 .8 8 3 7 .9 6 3 7 .7 4 3 4 .4 0 23 29 33 21 4 .8 0 6 .0 9 7 .1 0 4 .7 8 16 16 20 16 6 A 15 m in . p e rio d s 24 h r s . 14 h r s . 105 m in . 18 18 18 18 745 887 888 762 4 6 .7 2 4 3 .6 1 4 4 .9 4 3 7 .4 6 55 75 48 39 7 .3 8 8 .4 5 5 .4 0 5 .1 2 31 35 31 19 8 8 8 8 330 402 423 348 3 6 .8 3 4 6 .1 0 4 7 .2 1 3 8 .8 4 22 25 19 15 6 .6 7 6 .2 2 4 .4 9 4 .3 1 12 18 15 12 B C B C D 7 A B c D T r e a tm e n t None None None None Days P ro d u c tio n c h e c k e d T o t a l A v e .$ e g g s p ro d . B lo o d S p o ts T o t a l Ave. No. h e n s no. $ la y in g "blood s p o t s lig h t lig h t lig h t lig h t — - 100 - APPENDIX TABLE V III ANALYSES 0E VARIANCE EOR TOTAL BLOOD SPOTS S o u rc e D e g re e s fre e d o m Sum o f sq u ares T im e p e r i o d 1 T r e a tm e n ts E rro r T o ta l 3 84 87 5 5 .6 7 1 2 7 2 .2 2 7 3 2 7 .8 9 8 Tim e p e r i o d 2 T r e a tm e n ts E rro r T o ta l 3 36 39 9 .9 1 0 1 .2 1 1 1 .1 Tim e p e r i o d 3 T r e a tm e n ts E rro r T o ta l 3 28 31 3 3 .7 5 5 2 .2 5 8 6 .0 0 Tim e p e r i o d 4 T r e a tm e n ts E rro r T o ta l 3 34 37 Tim e p e r i o d 5 T r e a tm e n ts E rro r T o ta l Mean sq u are E 1 8 .5 5 7 3 .2 4 1 5 .7 2 6 * * 3 .3 0 2 .8 1 1 .1 7 4 1 1 .2 5 1 .8 7 6 .0 2 8 * * 3 9 .8 7 9 1 1 5 .7 0 0 1 5 5 .5 7 9 1 3 .2 9 3 3 .4 0 3 3 .9 0 6 * 3 40 43 8 .2 7 2 5 8 .3 6 4 6 6 .6 3 6 2 .7 5 7 1 .4 5 9 1 .8 9 6 Tim e p e r i o d 6 T r e a tm e n ts E rro r T o ta l 3 68 71 3 9 .0 4 2 1 4 9 .9 4 4 1 8 8 .9 8 6 1 3 .0 1 4 2 .2 0 5 5 .9 0 2 * * Tim e p e r i o d 7 T r e a tm e n ts E rro r T o ta l 3 28 31' 6 .8 4 4 1 2 9 .1 2 5 1 3 5 .9 6 9 2 .2 8 1 4 .6 1 2 .4 9 4 * S ig n if ic a n t a t th e 5 p e rc e n t le v e l. ** S i g n i f i c a n t a t t h e 1 p e r c e n t l e v e l . - 101 - APPENDIX TABLE IX ANALYSES 0E VARIANCE FOR PERCENTAGE BIOOD SPOTS S o u rc e E D e g re e s fre e d o m Sum o f sq u ares Mean sq u are Tim e p e r i o d 2 T r e a tm e n ts E rro r T o ta l 3 36 39 2 7 .2 3 9 2 9 6 .7 9 3 3 2 4 .0 3 2 9 .0 8 0 8 .2 4 4 1 .1 0 1 Tim e p e r i o d 5 T r e a tm e n ts E rro r T o ta l 3 40 43 3 9 .6 2 2 3 2 8 .0 1 1 3 6 7 .6 3 3 1 3 .2 0 7 8 .2 0 0 1 .6 1 6 - 102 - APPENDIX TABLE X ANALYSES OF VARIANCE TOR SHELL THICKNESS So tir e e D e g re e s fre e d o m Sum o f sq u ares Mean sq u are Tim e p e r i o d 1 Days T r e a tm e n ts E rro r T o ta l 6 3 18 27 1 .1 1 4 .2 2 4 1 .1 1 5 2 .4 5 3 .1 8 6 .0 7 5 .0 6 2 3 .0 0 0 1 .2 1 0 Tim e p e r i o d 3 D ays T r e a tm e n ts E rro r T o ta l 6 3 18 27 1 .7 5 3 .1 6 9 1 .6 1 4 3 .5 3 6 .2 9 2 .0 5 6 3 .0 8 9 6 3 .2 4 4 . 622 Tim e p e r i o d 4 T r e a tm e n t s E rro r T o ta l 3 30 33 .2 9 2 3 .4 7 6 3 .7 6 8 .0 9 7 3 .1 1 5 8 .8 4 0 Tim e p e r i o d 5 D ays T r e a tm e n ts E rro r T o ta l 4 3 12 19 . 666 . 161 3 .1 0 2 3 .9 2 9 .1 6 6 5 .0 5 3 6 .2 5 8 5 .6 4 4 .2 0 7 Tim e p e r i o d 6 D ays T r e a tm e n ts E rro r T o ta l 12 3 36 51 1 .2 7 0 .8 3 4 4 .2 6 8 6 .3 7 2 .1 0 5 8 .2 7 8 .1 1 8 .8 9 8 2 .3 5 6 E - 103 - APPENDIX TABLE XI ANALYSES 0 E VARIANCE EDR EGG WEIGHT S o u rc e D e g re e s fre e d o m Sum o f sq u ares Mean sq u are E Tim e p e r i o d 1 T r e a tm e n ts E rro r T o ta l 3 24 27 .4 3 1 2 1 .1 5 2 2 1 .5 8 3 .1 4 4 .8 8 1 .1 6 3 T im e p e r i o d 2 T r e a tm e n ts E rro r T o ta l 3 28 31 2 .6 9 4 6 4 .6 4 0 6 7 .3 3 4 .8 9 8 2 .3 0 8 .3 8 9 Tim e p e r i o d 3 T r e a tm e n ts E rro r T o ta l 3 24 27 7 .4 3 0 4 7 .1 9 6 5 4 .6 2 6 2 .4 7 7 1 .9 6 6 1 .2 6 0 Tim e p e r i o d 4 T r e a tm e n ts E rro r T o ta l 3 30 33 7 .5 9 1 5 3 .5 4 3 6 1 .1 3 4 2 .5 3 0 1 .7 8 5 1 .4 1 7 Time p e r i o d 5 T r e a tm e n ts E rro r To t a l 3 16 19 2 .0 7 5 1 7 .3 5 2 1 9 .4 2 7 .6 9 2 1 .0 8 4 .6 3 8 - 104 - APPENDIX TABLE XII ANALYSES OP VARIANCE TOR HAUGH SCORE S o u rc e D e g re e s fre e d o m Sam o f sq u ares Mean sq u are P Time p e r i o d 1 T r e a tm e n ts E rro r T o ta l 3 24 27 2 2 .8 4 6 6 5 .2 9 3 8 8 .1 3 9 7 .6 1 5 2 .7 2 0 Tim e p e r i o d 2 T r e a tm e n ts E rro r T o ta l 3 28 31 2 .8 7 0 1 4 6 .9 4 3 1 4 9 .8 1 3 .9 5 7 5 .2 4 8 .1 8 2 Tim e p e r i o d 3 T r e a tm e n ts E rro r T o ta l 3 24 27 1 3 .4 2 6 2 6 6 .7 3 5 2 8 0 .1 6 1 4 .4 7 5 1 1 .1 1 4 .4 0 3 Time p e r i o d 4 T r e a tm e n ts E rro r T o ta l 3 30 33 3 0 .5 3 9 1 1 0 .3 7 0 1 4 0 .9 0 9 1 0 .1 8 0 3 .6 7 9 2 .7 6 7 2 .8 0