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Genetic variation in the rate of early embryonic development of the chick and the relationship between… Crober, Donald Curtis 1971

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GENETIC VARIATION IN THE RATE OF EARLY EMBRYONIC DEVELOPMENT OF THE CHICK AND THE RELATIONSHIP BETWEEN PRE-HATCHING DEVELOPMENTAL RATE AND POST-HATCHING GROWTH by DONALD CURTIS CROBER B.Sc.(Agr.), McGill University, 1961 M . S c , McGill University, 196A  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the  Department of Poultry Science  We a c c e p t t h i s t h e s i s as required standard  c o n f o r m i n g to  THE UNIVERSITY OF BRITISH COLUMBIA January,  1971  the  In  presenting  an  advanced  the I  Library  further  for  degree shall  agree  scholarly  by  his  of  this  written  this  thesis  in  at  University  the  make  tha  it  purposes  for  may  be  It  financial  for  of  Poultry  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  February  1,  1971  of  of  Columbia,  British  by  the  understood  gain  Science Columbia  for  extensive  granted  is  fulfilment  available  permission.  Department  Date  freely  permission  representatives. thesis  partial  shall  Head  be  requirements  reference copying  that  not  the  of  agree  and  of my  I  this  or  allowed  without  that  study. thesis  Department  copying  for  or  publication my  ABSTRACT A study isted in  was c a r r i e d out to d e t e r m i n e i f g e n e t i c  v a r i a t i o n ex-  the r a t e o f embryonic development o f a l i n e of c h i c k embryos  the New Hampshire b r e e d ,  and to examine the e x t e n t to which v a r i a t i o n  e a r l y developmental r a t e ,  i n c l u d i n g that a t t r i b u t a b l e  to genotype,  be r e l a t e d to p o s t - h a t c h i n g growth o f s i b l i n g c h i c k s . mental r a t e examined i n i n d i v i d u a l embryos were  nervous system and  t a l p r o t e i n content,  tissues  within  o f embryonic b l o o d c e l l s ,  and  embryo d r y w e i g h t , Hamilton-Hamburger s t a g e , (LDH) a c t i v i t y and e x p r e s s i o n s  t o t a l a l k a l i n e phosphatase  (AP) a c t i v i t y and e x p r e s s i o n s  b)  total  t h e r e o f , at  existed  i n the a b s o l u t e  rate  96  of c e l l u l a r pro-  n e u r a l tube and metencephalon and, p o s s i b l y , the m y e l e n c e p h a l o n . i n the a c t i v i t y o f the mesencephalon,  the No  diencephalon or  embryonic b l o o d c e l l s were d e t e c t e d .  A l a r g e m a t e r n a l i n f l u e n c e on the  p r o l i f e r a t i v e a c t i v i t y of a l l tissues  examined was f e l t  differences  i n the p r o t e i n c o n t e n t  o f 112-hour  to e x i s t .  d r y w e i g h t but were masked by m a t e r n a l f a c t o r s . h o u r ' p r o t e i n c o n t e n t was e s t i m a t e d Corresponding values  were .294  and .344,  and embryo  The h e r i t a b i l i t y o f the 112-hour  f o r 96-hour and 112-hour  respectively.  e s t i m a t e s were v e r y h i g h .  to be . 4 1 2 ;  Sire  embryos were observed and  may a l s o have e x i s t e d w i t h r e s p e c t to 96-hour p r o t e i n c o n t e n t  .198.  to-  t h e r e o f , and  as d e t e r m i n e d by the t e c h n i q u e o f a u t o r a d i o g r a p h y , o f  such d i f f e r e n c e s  the  incubation.  Sire differences liferation,  could  a) . c e l l u l a r p r o l i f e r a t i o n  l a c t i c a c i d dehydrogenase  and 112 hours o f  in  Indices of develop-  r a t e between 77 and 98 hours o f i n c u b a t i o n o f s p e c i f i c embryonic c e n t r a l  of  96-  e s t i m a t e was  t o t a l LDH a c t i v i t y  Standard e r r o r s o f a l l h e r i t a b i l i t y  A s i r e e f f e c t on 96-hour  t o t a l LDH a c t i v i t y  was i n d i c a t e d and t h e r e was e v i d e n c e to support the e x i s t e n c e o f  a s i m i l a r e f f e c t on 112-hour  Hamilton-Hamburger s t a g e ; m a t e r n a l  factors  d i d not appear to have i n f l u e n c e d e i t h e r o f these embryonic t r a i t s either  i n c u b a t i o n s t a g e examined.  per u n i t o f s u p e r n a t a n t t i o n were o b s e r v e d .  Sire differences  i n t o t a l AP a c t i v i t y  p r o t e i n (AP/SP) determined a t 96 hours o f i n c u b a -  The h e r i t a b i l i t y of 96-hour t o t a l AP A c t i v i t y was  . 5 5 4 and the c o r r e s p o n d i n g 112-hour v a l u e was . 1 1 4 . i s t e d f o r b o t h the a b s o l u t e total protein. i t y decreased  Both the  Sire differences  l e v e l o f LDH^ a c t i v i t y and LDHj per u n i t  exof  p r o p o r t i o n a t e and a b s o l u t e amount o f LDH^ a c t i v -  between 96 and 112 hours o f i n c u b a t i o n .  isozyme was c o n c l u d e d to be d i r e c t l y a s s o c i a t e d at both i n c u b a t i o n s t a g e s examined. content  at  The a c t i v i t y o f  this  with p r o l i f e r a t i v e a c t i v i t y  I t was p o s i t i v e l y r e l a t e d to p r o t e i n  and t o t a l LDH a c t i v i t y and was i n v e r s e l y r e l a t e d to b o t h LDH c o n -  c e n t r a t i o n and LDH,- a c t i v i t y .  T o t a l LDH a c t i v i t y and p r o t e i n  content  were h i g h l y c o r r e l a t e d i n a p o s i t i v e f a s h i o n a t b o t h i n c u b a t i o n s t a g e s . P r o t e i n content  was a l s o p o s i t i v e l y c o r r e l a t e d  to Hamilton-Hamburger stage  a t b o t h s t a g e s w h i l e t o t a l LDH a c t i v i t y and Hamilton-Hamburger stage were c o r r e l a t e d a t 96 hours o f i n c u b a t i o n o n l y . A negative  l i n e a r a s s o c i a t i o n was found between  post-hatching  growth r a t e of males and the p r o l i f e r a t i v e a c t i v i t y o f a l l embryonic t r a l nervous system t i s s u e s  examined (except the d i e n c e p h a l o n ) ,  cen-  and between  female body w e i g h t to f o u r weeks o f age and the a c t i v i t y o f embryonic b l o o d cells.  There was a l s o a n e g a t i v e ,  between post<-hatching  though not always l i n e a r ,  relationship  growth performance and 96-hour Hamilton-Hamburger  stage, p r o t e i n content,  t o t a l LDH a c t i v i t y ,  s i o n s o f AP c o n c e n t r a t i o n ,  t o t a l AP a c t i v i t y , and  e s p e c i a l l y during early post-hatching growth.  P o s t - h a t c h i n g male growth was most c l o s e l y a s s o c i a t e d Hamburger s t a g e and A P / S P ;  expres-  w i t h 96-hour  Hamilton-  female growth was-most c l o s e l y r e l a t e d to  96-hour  p r o t e i n content  and AP a c t i v i t y per u n i t ; of t o t a l p r o t e i n ( A P / T P ) .  v e r s e r e l a t i o n s h i p between r a t e o f embryonic development at  An i n -  112 hours  of  i n c u b a t i o n and p o s t - h a t c h i n g growth was observed but i t was c o n s i d e r a b l y l e s s d e f i n i t e than was t h a t i n v o l v i n g 96-hour d e v e l o p m e n t a l r a t e .  Total  LDH a c t i v i t y and LDH a c t i v i t y per u n i t o f t o t a l p r o t e i n (LDH/TP) were 112-hour  the  embryonic t r a i t s most c l o s e l y a s s o c i a t e d w i t h p o s t - h a t c h i n g male  growth w h i l e female growth was most c l o s e l y r e l a t e d to 112-hour LDH/TP and A P / T P .  I t was c o n c l u d e d t h a t g e n e t i c  differences  i n e a r l y developmental  r a t e c o u l d be r e l a t e d to v a r i a t i o n i n p o s t - h a t c h i n g growth p e r f o r m a n c e .  T A B. L E  INTRODUCTION '  a_F  G • N T E KT S  •  REVIEW DF LITERATURE G e n e t i c D i f f e r e n c e s i n Developmental Rate o f C h i c k Embryos Measurement o f Embryonic Growth. R e l a t i o n s h i p o f D e o x y r i b o n u c l e i c A c i d (DNA) t o Growth o f t h e Chicle Embryo ' . M i t o t i c A c t i v i t y as an Index o f Rate of DNA I n c r e a s e The A p p l i c a t i o n o f A u t o r a d i o g r a p h y t o t h e Measurement o f • M i t o t i c Frequency Changes i n Snsyme A c t i v i t y A s s o c i a t e d w i t h Ontogeny Changes i n the C h i c k Embryo as a Whole Changes i n S p e c i f i c T i s s u e s o f t h e C h i c k Embryo B i o c h e m i c a l Changes D i r e c t l y R e l a t e d t o Changes i n S p e c i f i c I n d i c e s of,, Developmental Rate G e n e t i c V a r i a b i l i t y i n B i o c h e m i c a l A c t i v i t y o f C h i c k Embryos and t h e R e l a t i o n s h i p o f A c t i v i t y D i f f e r e n c e s t o P o s t - H a t c h i n g Growth L a c t i c A c i d Dehydrogenase (LDH) LDH: Changes i n A c t i v i t y D u r i n g Development LDH: S t r u c t u r e and G e n e t i c C o n t r o l LDH: Ontogenetic Changes i n Isozyme C o m p o s i t i o n LDH: I n f l u e n c e o f P y r u v a t e C o n c e n t r a t i o n as a B a s i s f o r O n t o g e n e t i c Changes i n . , I s o z y m e Composition' LDH: Thermal S t a b i l i t y o f Isozymes as a Means-'of D i f f e r e n t i a t i n g S p e c i f i c Forms . LDH: E v i d e n c e f o r More t h a n F i v e Bands A l k a l i n e Fnosphatase (AP) AP: Changes i n A c t i v i t y D u r i n g Development AP: I t s R e l a t i o n s h i p t o Performance T r a i t s i n C h i c k e n s AP: G e n e t i c V a r i a t i o n i n Serum L e v e l s AP: S t r u c t u r e and G e n e t i c C o n t r o l MATERIALS AND METHODS ' A. Growth T r i a l s S t a t i s t i c a l procedures B Embryonic S t u d i e s C o l l e c t i o n and i n c u b a t i o n p r o c e d u r e s 1) Study o f Comparative Rates o f C e l l u l a r P r o l i f e r a t i o n S t a t i s t i c a l procedures ' 2) S t u d i e s of P h y s i c a l , B i o c h e m i c a l and M o r p h o l o g i c a l I n d i c e o f Embryonic Development S t a t i s t i c a l procedures .continued  Page RESULTS  57  Growth T r i a l s ' G e n e t i c d i f f e r e n c e s i n growth t r a i t s examined R e l a t i o n s h i p • "between growth t r a i t s examined B Embryonic S t u d i e s 1) Rate o f C e l l u l a r P r o l i f e r a t i o n Genetic differences i n p r o l i f e r a t i v e a c t i v i t y of embryonic t i s s u e s examined R e l a t i o n s h i p between p r o l i f e r a t i v e a c t i v i t y and p o s t - h a t c h i n g development 2) P h y s i c a l , B i o c h e m i c a l and M o r p h o l o g i c a l I n d i c e s o f Emb r y o n i c Development (a) E x p r e s s i o n s of LDH A c t i v i t y , P r o t e i n C o n t e n t , Embryo Dry Weight and Hamilton-Hamburger Stage • G e n e t i c i n f l u e n c e on e x p r e s s i o n s o f LDH a c t i v i t y , p r o t e i n c o n t e n t , embryo d r y w e i g h t and Hamilton-Hamburger stage R e l a t i o n s h i p between e x p r e s s i o n s o f LDH a c t i v i t y , p r o t e i n c o n t e n t , embryo d r y w e i g h t and H a m i l t o n - H a m burger stage R e l a t i o n s h i p of e x p r e s s i o n s o f LDH a c t i v i t y , p r o t e i n c o n t e n t , embryo d r y w e i g h t and Hamilton-Hamburger s t a g e t o p o s t - h a t c h i n g development (b) E x p r e s s i o n s o f A l k a l i n e Phosphatase A c t i v i t y G e n e t i c i n f l u e n c e on e x p r e s s i o n s o f a l k a l i n e p h o s p h a .tase a c t i v i t y examined R e l a t i o n s h i p between e x p r e s s i o n s o f a l k a l i n e p h o s p h a tase a c t i v i t y R e l a t i o n s h i p between e x p r e s s i o n s o f a l k a l i n e p h o s p h a t a s e t o p o s t - h a t c h i n g development  57 57 59 65 65 65  A  70 74 74 74 91 54 127 127 130 133  DISCUSSICM  1  A  1 5 8  Growth T r i a l s G e n e t i c d i f f e r e n c e s i n growth t r a i t s examined R e l a t i o n s h i p between growth t r a i t s examined B G e n e t i c I n f l u e n c e on Embryonic Developmental Rate and t h e R e l a t i o n s h i p o f I n d i c e s of Developmental Rate t o P o s t - H a t c h i n g •• Growth 1) Rate o f C e l l u l a r P r o l i f e r a t i o n • G e n e t i c d i f f e r e n c e s i n p r o l i f e r a t i v e a c t i v i t y o f embryo n i c t i s s u e s examined R e l a t i o n s h i p of p r o l i f e r a t i v e a c t i v i t y t o ; p o s t - h a t c h i n g development 2) P h y s i c a l , , B i o c h e m i c a l and M o r p h o l o g i c a l I n d i c e s o f Embryonic Development ...continued  5  8  158 159 162 162 162 170 173  Page, DISCUSSION  '  1 7 8  (a)  P r o t e i n C o n t e n t , Embryo Dry W e i g h t , E x p r e s s i o n s o f LDH I A c t i v i t y and Hamilton-Hamburger Stage G e n e t i c i n f l u e n c e on p r o t e i n c o n t e n t • ' I R e l a t i o n s h i p between p r o t e i n c o n t e n t and embryo dry w e i g h t l G e n e t i c i n f l u e n c e on embryo d r y w e i g h t ,190 G e n e t i c ~ i n f l u e n c e on e x p r e s s i o n s o f t o t a l LDH a c t i v i t y 191 Gross changes i n isozyme c o m p o s i t i o n d u r i n g embryonic 195 development Changes i n LDH isozyme c o m p o s i t i o n o f i n d i v i d u a l s i r e 204 groups and t h e i r r e l a t i o n s h i p t o p r o t e i n c o n t e n t , t o t a l LDH a c t i v i t y and LDH c o n c e n t r a t i o n (LDH/TP) G e n e t i c i n f l u e n c e on LDH isozyme l e v e l s 219 - G e n e t i c i n f l u e n c e on Hamilton-Hamburger s t a g e 222 R e l a t i o n s h i p between d i r e c t ( p r o t e i n content, and embryo 224 d r y w e i g h t ) and i n d i r e c t (LDH a c t i v i t y and H a m i l t o n - H a m b u r g e r s t a g e ) i n d i c e s o f embryonic development R e l a t i o n s h i p between d i r e c t and i n d i r e c t i n d i c e s o f em23B b r y o n i c development and p o s t - h a t c h i n g growth (b) E x p r e s s i o n s o f A l k a l i n e Phosphatase A c t i v i t y 281 G e n e t i c i n f l u e n c e on e x p r e s s i o n s o f AP a c t i v i t y . 281 R e l a t i o n s h i p between e x p r e s s i o n s of AP a c t i v i t y 285 R e l a t i o n s h i p between e x p r e s s i o n s o f AP a c t i v i t y 289 and p o s t - h a t c h i n g growth General D i s c u s s i o n 303 7  7  8  SUMMARY AND CONCLUSIONS LITERATURE CITED ., APPENDIX  8  8  7  - . 3 1 7 •  323  LIST OF TABLES Table 1  Probabilities from the A n a l y s i s of V a r i a n c e o f Body Weights and Growth R a t e s , Growth T r i a l s One t o Four.  2  Simple . C o r r e l a t i o n C o e f f i c i e n t s Between Body Weights and Growth R a t e s , Growth T r i a l s One t o Four  3  P r o b a b i l i t i e s from the A n a l y s i s of V a r i a n c e o f P r o l i f e r a t i v e A c t i v i t y o f C e n t r a l Nervous System T i s s u e s P e r formed S e p a r a t e l y on A c t i v i t y V a l u e s of t h e ( l ) Upper and (2) Lower H a l v e s o f Each S e c t i o n , and (3) t h e Complete S e c t i o n  4  P r o b a b i l i t i e s from the A n a l y s i s o f V a r i a n c e of P r o l i f e r a t i v e A c t i v i t y o f C e n t r a l Nervous System T i s s u e s and B l o o d C e l l s ; A c t i v i t y E x p r e s s e d as Combined T o t a l Count, Data o f A n a l y s i s I I  . 5  Rank Order o f S i r e Means o f P r o l i f e r a t i v e A c t i v i t y o f C e n t r a l Nervous System T i s s u e s and B l o o d C e l l s , Comb i n e d Data o f A n a l y s i s I I  G  A n a l y s i s of V a r i a n c e o f P r o l i f e r a t i v e A c t i v i t y o f the N e u r a l Tube (Combined T o t a l Count), t o Determine the E f f e c t o f L e v e l on A c t i v i t y Data o f A n a l y s i s I I  7  Simple C o r r e l a t i o n Coefficients between'Selected Chick Growth T r a i t s and P r o l i f e r a t i v e A c t i v i t y (comb i n e d t o t a l count) o f Embryonic C e n t r a l Nervous S y s tem T i s s u e s and B l o o d C e l l s - Chick Data o f Growth T r i a l s One, Two and Three and Embryo Data o f A n a l y s i s II  8  P r o b a b i l i t i e s from the A n a l y s i s o f V a r i a n c e o f L a c t i c A c i d Dehydrogenase A c t i v i t y and E x p r e s s i o n s T h e r e o f , P r o t e i n C o n t e n t , Embryo Dry Weight and H a m i l t o n - H a m b u r g e r Stage at 96 and 112 Hours o f I n c u b a t i o n : A n a l y s i s o f G e n e t i c E f f e c t s , Experiment One ( i e . , R e p l i c ations One and Two)  9  Rank Order o f S i r e Means o f L a c t i c A c i d Dehydrogenase A c t i v i t y , and E x p r e s s i o n s T h e r e o f , P r o t e i n C o n t e n t , Embryo Dry Weight and Hamilton-Hamburger Stage a t ' 9 6 and 112 Hours o f I n c u b a t i o n , Experiment One ( i e . , R e p l i c a t i o n s One and Two)  .continued  Table 10  11  P r o b a b i l i t i e s from the A n a l y s i s o f V a r i a n c e o f L a c t i c A c i d Dehydrogenase and E x p r e s s i o n s T h e r e o f , P r o t e i n C o n t e n t , Embryo Dry Weight and Hamilton-Hamburger Stage a t 96 and 112 Hours o f I n c u b a t i o n : A n a l y s i s o f E x p e r i m e n t a l E f f e c t s , Experiment One ( i e . , R e p l i c a t i o n s One and Two) . P r o b a b i l i t i e s from t h e A n a l y s i s of V a r i a n c e o f L a c t i c A c i d Dehydrogenase A c t i v i t y and E x p r e s s i o n s T h e r e o f , P r o t e i n C o n t e n t , Embryo Dry Weight and H a m i l t o n - H a m b u r g e r Stage a t 96 and 112 Hours o f I n c u b a t i o n : A n a l y s i s o f G e n e t i c E f f e c t s , Experiment Two ( i e . , R e p l i c a t i o n Three)  12  Rank Order ( T r a i t s 1 t o 20) arid R e l a t i v e P r o p o r t i o n s • ( T r a i t s 18 t o 20) o f S i r e Means of L a c t i c A c i d D e h y d r o • genase A c t i v i t y and E x p r e s s i o n s T h e r e o f , P r o t e i n C o n . t e n t , Embryo Dry Weight and Hamilton-Hamburger Stage a t 96 Hours and 112 Hours o f I n c u b a t i o n , Experiment Two ( i e . , R e p l i c a t i o n Three)  13  P r o b a b i l i t i e s from the A n a l y s i s of V a r i a n c e o f L a c t i c A c i d Dehydrogenase A c t i v i t y and E x p r e s s i o n s T h e r e o f , and P r o t e i n Content,, a t 96 and 112 Hours of I n c u b a t i o n ; A n a l y s i s o f E x p e r i m e n t a l E f f e c t s , Experiment Two ( i e ; , R e p l i c a t i o n Three)  14  P r o b a b i l i t i e s from the A n a l y s i s o f V a r i a n c e o f L a c t i c A c i d Dehydrogenase A c t i v i t y and E x p r e s s i o n s T h e r e o f , P r o t e i n C o n t e n t , Embryo Dry Weight and H a m i l t o n - H a m b u r g e r Stage at 96 and 112 Hours o f I n c u b a t i o n : A n a l y s i s o f G e n e t i c E f f e c t s , E x p e r i m e n t s One and Two Comb i n e d ( i e . , R e p l i c a t i o n s One, Two and Three)  15  Rank Order o f S i r e Means o f L a c t i c A c i d Dehydrogenase A c t i v i t y and E x p r e s s i o n s T h e r e o f , P r o t e i n C o n t e n t , Emb r y o D r y Weight and Hamilton-Hamburger Stage a t 96 and • 112 Hours o f I n c u b a t i o n , E x p e r i m e n t s One and Two ( i e . , R e p l i c a t i o n s One, Two and T h r e e )  16.  ' P r o b a b i l i t i e s from the A n a l y s i s o f V a r i a n c e - o f L a c t i c A c i d Dehydrogenase A c t i v i t y and E x p r e s s i o n s T h e r e o f , P r o t e i n C o n t e n t , Embryo Dry Weight and H a m i l t o n - H a m b u r g e r Stage a t 96 and 112 Hours o f I n c u b a t i o n ; A n a l y s i s o f E x p e r i m e n t a l E f f e c t s , Experiments One and Two ( i e . , R e p l i c a t i o n s One, Two and Three)  .... continued'  E s t i m a t e s o f H e r i t a b i l i t y ( h ) arid t h e i r S t a n d a r d E r r o r s , o f L a c t i c A c i d Dehydrogenase A c t i v i t y and E x p r e s s i o n s T h e r e o f . P r o t e i n C o n t e n t , Embryo Dry Weight and Hamilton-Hamburger Stage a t 96 and 112 Hours o f I n c u b a t i o n . E x p e r i m e n t s One and Two ( i e . , • R e p l i c a t i o n s One, Two and Three) s  S i m p l e C o r r e l a t i o n C o e f f i c i e n t s Between E x p r e s s i o n s o f L a c t i c A c i d Dehydrogenase, T o t a l P r o t e i n , Embryo Dry Weight and Hamilton-Hamburger Stage Computed Sepa r a t e l y f o r 9 6 - and 112-Hour Embryos, Experiment One ( i e . , R e p l i c a t i o n s One and Two) C o r r e l a t i o n C o e f f i c i e n t s Between Embryonic T r a i t s E x amined at 96 Hours o f I n c u b a t i o n i n Experiments One and Two and Male Growth T r a i t s Measured i n Growth T r i a l s One. Two and Three Correlation Examined a t and Two and T r i a l s One,  C o e f f i c i e n t s Between Embryonic T r a i t s 96 Hours o f I n c u b a t i o n i n E x p e r i m e n t s One Female Growth T r a i t s Measured i n Growth Two and Three  C o r r e l a t i o n C o e f f i c i e n t s B e W e e n Embryonic T r a i t s E x amined a t 112 Hours o f I n c u b a t i o n i n E x p e r i m e n t s One and Two.and Male Growth T r a i t s Measured i n Growth T r i a l s One, Two and Three C o r r e l a t i o n C o e f f i c i e n t s Between Embryonic T r a i t s E x amined a t 112 Hours o f I n c u b a t i o n i n E x p e r i m e n t s One and Two and Female Growth T r a i t s Measured i n Growth T r i a l s One, Two and Three Simple R e g r e s s i o n C o e f f i c i e n t s (b) f r o m Simple L i n e a r R e g r e s s i o n A n a l y s e s o f Male Growth T r a i t s (Y) Measured i n Growth T r i a l s One, Two and Three on Embryonic T r a i t s (x) Examined a t 96 Hours o f I n c u b a t i o n i n E x p e r i m e n t s One and Two S i m p l e R e g r e s s i o n C o e f f i c i e n t s (b) From Simple L i n e a r R e g r e s s i o n A n a l y s e s o f Female Growth T r a i t s (Y) Measured i n Growth T r i a l s One, Two and Three on Embryonic T r a i t s (x) Examined a t 96 Hours o f I n c u b a t i o n i n ' E x p e r i m e n t s One and Two  ..continued  S i m p l e R e g r e s s i o n C o e f f i c i e n t s (b) f r o m Simple L i n e a r R e g r e s s i o n A n a l y s e s o f Male Growth T r a i t s (Y) Measured i n Growth T r i a l s One, Two and Three on Embryonic T r a i t s (x) Examined a t 112 Hours o f I n c u b a t i o n i n Experiments One and Two Simple R e g r e s s i o n C o e f f i c i e n t s (b) from Simple L i n e a r R e g r e s s i o n A n a l y s e s o f Female Growth T r a i t s (Y) Measured i n Growth T r i a l s One, Two.and Three on Embryonic T r a i t s (x) Examined a t 112 Hours o f I n c u b a t i o n i n Experiments One and Two C o e f f i c i e n t o f D e t e r m i n a t i o n (R^) and A s s o c i a t e d P r o b a b i l i t y , and the P r o b a b i l i t y A s s o c i a t e d w i t h the P a r t i a l R e g r e s s i o n C o e f f i c i e n t o f the H i g h e s t - O r d e r Independent V a r i a b l e , from N o n - L i n e a r R e g r e s s i o n s of Male Groxrth T r a i t s (Y) on 96-Hour Embryonic T r a i t s (x) Measured i n E x p e r i m e n t s One and Two w h i c h Y i e l d e d an R V a l u e f o r w h i c h the P r o b a b i l i t y was l e s s t h a n 0.10 2  C o e f f i c i e n t o f D e t e r m i n a t i o n (R2) and A s s o c i a t e d P r o b a b i l i t y ^ and t h e P r o b a b i l i t y A s s o c i a t e d w i t h the P a r t i a l R e g r e s s i o n C o e f f i c i e n t o f the H i g h e s t - O r d e r Independent V a r i a b l e , from N o n - L i n e a r R e g r e s s i o n s o f Female Growth T r a i t s (Y) on 96-Hour Embryonic T r a i t s (x) Measured i n . E x p e r i m e n t s One and Two w h i c h Y i e l d e d an R2 V a l u e f o r which' t h e P r o b a b i l i t y was l e s s t h a n 0.10 C o e f f i c i e n t o f D e t e r m i n a t i o n (R ) and A s s o c i a t e d P r o b a b i l i t y and t h e P r o b a b i l i t y A s s o c i a t e d w i t h the P a r t i a l R e g r e s s i o n C o e f f i c i e n t o f the H i g h e s t - O r d e r Independent V a r i a b l e , from N o n - L i n e a r R e g r e s s i o n s o f K a l e Growth T r a i t s (Y) on 112-Hour Embryonic T r a i t s (x) Measured i n E x p e r i m e n t s One and Two w h i c h Y i e l d e d an R V a l u e f o r w h i c h t h e P r o b a b i l i t y was l e s s t h a n 0.10 2  2  C o e f f i c i e n t of D e t e r m i n a t i o n (R ) and A s s o c i a t e d P r o b a b i l i t y , and the P r o b a b i l i t y A s s o c i a t e d w i t h t h e P a r t i a l R e g r e s s i o n C o e f f i c i e n t o f t h e H i g h e s t - O r d e r Independent V a r i a b l e ; from N o n - L i n e a r R e g r e s s i o n s o f Female Growth T r a i t s on 112-Hour Embryonic T r a i t s (x) Measured i n E x p e r i m e n t s One and Two w h i c h Y i e l d e d R2 V a l u e s f o r w h i c h t h e P r o b a b i l i t y was l e s s t h a n 0.10 2  2 C o e f f i c i e n t s o f D e t e r m i n a t i o n (R ) and A s s o c i a t e d P r o b a b i l i t y , and the P r o b a b i l i t y A s s o c i a t e d w i t h Each o f P a r t i a l Regression C o e f f i c i e n t s of M u l t i p l e Linear Regr e s s i o n s of Male and Female Growth T r a i t s (Y)on L a c t i c A c i d Dehydrogenase A c t i v i t y P r o t e i n Content (xi|,) and Hamilton-Hamburger Stage (xO w h i c h Y i e l d e d an R2 . V a l u e f o r w h i c h the P r o b a b i l i t y was Less t h a n 0.10 .continued  P r o b a b i l i t i e s f r o m t h e A n a l y s i s o f V a r i a n c e of A l k a l i n e Phosphatase A c t i v i t y and E x p r e s s i o n s t h e r e o f a t 95 and 112 Hours of I n c u b a t i o n : A n a l y s i s o f G e n e t i c E f f e c t s . Experiment One ( i e . R e p l i c a t i o n s One and Two) ;  Rank Order o f S i r e Means o f A l k a l i n e Phosphatase and E x p r e s s i o n s t h e r e o f , and T o t a l P r o t e i n a t 96 and 112 Hours o f I n c u b a t i o n , Experiment One ( i e . , R e p l i c a t i o n s One' and Two) P r o b a b i l i t i e s from the A n a l y s i s of V a r i a n c e o f - A l k a l i n e Phosphatase A c t i v i t y and E x p r e s s i o n s T h e r e o f a t 96 and 112 Hours of I n c u b a t i o n ; A n a l y s i s of E x p e r i m e n t a l E f f e c t s , Experiment One (ie. R e p l i c a t i o n s One and Two) t  E s t i m a t e s of H e r i t a b i l i t y ( h ) , and t h e i r S t a n d a r d E r r o r s , o f A l k a l i n e Phosphatase A c t i v i t y and E x p r e s s i o n s t h e r e o f a t 96 and 112 Hours o f I n c u b a t i o n , E x p e r i m e n t One ( i e . , R e p l i c a t i o n s One and Two) s  S i m p l e C o r r e l a t i o n C o e f f i c i e n t s Between E x p r e s s i o n s o f A l k a l i n e Phosphatase A c t i v i t y Computed S e p a r a t e l y f o r 9 6 - and 112-Hour Embryos, Experiment One ( i e R e p l i c a t i o n s One and Two) v  C o r r e l a t i o n C o e f f i c i e n t s Between E x p r e s s i o n s o f A l k a l i n e Phosphatase A c t i v i t y Examined at 96 Hours of I n c u b a t i o n i n E x p e r i m e n t One and Male Growth T r a i t s Measured i n Growth T r i a l s One, Two and .Three C o r r e l a t i o n C o e f f i c i e n t s B e W e e n E x p r e s s i o n s of A l k a l i n e Phosphatase A c t i v i t y Examined at 96 Hours o f I n c u b a t i o n i n Experiment One and Female Growth T r a i t s Measured i n Growth T r i a l s One, Two and Three C o r r e l a t i o n C o e f f i c i e n t s BeWeen Expressions of A l k a l i n e Phosphatase A c t i v i t y E x a m i n e d . a t 112 Hours o f I n c u b a t i o n i n Experiment One and Male Growth T r a i t s Measured i n Growth T r i a l s One, Two and T h r e e C o r r e l a t i o n C o e f f i c i e n t s BeWeen E x p r e s s i o n s o f A l k - . a l i n e Phosphatase A c t i v i t y Examined a t 112 Hours o f I n c u b a t i o n i n Experiment One- and Female Growth T r a i t s Measured i n Growth T r i a l s One, Two and Three Simple R e g r e s s i o n C o e f f i c i e n t s ( b ) f r o m Simple L i n e a r R e g r e s s i o n A n a l y s e s o f Male G o w t h T r a i t s (Y) Measured i n Growth T r i a l s One, Two and Three on E x p r e s s i o n s of A l k a l i n e Phosphatase A c t i v i t y (x) Examined at 96 Hours o f I n c u b a t i o n i n E x p e r i m e n t One r  .continued  S i m p l e R e g r e s s i o n C o e f f i c i e n t s (b) from Simple L i n ear R e g r e s s i o n A n a l y s e s o f Female Growth T r a i t s (T) Measured i n Growth T r i a l s One, Two and Three on E x p r e s s i o n s o f A l k a l i n e Phosphatase A c t i v i t y (x) Exami n e d a t 96 Hours o f I n c u b a t i o n i n Experiment One S i m p l e R e g r e s s i o n C o e f f i c i e n t s (b) f r o m Simple L i n ear . R e g r e s s i o n A n a l y s e s o f Male Growth T r a i t s (Y) Measured i n Growth T r i a l s One, Two and Three on E x p r e s s i o n s o f A l k a l i n e . Phosphatase A c t i v i t y (x) E x amined a t 112 Hours o f I n c u b a t i o n i n Experiment One Simple R e g r e s s i o n C o e f f i c i e n t s (b) f r o m Simple L i n ear R e g r e s s i o n A n a l y s e s o f Female Growth T r a i t s (Y) Measured i n Growth T r i a l s One, Two and Three on E x p r e s s i o n s o f A l k a l i n e Phosphatase A c t i v i t y (x) E x amined a t 112 Hours of I n c u b a t i o n i n Experiment One C o e f f i c i e n t s o f D e t e r m i n a t i o n ( R ) . and A s s o c i a t e d P r o b a b i l i t y , and the P r o b a b i l i t y A s s o c i a t e d w i t h Each of the P a r t i a l Regression C o e f f i c i e n t s of the H i g h e s t Order Independent V a r i a b l e from N o n - L i n e a r R e g r e s s i o n s o f Male and Female Growth T r a i t s (Y) on 96-Hour E x p r e s s i o n s o f A l k a l i n e Phosphatase A c t i v i t y (x) Measured i n E x p e r i m e n t One w h i c h Y i e l d e d an R V a l u e f o r w h i c h t h e P r o b a b i l i t y was l e s s t h a n 0.10 2  C o e f f i c i e n t s o f D e t e r m i n a t i o n ( R ) and A s s o c i a t e d P r o b a b i l i t y , and the P r o b a b i l i t y A s s o c i a t e d w i t h each o f t h e P a r t i a l R e g r e s s i o n C o e f f i c i e n t s o f the H i g h e s t Order Independent V a r i a b l e from N o n - L i n e a r R e g r e s s i o n s o f H a l e and Female Growth T r a i t s (Y) on 112-Hour E x p r e s s i o n s o f A l k a l i n e Phosphatase f o r w h i c h t h e P r o b a b i l i t y was l e s s t h a n 0.10 2  2  C o e f f i c i e n t s o f D e t e r m i n a t i o n (R ) and A s s o c i a t e d P r o b a b i l i t y , and the P r o b a b i l i t y A s s o c i a t e d w i t h each of the P a r t i a l Regression C o e f f i c i e n t of M u l t i p l e L i n ear R e g r e s s i o n s o f Male and Female Growth T r a i t s (Y) ' on A l k a l i n e Phosphatase A c t i v i t y ( x i ) , A P / T o t a l P r o t e i n ( x 2 ) , AP S u p e r n a t a n t P r o t e i n (2:3), and AP/Smbryo Dry -Weight (24)which Y i e l d e d an R V a l u e f o r w h i c h - t h e P r o b a b i l i t y v/as l e s s t h a n 0.10 2  L I S T OF F I G U R E S  1  Diagrammatic r e p r e s e n t a t i o n o f l o n g i t u d i n a l s e c t i o n o f a t y p i c a l 96-hour c h i c k embryo  2  H i s t o g r a m of 9 6 - and 112-hour means (Experiment Two) o f . t h e a b s o l u t e amounts o f l a c t i c a c i d dehydrogenase (LDH) a c t i v i t y and t o t a l p r o t e i n , and o f LDH]_, LDH5 and L D H 2 - 4 e x p r e s s e d b o t h i n a b s o l u t e terms and as a p r o p o r t i o n o f the- t o t a l LDH a c t i v i t y o f each s i r e  3  G e n e r a l forms o f the c u r v e s d e f i n e d by t h e polynomial  second-degree  4  R e g r e s s i o n o f t h r e e - w e e k body w e i g h t o f males on 96-hour l a c t i c a c i d dehydrogenase a c t i v i t y  5  G e n e r a l forms o f t h e c u r v e s d e f i n e d by the polynomial  6  T h i r d - d e g r e e p o l y n o m i a l r e g r e s s i o n b f t h r e e week, female body w e i g h t on 96-hour t o t a l p r o t e i n  7  T h i r d - d e g r e e p o l y n o m i a l r e g r e s s i o n of seven-week female body w e i g h t on 1 1 2 - h o u r . a l k a l i n e phosphatase a c t i v i t y per u n i t o f . t o t a l p r o t e i n (AP/TF)  third-degree  ACKNOWLE  D GEM  Completion o^ the project  BUT  S  on wliich tku> thefts  hcu> been ph.epax.ed  could not have been accomplished uuXhout the generous advi.ee and o^many {riends' and  associates.  The author Is extremely grateful excellent  supervision  the project  His encouragement and co-operation  OttavJa.  Division,  preparation  axe greatly  Canada Department o{ Agxicultuxe,  Statistics  to Dx. C W . Roberts under whose  and thesis  Thanks axe also due to personnel Section,  assistance  oat.  appreciated.  1  oi the Statistical  Research  Ottawa,and the Departmental  Vepaxtment o{ Indian Avoirs  In paxticulax,  was carried  and Northern Development,  the author wishes to thank Mrs. lhah.gan.et Nightman  and Wi. 8. Sakamoto. The awthox wishes to express his gratitude {or (vis encouragement during the course ol this alio  expressed lor the assistance  the poultry  to Processor J. Biely  study.  given by {ellow students  is  and members ojj  {arm sta{{.  Warmest thanks are aJUo extended to my wi{e, preparation  Appreciation  o{ the manuscript,  and {or her continuing  ragement throughout the course o{ both the project  both {or patience  and thesis  her  and encoupreparation.  1 INTRODUCTION The f i e l d of a p p l i e d q u a n t i t a t i v e  g e n e t i c s has advanced t o  p o i n t where i t has become a h i g h l y complex d i s c i p l i n e w i t h i n the a r e a of a p p l i e d b i o l o g y .  broader  The numerous s o p h i s t i c a t e d p r i n c i p l e s developed  over many y e a r s .\ hich have been used t o e x p l o i t the i n h e r e n t T  the  quantitative  T  v a r i a t i o n of a v a r i e t y of p r a c t i c a l c h a r a c t e r i s t i c s  of numerous p l a n t s and  a n i m a l s have y i e l d e d s i g n i f i c a n t rewards i n terms of the improvement made on many t r a i t s  of economic i m p o r t a n c e .  I n p a r t i c u l a r , improvements i n  b o t h growth and r e p r o d u c t i v e c h a r a c t e r i s t i c s  of p o u l t r y t h r o u g h the  s c a l e a p p l i c a t i o n o f c l a s s i c a l p r i n c i p l e s of q u a n t i t a t i v e  large-  g e n e t i c s have  been  q u i t e d r a m a t i c . ' I r o n i c a l l y , i t has become a p r o g r e s s i v e l y more: f o r m i d a b l e t a s k t o b r i n g about f u r t h e r improvement of many o f t h e s e because o f the e x t e n t t o w h i c h the g e n e t i c  characteristics  variation directly  associated  w i t h them has been f u r t h e r reduced w i t h the i n t r o d u c t i o n , a t the level,  practical  of each new p r i n c i p l e . Significantly,  have been s e l e c t e d  quantitative traits  f o r improvement, w i t h few e x c e p t i o n s ,  of t h e i r d i r e c t p r a c t i c a l s i g n i f i c a n c e , by. g e n e t i c i s t s  as attempts  the a p p l i c a t i o n o f q u a n t i t a t i v e merit consideration.  biological' variables,  p u r e l y on the  basis  l i t t l e or no r e g a r d h a v i n g been g i v e n  t o the b i o l o g i c a l f a c t o r s  I t seems apparent t h a t ,  measures  of domestic a n i m a l s i n g e n e r a l  involved i n their  determination.  t o f u r t h e r improve such t r a i t s  g e n e t i c s become more f u t i l e ,  through  alternate  S p e c i f i c a l l y , i n v e s t i g a t i o n i n t o the p u r e l y  i n terms of g e n e t i c  v a r i a t i o n , which influence  o m i c a l l y i m p o r t a n t t r a i t s w o u l d seem t o be a p r o m i s i n g a l t e r n a t i v e . w o u l d e x a m i n a t i o n o f the g e n e t i c  characteristics  econNot o n l y  of such v a r i a b l e s p r o v i d e a b e t t e r  2  u n d e r s t a n d i n g o f the p h y s i o l o g i c a l and b i o c h e m i c a l p r o c e s s e s w h i c h are m a n i p u l a t e d as the r e s u l t of g e n e t i c  change i n a p u r e l y economic  trait•  t h e r e . u o u l d a l s o e x i s t the r e a l p o s s i b i l i t y t h a t v a r i a t i o n r e m a i n i n g i n p h y s i o l o g i c a l and b i o c h e m i c a l f a c t o r s  d i r e c t l y associated with a particular  economic t r a i t w o u l d be s u f f i c i e n t t h a t c e s s e s would b e ,  g e n e t i c m a n i p u l a t i o n of such p r o -  i n economic t e r m s , . w o r t h w h i l e .  The r a t e of growth of meat-type i c a n c e i n the a f o r e m e n t i o n e d economic.importance, genetic  context  p o u l t r y i s of p a r t i c u l a r  in that,  because of i t s  fundamental  i t i s c h a r a c t e r i z e d by the f a c t t h a t q u i t e l i m i t e d  v a r i a t i o n remains a s s o c i a t e d w i t h i t i n ' m o s t improved s t o c k s .  a consequence,  d i r e c t attempts  t o f u r t h e r improve t h i s c h a r a c t e r i s t i c  become p r o g r e s s i v e l y more i n e f f e c t i v e .  variability,  and growth i t s e l f ,  means by w h i c h changes, teristic  would o b v i o u s l y represent  through s e l e c t i v e  might be p o s s i b l e .  an  It  genetic  b r e e d i n g , i n the l a t t e r  charac-  The e x t e n t t o w h i c h a procedure w o u l d be  i s a well-known fact that,  growth performance  ent i s e s p e c i a l l y i m p o r t a n t .  i n recent years,  trait  i t has become  c h i c k e n s a t a r e l a t i v e l y young age;  thus,  d u r i n g the e a r l y p e r i o d of growth w h i c h a t S i n c e growth o f the organism  pres-  theoretically  b e g i n s w i t h f e r t i l i z a t i o n o f the ovum, i t might be expected t h a t i n g growth o f c h i c k e n s ,  of  c o r r e l a t i o n between i t and growth r a t e .  p r a c t i c a l t o market meat-type is'the  have  alternative  s u c c e s s f u l w o u l d be dependent on the h e r i t a b i l i t y o f the i n d i r e c t and t h e degree o f g e n e t i c  As  Demonstration of the existence  a r e l a t i o n s h i p between an i n d i r e c t i n d e x of growth w h i c h d i s p l a y e d  it  signif-  post-hatch-  e s p e c i a l l y d u r i n g the f i r s t s e v e r a l weeks, w o u l d be  r e l a t e d i n some manner t o growth d u r i n g t h e p r e - h a t c h i n g p e r i o d .  In f a c t ,  3  s t u d i e s p r e v i o u s l y conducted have shown t h i s t o be the case w i t h r e s p e c t t o l a t e embryonic development.  The p r e s e n t  s t u d y was c a r r i e d out i n order  t o determine i f p o s t - h a t c h i n g growth was a l s o r e l a t e d t o e a r l y embryonic developmental r a t e ;  i t was h y p o t h e s i z e d t h a t g e n e t i c  p e r i o d w o u l d be more marked t h a n d u r i n g subsequent mental f a c t o r s tion.  differences  during t h i s  development as e n v i r o n -  e x e r t an i n c r e a s i n g i n f l u e n c e d u r i n g t h e course of i n c u b a -  . S p e c i f i c a l l y , the purpose of t h i s s t u d y was t h r e e - f o l d -  ermine i f g e n e t i c  differences  a) t o  det-  i n r a t e o f embryonic development c o u l d be  demonstrated p r i o r t o f i v e days of i n c u b a t i o n ,  b) t o determine the  extent  t o w h i c h any such d i f f e r e n c e s were a s s o c i a t e d w i t h p o s t - h a t c h i n g growth, and  c) t o p r o v i d e a d d i t i o n a l knowledge, of a q u a n t i t a t i v e n a t u r e ,  regarding  the p h y s i o l o g i c a l and b i o c h e m i c a l c o n t r o l mechanisms by w h i c h r a t e of emb r y o n i c development i s d e t e r m i n e d .  Developmental r a t e o f c h i c k embryos was  examined i n terms o f r a t e of c e l l u l a r p r o l i f e r a t i o n , embryonic s i z e ,  and  a c t i v i t y l e v e l s o f s p e c i f i c enzymes known t o change d u r i n g the course  of  development.  poten-  tial  The r e s u l t s  r e p o r t e d h e r e i n are f e l t t o demonstrate  the  v a l u e o f s t u d y i n g the r a t e o f e a r l y embryonic development o f the  as a means o f o b t a i n i n g a more complete u n d e r s t a n d i n g o f the  relationship  between p r e - and p o s t - h a t c h i n g development i n the c h i c k e n , and t o t h e p o s s i b l e rewards of e m p l o y i n g t r a d i t i o n a l q u a n t i t a t i v e  genetic  illustrate prin-  c i p l e s t o the a l t e r a t i o n of e a r l y developmental r a t e i n o r d e r t o b r i n g about improvement i n p o s t - h a t c h i n g growth i n t h a t  species.  chicken  4 REVIEW OF LITERATURE Genetic D i f f e r e n c e s  i n Developmental. Rate o f C h i c k Embryos  The i n f l u e n c e o f genotype on t h e r a t e o f development o f t h e c h i c k embryo has been examined by many, w o r k e r s , differences  B y e r l y (1930, 1932) observed b r e e d  i n embryonic w e i g h t among progeny from the Rhode I s l a n d Red ( R . I . R . )  and W h i t e Leghorn  (W.L.) breeds,  and c r o s s e s between t h e s e b r e e d s .  R.I.R.  and c r o s s b r e d embryos were f o u n d t o be h e a v i e r than W . L . embryos from the t e n t h day o f i n c u b a t i o n onward, a l t h o u g h W . L . and c r o s s b r e d embryos were h e a v i e r R.I.R.  embryos p r i o r t o t e n d a y s .  b r y o n i c w e i g h t , expressed e i t h e r  Henderson (1930) found no d i f f e r e n c e on a wet or d r y w e i g h t b a s i s ,  than  i n em-  between Dark  C o r n i s h and W . L . embryos from 96 h o u r s o f i n c u b a t i o n t o h a t c h i n g n o r d i d h e . d e t e c t any d i f f e r e n c e  i n t o t a l n i t r o g e n content  o f e m b r y o s . o f t h e two b r e e d s .  B l u n n and Gregory (1935) observed s i g n i f i c a n t d i f f e r e n c e s and W . L . breeds  i n favour of the.former  i n t h e r a t e o f c e l l p r o l i f e r a t i o n , as  r e p r e s e n t e d by m i t o t i c f r e q u e n c y o r t h e number o f c e l l s 72 h o u r s and f o u r t e e n days o f i n c u b a t i o n .  that,  p e r u n i t volume, a t  The number o f m i t o t i c f i g u r e s was  a l s o g r e a t e r a t n i n e t e e n days i n t h e R . I . R . f e r e n c e s , however,  between t h e R . I . R .  embryos.  e x i s t e d i n embryonic w e i g h t .  No s i g n i f i c a n t b r e e d  B y e r l y e_t al_. (1938) f o u n d  i n a comparison o f d i f f e r e n t breeds and c r o s s b r e d s ,  embryos f r o m h e a v i e r  p a r e n t s tended to be h e a v i e r t h a n t h o s e from l i g h t e r p a r e n t s between and seventeen  dif-  eleven  days o f i n c u b a t i o n .  Krzanowska (1959) s t u d i e d t h e e a r l y embryonic growth o f c h i c k emb r y o s a t 24 and 48 h o u r s o f development.  He o b t a i n e d s i g n i f i c a n t  differences  between two Brown Leghorn l i n e s i n d e o x y r i b o n u c l e i c a c i d (DNA) c o n t e n t ,  the  number o f n u c l e i i n t h e b l a s t o d e r m , Hamilton-Hamburger s t a g e , and b l a s t o d i s c diameter.  He a l s o n o t e d t h a t h e t e r o s i s  e x i s t e d f o r embryonic growth a t 24  5  hours with, cross-bred embryos being generally superior to either of the pure lines.  Somite number at 38 hours and embryo weight at one and too weeks of  incubation of inbred and crossbred embryos were compared by Mcl'Iary et al_. (i960). Two lines of W.L.,  an inbred line of R.I.R. and an inbred line of New Hamp-  shires (N.H.) were mated in all.combinations.  Significant genetic differences  were observed in a l l three traits at a l l stages and heterosis for weight was apparent for both weighing stages.  Also, there was evidence for the  existence of maternal effects on weight in one of the W.L. Si.  §A."  (1962)  lines.  Tolman  compared the growth rate of two- and three-way crossbred embryos  in terms of somite number at 38 hours,- and weight at six, ten and fifteen days. . It was found that hen differences were responsible for most of the variation in embryonic development at a l l stages. . At fifteen days there were genetic differences in embryo weight. Bray and Iton (1962) reported differences in embryo weight among"strains of chickens between nine and fourteen days of incubation. The correlation between embryonic growth rate over this period and chick weight to three weeks" of age was 0.3  (p<0.10).  Bray (1963) demonstrated  sire differences within strains for embryo weight at eighteen days of i n cubation in four different strains of turkeys.  'Genetic' correlations be-  tween eighteen-day weight and poult weight ranged between 0.4 and 0.7 in two of the strains but were very low in the Wo  other strains.  Ismail (1963)  reported a genetic correlation between fourteen-day embryo weight of chickens and eight-week old mala siblings of -0.39 while that between female siblings and the same embryos was 0.35.  Coleman et al_. (1964) observed that the  em-  bryo weight of a line selected for high body weight during the period from fourteen to nineteen days of incubatior- was significantly greater than -chat of another line selected for low body weight.  However, the low line had a s i g -  6 n i f i c a n t l y h i g h e r somite count a t 42 h o u r s o f i n c u b a t i o n . Deland (1965) made a comparison of t h e r a t e s of growth and body w e i g h t s between p r e - and p o s t - h a t c h i n g , s t a g e s of development o f t h e c h i c k e n as a f f e c t e d concentrated  by genotype.  S i g n i f i c a n t genetic  e f f e c t s on embryonic g r o w t h ,  m a i n l y i n t h e p e r i o d from e i g h t t o f o u r t e e n days o f i n c u b a t i o n ,  were o b s e r v e d .  An i n v e r s e r e l a t i o n s h i p e x i s t e d between t h e growth r a t e s o f  t h e e a r l y and l a t e embryonic stages'.  Embryonic growth between . e i g h t and  t w e l v e days o f i n c u b a t i o n was found t o be s i g n i f i c a n t l y c o r r e l a t e d w i t h b o t h • 6-week body w e i g h t and 6-7 week growth r a t e .  ' Z e r v a s and C o l l i n s  r e p o r t e d on a s t u d y o f t h e i n f l u e n c e o f genotype the c h i c k e n .  on 14-day embryo w e i g h t o f  They observed s i g n i f i c a n t l i n e and s i r e d i f f e r e n c e s  p a r i s o n i n v o l v i n g two d i f f e r e n t l i n e s and t h e i r c r o s s b r e d s . h e r i t a b i l i t y based on t h e s i r e •Maternal'effects  (19S5) have  i n a com-  Estimates  of  component were 0 . 0 2 and 0.31 f o r t h e two l i n e s .  were c l e a r l y e v i d e n t i n c r o s s - b r e d  embryos.  K o s i n and A r o r a (l96G) s t u d i e d e a r l y embryo' g e n e s i s i n W o l i n e s o f Broad B r e a s t e d Bronze t u r k e y s , one o f w h i c h had been s e l e c t e d mature body w e i g h t . tion,  for high  The r a t e o f development, b o t h b e f o r e and a f t e r  incuba-  as measured by t h e diameter o f t h e germ d i s c , l e n g t h o f t h e a r e a  pell-  u c i d a , number o f s o m i t e s and w i d t h o f one s i d e o f t h e a r e a v a s c u l o s a , was g e n e r a l l y h i g h e r i n t h e l i n e s e l e c t e d f o r h i g h body w e i g h t . Measurement o f Embryonic Growth ''Growth r a t e , increase  a l t h o u g h g e n e r a l l y d e f i n e d o p e r a t i o n a l l y as s i m p l y an  i n w e i g h t , i s , from t h e developmental s t a n d p o i n t ,  d i f f e r e n t i a t i o n and morphogenesis,  albeit  c h a r a c t e r i z e d by  involving biological synthesis.  r a t e of t h e embryo, t h e n , and p a r t i c u l a r l y t h e young embryo, can be i n t e r m s . o f v a r i o u s developmental • changes,  including weight.  Growth  represented  A considerable  number o f t h e s e have been employed as i n d i c e s o f growth r a t e o f t h e c h i c k embryo.  7 As a l r e a d y i n d i c a t e d (Henderson, 1930; B y e r l y e t a l . , 1938; McNary e t a l . , I 9 6 0 ; D e l a n d , 1965; and o t h e r s ) , e m b r y o prominent u s e .  w e i g h t i t s e l f has had by f a r t h e most  A comparison o f t h e wet and d r y w e i g h t growth c u r v e s o f the  c h i c k embryo has been g i v e n by Romanoff. little  difference  (1967) who p o i n t e d o u t t h a t t h e r e i s  i n the shape o f t h e two c u r v e s , p a r t i c u l a r l y d u r i n g e a r l y  development. B e r n i e r e t al_. (1951') r e p o r t e d t h a t t h e s i z e o f the embryo, as d e t e r m i n e d by t h e maximum diameter o f the b l a s t o d e r m , was a r e l i a b l e though somewhat i n e x a c t measure o f c e l l u l a r development.  T a y l o r (1939), Hamburger  and H a m i l t o n ( c i t e d i n O r l o v ) and O r l o v (1962) have employed t h e p r i n c i p l e o f comparison o f d i s c diameter i n s t u d i e s o f embryonic g r o w t h . p o r t e d t h a t b o t h embryo l e n g t h and a r e a , f o r m a t i o n on developmental s t a g e . an i n d e x o f p r o t e i n c o n t e n t ,  used t o g e t h e r ,  can p r o v i d e u s e f u l i n -  has a l s o been used as a measure o f developmental Protein constitutes  c h e m i c a l component o f t h e young embryo, a n d , i n g e n e r a l ,  t o t a l n i t r o g e n corresponds t o the increase e a r l y development  (Romanoff,  (1968) r e -  As a l r e a d y mentioned, t o t a l n i t r o g e n , as  r a t e o f the c h i c k embryo (Henderson, 1 9 3 0 ) . est  Elias  much t h e l a r g  the i n c r e a s e i n  i n p r o t e i n d u r i n g t h e course o f  1967).  R e l a t i o n s h i p o f D e o x y r i b o n u c l e i c A c i d (DNA) t o Growth o f t h e C h i c k Embryo Changes i n DNA as r e l a t e d t o embryonic development have been s t u d i e d by s e v e r a l w o r k e r s .  Solomon (1957a) observed an e x p o n e n t i a l i n c r e a s e  number and a s i m i l a r i n c r e a s e  in cell  i n DNA d u r i n g t h e f i r s t t h r e e days o f i n c u b a t i o n  i l l u s t r a t i n g t h e p r i n c i p l e o f c o n s t a n c y o f DNA p e r n u c l e u s .  He c o n c l u d e d t h a t  i n t h e young c h i c k embryo, t h e I n c r e a s e i n DNA p a r a l l e l e d t h e r a t e o f c e l l u l a r proliferation.  He a l s o found t h a t d u r i n g development t h e r a t e o f i n c r e a s e  p r o t e i n , wet w e i g h t and DNA a l l f o l l o w e d a s i m i l a r l o g a r i t h m i c c u r v e .  of  8  Emanuelsson (1961a), who e s t i m a t e d DNA i n c h i c k embryos from zero t o 48 h o u r s o f i n c u b a t i o n , r e p o r t e d t h a t t h e r e were a l t e r n a t i n g p e r i o d s o f p r o l i f e r a t i o n and d i f f e r e n t i a t i o n as i n d i c a t e d by a . v a r i a b l e r a t e o f i n c r e a s e N o v i k o f f and P o t t e r  (1948), Reddy e t a l . (1952) and M a r r i a n e t a l . (1957)  have a l l r e p o r t e d on the p a t t e r n o f i n c r e a s e the c o u r s e o f development. most r a p i d i n c r e a s e cubation.  o f DNA c o n c e n t r a t i o n  I n a l l these s t u d i e s ,  throughout  i t was observed t h a t t h e  i n c o n c e n t r a t i o n o c c u r r e d around the f i f t e e n t h day o f i n -  M a r r i a n e t al_. (1957) a l s o n o t e d an e x c e l l e n t  w e i g h t and t o t a l DNA c o n t e n t . t a l DNA i n c r e a s e d  o f DNA.  agreement between wet  Shimbayashi and Iwamoto (1964) r e p o r t e d t h a t t o -  i n d i r e c t r e l a t i o n t o t h e growth of the embryo from t h r e e t o  e i g h t e e n days o f i n c u b a t i o n .  They observed the p e r i o d o f most r a p i d  increase  t o be between the t w e l f t h and f i f t e e n t h d a y s , when t h e r a t e o f growth was a l s o the g r e a t e s t .  When t h e y e x p r e s s e d t h e change i n n u c l e i c a c i d on a m i l l i g r a m  per gram o f wet w e i g h t b a s i s ,  b o t h n u c l e i c a c i d s decreased s l i g h t l y from t h e  t h i r d t o t h e s e v e n t h days and t h e n began an i n c r e a s e teenth day.  Gluck and K u l o v i c h  t o a maximum by t h e t h i r -  (1964) observed a d e c l i n e i n p r o t e i n  concentra-  t i o n d u r i n g t h e f o u r t h day o f development o f the c h i c k embryo f o l l o w i n g a p a t t e r n o f i n c r e a s i n g c o n c e n t r a t i o n t h r o u g h days one t o t h r e e ,  b u t d i d n o t d e t e c t any  s i m i l a r d e c l i n e i n DNA c o n c e n t r a t i o n , w h i c h m a i n t a i n e d a g r a d u a l i n c r e a s e  from  two micrograms p e r m i l l i g r a m (u.g/mg) o f d r y w e i g h t a t two days t o t w e l v e ug/mg at s i x days. of increase  P r o t e i n c o n c e n t r a t i o n was observed t o r e g a i n i t s i n i t i a l t h r o u g h t h e f i f t h and s i x t h d a y s .  to the.entire  pattern,  The p r e c e d i n g i n f o r m a t i o n r e f e r s  embryo; t h e s e w o r k e r s emphasized t h a t d i f f e r e n t organs d i s p l a y e d  d i f f e r i n g DNA change p a t t e r n s .  The i n d i v i d u a l i t y o f p a t t e r n c h a n g e s - i n  partic-  u l a r organs has been i l l u s t r a t e d i n terms o f t h e n u c l e o t i d e changes i n t h e c h i c k embryonic b r a i n by W e g e l i n and M a n z o l i (1967); t h e y found t h a t ,  from t h e  s i x t h day o f i n c u b a t i o n t o h a t c h i n g , t h e p e r c e n t a g e v a l u e s f o r the t o t a l  9  n u c l e o t i d e c o n t e n t , i n terms o f d r y w e i g h t , r e g u l a r l y d i m i n i s h e d d u r i n g t h e entire  development p e r i o d , i n d i c a t i n g t h e a c c u m u l a t i o n o f o t h e r s o l i d m a t e r i a l  at a r a t e f a s t e r than t h a t of n u c l e o t i d e accumulation.  T h i s f i n d i n g was. i n  c o n t r a s t t o t h a t o f Shimbayashi and Iwamoto (1964) who observed a s l i g h t i n the concentration  increase  o f DNA i n t h e whole embryo over a s i m i l a r p e r i o d .  W h i l e t h e r a t e o f growth o f t h e embryo i s h i g h l y c o r r e l a t e d w i t h t h e r a t e o f DNA i n c r e a s e ( i e . , Solomon, 1 9 5 7 a ) , i t i s t h e c e l l u l a r r a t e and DNA i n c r e a s e r a t e t h a t are d i r e c t l y r e l a t e d  proliferation  ( i e . , Emmanuelson, 1 9 6 1 b ) .  An i n c r e a s e i n w e i g h t can occur w i t h o u t a c o r r e s p o n d i n g i n c r e a s e i n c e l l number as a r e s u l t o f c e l l u l a r enlargement  and a d d i t i o n o f e x t r a c e l l u l a r  C e l l s i z e and c e l l number, as s e p a r a t e components d i f f e r e n t l y by g e n e t i c a l ertson  o f growth, may be a f f e c t e d  and e n v i r o n m e n t a l f a c t o r s ,  (1959) i n D r o s o p h i l a .  material.  as has been shown b y R o b -  He found t h a t d u r i n g the e a r l y s t a g e s o f  selection  f o r l a r g e and s m a l l body s i z e , t h e predominant change was i n c e l l number, w h i l e a heterotic  e f f e c t was expressed m a i n l y t h r o u g h i n c r e a s e d c e l l s i z e .  On t h e  o t h e r h a n d , Krzanowska (1967) has found t h a t body w e i g h t , l i v e r w e i g h t and t h e number o f n u c l e i i n t h e l i v e r o f mouse embryos were s i g n i f i c a n t l y h i g h e r i n h y b r i d t h a n i n i n b r e d embryos d e v e l o p i n g i n t h e same u t e r u s f o l l o w i n g h e t e r ospermic i n s e m i n a t i o n .  He observed no s i g n i f i c a n t d i f f e r e n c e s  in cell  M i t o t i c ' A c t i v i t y as an Index o f Rate o f DNA I n c r e a s e To t h e e x t e n t t h a t c e l l u l a r p r o l i f e r a t i o n o f t h e embryo i s t o i t s growth r a t e , t h e l a t t e r  i s measurable  '  size. •  related  i n terms o f f r e q u e n c y o f mitoses  w h i c h i s d i r e c t l y a s s o c i a t e d w i t h DNA i n c r e a s e s i n c e each c e l l d i v i s i o n i n v o l v e s a d u p l i c a t i o n o f t h e DNA o f t h e mother c e l l .  A l e s c i o and D i M i c h e l e (1968) have  found t h a t e p i t h e l i a l c e l l p r o l i f e r a t i o n r a t e , as determined by m i t o t i c  count,  was h i g h l y c o r r e l a t e d w i t h t h e o v e r a l l amount o f e p i t h e l i a l growth i n mouse  10 embryonic l u n g t i s s u e .  •  The f i r s t attempt t o e x p r e s s m i t o t i c f r e q u e n c y n u m e r i c a l l y was a p p a r e n t l y t h a t o f M i n o t i n 1909; i t was f i r s t a p p l i e d t o c h i c k embryos i n 1910 by Schultz ( D e r r i c k , 1937).  D e r r i c k has d i s c u s s e d t h e s u i t a b i l i t y o f m i t o t i c i n -  dex, an e x p r e s s i o n o f m i t o t i c f r e q u e n c y , of the c h i c k .  i n the study o f the e a r l y  I t s a p p l i c a t i o n t o t h e measurement  of tissue  development  growth has been  d i s c u s s e d by Hoffman (1949) who p o i n t e d out t h e v a r i o u s s h o r t c o m i n g s o f t h e technique.  D e r r i c k (1937) employed m i t o t i c i n d e x , w h i c h he r e f e r r e d t o as  the number o f c e l l s  i n d i v i s i o n out o f 1 , 0 0 0 , i n a s t u d y o f t h e e a r l y d e v e l o p -  ment o f t h e c h i c k embryo. structure,  He noted t h a t ,  as w e l l as v a r y i n g from s t r u c t u r e  the index also v a r i e d w i t h i n a p a r t i c u l a r s t r u c t u r e .  to  A progressive  e  d e c l i n e i n the i n d e x was observed t o o c c u r w i t h a d v a n c i n g age and d i f f e r e n t i a tion.  He r e p o r t e d t h e v a l u e o f the i n d e x f o r t h e e n t i r e  be 8 . 0 4 ; a t 78 h o u r s i t was 4 . 5 . r a t e v a r i e d from t i s s u e t o t i s s u e . anuelsson  Bellairs  embryo a t 32 h o u r s t o  (1957) a l s o observed t h a t  mitotic  S i m i l a r o b s e r v a t i o n s have been made by Em-  (1961b). Bloom and Buss  (19G8) examined t h e e f f e c t o f age and k i n d o f t i s s u e  on m i t o t i c a c t i v i t y i n the c h i c k embryo.  A t r e n d toward d e c r e a s i n g m i t o t i c a c -  t i v i t y w i t h i n c r e a s i n g embryonic development was o b s e r v e d , w i t h the p a t t e r n o f d e c l i n e b e i n g more o r l e s s s i m i l a r f o r a l l t i s s u e s  studied.  During the p e r -  i o d from t h r e e t o f i v e d a y s , t h e m i t o t i c a c t i v i t y o f t h e b r a i n dropped r a p i d l y from a v a l u e o f 1 2 . 5 m i t o s e s p e r 100 c e l l s f i v e days. It  a t t h r e e days t o 9 . 2 m i t o s e s  The i n d e x o f the s p i n a l c o r d showed a somewhat d i f f e r e n t  i n c r e a s e d from 5.B m i t o s e s per 100 c e l l s  at  pattern.  a t t h r e e days t o 7 . 2 a t f o u r d a y s .  By f i v e days i t h a d dropped s l i g h t l y t o 7.0 and by day s i x i t s v a l u e was reduced to 4 . 0 .  T h u s , i t would appear t h a t the r a t e o f d e c l i n e o f a c t i v i t y o f t h e  s p i n a l c o r d i s out o f phase w i t h t h a t o f t h e b r a i n .  F r i e b o r a and J e l i n e k  (1963)  11  a l s o d e s c r i b e d a d e c r e a s i n g t r e n d i n m i t o t i c a c t i v i t y i n t h e c e n t r a l nervous system o f the c h i c k embryo from two t o s i x days o f i n c u b a t i o n , and p o i n t e d out t h a t the d u r a t i o n o f m i t o s i s was the same i n d i f f e r e n t p a r t s o f the s p i n a l cord at a given stage. Regional differences  i n the m i t o t i c a c t i v i t y o f s e v e r a l r e g i o n s  the c h i c k s p i n a l c o r d were r e p o r t e d by C o r l i s s and Robertson p e r i o d from one and o n e - h a l f t o f i v e days o f i n c u b a t i o n .  (1963) f o r  They observed  of  the that  t h e m i t o t i c p a t t e r n w i t h i n t h e n e u r a l e p i t h e l i u m changed d u r i n g the c o u r s e development.  The a c t i v i t y i n t h e b a s a l p l a t e ,  reasonably constant  of  or l o w e r h a l f o f the t u b e , was  and g r e a t e r t h a n t h a t of the a l a r  (upper) p l a t e  until  Hamilton-Hamburger s t a g e 19 ( t h r e e t o t h r e e and o n e - h a l f days of i n c u b a t i o n ) . The b a s a l p l a t e began t o d e c l i n e r a t h e r r a p i d l y from s t a g e 1 9 ,  r e a c h i n g a lower  a c t i v i t y t h a n t h a t o f t h e a l a r p l a t e by s t a g e 23 ( f o u r days o f i n c u b a t i o n ) .  It  c o n t i n u e d t o d e c l i n e to s t a g e 26 ( f i v e days o f i n c u b a t i o n ) so t h a t , by t h a t time, a considerable difference halves,  or p l a t e s .  i n m i t o t i c a c t i v i t y e x i s t e d between the two  Hamburger (1948) has s i m i l a r l y shown t h a t ,  from t h e  third  t o the e i g h t h day o f development, the m i t o t i c a c t i v i t y o f the a l a r p l a t e was c o n s i s t e n t l y h i g h e r t h a n t h a t o f the b a s a l p l a t e .  He n o t e d t h a t the  a t w h i c h the peak o f a c t i v i t y was reached d i f f e r e d i n the W o h a l v e s ;  stage the  a c t i v i t y o f the b a s a l p l a t e reached a maximum on the t h i r d day w h i l e t h a t the a l a r p l a t e occurred at s i x days.  Mitolo  (1957) a l s o examined the  of  distribu-  t i o n o f m i t o t i c a c t i v i t y a l o n g the v e n t r o - d o r s a l e x i s o f the n e u r a l t u b e ,  ob-  t a i n i n g r e s u l t s s i m i l a r t o t h o s e o f Hamburger (1948) and C o r l i s s and-Robertson (1963).  The p r o l i f e r a t i o n r a t e was h i g h e r i n the v e n t r a l ( b a s a l ) h a l f u n t i l  t h r e e and o n e - h a l f days o f i n c u b a t i o n ; the d o r s a l h a l f t h e n became the more a c t i v e area w i t h the p r o l i f e r a t i o n r a t e remaining higher i n t h a t h a l f stage  31.  to  C o r l i s s and R o b e r t s o n o f the upper and l o w e r p l a t e s s t a g e 26 no a n t e r i o r  (1963) a l s o examined the v a r i a t i o n i n a c t i v i t y  a l o n g the l e n g t h o f the n e u r a l t u b e .  t o p o s t e r i o r change was a p p a r e n t .  was a s t e a d y b u t n o n - s i g n i f i c a n t r i s e i n . t h e Hamburger (1948) and H i t o l o  P r i o r to  A t s t a g e 26,  a n t e r i o r to p o s t e r i o r  ( 1 9 6 7 ) , however, o b t a i n e d r e s u l t s  t h a t m i t o t i c a c t i v i t y was g r e a t e r i n more a n t e r i o r l o c a t i o n s .  to  direction.  indicate  Hamburger (1948)  observed a peak o f a c t i v i t y i n the p r e - b r a c h i a l and b r a c h i a l r e g i o n s alar plate,  of  the  a l t h o u g h the b a s a l p l a t e showed a more or l e s s even d i s t r i b u t i o n  t h r o u g h o u t the l e n g t h o f the embryo. a t i o n i n t h e b r a c h i a l and t h o r a c i c and o n e - h a l f  there  days, M i t o l o  I n an a n a l y s i s o f the p a t t e r n o f p r o l i f e r -  segments i n c h i c k embryos o f f o u r t o  seven  (1967) observed t h a t a peak o f m i t o t i c a c t i v i t y was  reached by s t a g e 27 ( f i v e and o n e - h a l f days)  i n b o t h segments.  I t then  decreased  p r o g r e s s i v e l y t h r o u g h s t a g e s 29 ( s i x t o s i x - and o n e - h a l f days o f i n c u b a t i o n ) 31  (seven t o seven and o n e - h a l f days o f i n c u b a t i o n ) .  a c t i v i t y between t h e b r a c h i a l and t h o r a c i c n o t s i g n i f i c a n t b u t i n a l l subsequent  The d i f f e r e n c e  and  i n mitotic  segments a t s t a g e 23 ( f o u r days) was  s t a g e s t o s t a g e 31, the n e u r a l tube  the b r a c h i a l a r e a e x h i b i t e d a s i g n i f i c a n t l y h i g h e r m i t o t i c a c t i v i t y ,  in  especially  a t s t a g e s 27 and 31. The A p p l i c a t i o n , o f A u t o r a d i o g r a p h y t o t h e Measurement o f M i t o t i c  Frequency  A most i m p o r t a n t method employed i n t h e s t u d y o f m i t o t i c a c t i v i t y that of autoradiography ( i e . , property of radiosotopes crystals  F u j i t a , 1962;  Wimber, 1 9 5 9 ) . .  It  is  i s based on the  t o emit r a d i a t i o n which, a c t s on the. s i l v e r . h a l i d e  of a photographic emulsion.  Proliferating tissue  i s exposed t o a  r a d i o a c t i v e p r e c u r s o r o f DNA, u s u a l l y t r i t i a t e d t h y m i d i n e ( H 3 - t h y m i d i n e ) . l a b e l l e d m a t e r i a l i s i n c o r p o r a t e d i n t o c e l l s w h i c h are s y n t h e s i z i n g DNA and t h e s e can be i d e n t i f i e d i n s e c t i o n e d m a t e r i a l w h i c h has been c o a t e d w i t h  The  13  p h o t o g r a p h i c e m u l s i o n and t h e n developed as an o r d i n a r y p h o t o g r a p h . of r a d i o a c t i v i t y i n the l a b e l l e d c e l l s s i o n o v e r - r i d i n g the s e c t i o n . i t s nucleus r e p l i c a t e s  appear as b l a c k e n e d areas i n the e m u l -  Thymidine i s i n c o r p o r a t e d i n t o a c e l l o n l y when  i t s DNA c o n t e n t  i n preparation for c e l l d i v i s i o n .  s t a g e o f c e l l d i v i s i o n i s r e f e r r e d to as t h e ' S ' phase c o m p l e t i o n o f t h i s phase,  e n t e r t h e p r e - d u p l i c a t i o n or G]_ phase;  t h i s phase w h i l e o t h e r s  (Wimber, 1-959).  This Upon  a c e l l e n t e r s t h e p o s t - d u p l i c a t i o n o r G 2 phase,  period of short duration, before mitosis begins. cells  Sites,  a  A f t e r m i t o s i s , t h e daughter  some o f the c e l l s never  leave  proceed a f t e r v a r i a b l e time i n t e r v a l s t o t h e S-phase i n  p r e p a r a t i o n f o r the next d i v i s i o n . p r e c u r s o r be i n c o r p o r a t e d .  Only d u r i n g the S-phase can t h e l a b e l l e d  However, a l l c e l l s w h i c h have passed t h r o u g h the  S-phase and on t o a f u r t h e r s t a g e d u r i n g t h e p e r i o d o f exposure t o t h e l a b e l w i l l be l a b e l l e d .  Thus, a t h e o r e t i c a l l y h i g h e r l a b e l l i n g i n d e x t h a n m i t o t i c  i n d e x would be expected o f t h e ' 5 ' phase.  i f t h e exposure p e r i o d was g r e a t e r t h a n t h e d u r a t i o n  The t e c h n i q u e  o f a u t o r a d i o g r a p h y can t h e r e f o r e  be a d a p t e d ,  by e m p l o y i n g an extended exposure p e r i o d , t o the t o t a l p r o l i f e r a t i o n over a g i v e n p e r i o d s i n c e c e l l s w h i c h have been l a b e l l e d i n f o r m a t i o n w i l l  retain a l l  o f t h e i r l a b e l i f t h e y do n o t proceed t o m i t o s i s . Examples o f t h e a p p l i c a t i o n o f a u t r a d i o g r a p h y t o t h e s t u d y o f c e l l p r o l i f e r a t i o n and b e h a v i o u r i n t h e c h i c k embryo a r e numerous.  Weston  (1963)  used t h e t e c h n i q u e  i n t h e s t u d y o f the m i g r a t i o n and l o c a l i z a t i o n o f t r u n k  neural crest c e l l s  and Gwatkin and B i g g e r s  i n v i v o and i n v i t r o DNA s y n t h e s i s graphy was employed by F u j i t a  (1963) have compared t h e r a t e s o f  i n developing chick t i b i o t a r s i .  (1965) i n a s t u d y o f t h e g l i o b l a s t c e l l s  embryonic c h i c k s p i n a l c o r d and J a n n e r s and S e a r l s in a characterization  Autoradioi n the  (1968) used t h e t e c h n i q u e  o f w i n g outgrowth i n the c h i c k embryo.  Somite d e v e l o p -  ment i n ' t h e c h i c k embryo has r e c e n t l y been s t u d i e d by Dohdua and Fedorova (1967)  14 and Langman and N e l s o n (i960) u s i n g t h i s t e c h n i q u e .  The former w o r k e r s found  t h a t w i t h a d v a n c i n g development o f t h e embryo t h e r e was a g r a d u a l d e c l i n e i n p r o l i f e r a t i v e a c t i v i t y w h i c h was d i f f e r e n t i n d i f f e r e n t somites and d i s t i n g u i s h e d them as t o t h e i r time o f o r i g i n and p o s i t i o n i n t h e system.  Kodak e t al_. (1968)  employed a u t o r a d i o g r a p h y i n a s t u d y o f t h e e a r l y development o f t h e c h i c k  lens  and o b s e r v e d t h a t the p a t t e r n o f l a b e l l i n g was s i m i l a r t o t h a t o f m i t o t i c a c tivity,  e x c e p t t h a t changes i n t h e d i s t r i b u t i o n o f a c t i v i t y became e v i d e n t  sooner w i t h the l a b e l l i n g t e c h n i q u e s . Dosages o f H ^ - t h y m i d i n e used by t h e aforementioned w o r k e r s ranged from 5 t o 25 m i c r o c u r i e s ( p c ) ; t h e mode o f a d m i n i s t r a t i o n f o l l o w e d depended l a r g e l y upon t h e age o f t h e embryos under s t u d y .  Sauer and Walker  (l95l)  have a d v i s e d t h a t r a d i a t i o n i n j u r y , r e s u l t i n g m a i n l y i n t h e p r o d u c t i o n o f D N A - c o n t a i n i n g c y t o p l a s m i c b o d i e s , can accompany a h i g h l e v e l o f i n c o r p o r a t i o n •of H g - t h y m i d i n e i n t o the young c h i c k embryo (45 t o 70 hours o f i n c u b a t i o n ) . Such b o d i e s can be m i s t a k e n f o r n u c l e i , g i v i n g r i s e t o an i n c r e a s e p a r e n t number of l a b e l l e d c e l l s . mitotic activity i t s e l f . the c o n c e n t r a t i o n ,  i n the a p - .  Sauer and Walker d i d n o t observe any change i n  I t was f o u n d t h a t t h e i n j u r y produced depended on  r a t h e r t h a n t h e t o t a l amount o f r a d i o a c t i v e m a t e r i a l  injected.  They demonstrated t h a t i n j u r y c o u l d be markedly reduced by d i l u t i n g t h e l a b e l l e d thymidine w i t h u n l a b e l l e d thymidine.  Weston (19B3) on t h e o t h e r h a n d , d e -  t e c t e d no r a d i a t i o n damage i n b l a s t o d e r m s , though the dosages used i n t h e W o s t u d i e s were n o t r e a d i l y comparable.  P o s t and Hoffman (1961), who have r e -  viewed much o f t h e l i t e r a t u r e r e l a t i n g t o m i t o t i c d i s t u r b a n c e s and c e l l d e a t h f o l l o w i n g r a d i a t i o n , r e p o r t e d an a l t e r a t i o n o f t h e p l o i d y c l a s s e s o f r a t l i v e r nuclei following radioactive l a b e l l i n g .  I t has been p o i n t e d out by Wand e t al_.  (1967) t h a t a g i n g H g - t h y m i d i n e undergoes d e c o m p o s i t i o n by s e l f - r a d i o l y s i s and t h a t the r e s u l t a n t breakdown p r o d u c t s do n o t l a b e l DNA b u t , r a t h e r e n t e r m a c r o -  15  molecules  o t h e r t h a n n u c l e i c a c i d v h i c h are c o n t a i n e d i n t h e c y t o p l a s m .  They  n o t e d , however, t h a t where gross l a b e l l i n g v/as b e i n g o b s e r v e d , no s i g n i f i c a n t e r r o r was i n t r o d u c e d .  They f o u n d t h a t use o f y e a r - o l d m a t e r i a l caused o n l y a  very s l i g h t a l t e r a t i o n i n l a b e l l i n g  patterns.  . Emanuelson (1367) observed t h a t F e u l g e n h y d r o l y s i s , used i n s t a i n i n g f o r DNA, can cause up t o a 50 p e r c e n t grains i n the n u c l e u s .  However,, t h i s  r e d u c t i o n i n t h e number of s i l v e r  i s not a matter  of p a r t i c u l a r  concern  when t h e t e c h n i q u e i s employed t o i n d i c a t e merely whether l a b e l l i n g w i t h i n a c e l l has or has n o t o c c u r r e d .  '  .  F u j i t a (1962) made an a u t o r a d i o g r a p h i c s t u d y of-'the of c e l l u l a r p r o l i f e r a t i o n i n the neural tube, o f o n e - t o s i x - d a y o l d c h i c k embryos. i n the t i s s u e s  mitosis.  and o p t i c  retina  He demonstrated t h a t t h e m a t r i x l a y e r  s t u d i e d v/as d i v i s i b l e i n t o a s e r i e s o f zones r e l a t e d t o the  degree o f m a t u r a t i o n o f t h e c e l l s layer,.the  mesencephalon  characteristics  5-zone,  c o m p r i s i n g them.  C e l l s o f t h e deepest  a r e t h o s e w h i c h are s y n t h e s i z i n g DNA i n p r e p a r a t i o n f o r  When t h e s e have undergone DNA s y n t h e s i s ,  enter i n t o the intermediate  I-zone  (corresponds  ing the p r e m i t o t i c p e r i o d .  D u r i n g subsequent  j a c e n t t o t h e i n t e r n a l l i m i t i n g membrane  t h e y b e g i n t o ascend and  t o Gg) w h i c h i s t r a v e r s e d d u r -  m i t o s i s , these c e l l s  (K-zone)  remain a d -  and d u r i n g t h e p o s t - m i t o t i c  p e r i o d t h e daughter c e l l s m i g r a t e back t o t h e I-zone and f i n a l l y r e a c h t h e S-zone t o s t a r t t h e g e n e r a t i v e  cycle again.  He e s t i m a t e d t h e d u r a t i o n o f  t h e various- s t a g e s i n t h e mesencephalon t o be as f o l l o w s : o f DNA s y n t h e s i s ,  length of period  a p p r o x i m a t e l y f i v e and o n e - h a l f h o u r s ; l e n g t h o f p r e - m i t o t i c  p e r i o d , two h o u r s ; complete g e n e r a t i o n t i m e o f t h e m a t r i x c e l l , s i x t e e n h o u r s ; duration of mitosis i t s e l f , the a l a r plate  one h o u r ; and p o s t - m i t o t i c p e r i o d , s i x h o u r s .  In  o f t h e s p i n a l c o r d t h e v a l u e s were s i m i l a r t o t h o s e o f t h e mesen  cephalon, v i z . two, s i r t e e n ,  one and n i n e h o u r s , r e s p e c t i v e l y .  In the basal  16 p l a t e o f the same s p i n a l c o r d l a b e l l i n g was s p a r s e ; t h e o n l y c o n s t a n t g i v e n was t h a t f o r g e n e r a t i o n t i m e , w h i c h was e s t i m a t e d to be two and o n e - h a l f days.  The m a t r i x c e l l s o f the f l o o r and r o o f p l a t e s were l a b e l l e d a t a v e r y  low r a t e and F u j i t a s u g g e s t e d t h a t t h e g e n e r a t i o n t i m e o f t h o s e c e l l s was f a r longer than t h a t of the basal p l a t e matrix c e l l s . .  The m i t o t i c i n d e x o f t h e  mesencephalon was 6.8 c e l l s i n m i t o s i s o u t o f 100, w h i l e t h e l a b e l l i n g i n d e x was 35 l a b e l l e d c e l l s p e r h u n d r e d . the s p i n a l c o r d were 6.5 and 25. o f the matrix c e l l s  Comparable v a l u e s f o r t h e a l a r p l a t e o f Both t h e m i t o t i c time and g e n e r a t i o n time  i n t h e one-day c h i c k embryo were much s h o r t e r t h a n i n t h e  s i x - d a y embryo; t h e m i t o t i c i n d e x o f t h e former was e i g h t m i t o s e s per hundred cells.  I n s o f a r as t h e y a r e comparable, F u j i t a ' s r e s u l t s agree w i t h those o f  o t h e r w o r k e r s who examined p r o l i f e r a t i o n o f t h e s p i n a l c o r d o f t h e c h i c k emb r y o i n terms o f m i t o t i c a c t i v i t y . (1963) and M i t o l o  Hamburger ( 1 9 4 8 ) , C o r l i s s and R o b e r t s o n  (1967) a l l observed a g r e a t e r  o f the cord than i n t h e ' b a s a l  a c t i v i t y i n the a l a r p o r t i o n  p o r t i o n a f t e r t h r e e and o n e - h a l f d a y s , t h o u g h  none o f t h e s e w o r k e r s observed as g r e a t a d i f f e r e n c e as t h a t r e p o r t e d by Fujita.  I t i s not u n l i k e l y t h a t the greater  d i f f e r e n c e observed by F u j i t a  was due t o t h e f a c t t h a t he examined s i x - d a y embryos w h i l e t h e o t h e r i n v o l v e d younger embryos.  studies  A l l s t u d i e s e m p l o y i n g m i t o t i c r a t e agreed t h a t t h e  d i f f e r e n c e between t h e two p l a t e s o f t h e n e u r a l tube i n c r e a s e d w i t h i n c r e a s i n g g age o f t h e embryo.  Changes i n Enzyme A c t i v i t y A s s o c i a t e d w i t h . Ontogeny There have been innumerable r e p o r t s o f changes d u r i n g the embryonic development o f many o r g a n i s m s .  i n enzyme a c t i v i t y  The p a t t e r n o f change  f o r i n d i v i d u a l enzymes i s o f t e n m u l t i p h a s i c i n t h a t a p a r t i c u l a r enzyme w h i c h l i a s been a c c u m u l a t i n g r a p i d l y may s h i f t t o a c o n s t a n t l e v e l o f a c t i v i t y or may  17  even r e g r e s s ,  and l a t e r  increase  a g a i n (Moog, 1 9 6 5 ) .  Changes i n t h e c h i c k embryo as a whole I n t h e c h i c k embryo, as i n most s i m i l a r o r g a n i s m s , enzymes o f g e n e r a l metabolic f u n c t i o n f o l l o w a v a r i e t y of d i f f e r e n t patterns, enzyme b e i n g more or l e s s independent o f t h e a c t i v i t i e s o f - o t h e r ent.  For i n s t a n c e ,  (Moog and S t e i n b a c h ,  t h a t f o r each enzymes  t h e e a r l y c h i c k embryo p o s s e s s e s l o w phosphatase  pres-  activity,  1 9 4 6 ) , r e l a t i v e l y h i g h p e p t i d a s e a c t i v i t y (Levy and P a l -  mer, 1943) and v e r y h i g h l e v e l s o f cytochrome o x i d a s e and s u c c i n i c  oxidase  (Albaura et a l . , 1 9 4 6 ) . Solomon (1959)  found t h a t t h e s p e c i f i c a c t i v i t y o f m a l i c a c i d d e -  hydrogenase o f whole c h i c k embryos was