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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 . S c . ( A g r . ) , M c G i l l U n i v e r s i t y , 1961 M . S c , M c G i l l U n i v e r s i t y , 196A A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f P o u l t r y Sc ience We accept t h i s t h e s i s as conforming to the r e q u i r e d s tandard THE UNIVERSITY OF BRITISH COLUMBIA January , 1971 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i 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 an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e H e a d o f my D e p a r t m e n t o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f P o u l t r y S c i e n c e The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e February 1, 1971 ABSTRACT A s tudy was c a r r i e d out to determine i f g e n e t i c v a r i a t i o n ex-i s t e d i n the r a t e of embryonic development of a l i n e of c h i c k embryos of the New Hampshire breed , and to examine the extent to which v a r i a t i o n i 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, c o u l d be r e l a t e d to p o s t - h a t c h i n g growth of s i b l i n g c h i c k s . I n d i c e s o f d e v e l o p -mental r a t e examined i n i n d i v i d u a l embryos were a) . c e l l u l a r p r o l i f e r a t i o n r a t e between 77 and 98 hours o f i n c u b a t i o n of s p e c i f i c t i s s u e s w i t h i n the embryonic c e n t r a l nervous system and of embryonic b lood c e l l s , and b) t o -t a l p r o t e i n content , embryo dry w e i g h t , Hamilton-Hamburger s tage , t o t a l l a c t i c a c i d dehydrogenase (LDH) a c t i v i t y and express ions t h e r e o f , and t o t a l a l k a l i n e phosphatase (AP) a c t i v i t y and express ions t h e r e o f , at 96 and 112 hours o f i n c u b a t i o n . S i r e d i f f e r e n c e s e x i s t e d i n the a b s o l u t e r a t e o f c e l l u l a r p r o -l i f e r a t i o n , as determined by the technique o f a u t o r a d i o g r a p h y , o f the n e u r a l tube and metencephalon and, p o s s i b l y , the myelencephalon. No such d i f f e r e n c e s i n the a c t i v i t y of the mesencephalon, d iencepha lon 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 maternal 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 o f a l l t i s s u e s examined was f e l t to e x i s t . S i r e d i f f e r e n c e s i n the p r o t e i n content of 112-hour embryos were observed and may a l s o have e x i s t e d w i t h respect to 96-hour p r o t e i n content and embryo d r y weight but were masked by maternal f a c t o r s . The h e r i t a b i l i t y o f 96-hour ' p r o t e i n content was es t imated to be .412 ; the 112-hour es t imate was .198 . Corresponding v a l u e s f o r 96-hour and 112-hour t o t a l LDH a c t i v i t y were .294 and .344, r e s p e c t i v e l 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 es t imates were v e r y h i g h . 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 there was evidence 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 tage ; maternal f a c t o r s d i d not appear to have i n f l u e n c e d e i t h e r of these embryonic t r a i t s at e i t h e r i n c u b a t i o n stage examined. S i r e d i f f e r e n c e s i n t o t a l AP a c t i v i t y per u n i t o f supernatant p r o t e i n (AP/SP) determined a t 96 hours o f i n c u b a -t i o n were observed . 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 .554 and the c o r r e s p o n d i n g 112-hour v a l u e was .114. S i r e d i f f e r e n c e s ex-i s t e d f o r both the a b s o l u t e l e v e l of LDH^ a c t i v i t y and LDHj per u n i t o f t o t a l p r o t e i n . Both the 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 -i t y decreased between 96 and 112 hours of i n c u b a t i o n . The a c t i v i t y o f t h i s isozyme was concluded to be d i r e c t l y a s s o c i a t e d w i t h p r o l i f e r a t i v e a c t i v i t y at both i n c u b a t i o n stages examined. 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 content 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 both LDH con-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 both 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 both stages 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 at 96 hours o f i n c u b a t i o n o n l y . A n e g a t i v e l i n e a r a s s o c i a t i o n was found between p o s t - h a t c h i n g 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 cen-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 ) , and between female body weight to four weeks o f age and the a c t i v i t y o f embryonic b lood c e l l s . There was a l s o a n e g a t i v e , though not always l i n e a r , r e l a t i o n s h i p between post<-hatching growth performance and 96-hour Hamilton-Hamburger s tage , p r o t e i n content , t o t a l LDH a c t i v i t y , t o t a l AP a c t i v i t y , and expres-s i o n s o f AP c o n c e n t r a t i o n , e s p e c i a l l y d u r i n g e a r l y p o s t - h a t c h i n g 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 w i t h 96-hour H a m i l t o n -Hamburger stage and A P / S P ; 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 ) . An i n -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 112 hours o f 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 that i n v o l v i n g 96-hour developmental r a t e . T o t a l 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 the 112-hour 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 concluded that g e n e t i c d i f f e r e n c e s 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 performance. T A B. L E a_F G • N T E K T S INTRODUCTION ' • REVIEW DF LITERATURE Genet ic D i f f e r e n c e s i n Developmental Rate of Chick 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 the Chicle Embryo ' . M i t o t i c A c t i v i t y as an Index of Rate of DNA Increase The A p p l i c a t i o n of Autoradiography t o the Measurement of • 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 Chick Embryo as a Whole Changes i n S p e c i f i c T i s s u e s of the Chick 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 Genet ic 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 of Chick Embryos and the R e l a t i o n s h i p of 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 Genet ic C o n t r o l LDH: Ontogenetic Changes i n Isozyme Composi t ion LDH: I n f l u e n c e of Pyruvate C o n c e n t r a t i o n as a B a s i s f o r Ontogenetic Changes i n . , I s o z y m e Composit ion' 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: Evidence f o r More than F i v e Bands A l k a l i n e Fnosphatase (AP) A P : Changes i n A c t i v i t y D u r i n g Development A P : 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 Chickens A P : Genet ic V a r i a t i o n i n Serum L e v e l s A P : S t r u c t u r e and Genet ic 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 procedures 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 . c o n t i n u e d Page RESULTS 57 A Growth T r i a l s ' 57 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 57 R e l a t i o n s h i p • "between growth t r a i t s examined 59 B Embryonic S t u d i e s 65 1) Rate of C e l l u l a r P r o l i f e r a t i o n 65 Genet 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 65 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 70 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 Indices of Em- 74 b 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 Content , Embryo Dry 74 Weight and Hamilton-Hamburger Stage • Genet 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 - 74 t e i n c o n t e n t , embryo dry weight and Hamilton-Hamburger s tage 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 , 91 p r o t e i n c o n t e n t , embryo dry weight and Hami l ton-Ham-burger s tage R e l a t i o n s h i p of 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 54 c o n t e n t , embryo dry weight and Hamilton-Hamburger stage 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 of A l k a l i n e Phosphatase A c t i v i t y 127 Genet i c i n f l u e n c e on e x p r e s s i o n s of a l k a l i n e phospha- 127 .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 phospha- 130 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 phospha- 133 tase t o p o s t - h a t c h i n g development D I S C U S S I C M 1 5 8 A Growth T r i a l s 1 5 8 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 158 R e l a t i o n s h i p between growth t r a i t s examined 159 B Genet i c I n f l u e n c e on Embryonic Developmental Rate and the R e - 162 l a t i o n s h i p of 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 162 • Genet ic 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 embry- 162 o 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 170 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 of Embryonic 173 Development . . . c o n t i n u e d Page, DISCUSSION ' 1 7 8 (a) P r o t e i n Content , Embryo Dry Weight , E x p r e s s i o n s of LDH I 7 8 A c t i v i t y and Hamilton-Hamburger Stage Genet i c i n f l u e n c e on p r o t e i n content • ' I 7 8 R e l a t i o n s h i p between p r o t e i n content and embryo dry w e i g h t l 8 7 Genet i c i n f l u e n c e on embryo d r y weight , 1 9 0 G e n e t i c ~ i n f l u e n c e on express ions 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) Genet ic i n f l u e n c e on LDH isozyme l e v e l s 219 -Genet i c i n f l u e n c e on Hamilton-Hamburger stage 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 weight ) and i n d i r e c t (LDH a c t i v i t y and Hamil ton-Ham-burger stage) 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 em- 23B 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 Genet 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 SUMMARY AND CONCLUSIONS - . 3 1 7 LITERATURE CITED ., • 323 APPENDIX LIST OF TABLES Table 1 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 of Body Weights and Growth Rates , 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 of P r o l i f -e r a t i v e A c t i v i t y of 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 Va lues of the ( l ) Upper and (2) Lower Halves of Each S e c t i o n , and (3) the 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 of 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 of C e n t r a l Nervous System T i s s u e s and B lood C e l l s ; A c t i v i t y Expressed as Combined T o t a l Count, Data of A n a l y s i s I I . 5 Rank Order of S i r e Means of P r o l i f e r a t i v e A c t i v i t y of 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 , Com-b 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 of P r o l i f e r a t i v e A c t i v i t y of 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 of L e v e l on A c t i v i t y Data of A n a l y s i s I I 7 Simple C o r r e l a t i o n C o e f f i c i e n t s be tween 'Se lec ted 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 (com-b i n e d t o t a l count) of Embryonic C e n t r a l Nervous S y s -tem T i s s u e s and B lood C e l l s - Chick Data of Growth T r i a l s One, Two and Three and Embryo Data o f A n a l y s i s I I 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 of 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 Thereof , P r o t e i n Content , Embryo Dry Weight and Hamil ton-Ham-burger Stage at 96 and 112 Hours of I n c u b a t i o n : A n a l -y s i s of Genet ic E f f e c t s , Experiment One ( i e . , R e p l i c -at ions One and Two) 9 Rank Order of S i r e Means of 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 Thereof , P r o t e i n Content , Embryo Dry Weight and Hamilton-Hamburger Stage a t ' 9 6 and 112 Hours of 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) . c o n t i n u e d Table 10 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 of L a c t i c A c i d Dehydrogenase and Express ions Thereof , P r o t e i n Content , 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 of 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) 11 . 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 Thereof , P r o t e i n Content , Embryo Dry Weight and Hamil ton-Ham-burger Stage at 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 Genet ic 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 Dehydro-• genase A c t i v i t y and E x p r e s s i o n s Thereof , P r o t e i n C o n -. t e n t , Embryo Dry Weight and Hamilton-Hamburger Stage at 96 Hours and 112 Hours of 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 Var iance of 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 Thereof , 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 of V a r i a n c e of 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 Thereof , P r o t e i n Content , Embryo Dry Weight and Hamil ton-Ham-burger Stage at 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 Genet i c E f f e c t s , Experiments One and Two Com-b 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 of 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 Content , Em-bryo Dry Weight and Hamilton-Hamburger Stage at 96 and • 112 Hours o f I n c u b a t i o n , Experiments One and Two ( i e . , R e p l i c a t i o n s One, Two and Three) 16. ' 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 Thereof , P r o t e i n Content , Embryo Dry Weight and Hamil ton-Ham-burger 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 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) . . . . cont inued' E s t i m a t e s o f H e r i t a b i l i t y ( h s ) arid t h e i r Standard 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 Thereof . P r o t e i n Content , 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 . Experiments One and Two ( i e . , • R e p l i c a t i o n s One, Two and Three) 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 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 Sep-a r a t e l y f o r 96- 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 of 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 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 Examined a t 96 Hours of I n c u b a t i o n i n Experiments 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 C o r r e l a t i o n C o e f f i c i e n t s BeWeen 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 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 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 112 Hours o f I n c u b a t i o n i n Experiments 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) from Simple L i n e a r R e g r e s s i o n Analyses 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 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 of 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 at 96 Hours of I n c u b a t i o n i n ' Experiments One and Two . . c o n t i n u e d 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 rom 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 at 112 Hours of I n c u b a t i o n i n Experiments One and Two Simple Regress ion 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 at 112 Hours of 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 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 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 Regress ions of Male Groxrth T r a i t s (Y) on 96-Hour Embryonic T r a i t s (x) Measured i n Experiments One and Two w h i c h Y i e l d e d an R 2 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 than 0.10 C o e f f i c i e n t of Determinat ion (R2) 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 Non-Linear Regress ions of Female Growth T r a i t s (Y) on 96-Hour Embryonic T r a i t s (x) Measured i n . Experiments One and Two which Y i e l d e d an R2 V a l u e f o r which ' the P r o b a b i l i t y was l e s s than 0.10 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 2 ) 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 Regress ions of 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 Experiments One and Two w h i c h Y i e l d e d an R 2 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 than 0.10 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 2 ) 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 of 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 Regress ions of Female Growth T r a i t s on 112-Hour Embryonic T r a i t s (x) Measured i n E x -per iments One and Two w h i c h Y i e l d e d R2 Va lues 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 than 0.10 2 C o e f f i c i e n t s 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 Each of 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 of M u l t i p l e L i n e a r Reg-r 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 than 0.10 . c o n t i n u e d P r o b a b i l i t i e s f rom the A n a l y s i s of 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 at 95 and 112 Hours of I n c u b a t i o n : A n a l y s i s of Gen-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 of 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 at 96 and 112 Hours of 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 Thereof at 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.t R e p l i c a t i o n s One and Two) E s t i m a t e s of H e r i t a b i l i t y ( h s ) , and t h e i r Standard 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 at 96 and 112 Hours o f I n c u b a t i o n , E x -per iment One ( i e . , R e p l i c a t i o n s One and Two) 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 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 96- and 112-Hour Embryos, Experiment One ( i e v 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 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 Experiment 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 BeWeen 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 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 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 at 112 Hours of 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)from Simple L i n e a r R e g r e s s i o n Analyses of Male G r owth 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 Experiment One . c o n t i n u e d 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 -ear R e g r e s s i o n Analyses of 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) Exam-i n e d a t 96 Hours of 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) from Simple L i n -ear . R e g r e s s i o n Analyses of 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 at 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) from Simple L i n -ear R e g r e s s i o n Analyses of 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 of A l k a l i n e Phosphatase A c t i v i t y (x) E x -amined at 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 of D e t e r m i n a t i o n ( R 2 ) . 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 R e g r e s s i o n 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 Regress ions of 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 of A l k a l i n e Phosphatase A c t i v i t y (x) Measured i n Experiment One which Y i e l d e d an R V a l u e f o r which the 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 s of Determinat ion (R 2 ) 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 the P a r t i a l Regress ion 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 Regress ions o f Hale and Female Growth T r a i t s (Y) on 112-Hour E x -p r e s s i o n s of A l k a l i n e Phosphatase f o r which the P r o b -a b i l i t y was l e s s than 0.10 2 C o e f f i c i e n t s 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 each o f 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 M u l t i p l e L i n -ear Regress ions 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 Supernatant 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 2 Va lue 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 than 0.10 L I S T OF FIGURES 1 Diagrammatic r e p r e s e n t a t i o n of 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 His togram of 96 - and 112-hour means (Experiment Two) o f . the a b s o l u t e amounts of 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 of LDH]_, LDH5 and L D H 2 - 4 expressed both 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 of each s i r e 3 General forms of the curves d e f i n e d by the second-degree p o l y n o m i a l 4 R e g r e s s i o n of three-week body weight 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 Genera l forms of the curves d e f i n e d by the t h i r d - d e g r e e p o l y n o m i a l 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 three 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 weight 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) ACKNOWLE D GEM BUT S Completion o^ the project on wliich tku> thefts hcu> been ph.epax.ed could not have been accomplished uuXhout the generous advi.ee and assistance o^many {riends' and associates. The author Is extremely grateful to Dx. C W . Roberts under whose excellent supervision the project and thesis preparation was carried oat. His encouragement and co-operation axe greatly appreciated. 1 Thanks axe also due to personnel oi the Statistical Research Section, Canada Department o{ Agxicultuxe, Ottawa,and the Departmental Statistics Division, Vepaxtment o{ Indian Avoirs and Northern Development, OttavJa. In paxticulax, the author wishes to thank Mrs. lhah.gan.et Nightman and Wi. 8. Sakamoto. The awthox wishes to express his gratitude to Processor J. Biely {or (vis encouragement during the course ol this study. Appreciation is alio expressed lor the assistance given by {ellow students and members ojj the poultry {arm sta{{. Warmest thanks are aJUo extended to my wi{e, both {or her preparation o{ the manuscript, and {or her continuing patience and encou-ragement throughout the course o{ both the project and thesis preparation. 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 gene t i c s has advanced t o the 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 broader area of a p p l i e d b i o l o g y . 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 T.\Thich have been used t o e x p l o i t the i n h e r e n t q u a n t i t a t i v e 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 animals 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 importance . In p a r t i c u l a r , improvements i n both 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 through the l a r g e -s c a l e a p p l i c a t i o n of 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 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 task t o b r i n g about f u r t h e r improvement of many of these c h a r a c t e r i s t i c s because of the ex tent t o which the g e n e t i c v a r i a t i o n d i r e c t l y a s s o c i a t e d 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 p r a c t i c a l l e v e l , of each new p r i n c i p l e . S i g n i f i c a n t l y , q u a n t i t a t i v e t r a i t s of domestic animals i n general have been s e l e c t e d f o r improvement, w i t h few e x c e p t i o n s , p u r e l y on the b a s i 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 , l i t t l e or no r e g a r d h a v i n g been g iven by. g e n e t i c i s t s t o the b i o l o g i c a l f a c t o r s i n v o l v e d i n t h e i r d e t e r m i n a t i o n . I t seems apparent t h a t , as attempts t o f u r t h e r improve such t r a i t s through the a p p l i c a t i o n of q u a n t i t a t i v e gene t i c s become more f u t i l e , a l t e r n a t e measures m e r i t c o n s i d e r a t i o n . 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 b i o l o g i c a l ' v a r i a b l e s , i n terms of g e n e t i c v a r i a t i o n , which i n f l u e n c e econ-o m i c a l l y important t r a i t s would 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 . Not o n l y would examinat ion of the g e n e t i c c h a r a c t e r i s t i c s 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 of 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 processes which are manipula ted 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 t r a i t • 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 remain ing 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 a s s o c i a t e d w i t h a p a r t i c u l a r economic t r a i t would be s u f f i c i e n t t h a t g e n e t i c m a n i p u l a t i o n of such p r o -cesses would be , 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 p o u l t r y i s of p a r t i c u l a r s i g n i f -i cance i n the aforementioned context i n t h a t , because of i t s fundamental economic . importance , 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 genet i c 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 . As 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 have 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 . Demonstrat ion of the e x i s t e n c e of a r e l a t i o n s h i p between an i n d i r e c t index of growth which d i s p l a y e d g e n e t i c v a r i a b i l i t y , and growth i t s e l f , would o b v i o u s l y represent an a l t e r n a t i v e means by which changes, through s e l e c t i v e b r e e d i n g , i n the l a t t e r c h a r a c -t e r i s t i c might be p o s s i b l e . The extent t o which a procedure would be s u c c e s s f u l would be dependent on the h e r i t a b i l i t y of the i n d i r e c t t r a i t and the degree of g e n e t i c c o r r e l a t i o n between i t and growth r a t e . I t i s a w e l l - k n o w n f a c t t h a t , i n recent y e a r s , i t has become p r a c t i c a l t o market meat-type ch ickens at a r e l a t i v e l y young age; t h u s , i t i s ' t h e growth performance 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 p r e s -ent i s e s p e c i a l l y i m p o r t a n t . S ince growth o f the organism t h e o r e t i c a l l y begins w i t h f e r t i l i z a t i o n of the ovum, i t might be expected t h a t p o s t - h a t c h -i n g growth of c h i c k e n s , 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, would be r e l a t e d i n some manner t o growth d u r i n g the 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 to be the case w i t h r e s p e c t t o l a t e embryonic development. The present s tudy 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 d i f f e r e n c e s d u r i n g t h i s p e r i o d would be more marked than d u r i n g subsequent development as e n v i r o n -mental f a c t o r s e xe 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 the course of i n c u b a -t i o n . . S p e c i f i c a l l y , the purpose of t h i s s tudy was t h r e e - f o l d - a) t o d e t -ermine i f g e n e t i c d i f f e r e n c e s i n r a t e of 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 to which 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 , r e g a r d i n g 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 em-b r y o n i c development i s determined. Developmental r a t e of c h i c k embryos was examined i n terms of 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 of s p e c i f i c enzymes known to change d u r i n g the course of development. 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 p o t e n -t i a l v a l u e of s t u d y i n g the r a t e of e a r l y embryonic development of the c h i c k e n as a means of 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 of the r e l a t i o n s h i p 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 i l l u s t r a t e the p o s s i b l e rewards of employing t r a d i t i o n a l q u a n t i t a t i v e g e n e t i c p r i n -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 order 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 s p e c i e s . 4 REVIEW OF LITERATURE  Genet ic D i f f e r e n c e s i n Developmental. Rate of Chick Embryos The i n f l u e n c e o f genotype on the r a t e o f development o f the c h i c k embryo has been examined by many, w o r k e r s , B y e r l y (1930, 1932) observed breed d i f f e r e n c e s i n embryonic weight among progeny from the Rhode I s l a n d Red ( R . I . R . ) and White Leghorn (W.L. ) breeds , and crosses between these b r e e d s . R . I . R . and c r o s s b r e d embryos were found to 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 though W . L . and c r o s s b r e d embryos were h e a v i e r than R . I . R . embryos p r i o r to ten days. Henderson (1930) found no d i f f e r e n c e i n em-b r y o n i c w e i g h t , expressed e i t h e r on a wet or dry weight b a s i s , between Dark C o r n i s h and W . L . embryos from 96 hours of i n c u b a t i o n t o h a t c h i n g nor d i d he. de tec 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 embryos.of the two breeds . B lunn 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 between the R . I . R . and W.L . breeds i n favour of t h e . f o r m e r i n the r a t e o f c e l l p r o l i f e r a t i o n , as represented by m i t o t i c f requency or the number of c e l l s per u n i t volume, at 72 hours and f o u r t e e n days o f i n c u b a t i o n . The number o f m i t o t i c f i g u r e s was a l s o grea ter at n i n e t e e n days i n the R . I . R . embryos. No s i g n i f i c a n t breed d i f -f e r e n c e s , however, e x i s t e d i n embryonic w e i g h t . B y e r l y e_t al_. (1938) found t h a t , i n a comparison of d i f f e r e n t breeds and c r o s s b r e d s , embryos from h e a v i e r parents tended to be h e a v i e r than those from l i g h t e r parents between e leven and seventeen days o f i n c u b a t i o n . Krzanowska (1959) s t u d i e d the e a r l y embryonic growth o f c h i c k em-bryos at 24 and 48 hours o f development. He obta ined s i g n i f i c a n t d i f f e r e n c e s 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 the 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 d iameter . He a l s o noted 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 all 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. lines. Tolman Si. §A." (1962) 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 all 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 in-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 sig-6 n i f i c a n t l y h i g h e r somite count at 42 hours of i n c u b a t i o n . Deland (1965) made a comparison of the 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 , stages of development of the c h i c k e n as a f f e c t e d by genotype. S i g n i f i c a n t g e n e t i c e f f e c t s on embryonic growth, concentra ted m a i n l y i n the p e r i o d from e i g h t t o f o u r t e e n days of i n c u b a t i o n , were observed. 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 the growth r a t e s of the e a r l y and l a t e embryonic stages'. Embryonic growth between . e i g h t and twelve days of 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 both • 6-week body w e i g h t and 6-7 week growth r a t e . ' Zervas and C o l l i n s (19S5) have r e p o r t e d on a s tudy of the i n f l u e n c e of genotype on 14-day embryo w e i g h t o f the c h i c k e n . 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 i n a com-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 . Es t imates of h e r i t a b i l i t y based on the s i r e component were 0 .02 and 0.31 f o r the two l i n e s . • M a t e r n a l ' e f f e c t 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' genesis i n W o l i n e s of Broad Breas ted Bronze t u r k e y s , one of which had been s e l e c t e d f o r h i g h mature body w e i g h t . The r a t e of development, both before and a f t e r i n c u b a -t i o n , as measured by the diameter of the germ d i s c , l e n g t h of the area p e l l -u c i d a , number of somites and w i d t h of one s i d e of the area 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 the 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 of Embryonic Growth ' 'Growth r a t e , 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 c r e a s e i n w e i g h t , i s , from the developmental s t a n d p o i n t , c h a r a c t e r i z e d by d i f f e r e n t i a t i o n and morphogenesis , a l b e i t i n v o l v i n g b i o l o g i c a l s y n t h e s i s . Growth r a t e of the embryo, t h e n , and p a r t i c u l a r l y the young embryo, can be r e p r e s e n t e d i n t e r m s . o f v a r i o u s developmental • changes, i n c l u d i n g w e i g h t . A c o n s i d e r a b l e number of these have been employed as i n d i c e s of growth r a t e of the 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 et a l . , 1938; McNary e t a l . , I 960 ; Deland, 1965; and others) ,embryo weight i t s e l f has had by f a r the most prominent use . A comparison of the wet and d r y weight growth curves of the c h i c k embryo has been g iven by Romanoff. (1967) who p o i n t e d out t h a t there i s l i t t l e d i f f e r e n c e i n the shape of the 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 et al_. (1951') r e p o r t e d t h a t the s i z e o f the embryo, as d e -termined by the 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 Orlov) and Orlov (1962) have employed the p r i n c i p l e o f comparison of d i s c diameter i n s t u d i e s o f embryonic growth. E l i a s (1968) r e -p o r t e d t h a t bo th embryo l e n g t h and a r e a , 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 -f o r m a t i o n on developmental s t a g e . As a l r e a d y mentioned, t o t a l n i t r o g e n , as an index o f p r o t e i n c o n t e n t , has a l s o been used as a measure o f developmental r a t e o f the c h i c k embryo (Henderson, 1930) . P r o t e i n c o n s t i t u t e s much the l a r g es t chemical component o f the young embryo, and, i n g e n e r a l , the i n c r e a s e i n t o t a l n i t r o g e n corresponds t o the i n c r e a s e i n p r o t e i n d u r i n g the course of e a r l y development (Romanoff, 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 the Chick 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 i n c e l l number and a s i m i l a r i n c r e a s e i n DNA d u r i n g the f i r s t three 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 the p r i n c i p l e o f constancy o f DNA per n u c l e u s . He concluded t h a t i n the young c h i c k embryo, the Increase i n DNA p a r a l l e l e d the r a t e o f c e l l u l a r p r o l i f e r a t i o n . He a l s o found t h a t d u r i n g development the r a t e o f i n c r e a s e o f 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 . 8 Emanuelsson (1961a), who es t imated DNA i n c h i c k embryos from zero to 48 hours o f i n c u b a t i o n , r e p o r t e d t h a t there 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 o f DNA. N o v i k o f f and P o t t e r (1948), Reddy et 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 o f DNA c o n c e n t r a t i o n throughout the course o f development. I n a l l these s t u d i e s , i t was observed t h a t the most r a p i d i n c r e a s 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 -c u b a t i o n . M a r r i a n et al_. (1957) a l s o noted an e x c e l l e n t agreement between wet w e i g h t and t o t a l DNA content . Shimbayashi and Iwamoto (1964) r e p o r t e d t h a t t o -t a l DNA i n c r e a s e d i n d i r e c t r e l a t i o n t o the growth of the embryo from three t o e ighteen days of i n c u b a t i o n . They observed the p e r i o d of 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 days , when the r a t e of growth was a l s o the g r e a t e s t . When they expressed the 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 weight b a s i s , both n u c l e i c a c i d s decreased s l i g h t l y from the t h i r d to the seventh days and then began an i n c r e a s e to a maximum by the t h i r -t e e n t h day. Gluck and K u l o v i c h (1964) observed a d e c l i n e i n p r o t e i n c o n c e n t r a -t i o n d u r i n g the 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 through days one to t h r e e , but d i d not detec 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 mainta ined a gradual i n c r e a s e from two micrograms per m i l l i g r a m (u.g/mg) of d r y weight a t two days to twelve ug/mg at s i x days . 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 p a t t e r n , o f i n c r e a s e through the f i f t h and s i x t h days . 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 t o t h e . e n t i r e embryo; these workers 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 changes - in p a r t i c -u l a r organs has been i l l u s t r a t e d i n terms o f the n u c l e o t i d e changes i n the c h i c k embryonic b r a i n by Wegel in and M a n z o l i (1967); they found t h a t , from the s i x t h day o f i n c u b a t i o n to h a t c h i n g , the percentage va lues 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 dry 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 the e n t i r e development p e r i o d , i n d i c a t i n g the accumulat ion of other s o l i d m a t e r i a l a t a r a t e f a s t e r than t h a t o f n u c l e o t i d e a c c u m u l a t i o n . 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 c r e a s e i n the c o n c e n t r a t i o n o f DNA i n the whole embryo over a s i m i l a r p e r i o d . W h i l e the r a t e o f growth o f the embryo i s h i g h l y c o r r e l a t e d w i t h the r a t e o f DNA i n c r e a s e ( i e . , Solomon, 1957a) , i t i s the c e l l u l a r p r o l i f e r a t i o n 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 ( i e . , Emmanuelson, 1961b). An i n c r e a s e i n weight 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 m a t e r i a l . C e l l s i z e and c e l l number, as separate components o f growth, may be a f f e c t e d d i f f e r e n t l y by g e n e t i c a l and environmenta l f a c t o r s , as has been shown by Rob-e r t s o n (1959) i n D r o s o p h i l a . He found t h a t d u r i n g the e a r l y s tages of s e l e c t i o n f o r l a r g e and s m a l l body s i z e , the predominant change was i n c e l l number, w h i l e a h e t e r o t i c e f f e c t was expressed m a i n l y through i n c r e a s e d c e l l s i z e . On the o ther hand, Krzanowska (1967) has found t h a t body w e i g h t , l i v e r weight and the number of n u c l e i i n the 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 than i n i n b r e d embryos d e v e l o p i n g i n the 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 i n c e l l s i z e . M i t o t i c ' A c t i v i t y as an Index o f Rate o f DNA Increase ' • To the extent 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 of the embryo i s r e l a t e d t o i t s growth r a t e , the l a t t e r i s measurable i n terms of frequency 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 the DNA o f the 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 the 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 to express m i t o t i c f requency 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 Minot 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 S c h u l t z ( 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 the 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 , i n the s tudy o f the e a r l y development o f the c h i c k . I t s a p p l i c a t i o n t o the measurement o f t i s s u e growth has been d i s c u s s e d by Hoffman (1949) who p o i n t e d out the v a r i o u s shortcomings of the t e c h n i q u e . D e r r i c k (1937) employed m i t o t i c i n d e x , which he r e f e r r e d t o as the number of c e l l s i n d i v i s i o n out of 1 ,000, i n a s tudy o f the e a r l y d e v e l o p -ment o f the c h i c k embryo. 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 to s t r u c t u r e , the index a l s o 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 . A p r o g r e s s i v e e d e c l i n e i n the index was observed to occur w i t h advancing age and d i f f e r e n t i a -t i o n . He r e p o r t e d the value o f the index f o r the e n t i r e embryo at 32 hours to be 8 .04; a t 78 hours i t was 4 . 5 . B e l l a i r s (1957) a l s o observed t h a t m i t o t i c r a t e v a r i e d from t i s s u e to t i s s u e . S i m i l a r o b s e r v a t i o n s have been made by Em-anuelsson (1961b). Bloom and Buss (19G8) examined the e f f e c t o f age and k i n d of 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 observed, 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 or l e s s s i m i l a r f o r a l l t i s s u e s s t u d i e d . D u r i n g the p e r -i o d from t h r e e to f i v e days , the m i t o t i c a c t i v i t y o f the b r a i n dropped r a p i d l y from a va lue o f 12 .5 mitoses per 100 c e l l s at t h r e e days to 9 .2 mitoses a t f i v e days . The index of the s p i n a l cord showed a somewhat d i f f e r e n t p a t t e r n . I t i n c r e a s e d from 5.B mitoses per 100 c e l l s at three days t o 7 .2 at f o u r days. By f i v e days i t had dropped s l i g h t l y to 7.0 and by day s i x i t s va lue was reduced t o 4 . 0 . Thus, i t would appear t h a t the r a t e of d e c l i n e of a c t i v i t y o f the 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 the 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 the 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 of 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 g i v e n s t a g e . R e g i o n a l d i f f e r e n c e s i n the m i t o t i c a c t i v i t y of s e v e r a l reg ions of the c h i c k s p i n a l cord were r e p o r t e d by C o r l i s s and Robertson (1963) f o r the p e r i o d from one and o n e - h a l f to f i v e days o f i n c u b a t i o n . They observed t h a t the m i t o t i c p a t t e r n w i t h i n the n e u r a l e p i t h e l i u m changed d u r i n g the course of development. The a c t i v i t y i n the b a s a l p l a t e , or lower h a l f o f the t u b e , was reasonably cons tant and greater than t h a t of the a l a r (upper) p l a t e u n t i l Hamilton-Hamburger stage 19 ( three t o three 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 stage 19 , r e a c h i n g a lower a c t i v i t y than t h a t of the a l a r p l a t e by stage 23 ( four days o f i n c u b a t i o n ) . I t c o n t i n u e d t o d e c l i n e to stage 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 t i m e , a c o n s i d e r a b l e d i f f e r e n c e 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 h a l v e s , or p l a t e s . Hamburger (1948) has s i m i l a r l y shown t h a t , from the t h i r d 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 than t h a t o f the b a s a l p l a t e . He noted t h a t the stage a t which the peak of 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 ; the a c t i v i t y of 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 of the a l a r p l a t e o c c u r r e d at s i x days . M i t o l o (1957) a l s o examined the d i s t r i b u -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 , o b -t a i n i n g r e s u l t s s i m i l a r t o those 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 (basal ) h a l f u n t i l three and o n e - h a l f days of i n c u b a t i o n ; the d o r s a l h a l f then 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 remain ing h i g h e r i n t h a t h a l f to s tage 31. C o r l i s s and Robertson (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 o f the upper and lower p l a t e s 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 . P r i o r to stage 26 no a n t e r i o r to p o s t e r i o r change was apparent . A t s tage 26, there was a s teady but n o n - s i g n i f i c a n t r i s e i n . t h e a n t e r i o r to p o s t e r i o r d i r e c t i o n . Hamburger (1948) and H i t o l o (1967), however, obta ined r e s u l t s to i n d i c a t e t h a t m i t o t i c a c t i v i t y was grea ter i n more a n t e r i o r l o c a t i o n s . 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 reg ions of the a l a r p l a t e , 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 throughout the l e n g t h o f the embryo. 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 -a t i o n i n the b r a c h i a l and t h o r a c i c segments i n c h i c k embryos of f o u r t o seven and o n e - h a l f days, M i t o l o (1967) observed t h a t a peak of m i t o t i c a c t i v i t y was reached by stage 27 ( f i v e and o n e - h a l f days) i n both segments. I t then decreased p r o g r e s s i v e l y through stages 29 ( s i x to s i x - and o n e - h a l f days of i n c u b a t i o n ) and 31 (seven t o seven and o n e - h a l f days of i n c u b a t i o n ) . The d i f f e r e n c e i n m i t o t i c a c t i v i t y between the b r a c h i a l and t h o r a c i c segments a t s tage 23 ( four days) was not s i g n i f i c a n t but i n a l l subsequent s tages to stage 31, the n e u r a l tube i n 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 , e s p e c i a l l y a t s tages 27 and 31. The A p p l i c a t i o n , o f Autoradiography to the Measurement of M i t o t i c Frequency A most important method employed i n the s tudy o f m i t o t i c a c t i v i t y i s t h a t o f a u t o r a d i o g r a p h y ( i e . , F u j i t a , 1962; Wimber, 1959). . I t i s based on the p r o p e r t y o f r a d i o s o t o p e s to emit r a d i a t i o n which, ac ts on the. s i l v e r . h a l i d e c r y s t a l s o f a photographic e m u l s i o n . P r o l i f e r a t i n g t i s s u e 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 thymidine ( H 3 - t h y m i d i n e ) . The 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 which are s y n t h e s i z i n g DNA and these 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 coated w i t h 13 photographic emuls ion and then developed as an o r d i n a r y photograph. S i t e s , o f 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 appear as b lackened areas i n the emul -s i o n o v e r - r i d i n g the s e c t i o n . 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 n u c l e u s r e p l i c a t e s i t s DNA content i n p r e p a r a t i o n f o r c e l l d i v i s i o n . This s tage of c e l l d i v i s i o n i s r e f e r r e d to as the ' S ' phase (Wimber, 1-959). Upon c o m p l e t i o n of t h i s phase, a c e l l enters the p o s t - d u p l i c a t i o n o r G 2 phase, a p e r i o d o f s h o r t d u r a t i o n , be fore m i t o s i s b e g i n s . A f t e r m i t o s i s , the daughter c e l l s enter the p r e - d u p l i c a t i o n or G]_ phase; some o f the c e l l s never l e a v e t h i s phase w h i l e o thers proceed a f t e r v a r i a b l e t ime i n t e r v a l s to the 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 . Only d u r i n g the S-phase can the l a b e l l e d p r e c u r s o r be i n c o r p o r a t e d . However, a l l c e l l s which have passed through the S-phase and on to a f u r t h e r stage d u r i n g the p e r i o d o f exposure to the 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 index than m i t o t i c index would be expected i f the exposure p e r i o d was g r e a t e r than the d u r a t i o n o f the ' 5 ' phase. The technique o f autoradiography can t h e r e f o r e be adapted, by employing an extended exposure p e r i o d , to 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 which 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 r e t a i n a l l o f t h e i r l a b e l i f they do not proceed t o m i t o s i s . Examples o f the a p p l i c a t i o n of autradiography t o the s tudy o f c e l l p r o l i f e r a t i o n and behaviour i n the c h i c k embryo are numerous. Weston (1963) used the technique i n the study of 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 runk n e u r a l c r e s t c e l l s and Gwatkin and Biggers (1963) have compared the r a t e s o f i n v i v o and i n v i t r o DNA s y n t h e s i s i n d e v e l o p i n g c h i c k t i b i o t a r s i . A u t o r a d i o -graphy was employed by F u j i t a (1965) i n a s tudy of the g l i o b l a s t c e l l s i n the embryonic c h i c k s p i n a l c o r d and Janners and S e a r l s (1968) used the technique i n a c h a r a c t e r i z a t i o n 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 Nelson (i960) u s i n g t h i s t e c h n i q u e . The former workers found t h a t w i t h advancing development o f the embryo there was a gradual 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 which 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 of o r i g i n and p o s i t i o n i n the system. Kodak et al_. (1968) employed autoradiography i n a s tudy o f the e a r l y development o f the c h i c k l e n s and observed 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 to t h a t o f m i t o t i c a c -t i v i t y , except t h a t changes i n the d i s t r i b u t i o n o f a c t i v i t y became ev ident 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 of H^- thymidine used by the aforementioned workers ranged from 5 to 25 m i c r o c u r i e s ( p c ) ; the 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 the age of the embryos under s t u d y . Sauer and Walker ( l 9 5 l ) 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 mainly i n the p r o d u c t i o n of 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 Hg- thymidine 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 mistaken 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 i n the a p - . parent number of l a b e l l e d c e l l s . Sauer and Walker d i d not observe any change i n m i t o t i c a c t i v i t y i t s e l f . I t was found t h a t the i n j u r y produced depended on the c o n c e n t r a t i o n , r a t h e r than the 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 i n j e c t e d . 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 the l a b e l l e d thymidine w i t h u n l a b e l l e d t h y m i d i n e . Weston (19B3) on the other hand, 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 the W o -s t u d i e s were not r e a d i l y comparable. Post and Hoffman (1961), who have r e -viewed much o f the l i t e r a t u r e r e l a t i n g to m i t o t i c d i s t u r b a n c e s and c e l l death 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 the p l o i d y c l a s s e s of r a t l i v e r n u c l e i f o l l o w i n g r a d i o a c t i v e l a b e l l i n g . I t has been p o i n t e d out by Wand et al_. (1967) t h a t a g i n g Hg- thymidine undergoes decomposi t ion 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 products do not l a b e l DNA b u t , r a t h e r enter macro-15 molecules other than n u c l e i c a c i d v h i c h are conta ined i n the cy top lasm. 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 observed, 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 found 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 v e r y 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 p a t t e r n s . . Emanuelson (1367) observed t h a t Feulgen 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 percent r e d u c t i o n i n the number of s i l v e r g r a i n s i n the n u c l e u s . However,, t h i s i s not a matter o f p a r t i c u l a r concern when the technique 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 not 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 tudy of- ' the c h a r a c t e r i s t i c s o f c e l l u l a r p r o l i f e r a t i o n i n the n e u r a l t u b e , mesencephalon and o p t i c r e t i n a o f one- t o s i x - d a y o l d c h i c k embryos. He demonstrated t h a t the m a t r i x l a y e r i n the t i s s u e s 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 of zones r e l a t e d t o the degree o f m a t u r a t i o n of the c e l l s c o m p r i s i n g them. C e l l s o f the deepest l a y e r , . t h e 5 -zone , are those which 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 m i t o s i s . When these have undergone DNA s y n t h e s i s , they b e g i n t o ascend and enter i n t o the i n t e r m e d i a t e I -zone (corresponds t o Gg) w h i c h i s t r a v e r s e d d u r -i n g the p r e m i t o t i c p e r i o d . D u r i n g subsequent m i t o s i s , these c e l l s remain a d -j a c e n t t o the i n t e r n a l l i m i t i n g membrane (K-zone) and d u r i n g the p o s t - m i t o t i c p e r i o d the daughter c e l l s migrate back t o the I-zone and f i n a l l y reach the S-zone t o s t a r t the g e n e r a t i v e c y c l e a g a i n . He es t imated the d u r a t i o n of the various- s tages i n the mesencephalon t o be as f o l l o w s : l e n g t h of p e r i o d o f DNA s y n t h e s i s , 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 of 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 ime o f the m a t r i x c e l l , s i x t e e n h o u r s ; d u r a t i o n of m i t o s i s i t s e l f , 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 the a l a r p l a t e of the s p i n a l cord the v a l u e s were s i m i l a r t o those of the mesen c e p h a l o n , 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 b a s a l 16 p l a t e of the same s p i n a l cord l a b e l l i n g was s p a r s e ; the o n l y constant g iven was t h a t f o r g e n e r a t i o n t i m e , which was es t imated to be two and o n e - h a l f days . The m a t r i x c e l l s of 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 at a very low r a t e and F u j i t a suggested t h a t the genera t ion t ime of those c e l l s was f a r l o n g e r than t h a t of the b a s a l p l a t e m a t r i x c e l l s . . The m i t o t i c index o f the mesencephalon was 6.8 c e l l s i n m i t o s i s out o f 100, w h i l e the l a b e l l i n g index was 35 l a b e l l e d c e l l s per hundred. Comparable va lues f o r the a l a r p l a t e o f the s p i n a l cord were 6.5 and 25. Both the m i t o t i c t ime and g e n e r a t i o n time o f the m a t r i x c e l l s i n the one-day c h i c k embryo were much s h o r t e r than i n the s i x - d a y embryo; the m i t o t i c index of the former was e i g h t mitoses per hundred c e l l s . I n s o f a r as they are comparable, F u j i t a ' s r e s u l t s agree w i t h those of o ther workers who examined p r o l i f e r a t i o n of the s p i n a l c o r d o f the c h i c k em-bryo i n terms o f m i t o t i c a c t i v i t y . Hamburger (1948), C o r l i s s and Robertson (1963) and M i t o l o (1967) a l l observed a grea ter a c t i v i t y i n the a l a r p o r t i o n o f the cord than i n t h e ' b a s a l p o r t i o n a f t e r three and o n e - h a l f days , though none of these workers observed as great 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 F u j i t a . 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 to the f a c t t h a t he examined s i x - d a y embryos w h i l e the other s t u d i e s i n v o l v e d younger embryos. A l l s t u d i e s employing m i t o t i c r a t e agreed t h a t the d i f f e r e n c e between the two p l a t e s o f the 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 the 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 i n enzyme a c t i v i t y d u r i n g the embryonic development o f many organisms. 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 which l ias been accumula t ing r a p i d l y may s h i f t to a constant 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 i n c r e a s e aga in (Moog, 1965) . Changes i n the c h i c k embryo as a whole In the c h i c k embryo, as i n most s i m i l a r organisms, enzymes o f gen-e r a l metabo l i c 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 p a t t e r n s , t h a t f o r each enzyme b e i n g more or l e s s independent of the a c t i v i t i e s o f - o t h e r enzymes p r e s -ent . For i n s t a n c e , the e a r l y c h i c k embryo possesses low phosphatase a c t i v i t y , (Moog and S t e i n b a c h , 1946), r e l a t i v e l y h i g h pept idase a c t i v i t y (Levy and P a l -mer, 1943) and very h i g h l e v e l s of cytochrome oxidase and s u c c i n i c oxidase (Albaura et a l . , 1946) . Solomon (1959) found t h a t the s p e c i f i c a c t i v i t y of m a l i c a c i d de -hydrogenase of whole c h i c k embryos was greater t h a n t h a t ' of l a c t i c a c i d dehy-drogenase (LDH) d u r i n g the p e r i o d from one and o n e - h a l f t o s i x t e e n days of i n c u b a t i o n , and t h a t from two t o f o u r days and from ten t o s i x t e e n days there was about t w i c e as much of the l a t t e r as of the f o r m e r , Rudnick et_ a l . (1953) s i m i l a r l y observed t h a t g lu tamotrans ferase a c t i v i t y of the c h i c k embryo, i n -c l u d i n g the membrane system, i n c r e a s e d d u r i n g i n c u b a t i o n i n a manner d i r e c t l y p r o p o r t i o n a l t o p r o t e i n growth of the whole system. Solomon (1957c) r e p o r t e d g lu tamic dehydrogenase a c t i v i t y t o i n c r e a s e a t a r a t e c o i n c i d e n t w i t h the r a t e of change o f t o t a l p r o t e i n i n the whole c h i c k e n embryo from the f o u r t h day of i n c u b a t i o n onward. .„ • A c c o r d i n g t o Mahler et_ al_. (1958) the s p e c i f i c a c t i v i t i e s o f many enzymes of the c i t r i c a c i d c y c l e , and LDH, i n the whole c h i c k embryo r i s e t o t h r e e days of i n c u b a t i o n when they reach a t u r n i n g p o i n t and proceed t o d e c l i n e . g r a d u a l l y t o the t e n t h day. Cytochrome oxidase and s u c c i n i c dehydrogenase a c t i v -i t i e s r i s e s t e a d i l y from seven t o t e n days (Davidson, 1957) . By c o n t r a s t , t h e a c t i v -i t y of adenosine deaminase i s reasonably constant from the t h i r d day t o the e l e v e n t h day o f i n c u b a t i o n and then i n c r e a s e s t h r e e - f o l d , b e t w e e n the e l e v e n t h and f i f t e e n t h 18 days , ( F i s h e r et a l . , 1962) . Changes i n s p e c i f i c t i s s u e s of the c h i c k embryo W i t h i n the same t i s s u e and organ, enzymes tend to i n c r e a s e or d e -crease i n a c t i v i t y independent ly of each o t h e r , a l t h o u g h p a r a l l e l changes may o c c u r . Such v a r i a t i o n d u r i n g the development of the c h i c k embryo h e a r t has been demonstrated f o r s u c c i n o x i d a s e ( S i p p e l , 1954), f o r cytochrome ox idase and s u c c i n i c dehydrogenase (Davidson, 1957), and f o r l a c t i c a c i d and•malic -ac id dehydrogenase (Solomon, 1958b). C o r r e l a t e d enzymatic and s t r u c t u r a l changes i n the d e v e l o p i n g t i s s u e s of the c h i c k embryo have been r e p o r t e d by s e v e r a l w o r k e r s . Both g l u t a m o t r a n s f e r a s e and glutamine synthetase a c t i v i t y o f the r e t i n a undergo a r a p i d i n c r e a s e i n a c t i v i t y a t approx imate ly seventeen days o f i n c u b a t i o n a t the t ime t h a t the r e t i n a i s becoming v i s u a l l y f u n c t i o n a l (Hoscona and Hubby, 1963; Rudnick and Waelsch , 1953) . A c i d phosphatase, a l k -a l i n e phosphatase ( A P ) , g lucgse-6-phosphate dehydrogenase, 5 - n u c l e o t i d a s e , s u c c i n i c dehydrogenase, LDH and B - g l u c u r o n i d a s e have a l l been found t o be l o c -a l i z e d i n nephons of the c h i c k embryo c o i n c i d e n t w i t h the onset o f k i d n e y f u n c t i o n (Moog, 1944) . R i c h a r d s o n et al_. (1955) found t h a t n o n - s p e c i f i c e s te rase underwent a sharp r i s e i n a c t i v i t y i n the c h i c k embryo duodenum a t seventeen days and concluded t h a t i t was i n v o l v e d i n m a t u r a t i o n of t h a t s t r u c t u r e . Numerous workers have demonstrated t h a t a r a p i d i n c r e a s e i n a c e t y l c h o l i n e s t e r a s e a c t i v i t y i s . a s s o c i a t e d w i t h the f u n c t i o n a l d i f f e r e n t i a t i o n , of. the b r a i n and s p i n a l c o r d o f the c h i c k embryo (Wenger and Lowry , 1951; Rogers , i 9 6 0 ; B u r t , 1968; Turbow and B u r k h a l t e r , 1968) . F i s h e r et a l . (1962) r e p o r t e d developmental changes i n the adenosine deaminase a c t i v i t y of the c h i c k embryo duodenum as 19 N u n n a l l y (1962) has done f o r s u c c i n i c dehydrogenase. Changes i n l i v e r enzyme l e v e l a s s o c i a t e d w i t h ontogeny have been- r e p o r t e d f o r p e p t i d a s e (Dumm and L e v y , 1949), cytochrome oxidase and s u c c i n i c dehydrogenase (Davidson, 1957), g l u t a m i c dehydrogenase and m a l i c a c i d dehydrogenase (Solomon, 1959) , and s u c -c i n i c dehydrogenase, d iaphorases and n o n - s p e c i f i c e s t e r a s e s , g lycerophosphate dehydrogenase and a l k a l i n e phosphatase ( C o n k l i n , 1966) . Neuramidase a c t i v i t y was determined i n s e v e r a l t i s s u e s o f d e v e l o p i n g c h i c k and duck embryos by Cook and Ada (1963) who observed t h a t , i n those t i s s u e s which were p a r t i a l l y or c o m p l e t e l y d e r i v e d from the ectodermal l a y e r , there was a marked i n c r e a s e i n a c t i v i t y as the embryo grew o l d e r w h i l e other t i s s u e s showed low a c t i v i t y a t a l l s t a g e s . Mot te t (1967) found the m o r p h o l o g i c a l stages i n the d i f f e r e n -e t i a t i o n o f the c h i c k embryo l i m b bud from Hamilton-Hamburger s tages 19 to 34 to be c o r r e l a t e d w i t h aminopeptidase a c t i v i t y . 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 o f d e v e l o p -mental r a t e \ . B u r t and Wenger ( l 9 6 l ) observed a r e l a t i o n s h i p between the a c t i v i t y o f g lucose -6-phosphate dehydrogenase (G-6-p-d) and the m i t o t i c a c t i v i t y o f b r a i n t i s s u e o f the c h i c k embryo. The r e l a t i v e r e l a t i o n s h i p s o f G-6~p-d and i s o c i t r i c dehydrogenase t o the growth r a t e o f the t o t a l c h i c k embryo were com-pared by Newburgh et a l . (1962). They observed a h i g h c o r r e l a t i o n of bo th e n -zymes w i t h DNA content and t o t a l p r o t e i n o f the embryo d u r i n g the two-day p e r -i o d between 40 and 88 hours o f i n c u b a t i o n . The c o r r e l a t i o n between enzyme a c t i v i t y and DNA content was g r e a t e r than t h a t between enzyme a c t i v i t y and p r o t e i n c o n t e n t , i n d i c a t i n g a c l o s e r e l a t i o n s h i p between these enzymes and p r o l i f e r a t i o n r a t e . DNA, p r o t e i n and i s o c i t r i c dehydrogenase a c t i v i t y i n c r e a s e d s e v e n - f o l d d u r i n g the 40 to 60 hour p e r i o d w h i l e G-6~p-d i n c r e a s e d f o u r - f o l d . 20 The a c t i v i t y of 6 -phosphofruc tok inase of the c h i c k embryo was r e p o r t e d by Bur t (1967) t o i n c r e a s e more than n i n e - f o l d from f o u r and o n e - h a l f t o s i x days ; i t a l s o i n c r e a s e d between t e n and s i x t e e n days . The same worker obs-erved a n e a r l y t w o - f o l d i n c r e a s e i n adolase a c t i v i t y from f o u r and o n e - h a l f days of i n c u b a t i o n t o h a t c h i n g , w i t h a 50 percent i n c r e a s e i n a c t i v i t y o c c u r -r i n g between t e n and twelve days . Genet ic 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 of Chick Embryos and the R e l a -t i o n s h i p of 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  G l u t a t h i o n e , a p o l y p e p t i d e o f lm.; molecular w e i g h t , i s w i d e l y d i s -t r i b u t e d i n l i v i n g t i s s u e and i s mainta ined t h e r e i n i n r e l a t i v e l y h i g h c o n -c e n t r a t i o n , presumably because i t i s o f fundamental importance to the o r g a n -i s m . I t s v a r i o u s r o l e s have been reviewed by Charkey et al_. (1965) and S t u t t s et a l . (1956). The l a t t e r workers found g l u t a t h i o n e l e v e l t o be c o r r e l a t e d w i t h egg p r o d u c t i o n i n c h i c k e n s , and have a l s o demonstrated g e n e t i c d i f f e r -ences between l i n e s i n b l o o d l e v e l s o f the compound. I t i s r e c a l l e d t h a t the c e l l s o f R . I . R . embryos have been shown t o p r o l i f e r a t e at a f a s t e r r a t e than those o f W.L. embryos (Blunn and Gregory, : 1935) . Gregory et al_. (1935) demonstrated t h a t the mean g l u t a t h i o n e c o n c e n t r a t i o n of R . I . R . embryos was a l s o g r e a t e r than t h a t o f W.L. embryos at f o u r t e e n days of i n c u b a t i o n . They a l s o r e p o r t e d t h a t g l u t a t h i o n e c o n c e n t r a t i o n was r e l a t e d t o p o s t - h a t c h i n g growth r a t e and t o a d u l t w e i g h t . In a subsequent comparison between the W.L. and B a r r e d Plymouth Rock ( B . P . R . ) b r e e d s , Gregory e t al_. (1936) demonstrated t h a t embryonic d i f f e r e n c e s i n g l u t a t h i o n e l e v e l between breeds of ch ickens were a s -s o c i a t e d w i t h a d u l t breed s i z e , even though embryonic weight, d i f f e r e n c e s d i d not e x i s t between the W o breeds compared. The Gregory group concluded t h a t g l u t a t h i o n e c o n c e n t r a t i o n was c o r r e l a t e d w i t h the r a t e o f c e l l p r o l i f e r a t i o n 21 (Gregory et a l . , 1.936). More r e c e n t l y , Charkey et a l . , (1965) found t h a t c o c k e r e l s of grea ter i n h e r e n t e a r l y growth r a t e possessed h i g h e r b l o o d and l i v e r l e v e l s of g l u t a t h i o n e than d i d s l o w e r - g r o w i n g b i r d s . Rudnick e t , a l . . (1953) and Rudnick and Waelsh (1955) r e p o r t e d t h a t a c o r r e l a t i o n e x i s t e d b e -tween the l e v e l o f g l u t a m o t r a n s f e r a s e , an enzyme w h i c h c a t a l y z e s the t r a n s f e r of the g l u t a m i c a c i d r a d i c a l o f g l u t a t h i o n e and glutamine t o other amines, and p r o t e i n c o n t e n t , but were unable t o show a d i r e c t r e l a t i o n s h i p between the a c t i v i t y of the enzyme and p r o t e i n s y s t h e s i s . Genet i c d i f f e r e n c e s i n enzyme l e v e l of the c h i c k embryo have been demonstrated f o r c y s t e i n e desulphydrase by Solomon (1963). He found t h a t , at the same stage o f development, the y o l k sac of White Leghorns c o n t a i n e d t w i c e as much of the enzyme as d i d Brown Leghorns . L a c t i c . A c i d Dehydrogenase (LDH) L a c t i c a c i d dehydrogenase (LDH) i s one o f a l a r g e number of enzymes i n v o l v e d i n * i n t e r m e d i a r y metabol ism. I t s p a r t i c u l a r r o l e i s t w o - f o l d - i t c a t a l y z e s the r e d u c t i o n of p y r u v i c a c i d by reduced d i p h o s p h o p y r i d i n e n u c l e o -t i d e ( D P M ) , and i t a l s o a i d s i n m a i n t a i n i n g the l e v e l o f d i p h o s p h o p y r i d i n e n u c l e o t i d e (DPNH) by the o x i d a t i o n o f the DPN so t h a t anaerobic g l y c o l y s i s may cont inue t o proceed (White et_ a l . , 1959) . LDH:~' Changes i n a c t i v i t y d u r i n g development Solomon (1958b) de tec ted LDH a c t i v i t y i n unincubated egg y o l k and i n y o l k taken from embryonated eggs up t o e i g h t e e n days of i n c u b a t i o n . A c -t i v i t y remained very low u n t i l f o u r t e e n days o f i n c u b a t i o n , except f o r a tem-p o r a r y r i s e at e leven days . At e ighteen days a v e r y l a r g e i n c r e a s e i n a c t i v i t y o c c u r r e d . The LDH a c t i v i t y of the y o l k sac was found t o be low at two days and 22 t o r i s e t o a maximum at seven days when i t d e c l i n e d to n e a r l y i t s W o - d a y l e v e l by e i g h t e e n days . The a c t i v i t y o f the v a s c u l a r area of the y o l k sac showed a s i m i l a r , but e a r l i e r , r i s e and f a l l , r e a c h i n g a maximum at three days. . The l e v e l o f a c t i v i t y i n the whole embryo, expressed per gram o f wet w e i g h t , i n c r e a s e d approx imate ly t w o - f o l d from twelve hours to -one and o n e - h a l f days and cont inued to r i s e i n an apparent l o g a r i t h m i c f a s h i o n to f o u r t e e n days , when i t l e v e l l e d o f f . Solomon p o i n t e d out t h a t the sharp i n c r e a s e i n a c t i v i t y a f t e r the t e n t h day was the r e s u l t o f a general i n c r e a s e i n p r o t e i n per gram o f wet weight and t h a t when expressed per m i l l i g r a m o f p r o t e i n , no s i g n i f i c a n t change d u r i n g subsequent development o c c u r r e d . Mahler et al_. (1958) r e p o r t e d t h a t the s p e c i f i c a c t i v i t y of LDH i n whole c h i c k embryo homo-genates and i n v a r i o u s f r a c t i o n s obta ined by d i f f e r e n t i a l c e n t r i f u g a t i o n reached a maximum d u r i n g the p e r i o d between three and f o u r days . Adams and Finnegan (1965) r e p o r t e d developmental changes i n t o t a l LDH a c t i v i t y i n two spec ies of amphibians . Solomon (1958b) observed a gradual i n c r e a s e i n LDH a c t i v i t y per m i l -l i g r a m o f p r o t e i n i n c h i c k embryonic h e a r t t i s s u e from seven t o e ighteen days , f o l l o w e d by a s l i g h t d e c l i n e t o three days a f t e r h a t c h i n g . The a c t i v i t y of b r a i n t i s s u e , expressed on the same b a s i s , underwent no change p r i o r t o e i g h t -een days when i t began to i n c r e a s e g r a d u a l l y . A c t i v i t y i n muscle t i s s u e from f o u r t e e n days t o three hours a f t e r h a t c h i n g showed a s l i g h t decrease . L i v e r t i s s u e showed a c o n s i d e r a b l e change i n a c t i v i t y d u r i n g i n c u b a t i o n , r i s i n g r a t h e r r a p i d l y from seven days to s i x t e e n days , dropping s h a r p l y t o twenty-days , and then c o n t i n u i n g to d e c l i n e somewhat more g r a d u a l l y . 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 Wide b i o c h e m i c a l v a r i a t i o n e x i s t s among the v e r t e b r a t e s ; a l i s t 23 of s p e c i f i c , v a r i a n t s w h i c h have a q u a l i t a t i v e genet i c b a s i s has been, com-p i l e d by Lush (1966). W i t h i n the c l a s s Aves , enzymes which have been s t u d i e d i n c l u d e l a c t i c a c i d dehydrogenase (Zinkman et_ al_. , 1966) , a l k a l i n e phosphatase ( W i l c o x , 1966a; Law and Hunro, 1965) , a c i d phosphatase (Okada and Hachinohe, 1968) , and serum es terase (Grunder, 1968) . Many o f these compounds e x i s t i n polymorphic f o r m - w h i c h , when i n c o m b i n a t i o n , are separab le by v a r i o u s p h y s -i o c h e m i c a l p r o c e d u r e s . The term ' i s o z y m e ' was i n t r o d u c e d by Marker t and H o l l e r (1959) t o d e s c r i b e p r o t e i n s which have s i m i l a r enzymatic a c t i v i t y but d i f f e r i n m o l e c u l a r s t r u c t u r e which render them separable from one another by e l e c t r o p h o r e s i s . These workers have reviewed many of those enzymes w h i c h have been r e p o r t e d t o e x i s t i n m u l t i m o l e c u l a r forms . Among the f i r s t t o draw a t t e n t i o n t o . t h e m u l t i m o l e c u l a r nature of LDH were V e s s e l l and B e a m (1957). A t l e a s t f i v e d i s t i n c t forms of LDH have been i d e n t i f i e d i n the somatic t i s s u e s of most mammalian and a v i a n s p e c i e s , (Cahn et a l . , 1962; M a r k e r t , 19B3) . The molecular weight of a s p e c i f i c LDH isozyme i s a p p r o x i m a t e l y 135,000 (Markert and A p p e l l a , 1961; Pesce et a l . . ' 1964) . I t was f i r s t proposed by the Markert group ( A p p e l l a and M a r k e r t , 19Sl) t h a t each LDH m o l e c u l e , r e p r e s e n t i n g a s i n g l e f o r m , i s composed of f o u r p o l y -p e p t i d e s u b u n i t s o f equal s i z e . These s u b u n i t s are r e s o l v a b l e i n t o two e l e c t r p h o r e t i c a l l y d i s t i n c t v a r i e t i e s , A and B (Markert and Ursprung , 1962) . Each isozyme i s a te t ramer formed by the, a s s o c i a t i o n of f o u r s u b u n i t s . LDH-^, c o n -s i s t i n g o f f o u r i d e n t i c a l B s u b u n i t s , has t h e . g r e a t e s t n e g a t i v e charge and migra tes most r a p i d l y towards the anode d u r i n g e l e c t r o p h o r e s i s - . L D H 5 , made up e n t i r e l y of A s u b u n i t s , has the l e a s t n e g a t i v e charge and consequent ly migrates l e a s t r a p i d l y . The t h r e e remain ing i sozymes , which are h y b r i d s com-posed of bo th A and B s u b u n i t s , possess a charge and m i g r a t i o n r a t e dependent 24 upon the r e l a t i v e p r o p o r t i o n s of each of the sub-units which they c o n t a i n . LDH2, w h i c h migrates more r a p i d l y than e i t h e r of the other two h y b r i d s , c o n s i s t s of one A s u b u n i t and three B s u b u n i t s ; LDK^, which migrates a t a r a t e i n t e r m e d i a t e between those o f . L D H and bDH^, c o n t a i n s equal p r o p o r -t i o n s o f the two s u b u n i t s j and LDH^, c o n s i s t i n g of three A s u b u n i t s and one B s u b u n i t , migrates l e a s t r a p i d l y of the h y b r i d s but more r a p i d l y than L D H ^ . The A and B s u b u n i t s of v a r i o u s a n i m a l s , i n c l u d i n g the c h i c k e n , have been shown t o d i f f e r i n t h e i r amino a c i d c o m p o s i t i o n ( l i a r k e r t , 1953 ; 'Pesce et a l . . 1964) , p e p t i d e p a t t e r n of t r y p s i n d i g e s t s ( l i a r k e r t , 1963), and immunologica l p r o p e r t i e s ( l i a r k e r t and A p p e l l a , 1961; Cahn et a l . , 1962; L i n d s a y , 1963) . e The s y n t h e s i s of the t w o ' p o l y p e p t i d e s u b u n i t s , A and B, i s regarded as b e i n g under the c o n t r o l of two separate f i x e d genet i c l o c i , the r e s u l t a n t isozyme p a t t e r n s b e i n g dependent on the r e l a t i v e amounts of A and B p o l y p e p t i d e p r e s e n t , as determined by the r e l a t i v e a c t i v i t i e s of these genes ( l i a r k e r t and U r s p r u n g , 1962; Cahn et, a l _ . , 1962; Shaw and B a r t o , 1963) . For example, i f the A and B genes were e q u a l l y a c t i v e and i f a s s o c i a t i o n of the monomers was assumed t o be random, a l l f i v e i sozymes, LDH^ through LDH^, would be present i n the r a t i o o f 1 : 4 : 6 : 4 : 1 . Zinkham et a l . , (1966) have r e p o r t e d a v a r i a n t , B ^ , o f the B p o l y p e p t i d e i n - p i g e o n s which was produced by a second a l l e l e at the B l o c u s . The B^ " v a r i a n t possessed a r a t e o f e l e c t r o p h o r e t i c m i g r a t i o n i d e n t i c a l t o t h a t of the A p o l y p e p t i d e . In a d d i t i o n t o the f i v e somatic i sozymes , a f u r t h e r t y p e , LDH , has been found i n the t e s t e s o f s e x u a l l y mature mammals and b i r d s (Blanco and Zinkham, 1953; G o l d b e r g , 1963). . A c c o r d i n g t o Goldberg (1955), i t i s under the c o n t r o l of a t h i r d gene, C, w h i c h i s r e s p o n s i b l e f o r a spermatozoan-spec-i f i c LDH. He has suggested t h a t a C p o l y p e p t i d e r e s u l t s when t h e r e i s no r e p r e s s i o n of the C - l o c u s i n ' mature t e s t e s and t h a t r e p r e s s i o n ' o f the- gene i n 25 immature t e s t e s and i n a l l somatic t i s s u e i s r e s p o n s i b l e f o r the absence o f L B H X i n those t i s s u e s . He has demonstrated (Goldberg and Hawtrez , 1967) , t h a t LDH X appears i n mice at the t ime t h a t spermatocytes f i r s t b e g i n to mature. How-ever,, Stambaugh and Buckley (1967) have r e c e n t l y r e p o r t e d on experiments on LDH X from human and r a b b i t t e s t e s which suggested to them t h a t the somatic and t e s t i c u l a r isozymes share a common molecular s t r u c t u r e . They suggested t h a t the s y n t h e s i s of r a b b i t LDH X i s c o n t r o l l e d by the same genes w h i c h r e g u l a t e the s y n t h e s i s of the somatic isozymes. LDH: Ontogenet ic changes i n isozyme compos i t ion A c c o r d i n g t o Markert and Ursprung (1962) isozymes are a r e f i n e d e x p r e s s i o n of the enzymatic d i f f e r e n t i a t i o n of c e l l s . I n the c h i c k e n , as i n many other v e r t e b r a t e s , changes i n the r e l a t i v e p r o p o r t i o n s o f the d i f f e r -ent molecular v a r i e t i e s o f LDH occur d u r i n g embryonic l i f e and once adul thood i s reached no f u r t h e r change i s observed (Cahn et a l _ . , 1962) . F l e x n e r et a l . , ( i960) were among the f i r s t t o r e p o r t ontogenic changes i n LDH isozyme compos-i t i o n . They observed d i f f e r e n c e s between b r a i n LDH of a d u l t and newborn mice and between l i v e r LDH o f a d u l t and newborn guinea p i g s . In an e x t e n s i v e s tudy on the ontogeny o f the isozyme p a t t e r n o f LDH i n the mouse, Marker t and U r -sprung (1962) found t h a t i n a l l organs s t u d i e d the p r i n c i p a l LDH a c t i v i t y was i n i t i a l l y a s s o c i a t e d w i t h LDH<- and t h a t a t b i r t h LDH was o f t e n c o m p l e t e l y a b -D 1 s e n t . W i t h i n c r e a s i n g age, a gradual displacement of LDH a c t i v i t y occurred w i t h a p r o g r e s s i v e i n c r e a s e i n the r e l a t i v e amounts of the more anodal i s o -zymes, LDH , LDH , and LDH , w h i c h predominate i n the a d u l t t i s s u e s . They X 2 3 made the p o i n t t h a t a l though the general p a t t e r n was as j u s t d e s c r i b e d , the t r a n s p o s i t i o n o f a c t i v i t y from one end o f the spectrum (LDH^) t o the other iDH-j) d i d not occur synchronously throughout a l l d e v e l o p i n g t i s s u e but r a t h e r 26 each t i s s u e matured at i t s own c h a r a c t e r i s t i c r a t e . Thus, w h i l e i n the k i d n e y t h e - p a t t e r n found i n the t h r e e - d a y o l d animal was e s s e n t i a l l y t h a t o f the a d u l t , f u l l development of the c h a r a c t e r i s t i c a d u l t p a t t e r n of the-stomach o c -c u r r e d about three .weeks l a t e r . B r i n s t e r . and Auerbach (1968) have r e c e n t l y n o t i c e d a reverse s h i f t i n LDH isozyme p a t t e r n of young mouse embryos t o t h a t observed f o l l o w i n g b i r t h . They r e p o r t e d t h a t the a c t i v i t y i n the p r e - i m p l a n t a t i o n mouse embryo was m a i n -l y ' a s s o c i a t e d w i t h LDH a n d . s i m i l a r t y p e s . I n contrast ,•embryos which had been implanted had a p a t t e r n c o n s i s t i n g of a l a r g e p r o p o r t i o n of LDH^. About 98 percent of the s u b u n i t s produced before i m p l a n t a t i o n were of the B type w h i l e , f o l l o w i n g t h a t event , approx imate ly 90 percent were A s u b u n i t s . I n the r a t the s i t u a t i o n i s s i m i l a r - t o t h a t i n the mouse w i t h LDH^ a p p e a r i n g f i r s t i n the h e a r t and LDH-^ r e p l a c i n g i t as the predominant form soon a f t e r b i r t h (iCaplan and C i o t t i , 1961; F i n e et a l . , 1963; Latner and S k i l -l e n , 1964) . However, t h e r e i s l i t t l e d i f f e r e n c e between the l i v e r p a t t e r n of f o e t a l and a d u l t r a t s . In the human, a l l f o e t a l t i s s u e s show a great p r e p o n -derance o f LDHg b u t , w i t h development, the p r o p o r t i o n s of LDH^ and LDH^ i n -crease p r o g r e s s i v e l y ( F f l e i d e r e r and Wachsmuth, 1961; Latner and S k i l l e n , 1964) . I n the c h i c k e n , some c o n f u s i o n e x i s t s r e g a r d i n g the p a t t e r n of i s o z y m i c change i n the d e v e l o p i n g embryo. P h i l i p and V e s e l l (1962) found t h a t , i n the young embryo, LDH^ was the most abundant form present i n the l i v e r , muscle and h e a r t a l t h o u g h LDHg and LDK3 were a l s o present i n s m a l l amounts. S i m i l a r l y , Cahn et_ a l . (1962) found t h a t LDH^ was the predominant isozyme i n the v e r y young b las toderm but t h a t a t r a c e amount of LDH was a l s o . present at t h i s s t a g e . Between the second and f i f t h day of i n c u b a t i o n they f i r s t de tec ted LDH„ a c t i v i t y i n b r e a s t muscle w h i c h , t o g e t h e r w i t h LDHo, 27 subsequent ly i n c r e a s e d p r o g r e s s i v e l y up to s i x t e e n days o f i n c u b a t i o n at the exnense o f LDH . Trace amounts o f LDH f i r s t appeared at s i x t e e n days 1 4 of i n c u b a t i o n .and by the t w e n t i e t h day, LDH^ and LDH^ were the predominant forms , a l though r e l a t i v e l y s m a l l amounts o f the more ano'dal forms were a l s o d e t e c t a b l e . The t r a n s i t i o n from B to A s u b u n i t p r o d u c t i o n was not the same f o r a l l t i s s u e s . ; s k e l e t a l muscle c e l l s began t o s y n t h e s i z e A s u b u n i t s at-approx imate ly s i x to ten days of i n c u b a t i o n w h i l e h e a r t c e l l s d i d not undergo any change-over and cont inued t o produce o n l y B subuni t s throughout embryonic development (Cahri, 1964). L i n d s a y (1963) r e p o r t e d t h a t LDH]_ and, to a l e s s e r e x t e n t , LDHg were the o n l y isozymes present i n v a r i o u s t i s s u e s of the young embryo and t h a t , w i t h the e x c e p t i o n of h e a r t muscle i n which LDH^ was the o n l y form present between-three days o f i n c u b a t i o n and a d u l t h o o d , there was a g radual i n c r e a s e i n the r e l a t i v e p r o p o r t i o n o f A s u b u n i t s as development proceeded. The time o f f i r s t appearance i n v a r i o u s t i s s u e s o f isozymes other than LDH and LDH was c o n s i d e r a b l y l a t e r than t h a t observed f o r b r e a s t 1 2 muscle by Cahn et a l . (1962). However, s i n c e L i n d s a y (1963) d i d de tec t L-DHg, which i s compr ised , i n s m a l l p a r t , o f A monomers, i n both h e a r t and l e g mus-c l e t i s s u e p r i o r to the f o u r t h day o f i n c u b a t i o n , i t may be assumed t h a t A s u b u n i t s were present as e a r l y as t h i s stage of development. That he d i d not d e t e c t A s u b u n i t a c t i v i t y from any other isozyme source p r i o r to ha tch ing 'may p o s s i b l y have been due to the m o d i f i c a t i o n he i n t r o d u c e d i n t o h i s procedure o f isozyme s e p a r a t i o n by s t a r c h - g e l e l e c t r o p h o r e s i s . W h i l e a l l o f the aforementioned groups r e p o r t e d a g r a d u a l t r a n s f e r o f LDH a c t i v i t y i n the c h i c k embryo from the anodal to the c a t h o d a l i sozymes , Nebel and C o n k l i n (1964, i n c o n t r a s t , r e p o r t e d t h a t LDH3 and LDH4 were the o n l y isozymes present i n the v e r y young embryo ( i e . . , one to two days of 28 i n c u b a t i o n ) and t h a t they were the o n l y ones present i n the l i v e r , sp leen and b r a i n u n t i l the s i x t h , tenth and t w e l f t h day of development r e s p e c t i v e l y . The o b s e r v a t i o n s o f Nebel and C o n k l i n (1964) a r e , however, not r e a d i l y com-p a r a b l e to those o f other workers because o f the f a c t t h a t they examined s p e c i f i c organs f o r which the p a t t e r n o f change i n isozyme c o m p o s i t i o n might be expected t o d i f f e r from t h a t f o r i n d i v i d u a l t i s s u e s examined by others ( i e . , Cahn et a l . , 1962; L i n d s a y , 1963), or from the p a t t e r n o f the embryo as a whole as s t u d i e d by P h i l i p and V e s e l l (1962) and Cahn et a l . (1962). The most recent r e p o r t on the developmental changes i n LDH isozyme p a t t e r n i n the c h i c k embryo supports the view t h a t LDH]_-type s u b u n i t s are r e s p o n s i b l e f o r the major i s o z y m i c forms seen d u r i n g e a r l y development. A l -corn and M a i s e l (1967), who s t u d i e d the embryonic c h i c k cornea , observed t h a t , u n t i l the f o u r t e e n t h day, LDH]_, LDH2, and LDH3 were the main forms present i n t h i s s t r u c t u r e . D u r i n g subsequent development a marked i n c r e a s e i n LDH^ and LDH,- o c c u r r e d , r e f l e c t i n g a change-over i n the r e l a t i v e , producton o f the A and B p o l y p e p t i d e s . Adams and Finnegan (1965) r e p o r t e d a change d u r i n g development i n the number and p r o p o r t i o n s o f LDH isozymes i n spec ies o f two a m p h i b i a n s : ' -Amblystoma g r a c i l e and. Rana a u r o t a ; a s i m i l a r r e p o r t has been made by Kunz and Hearn (1967) f o r Xenopus l a e v i s . Balek and Snow (1967) observed t h a t the LDH isozyme content o f d e v e l o p i n g Amblystoma embryos changed from one composed predominant ly o f f a s t isozymes to one composed l a r g e l y o f isozymes w i t h s lower e l e c t r o p h o r e t i c m o b i l i t y . Chen (1968) r e p o r t e d three molecu lar forms o f LDH i n B o m b i n i , T r i t u r u s and Rana, a l l amphibians , and noted changes i n the p r o p o r t i o n s of these isozymes a s s o c i a t e d w i t h changes i n development. 29 LDH: I n f l u e n c e of pyruvate c o n c e n t r a t i o n as a b a s i s f o r .ontogenetic changes i n i s o z y m e c o m p o s i t i o n ; The isozymes o f LDH d i f f e r i n t h e i r degree of i n h i b i t i o n by e x -cess p y r u v a t e ; t h i s i s a f e a t u r e which i s o f c o n s i d e r a b l e s i g n i f i c a n c e t o the d e v e l o p i n g embryo. LDH^ i s s t r o n g l y i n h i b i t e d by q u i t e low c o n c e n t r a -t i o n s o f pyruvate w h i l e LDH^, on the other hand, i s r e l a t i v e l y u n a f f e c t e d by r e l a t i v e l y h i g h c o n c e n t r a t i o n s of pyruvate (Cahn et a_ l . , 1962; Marker t and U r s p r u n g , 1962) . The i n f l u e n c e o f pyruvate c o n c e n t r a t i o n on the v a r -i o u s h y b r i d forms i s d i r e c t l y r e l a t e d t o t h e i r degree of s i m i l a r i t y i n compos i t ion t o e i t h e r of the pure forms , LDEj_ and ^DH^.. The former i s adapted t o a e r o b i c c o n d i t i o n s s i n c e i t would be prevented from c o n v e r t i n g l a r g e q u a n t i t i e s of pyruvate t o l a c t a t e by v i r t u e o f the p r o g r e s s i v e i n -h i b i t i o n o f the isozyme by pyruvate which would i n s t e a d be m e t a b o l i z e d v i a the c i t r i c a c i d c y c l e . LDH5, however, would c a r r y out the r a p i d c o n v e r s i o n of pyruvate i n t o l a c t a t e w i t h the r e s u l t a n t es tab l i shment of an oxygen debt under anaerobic c o n d i t i o n s . DPN would a l s o be generated from DPNH. Embryonic t i s s u e s i n g e n e r a l , and those of the mouse i n p a r t i c u l a r , f u n c t i o n under c o n d i t i o n s of r e l a t i v e l y low oxygen supply and produce s u b s t a n t i a l q u a n t i t i e s of l a c t a t e ; they c o n t a i n predominant ly those isozymes at the LDH^ end o f the spectrum. H i g h l y oxygenated t i s s u e s , the p r o p o r t i o n of w h i c h xrould be expected t o i n c r e a s e as development proceeds , c o n t a i n most ly LDH^-type isozymes ( P f l e i d e r e r and Wachsmuth, 1961; Marker t and Ursprung , 1962) . Cahn ejb a l . (1962) who, a l o n g w i t h o t h e r s , found LDH^ t o be the i n i t i a l form i n the c h i c k embryo,account f o r t h i s o b s e r v a t i o n i n s o f a r as pyruvate t o l e r a n c e i s concerned by s u g g e s t i n g t h a t the embryonic t i s s u e s 30 o f the c h i c k i n p a r t i c u l a r depend upon a e r o b i c metabolism of p y r u v a t e , n o t i n g t h a t l a c t i c a c i d has been r e p o r t e d t o be t o x i c t o the c h i c k embryo. I t i s r e c a l l e d , however, t h a t Nebal and C o n k l i n (1964) have found t h a t LDHg and LDH were the f i r s t isozymes t o appear i n embryonic t i s s u e s of the c h i c k . P a r e n t a l i n f l u e n c e s cn LDH isozyme c o n s t i t u t i o n d u r i n g the e a r l y development of amphibians have been demonstrated by Wright and lioyer (1966) who made r e c i p r o c a l crosses between f r o g s v / i t h d i f f e r e n t LDH p a t t e r n s . The maternal p a t t e r n p e r s i s t e d i n the embryo u n t i l the. h e a r t - b e a t stage'when, both the h y b r i d and maternal bands were apparent . LDH: Thermal s t a b i l i t y of 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 ; Wroblewski and Gregory ( l 9 6 l ) r e p o r t e d t h a t LDH^ from human h e a r t t i s s u e was much more r e s i s t a n t t o temperature i n a c t i v a t i o n than LDH^ from human l i v e r t i s s u e . That a greater heat s t a b i l i t y i s possessed by the more anodal isozymes as.compared t o the more c a t h o d a l forms of a l l mammals and b i r d s has been p o i n t e d out by L i n d s a y (1963), who observed t h a t a d d i t i o n of DPNH t o the emzyme p r e p a r a t i o n s p r i o r t o h e a t i n g reduced the e f f e c t o f the heat t reatment on both LDH^ and bDH^. Wroblewski and Gregory ( l 9 5 l ) have a d v i s e d t h a t h e i r d i f f e r i n g heat s t a b i l i t y c h a r a c t e r i s t i c s can be u t i l i z e d i n d i f f e r e n t i a t i n the a c t i v i t i e s o f the component isozymes i n a m i x t u r e . Separate samples c o n -o o t a i n i n g the d i f f e r e n t isozymes are heated at 57 and 65 C a f t e r NADH^ has been added and the a c t i v i t y remain ing a f t e r t reatment i s compared w i t h t h a t o f an unheated sample. The c o n t r o l sample represents the t o t a l LDH a c t i v i t y . . 3 1 The d i f f e r e n c e between the c o n t r o l sample and the one heated at 57°C g ives a measure of the h e a t - l a b i l e f r a c t i o n which i s p r i n c i p a l l y LDH^. The a c t i v i t y o f the h e a t - s t a b l e f r a c t i o n , LDH^, i s represented by the d i f f e r e n c e between the sample heated a t 65°C and the unheated c o n t r o l , and the d i f f e r e n c e between the two heated samples i s an index o f the t o t a l a c t i v i t y o f the h y b r i d enzymes w h i c h are i n t e r m e d i a t e i n heat s t a b i l i t y . LDH: Evidence f o r L more than f i v e bands D u r i n g recent years t h e r e have been r e p o r t s to the e f f e c t t h a t more than f i v e e l e c t r o p h o r e t i c bands o f LDH may be r e s o l v e d i n the t i s s u e s o f some organisms. A l l e n ( l 9 6 l ) r e p o r t e d a t o t a l of n ine f r a c t i o n s separated by s t a r c h ge l e l e c t r o p h o r e s i s i n v a r i o u s t i s s u e s of the mouse. F r i t z and Jac~ obson 0-963) found t h a t the f i v e r e g u l a r l y o c c u r r i n g major bands of a c t i v i t y i n e x t r a c t s of mouse t i s s u e s c o u l d be s p l i t i n t o a t o t a l of f i f t e e n bands d u r i n g e l e c t r o p h o r e s i s on p o l y a c r y l a m i d e gels when 0.005 M B-mercaptoethanol was i n c o r p o r a t e d i n t o the g e l . Kunz and Hearn (1967) obta ined a t o t a l of e i g h t bands employing agar gel e l e c t r o p h o r e s i s on Xenopus l a e v i s t i s s u e and a t o t a l o f seven bands when the s t a r c h ge l technique was u s e d . Adams and F i n -negan (1965) ob ta ined t e n bands from Amblystoma p r a c i l e embryos u s i n g s t a r c h gel e l e c t r o p h o r e s i s . In f i s h the l i t e r a t u r e i s s t r o n g l y s u g g e s t i v e t h a t more than f i v e isozymes are produced by somatic t i s s u e . Goldberg (1965) p r e -sented evidence f o r the occurrence of a t h i r d a c t i v e l o c u s , C, i n a l l t i s s u e s o f the s p e c k l e d t r o u