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The relationship of cleft palate to riboflavin deficiency and genotype in chickens Juriloff, Diana Marie 1973

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C.I THE RELATIONSHIP OF CLEFT PALATE TO RIBOFLAVIN DEFICIENCY AND GENOTYPE IN CHICKENS by DIANA MARIE JURILOFF B.Sc. (Agric), University of British Columbia 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of Poultry Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA July, 1973 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that publication, in part or in whole, or the copying of this thesis for financial gain shall not be allowed without my written permission. Diana M. J u r i l o f f Department of Poultry Science The University of British Columbia Vancouver 8, Canada Date Xt, /f 75 i i ABSTRACT The incidence of cl e f t palate was observed in 1361 F-|, 1531 F^, and 2275 backcross embryos and chicks from a reciprocal cross between an inbred Leghorn line selected for high incidence (30 to 50%) of c l e f t palate and a non-cleft palate (New Hampshire; zero %) li n e . Cleft palate appeared in the F^ at frequencies less than 1%, in the F^ at approximately 1%, and in the backcrosses. at approximately 8%. When dams were fed a diet deficient in riboflavin, the incidence of cle f t palate was shown to increase for the F^ and backcross progeny to 4% and 12% respectively. The response of cl e f t palate incidence to riboflavin deficiency was shown to be in large part a genetic characteristic of the embryo i t s e l f and not the dam. The reduced hatchability of eggs during maternal riboflavin deficiency was shown to be similar to earlier reports. No evidence of any unusual response of c l e f t palate line hens to riboflavin deficiency was found for hatchability, chick body weight, nor maternal effect on cl e f t palate. The c l e f t palate condition was shown to be semi-lethal, the lethality being partially due to severe expression of the t r a i t . Genetic models were considered and i t was suggested that the model to be further tested should be that of 3 recessive l o c i , one of which involves a fault in the normal metabolism of riboflavin, and a few additive loci controlling penetrance and expressivity of the t r a i t . i i i TABLE OF CONTENTS Page INTRODUCTION . 1 REVIEW OF LITERATURE 2 A. Cleft Lip and/or Palate in Mammals Other than Man 2 B. Cleft Lip and/or Palate in Man 7 C. The 'Quasi-Continuous Variation' Model or Multi-factoral Concept as Applied to Mammalian CL(P) 10 D. Cleft Palate in Chickens . 12 E. Riboflavin Requirement and Effects of Deficiency in Chickens 23 MATERIALS AND METHODS 33 A. The New Hampshire Line 33 B. The Cleft Palate Line 34 C. The Breeding Design 36 1. Production of the F^ 36 2. Selection Criteria for Parents of the F^ 37 3. Production of the F 2 and the BC 38 4. Selection Criteria for Parents of the F? and BC . . . 39 D. The Data Collected for the CP l i n e , F-j, F 2 and BC . . . . 40 1. The Cleft Palate Line: G2 and G3 40 2. The F-j Generation 40 3. The F 0 and BC Generation 41 i v Page E. Special Studies 42 1. Crosses of Phenotypically Normal Cleft Palate Line Individuals 42 2. The New Hampshire x Cleft Palate Cross Tested on the Riboflavin Deficient Diet 43 3. The Selected New Hampshire Male Study 43 F. General Considerations for Data Analysis 44 RESULTS AND DISCUSSION 47 A. Effect of Riboflavin Deficient Diet on Hatchability . . . 47 B. Effect of Riboflavin Deficient Diet on Embryonic Mortality 53 C. Incidence of Cleft Palate on Normal and Riboflavin Deficient Diets 56 1. The F-j Data 56 2. The F 2 and BC Data 62 3. Facial Coloboma . . 68 4. Palatine Grooves 69 D. -Abnormalities Other than Cleft Palate 71 1. Curled Toe Paralysis and Club Down 71 2. Mai positioned and Strangled Embryos 74 3. The Exencephaly Syndrome 79 4. Palatal Pits 83 E. Sex Ratios in the Cleft Palate Progeny in the F ? and BC Generations 91 F. The Effect of Cleft Palate, Diet and Maternal Body Weight on Hatch Weight of Chicks 93 V Page G. Liveability of Embryos Related to the Severity of Cleft Palate in the and BC Generations 102 H. Embryonic Mortality of the F0 and BC Generations Associated with Cleft Palate 108 I. The Frequency of Different Types of Clefts Observed in the F? Generation and BC and the Response of the Frequency of Each Type to Riboflavin Deficiency 113 J. Multiple Regression Models 121 K. Incidence of Cleft Palate in the Fo Generation;.and BC Compared to Expectations for Multiple Recessive Loci 129 L. The Additive Model Applied to the Incidence of Cleft Palate in the Generation and BC 140 M. Additional Considerations 144 SUMMARY 154 REFERENCES 1 5 0 APPENDIX 171 vi LIST OF TABLES Table Page 1. Breeding I d e n t i f i c a t i o n Used Throughout the Study of C l e f t Palate (CP) and New Hampshire (NH) Matings Producing the F-j, F^ and Backcross Generations 46 2. Mean Percent Hatchability f o r Riboflavin Normal (+) and Deficient (-) Dietary Periods for the F-|, F2 and Backcross Generations 48 3. Mean Percent Embryonic Mortality of Hatches 7 Through 16 that Exceeded that of the Mean of Hatches 1-6 and Twice i t s Standard Deviation (SD) for Riboflavin (R) Normal ( + ) and Deficient (-) Diets 54 4. Incidence of Cleft Palate (CP) in the F] and F-j Progeny Over 10 Weekly Hatches of the CPxNH Cross and i t s Reciprocal on a Riboflavin (R) Normal (+) or Deficient (-) Diet 57 5. Incidence of Cle f t Palate Progeny and Total Observa-tions ( ) from New Hampshire Males (With Palate Grooves) Mated to Cl e f t Palate Females of G3, on Riboflavin Normal (+) and Deficient (-) Diets 60 6. Incidence of Cl e f t Palate Progeny and Total Observa-tions ( ) from the Remated NHxCP Cross on Riboflavin Normal (+) and Deficient (-) Diets 6 1 7. Mean Percent C l e f t Palate Progeny and Total Observa-tions ( ) from Matings Producing the F? and 4 Types of Backcross Generations on Riboflavin Normal (+) and Deficient (-) Diets 63 8. Incidence of Club Down (CD), C l e f t Palate (CP) and the Combination of Both in the Progeny of Hen 549 on a Riboflavin (R) Normal (+) and Deficient Diet 67 9. Incidence of Club Down (CD) i n Fi, F2 and Fo by Developmental Stages and by Sires and Dams [Producing CD) on Riboflavin Normal (+) and Deficient (-) Diets . . 72 10. Incidence of Club Down (CD) i n the Backcross Progeny by Developmental Stages and by Sires and Dams (Producing CD) on Riboflavin Normal (+) and Deficient (-) Diets 73 V Table Page 11. Mai positioned Embryos (M) of the F2 and Backcross Progeny for 3 Hatch Periods on Riboflavin Normal ( + ) and Deficient (-) Diets 75 12. Strangled Embryos (S) of the F? and Backcross Progeny for 3 Hatch Periods on Riboflavin Normal ( + ) and Deficient (-) Diets 77 13. Observed and Expected Frequencies of the "Exencephaly Syndrome T r a i t " Based on a Total Sample of 5169 Observations 80 14. Population Incidence of the Exencephaly Syndrome (En) and Total Observations (0) for Each Type of Cross on a Riboflavin Normal ( + ) and Deficient (-) Diet . . . 82 15. Incidence of Palatal P i t s in the Backcross Popu-lations for Riboflavin Normal (+) and Deficient (-) Diets / 86' 16. Percent C l e f t Palate for Hens that Did and Did Not Produce Palatal Pits f o r Riboflavin Normal (+) and Deficient (-) Diets 87 17. Number of Palatal Pits for Males (M) and Females (F) and Their.Palate Location 89 18. Sex Differences in the Backcross and F2 Generations for Hatched and Dead Progeny for Riboflavin Normal ( + ) and Deficient (-) Diets 92 19. Mean Hatch Body Weight (Gms.) of Cle f t Palate (CP) and Non-Cleft Palate (N) Chicks for the F2 Generation for Riboflavin Normal (+) and Deficient (-) Diets . . . 94 20. Mean Hatch Body Weight (Gms.) of C l e f t Palate (CP) and Non-Cleft Palate (N) Chicks for the Backcross Generation for Riboflavin Normal (+) and Deficient (-) Diets 95 21. Analysis of Variance of Mean Hatch Body Weight (Gms.) of Non-Cleft Palate Chicks of the Fg Generation f o r Riboflavin Normal (+) and Deficient (-) Diets 97 22. Mean Body Weight (Gms.) of the F 1, F] and C l e f t Palate (CP) Dams and the Mean Hatch Weight of Their Respective F 9, and Backcross Progeny 99 vi i i Table Page 23. Analysis of Variance of Mean Hatch Body Weight (Gms.) of C l e f t Palate and Non-Cleft Palate Chicks for the backcross Generation for Riboflavin Normal-(+) and Deficient (-) Diets TOO 24. Mean Sum of Right and Left Palate Scores of Hatched and Non-Hatched Chicks for the Backcrosses Pooled for Hen Genotype (CPxF-j and F^xCP) and for the Pooled Fp for Riboflavin Normal (+) and Deficient (-) Diets 103 25. Mean Strong Side Palate Scores for Hatched and Non-Hatched ( ) Chicks f o r the Backcrosses Pooled for Hen Genotype (CP x F] and F-| x CP) and for the Pooled Fp for Riboflavin Normal (+) and Deficient (-) Diets 104 26. Analysis of Variance of Mean Sum of Right and Left Palate Scores of Hatched and Non-Hatched Chicks f o r the Backcrosses Pooled for Hen Genotype (CPxF] and F-jxCP) and for the Pooled F 2 for Riboflavin Normal and Deficient Diets 105 27. Analysis of Variance of Mean "Strong Side" Palate Scores of Hatched and Non-Hatched Chicks for the Backcrosses Pooled for Hen Genotype, (CPxF] and FixCP) and for the Pooled F2 for Riboflavin Normal and Deficient Diets 106 28. C l e f t Palate as Percent of Hatched and Dead F 2 Generation Progeny, Across Maternal Riboflavin (R) Normal (+) and Deficient (-) Diets 109 29. C l e f t Palate as Percent of Hatched and Dead Backcross Progeny Across Maternal Riboflavin (R) Normal (+) and Deficient (-) Diets 110 30. Percent Hatched and Non-Hatched C l e f t Palate Types for the Pooled Fg and Backcross Pooled by Dam Genotype for Riboflavin Normal (+) and Deficient (-) Diets 117 31. Average Percent Hatch and Non-Hatched C l e f t Palate Types for the Backcross Data of Table 30 118 Table Page 32. Relative Change in Percent C l e f t Palate Frequencies of Hatches 7-10 and 11-16 Compared to Hatches 1-6 for Each Palate Type for the Average Backcross and F2 Data of Tables 30 and 31 1 2 0 33. Percent Mortality of Total Percent of C l e f t Palate Type for Combined Backcross Means of Table 31 and the F 2 of Table 30 With Riboflavin Normal (+) and Deficient (-) Diets 1 2 2 34. Coefficients of Determination (R2xl00) for Percent C l e f t Palate Progeny Per Dam (Y) Related to C l e f t Palate Severity of the C l e f t Palate Line F-j Parents (Xi) and the C l e f t Palate Line Backcross Parents (Xg) for Riboflavin Normal (+) and Deficient (-) Diets for 3 Multiple Regression Models ^ 35. Coefficients of Determination (R xlOO) for Percent C l e f t Palate Progeny Per Dam (Y) Related to C l e f t Palate Scores of the C l e f t Palate Line F] Parents (Right Side: X ] , Left Side: X2) and the C l e f t Palate Line Backcross Parents (Right Side: X3, Left Side: X4) for Riboflavin Normal (+) and Deficient (-) Diets for 3 Multiple Regression Models ^ 36. Coefficients of Determination (R2xl00) for Cl e f t Palate Progeny Scores for Right Side ( Y i ) , Left Side (Y 2) and Sum of Sides (Y3) Related to Original Parent C l e f t Palate Line Scores (Right Side: X]: Left Side: X2) and C l e f t Palate Line Backcross Parent Palate Scores (Right •Side: X3: Left Side: X.) for Riboflavin Normal (+) and Deficient (-) Diets Using the Model Y = a + b,X, + b 0X 0 x k v x k v ' > 2 2 128 + b3X3 + b4X4 • ' 37. Number of C l e f t Palate and Normal Progeny in the Fo and Backcross Generations for Riboflavin Normal (+] and Deficient (-) Diets 1 3 0 38. Number of C l e f t Palate and Normal Progeny of the Back-cross Generation for Riboflavin Normal (+) and Deficient (-) Diets 1 3 1 39. Number, of C l e f t Palate and Normal Progeny of the F2 Generation for Riboflavin Normal (+) and Deficient (-) Diets 1 3 2 x Table Page 40. Chi-Square for Goodness of F i t to Expected Fre-quencies for Multiple Recessive Loci Assuming 100 Percent Penetrance for the F2 and Backcross C l e f t Palate Progeny When Their Dams Were on Riboflavin Normal ( + ) and Deficient (-) Diets 134 41. Number and Percent of C l e f t Palate Progeny from Matings of Normal Progeny of Cle f t Palate Parents of Generation 3, on Riboflavin S u f f i c i e n t (+) and Low (-) Diets U b 42. Chi-Squares for Goodness of F i t to Expected Fre-quencies for Multiple Recessive Loci Assuming 50 Percent Penetrance for the F 2 and the Backcross C l e f t Palate Progeny When Their Dams Were on Riboflavin Normal (+) and Deficient (-) Diets 137 43. Number of Loci Controlling the C l e f t Palate T r a i t Based on the Goodness of F i t Chi-Square Tests for Riboflavin Normal (+) and Deficient (-) Diets 138 44. Estimation of Number of Loci (n) for an Additive Genetic Model Using Wright's Formulae for F 2 and Backcross Data 142 XI LIST OF FIGURES Figure Page 1. Plot of response of percent hatchability to the r i b o f l a v i n d e f i c i e n t diet 50 2. Facial coloboma and palatine grooves 70 3. Palatal p i t s 84 4. P a r t i a l pedigree of matings giving incidence of pi t s 90 5. Palates of 8 to 12 day dead embryos 114 6. Palates of 8 to 12 day dead embryos 115 7. Adult birds with c l e f t palate 116 x i i ACKNOWLEDGEMENTS The author i s grateful to Dr. C.W. Roberts, who provided supervision, suggestions, and f a c i l i t i e s for this study. Apprec-i a t i o n i s also expressed to Dr. J . Biely who formulated the rations used. Appreciation i s expressed to those members of the Faculty who read the manuscript and offered t h e i r advice, and to other members of the Poultry Science Department for t h e i r various contributions. The author would l i k e to express her gratitude to Mr. J . T o t h i l l , Dr. J . B i e l y , and Dr. C.W. Roberts, who provided the opportunity for the author to obtain her Bachelor's degree, and thereby enabled t h i s thesis to be begun. INTRODUCTION Cl e f t l i p and/or palate (CLP) has been observed in a wide variety of species including man, c a t t l e , sheep, dogs, ra b b i t s , mice, r a t s , and l i o n s . Of the species affected, the laboratory mouse and rat have been most extensively studied, for both genetic and environmental causes of the t r a i t . Many chemical and environmental f a c t o r s , including r i b o -f l a v i n deficiency, have been shown to increase .the incidence of CLP in mice, r a t s , chickens, but the mechanism of t h e i r action i s not known. Studies of inbred populations of mice have shown there i s a genetic basis of the t r a i t , but the nature has not been c l e a r l y elucidated. This study was based on the recent development, at the University of B r i t i s h Columbia, of an inbred l i n e of chickens having a high occurrence of c l e f t palate and a s e n s i t i v i t y in that frequency to maternal r i b o f l a v i n deficiency. I t was designed to test the genetic control of the t r a i t by observing the incidence of c l e f t palate i n the segregating generation of a cross to a non-susceptible l i n e , and the backcross to the susceptible l i n e , and to determine the extent of continuation of the r i b o f l a v i n deficiency effect across these generations. The p o s s i b i l i t i e s of maternal inheritance, maternal i n -fluence, and lethal effects were examined and both additive and non-additive models of inheritance were considered with respect to incidence, penetrance, and expressivity of the t r a i t . 2 REVIEW OF LITERATURE A. C l e f t Lip and/or Palate in Mammals Other than Man C l e f t l i p and/or palate (CLP) has been observed in a wide variety of species including dogs, horses, c a t t l e , pigs, l i o n s , jaguars, t i g e r s , domestic cats ( l i s t e d by Loevy and Fenyes, 1968), hamsters (Shenefelt, 1972), baboons (Steffek and Hendrickx, 1972), sheep (Hughes et. al _ . , 1972), rabbits (Robertson, 1966), rats (Warkany et al_ . , 1943), and mice ( K a l t e r , 1954). Of the species affected, the laboratory mouse and rat have been most extensively studied with respect to the t r a i t . Both genetic and environmental causes have been sought in an attempt to provide models for the human CLP condition. A large number of environmental factors have been shown to increase the incidence of CLP in mice and/or rats when the pregnant female i s exposed to them pr i o r to the closure of the primary and/or secondary palates in the embryos being c a r r i e d . Among these factors are: cortisone ( K a l t e r , 1954); cortisone related substances such as triamcinolone (Walker, 1965), hydrocortisone (Loevy and Wade, 1972), and corticosterone (Hackman and Brown, 1972); adrenocorticotropic hormone (ACTH) (Heiberg et al_ . , 1959); "stress," either by commercial mouse transport methods (Brown et al_., 1972), r e s t r a i n t (Rosenzweig et a]_., 1970) or deprivation of food and water (Rosenzweig and Blaustein, 1970); and pyridoxine deficiency, (whose supplements can counteract the effect of cortisone) ( M i l l e r , 1972; Davis ejt aj_., 1970). A l l of these factors can be related to cortisone i n that the authors indicated that "stress" has been demonstrated to increase ACTH production which in turn increases production of adrenocorticoid products such- as cortisone in some species, and corticosterone in mice. One of the functions of cortisone appears to be the retarda-tion of release of lysosomal enzymes and perhaps of other secreted c e l l products such as glycoproteins (Weissmann and Thomas, 1963; De Duve, 1969). Other glycoprotein release i n h i b i t o r s , the antihistamines meclozine, buclizine and hydroxyzine have been shown to induce c l e f t palate in embryonic rats (Harris and Morgan, 1969). Excess of vitamin A, which i s thought to disrupt glyco-protein synthesis (Wolf and Varandani, 1960) has been shown to increase incidence of CLP in both mice and rats (Walker and Crain, I960; Kochhar and Johnson, 1965). Throxine excess, which could disrupt glycoprotein synthesis through disturbed carbohydrate metabolism has been shown to increase CLP incidence in mice (Woollam and Mi 11 en, 1960). Salicylate-induced CLP has been reported (Trasler, 1965.)-S a l i c y l a t e has been reported to inte r f e r e with glycoprotein synthesis (Allen and Kent, 1966). Chemicals which affect the synthesis and metabolism of nucleic acids such as procarbazine and 5-fluoro-2-deoxycytidine have been reported to produce CLP i n rats and mice (Chaube and Murphy, 1969; Franz and Degenhardt, 1969). Lathyrogens, (which could disrupt the structure of some glycoproteins by tying up divalent cations) have been shown to produce CLP in rats (Wilk et a l . , 1972; Steffek et a l _ . , 1972). Nicotinamide-, f o l i c a c i d - , and rib o f l a v i n - d e f i c i e n c y -producing chemicals or circumstances form an interrelated grouping of factors which have been reported to increase incidence of c l e f t palate. Tamburro et al_. (1971) reported that nicotinate deficiency in rats causes depletion of r i b o f l a v i n tissue stores which in turn diminishes tetrahydrofolate biosynthesis, that r i b o f l a v i n deficiency 5 may i n h i b i t nicotinate co-enzymes and N - methyltetrahydrofolates from catalyzing synthesis of reduced folates in t i s s u e , and that deficiencies of any of nicotinate', folate or r i b o f l a v i n gives depressed tetrahydrofolates in the l i v e r . The teratogenic action of 6-aminonicotinamide (6-AN), a nicotinamide antagonist, has been suggested to be the i n h i b i t i o n of energy exchange reactions which in turn s e l e c t i v e l y i n t e r f e r e with synthesis of mucopolysaccharides (glycoproteins)(Overman et_ aj_., 1972; Seegmiller et al_. ,1972). I t i s reasonable to i n f e r that the effects of r i b o f l a v i n or f o l i c acid deficiency, through the interrelationships with nicotinamide may also i n t e r f e r e with glycoprotein synthesis. Evidence has been presented that l i k e 6-AN, r i b o f l a v i n deficiency reduces the a c t i v i t y of the terminal electron transport system in embryos, and in a preferential manner--i.e. the mother i s not affected (Asku et a_]_., 1968). Treatment of pregnant mice with 6-AN produced CLP in mice (Pollard and Fraser, 1968). Various anticonvulsants such as diphen-ylhydantoin and pteroylglutamic acid analogs have been shown to produce CLP i n rats and mice (Gibson and Becker, 1968; Evans et a l . , 1951). These chemicals are generally believed to be f o l i c 5 acid antagonists, perhaps at the level of absorption of dietary f o l i c acid polyglutamates (Schardein et al_., 1973). Dietary r i b o f l a v i n deficiency in rats (Warkany and Schraffenberger, 1943; Warkany et a l . , 1943) and the r i b o f l a v i n antagonist galactof1avin in mice (Walker and Crain, 1961) have been reported to produce increased frequencies of CLP. Though by no means a complete l i s t of factors producing increased frequencies of CLP, those l i s t e d above are representative and consistent in t h e i r reported e f f e c t s . It i s notable that a l l can be d i r e c t l y or circumstantially related to glycoprotein (mucopolysaccharide) metabolism or structure. I t has been suggested (Walker, 1961) that there i s a relationship between palate closure and synthesis of mucopolysaccharides, though some researchers have not agreed (Zimmerman and Bowen, 1972). The mechanism by which any of these teratogens brings about CLP i s d i f f i c u l t to separate from i t s other e f f e c t s . For example, both cortisone and 6-AN produce CLP and reduced body weight of CLP embryos only, while cortisone has the additional effect of reducing amniotic f l u i d volume for both normal and CLP embryos (Fraser et a l . , 1967). The differences in s u s c e p t i b i l i t y to induced CLP in'inbred strains of mice are well known. From the review of Smithberg (1967) and from the work of Brown et al_. (1972), Verrusio et. al_. (1968), Walker and Crain (1960), and Gibson and Becker (1968), i t can be concluded that the spontaneous incidence of CLP in an inbred s t r a i n 6 of mice i s a good predictor of i t s r e l a t i v e rank in production of "induced" CLP by any of, for example, cortisone, "stress," 6-AN, hypervitaminosis A, or f o l i c acid antagonists. Furthermore, i f a l i n e produces high incidences of CLP on one CLP producing-teratogen, i t i s l i k e l y to do so on most others: the A/J or A/Jax s t r a i n of mice, which produces up to 100% CLP during cortisone treatment also produces high frequencies during "stress," 6-AN, hypervitaminosis A, or f o l i c acid antagonist treatment. The apparent greater s e n s i t i v i t y of the DBA s t r a i n to r i b o f l a v i n deficiency compared to the A-Jax s t r a i n suggests that the ranking may be broken by strains having s p e c i f i c s u s c e p t i b i l i t i e s to some factors (Kalter and Warkany 1957). Studies to determine the mode of inheritance of both spontaneous and induced CLP have been conducted. Maternal effects consistent with s t r a i n differences in incidence of spontaneous CLP have been demonstrated in reciprocal crosses (Bornstein et aj_., 1970; Davidson et al_., 1969). Maternal effects have been s i m i l a r l y demonstrated for the incidence i n response to 6-AN (Verrusio et a]_., 1968) or cortisone ( K a l t e r , 1954). Cytoplasmic inheritance has been suggested to explain the lack of equal incidence between the progeny of reciprocal F^ females backcrossed to susceptible l i n e males (Pollard and Fraser, 1968). The data, however, are not convincing. The eff e c t did not persist beyond the F-j females and could be attributed to differences in t h e i r reproductive physiology re s u l t i n g from t h e i r embryonic environments. 7 Davidson et al_., (1969) suggested from the observation that 5 backcrosses to an inbred susceptible s t r a i n were required to bring the spontaneous incidence of CLP back to the o r i g i n a l level (14%) after a cross to a non-CLP s t r a i n , that at least three or four genes were involved, but that the data was not testable. Trasler (1968) suggested, based on observed differences in the morphology of the faces of A/J (susceptible) and C57BL/6J s t r a i n (non-susceptible) developing embryos, that the differences observed were consistent with a predisposition to CLP being controlled as a quasi-continuous variable (Gruneberg, 1952). Loevy and Wade (1972) reported no s i g n i f i c a n t difference in numbers of males and females having CLP induced by hydrocortisone. In t h e i r review of the sex r a t i o information, i t was reported that a s i m i l a r lack of difference was found for cortisone-induced or 6AN-induced CLP, while for 5-f1uorouraci1 and stressed mice, there was a s i g n i f i c a n t l y greater number of females than males. Time of treatment was indicated to be a possible f a c t o r . B. C l e f t Lip and/or Palate i n Man Volumes of data have been collected in the study of possible causes of c l e f t palate in humans, but no single environmental or genetic explanation has been found. Approximately 3% (Fraser, 1970) of the c l e f t l i p and/or palate observed can be accounted for by 50 (Fraser, 1970) to 100 (Burdi et al_., 1972) recognized syndromes of which Fraser (1970) estimated 60% to be accounted for by mutant 8 recessive or dominant genes and about 40% to be the re s u l t of various chromosomal aberrations. This leaves about 97% of the occurrence unaccounted f o r . The current view expressed by Fraser (1970) and apparently widely accepted among human geneticists (Ross and Johnson, 1972; Burdi et al_., 1972; Stark, 1968), i s that isolated c l e f t palate is a d i f f e r e n t genetic entity than c l e f t l i p with or without c l e f t palate, (CL(P)). Fraser (1970) reported that this view was based on e a r l i e r work byTrasler and Fraser in 1963 where i t was found in mice that c l e f t s of the secondary palate could be induced after the primary palate (lip)(which closes f i r s t ) had closed, and that severe c l e f t s of the primary palate could cause c l e f t s in the secondary palate. Fraser's' review (1970) also reported the observations of several other workers as further evidence of the separateness of the t r a i t s . It had been noted that s i b l i n g s of individuals having CL(P) have an increased frequency of CL(P) but not of isolated c l e f t palate, and that s i b l i n g s of individuals having isolated c l e f t palate show increased frequencies of isolated c l e f t palate but not of CL(P). Fraser stated that this pattern decreases as the degree of relationship to the proband decreased, and further noted "exceptions to the rule" in two genetically i s o -lated populations. It seems to the author that such a separation of c l e f t palate from CL(P) should be reconsidered. No evidence was reported which would not support the view that isolated c l e f t palate and 9 CL(P) are alternate phenotypes of the same genetic entity that are under the control of modifying a l l e l e s . Such a l l e l e s would tend to be most sim i l a r in closest r e l a t i v e s , explaining the s i m i l a r i t y of s i b l i n g s in phenotype. The causation of c l e f t palate by the c l e f t l i p alternate in some cases presents l i t t l e problem in such a model. Estimates of frequency per thousand births CL(P) range between 0.6 to 1.8, and for c l e f t palate range between 0.4 and 0.5. Differences between environments, r a c i a l gene frequencies, and report-ing of the t r a i t s are confounded (Fraser, 1970). I t i s thought, regardless of these d i f f i c u l t i e s , that the observed higher incidences of CL(P) in the order Mongoloid races > Caucasian > Negro, and intermediate frequencies in i n t e r r a c i a l populations (Hawaii) represent underlying genetic differences. The incidences of c l e f t palate are considered to be s i m i l a r between races (Fraser, 1970). Fraser (1970) found that for c l e f t palate and CL(P) combined, between 60% and 80% males has been reported. I t was also reported that for u n i l a t e r a l c l e f t l i p , CL(P) or c l e f t palate, about two-thirds were on the l e f t side. A large number of environmental factors and drugs have been correlated, after the f a c t , to c l e f t palate using mother surveys, but none have shown any clear relationship to the anomaly. The genetic control of CL(P) has been believed to be. consistent with a "quasi-continuous variant" model (Gruneberg, 1952), while that of c l e f t palate, due to lack of data, has not been agreed upon. The "quasi-continuous variant" model was f i r s t discussed at length with r e l a t i o n to CL(P) i n humans by Fraser (1970). 10 C. The 'Quasi-Continuous Variation' Model or Multifactoral  Concept as Applied to Mammalian CL(P) ~ The "quasi-continuous variation" concept was f i r s t described by Gruneberg (1952). Wright (1934) had explained the appearance or non-appearance of a q u a l i t a t i v e character (Polydactyly in guinea pigs) on the basis of an underlying s t r i c t l y additive genetic model where a threshold number of a l l e l e s had to be present in order for the t r a i t to appear. Gruneberg (1952) considered that environmental factors could influence the t r a i t (skeletal abnormalities) in addition to the underlying additive genetic e f f e c t , and that additive t r a i t s would be p a r t i c u l a r l y sensitive to environment because, as he saw i t , i t was in additive type t r a i t s that- the genes were furthest removed from t h e i r ultimate e f f e c t s . At this point the s i m i l a r i t y between Gruneberg's model and Wright's model stops. Gruneberg proposed a physiological threshold of development—that i s , that although due to environmental and genetic factors various components of an organ or structure could develop at d i f f e r e n t r a t e s , at some par t i c u l a r stage of development (threshold) the whole series of developments had to be complete in order for the next series to begin. If the tissue was not at that threshold of development, the next step would not take place and an abnormality would r e s u l t . In contrast to this developmental or physiological threshold of Gruneberg's, Wright's threshold consisted of a s p e c i f i c number of genes only. Another apparent major area of difference between Wright's model and Gruneberg's i s the number of l o c i involved. Wright's model could conceivably work for any number of l o c i , while Gruneberg's model could not accommodate small numbers of genes and a consequent small number of steps i n the development of an organ and s t i l l maintain the opportunity for environmental factors to i n f l u -ence development in an additive fashion. Furthermore, using the method of Falconer (1965) which calculates the h e r i t a b i l i t y of " a l l or none" characters from the correlation of l i a b i l i t y between r e l a t i v e s , gives h e r i t a b i l i t y e s t i -mates of l i a b i l i t y of 78% to 83% for CL(P) (Ross and Johnson, 1972). It should be pointed out that this system assumes only additive genetic v a r i a t i o n , ignores the p o s s i b i l i t y of dependence between environment and family, and assumes l i a b i l i t y to be a continuously distri b u t e d variable of l i k e l i h o o d to develop the t r a i t in question due to a combination of innate tendencies and external circumstances. These high h e r i t a b i l i t y estimates suggest that for the human population, where gene frequencies are probably low f o r c l e f t palate, the majority of control of the t r a i t i s genetic, not environmental, and that although environment plays a r e l a t i v e l y small part i n the formation of the t r a i t , the c r i t i c a l "threshold" under consideration may not be so much physiological or develop-mental timing as suggested by Gruneberg's quasi-continuous variation model, but rather mainly a genetic threshold of additive genes as described by Wright. The report by Walker and Fraser (1956) that the palate closes l a t e r i n inbred mouse lines most susceptible to teratogen-induced c l e f t palate, and the reports that f a c i a l shape in both humans (Pashayan and Fraser, 1969) and mice (Trasler, 1968) may 12 influence s u s c e p t i b i l i t y , and the p o s s i b i l i t y of genetically con-t r o l l e d maternal influences (Fraser, 1970) can a l l be viewed as consistent with the effects of underlying additive genetic c o n t r o l . The effects of teratogens in such cases may be to mimic the effects of additional a l l e l e s required for the t r a i t to appear; that i s to move the individual across a gene-number threshold rather than to move i t r e l a t i v e to a developmental stage threshold. In summary, with view to the application of Gruneberg's "quasi-continuous v a r i a t i o n " model to the c l e f t palate t r a i t in mammals, i t i s possible that the control of the t r a i t i s more under additive genetic control than that model implies, and that a d d i t i v e ' genetic models such as that of Wright (1934) might be more appropriate i f methods to overcome the " a l l or none" quality of the data are used. D. C l e f t Palate in Chickens C l e f t palate does not seem to have been previously studied in poultry, although i t has been reported by several workers. It i s possible that the c l e f t palate t r a i t in poultry i s the same as that named "Abnormal Upper Mandible" and described as a recessive " l e t h a l " by Asmundsen in 1936. The verbal description by Asmundsen that "the maxillae of the upper mandible are absent or much reduced while the premaxillae (and egg tooth) are present and normal or nearly so . . !' i s an appropriate description of the t r a i t l a t e r termed " c l e f t palate." In addition some of Asmundsen's photographs of the side views of "abnormal upper mandible" chicks bear a s t r i k i n g resemblance to the side views of " c l e f t palate" chicks presented by Landauer (1947) and Shoffner et_ al_. (1953). A l l of Asmundsen's data were based on the f u l l - s i b progeny from one o r i g i n a l White Leghorn hen, and the " l e t h a l i t y " (not a l l chicks died before hatch) becomes subject to the p o s s i b i l i t y of " l e t h a l , " l i n k e d , genes. Asmundsen reported the presence of a phenotypically s i m i l a r t r a i t in domestic bronze turkeys, Eastern Chinese Ring Neck pheasants, Mongolian pheasants, and Indian Chukar partridges. It i s unfortunate that there i s no certainty that Asmundsen's t r a i t was c l e f t palate, but i t does remain a p o s s i b i l i t y . C l e f t palate as such in poultry has not been the subject of genetic studies published to date, but has been reported as a minor aspect of teratogen studies. Landauer (1951) reported i n 59 observations an incidence of 3.4% of c l e f t palate in 13 day old duck embryos injected with 2 units of i n s u l i n into the yolk sac at 96 hours of incubation. Neither 1072 embryos injected at or before 72 hours, nor 332 embryos injected a f t e r 96 hours, nor 363 control embryos showed any occurrence of c l e f t palate. Landauer and Rhodes (1952) reported that the "developmental defect of the base of the beak" e a r l i e r reported by Landauer (1947) as an insulin-induced abnormality i n White Leghorn embryos was " c l e f t palate and f a c i a l coloboma." The frequency of this t r a i t i n the 1947 paper was not reported independently of other beak abnormalities. Landauer and Rhodes in 1952 restated that occurrence of c l e f t palate in chickens was dependent on i n s u l i n effects but did not provide s p e c i f i c data to that e f f e c t . They further reported that another researcher, Ancel,(1950 publication not a v a i l a b l e ) , had produced c l e f t palate in response to t r y p a f l a v i n . Landauer (1952) reported that the combined i n j e c t i o n of i n s u l i n and r i b o f l a v i n into White Leghorn eggs produced a d i f f e r e n t i a l potentiation of the i n s u l i n e f f e c t , the increase in occurrence being the greater the less common the p a r t i c u l a r abnormality after i n s u l i n alone. Since c l e f t palate was one of the o r i g i n a l , r a r e r , effects of i n s u l i n , i t can be speculated that c l e f t palate was among the "beak defects" j o i n t l y reported to be enhanced by r i b o f l a v i n . Landauer concluded that r i b o f l a v i n put an additional burden upon the metabolic processes interfered with by i n s u l i n . Landauer (1952) reported the results of i n j e c t i o n of boric acid into the yolk sac of White Leghorn embryos. The most frequent beak defect was reported to be a combination of shortened lower beak, c l e f t palate, and b i l a t e r a l f a c i a l coloboma a l l of which varied considerably in extent. Coloboma and c l e f t palate.were sometimes found without shortened lower beak, and c l e f t palate was occasionally found without f a c i a l coloboma. An incidence of 0.08% c l e f t palate and coloboma was reported for 1244 control observations. In contrast 4.2% c l e f t palate and coloboma was reported for 72 hour-injected embroyos and 48.6% c l e f t palate with or without coloboma or shortened lower beak, for 96-hour-injected embryos. 15 A high frequency (maximum 70%) of "curled toe paralysis" in newly hatched chicks from boric-acid injected eggs was also reported by Landauer. Because this paralysis was believed to be r i b o f l a v i n -d e f i c i e n c y - s p e c i f i c , the boric acid treatment was amended to be preceded, accompanied, or followed by a supplement of r i b o f l a v i n with or without nicotinamide. Riboflavin and nicotinamide were also injected together in absence of boric acid. Nicotinamide was also used along with the boric acid treatment. In none of the tests did r i b o f l a v i n have a beneficial e f f e c t on embryo s u r v i v a l . But malformations were reduced by a t h i r d with r i b o f l a v i n and by nearly half with r i b o f l a v i n and nicotinamide. Nicotinamide alone reduced beak defects, but increased curled toe p a r a l y s i s . The possible mechanism of boric acid action as a teratogen was discussed by Landauer. Based on the known formation of a complex when r i b o f l a v i n i s in the presence of boric a c i d , i t was suggested that boric acid i n h i b i t e d a r i b o f l a v i n - c o n t a i n i n g , or riboflavin-dependent enzyme system. The function of the supplementary r i b o f l a v i n was thought to be to reduce the amount of b i o l o g i c a l l y active boric acid present in the embryo by undergoing complex formation with i t . Further evidence of the r i b o f l a v i n deficiency postulated to be caused by boric acid was the finding that boric acid-treated embryos' l i v e r s were "grossly" d e f i c i e n t in r i b o f l a v i n . Landauer (1954) reported that substances such as ribose and s o r b i t o l , known to form complexes with boric a c i d , i f added to boric acid prior to i n j e c t i o n , or injected simultaneously, rendered boric acid non-teratogenic. 16 In view of the greater s u s c e p t i b i l i t y of pigmented-downed embryos to r i b o f l a v i n deficiency reported by Bernier and Cooney (1954), i t i s interesting to note the report by Landauer (1954) that black embryos showed a higher incidence of boric acid-induced malform-ations. Using Minorca fowl segregating with respect to plumage colour, Landauer showed a s t a t i s t i c a l l y s i g n i f i c a n t (p ^  0.01) difference in boric acid induced malformations between white and black progeny. C l e f t palate was among these, but was not reported separately. The values for "complex beak defects" which appear to have been mainly c l e f t palate were 17% and 38% for white and black chicks, respectively. Black embryos showed a sharper decline i n defects than white embryos during the spring months. These seasonal effects were s t a t i s t i c a l l y s i g n i f i c a n t (p <_ 0.01). Cleft palate was among these defects. In addi t i o n , the color of the hen affected frequency of defects, including "complex beak defects." White progeny from white hens showed s i g n i f i c a n t l y (p <^0.01) lower incidences of defects than white chicks from black hens. Landauer (1953) reported that pilocarpine, an imidazole d e r i v a t i v e , injected into the yolk sac of White Leghorn embryos produced, among other defects, c l e f t palate and f a c i a l coloboma. A maximum incidence of 14% for c l e f t palate, f a c i a l coloboma and shortened lower beak was reported. Whether the l a t t e r always accompanied the f i r s t two t r a i t s was not c l e a r . Dagg and Karnofsky (1955) using White Leghorn embryos, reported that azaserine, which was believed to interfere with de novo purine synthesis by blocking the transfer of an amino group to formylglycine amide ribotide (Karnofsky and Lacon, 1964), caused an incidence, among other abnormalities, of c l e f t palate and f a c i a l coloboma. The incidence ranged between 18% and 80%, based on samples ranging between 2 and 37 observations. Dosages of nicotinamide, r i b o f l a v i n , pyridoxine, b i o t i n , or ascorbic acid were not found to decrease the t o x i c i t y or the teratogenicity of azaserine. This was interpreted to indicate that azaserine may act at a l a t e r s i t e than other s i m i l a r l y acting agents. Karnofsky et al_. (1958) reported that, among the effects of the 5-fluoro substituted pyrimi.dines, 5-f 1 uorouraci 1 and 5-fluoro-orot i c a c i d , when injected into the yolk sac of 3 and 4 day embryos, were f a c i a l coloboma and c l e f t palate. Frequencies were not reported. Kury and Crosby (1967) reported that the fluoridated pyrimidine, 5-trifluoromethyl-2'-deoxyuridine, when injected into White Plymouth Rock embryo yolk sacs at or before 4 days of incubation produced 1 c l e f t palate out of 1230 observations, compared with zero out of 556 control observations. This could hardly be interpreted as a treatment e f f e c t , but does indicate that the naturally occurring incidence of c l e f t palate in White Plymouth Rock embryos i s very low. Karnofsky and Lacon (1961) reported incidences of c l e f t palate and f a c i a l coloboma f o r various deoxyribosyl derivatives of physiological purines injected into the yolk sacs of 4 day White Leghorn embryos. They were (maximum frequencies reported for most ef f e c t i v e dosage l e v e l ) : 2'-deoxyguanosine, 9/20; 2'-deoxyadenosine, 18 1/20; 2'-deoxyinosine, 2/20; 2-deoxyguanosine, 41/85. The l a t t e r , 2-deoxyguanosine, was reported to act by in t e r f e r i n g with formation of deoxycytidine (Karnofsky and Lacon, 1964). Further along the same metabolic pathway, 1-3-D-arabinofuranosyl-cytosine, thought to block the reduction of c y t i d y l i c acid diphosphate to i t s "deoxy-" form, was also found to produce c l e f t palate in White Leghorn embryos (Karnofsky and Lacon, 1966). The maximum frequency of c l e f t palate produced was 11/17 observations. If treatment days up to and including 4 days of incubation were combined, totals of 18 c l e f t palates with f a c i a l coloboma and 4 isolated c l e f t palate cases out of 47 observations were reported. Landauer and Wakasugi (1967) using White Leghorn 96 hour embryos and injections into the yolk sac of acetazolamide or N-ethylnicotinamide reported the following incidences of c l e f t palate: at 5 mg dosages- acetazolamide produced 0%; N-ethynicotinamide, 0.5%; at 10 mg dosages-acetazolamide produced 0.5%; N-ethylnicotinamide, 0.9%. Combined 5 mg dosages of each produced apparent synergism and 16.8% c l e f t palate. The sample sizes were between 105 and 134 for each group. Both chemicals tested were thought to interfere with nicotinamide adenine dinucleotide (NAD) functions and oxidative phosphorylation. Romanoff (1972) cit e d the 1970 work of Kocher as indicating that triethylene melamine produced c l e f t palate in chick embryos. Cortisone acetate applied to the a l l a n t o i c vesicle or chorioallantoic membrane of White Leghorn embryos at 4 or 5 days of incubation can be interpreted to have produced 32% and 30% c l e f t 19 palate with f a c i a l coloboma for 2 dosage levels and 22 and 27 observations respectively (Moscona and Karnofsky, 1960). A descrip-tio n of the t r a i t was given as follows: In most of the cases the coloboma was b i l a t e r a l , but occasionally a u n i l a t e r a l coloboma, combined with a c l e f t palate was observed; in such cases the beak was crossed toward the colobomatous side. These defects were brought about by changes in s i z e , shape, and orientation of several membranous bones of the upper beak . . . . The premax-i l l a e developed i n t h e i r appropriate position but th e i r l a t e r a l extensions were considerably shortened. The maxillae were smaller and t h e i r anterior extensions were missing. There was thus no continuity and contact between the pre-maxillae and the maxillae, and they were separated by a gap. The nasal bones which normally curve a n t e r i o r l y , curved here in a posterior direction to j o i n the reduced and displaced maxillae. The palatal bones were considerably shortened and f l a t t e n e d , and instead of extending anteriorly to j o i n the premaxillae, they were directed sideways and met the maxillae. In addition to chemical teratogenic a c t i v i t y producing c l e f t palate, 2 environmental factors of incubation have been shown to be capable of producing the defect. Ancel (1958, 1959) reported the results of cooling White Leghorn incubating eggs to 17 to 24°C from the normal 38°C for a 2 day period between 4 and 10 days of incubation. In 2 experiments the maximum frequencies of c l e f t palate were found for eggs cooled from days 4 to 6, being 12.0% and 5.7% respectively. Grabowski and Paar (1958) applied hypoxia to embryos of a Mount Hope l i n e of White Leghorns. The hypoxia period provided from approximately 79% to 2% of normal oxygen levels for approxi-mately 6 hours to 18 hours to 9 day incubating embryos. Untreated con-t r o l s produced 1 c l e f t palate i n 284 observations (0.4%). Of 954 20 experimental embryos, 11 showed " c l e f t l i p and palate" ( f a c i a l coloboma? and c l e f t p alate), or 1.2% incidence. Three of these were b i l a t e r a l and 8 on the l e f t side only. This experimental r e s u l t i s probably an underestimate of the incidence of c l e f t palate occurring as a r e s u l t of the treatment, because those embryos treated a f t e r 5 days 1development were probably not inducible to c l e f t palate, but they were included in the estimate. These researchers provided some insight into the develop-ment of the palate of the chicken, st a t i n g : C l e f t l i p and palate can.be considered as a defect caused by an underdevelopment of the maxillary and fronto-nasal processes. Neither the maxillary nor the palatine bones normally fuse in the midline in the chicken, but are joined together by a bridge of soft t i s s u e . But in these abnormal embryos the c l e f t between the bones i s larger than normal, open, and continuous with a c l e f t in the upper beak which extends to the external nares. The cause of malformations in the presence of oxygen deficiency was not readily apparent to these researchers, since the embryo was believed to have a considerable capacity for anaerobic metabolism. In general i t can be deduced from the teratogen studies producing c l e f t palate in poultry that: 1. C l e f t palate can be induced in embryos treated prior to the s i x t h day of incubation but not a f t e r . 2. The frequency of c l e f t palate induced i s generally low, and when high has not exceeded 50%. 3. Experiments using large numbers of controls tend to show a spontaneous incidence of c l e f t palate of less than 1%. 21 4. When c l e f t palate i s induced by a teratogen, there i s a tendency for some or a l l of the following t r a i t s to also occur: micromelia (shortened limbs), shortened lower beak, "parrot beak," down defects, and edema. Aside from the genetic analysis of "Abnormal Upper Mandible" by Asmundsen, there seems to be no published work on the c l e f t palate t r a i t from a genetic point of view. That there i s a genetic base for the occurrence of the t r a i t was suggested by Shoffner et aj_. (1953), who reported the c l e f t palate condition in an inbred l i n e of White Leghorns. I t was thought to be a t r a i t exposed by inbreeding and controlled by a gene or genes of a recessive nature with low penetrance. The t r a i t was noted when inbreeding had' reached more than 60%. The 420 l i n e in which the t r a i t appeared was also reported to have been derived from a l i n e selected for s u s c e p t i b i l i t y to avian leukosis, and to have been unusually responsive (as measured by hatchability) to low or threshold levels of vitamins A, and D, and r i b o f l a v i n . The photographs provided with the publication of the c l e f t palate condition show that the c l e f t palate was sometimes accompanied by the " f a c i a l coloboma" described by e a r l i e r workers, and that both the c l e f t s and the coloboma could be un i l a t e r a l or b i l a t e r a l . Further work on the c l e f t palate t r a i t in the 420 l i n e described by Shoffner has been carried out at the University of B r i t i s h Columbia. Roberts e_t aj_. (1973) in a study whose purpose was to develop a l i n e of chickens with a high natural occurrence of c l e f t palate found that d e f i c i e n t or marginal levels of r i b o f l a v i n in the diet of hens increased the frequency of c l e f t palate from 0.4% to 2.0%, and that survival of these chicks was s u f f i c i e n t f o r sel ection to be undertaken. Facial coloboma was disregarded in that i t had been observed by the authors that i t occurs only i f c l e f t palate occurs, and not by i t s e l f . These selected birds produced when intermated, and the hens placed on a marginal r i b o f l a v i n d i e t , progeny with a frequency of 30% c l e f t palate. These chicks when selected and reproduced did not show response to marginal r i b o f l a v i n regimes in the incidence of c l e f t palate in t h e i r progeny, but did show a further increase above the previous generation to 42% to 53% incidence of c l e f t palate. This response to selection was taken to indicate that the t r a i t was d e f i n i t e l y under genetic c o n t r o l , that the genetics had been "unmasked" for selection by the r i b o f l a v i n deficiency, that the number of genes involved was probably approxi-mately 3 or 4, and that the experiments had been an example of the genetic control of the response to what might be termed a "teratogenic treatment. 23 E. Riboflavin Requirement and Effects of Deficiency i n Chickens The dietary requirement for r i b o f l a v i n in poultry has been known for many, years and the effects of deficiency in hens, growing chicks and embryos has been well documented. The structure of r i b o f l a v i n i s known, and the synthesis of r i b o f l a v i n was accomplished i n 1935 (Scott et al_., 1969). Ribo-f l a v i n i s a component of the coenzymes f l a v i n mononucleotide, r i b o f l a v i n 5'-phosphate (FMN), and f l a v i n adenine dinucleotide (FAD), and i s a prosthetic part of more than a dozen enzymes in the animal body, among which are cytochrome reductase, lipoamide dehydrogenase, xanthine oxidase, 1- and d- amino acid oxidases, and histaminase. The main functions of FMN and FAD are to transfer hydrogen between the n i c o t i n i c acid-containing coenzymes, NAD and NADP, and the iron porphyrin cytochromes. Thus these co-enzymes are a part of the chain which carries hydrogen from substrates such as carbohydrates, amino acids and l i p i d s to molecular oxygen, forming water. Other r i b o f l a v i n -containing enzymes may be c l a s s i f i e d into three groups: reduced pyridine nucleotide dehydrogenases; mitochondrial metabolic dehydro-genases coupled to the respiratory chain; and oxidases. Through these enzymes, r i b o f l a v i n i s essential for c e l l u l a r r e s p i r a t i o n , growth and tissue repair in a l l animals (Scott et al_ . , 1969). According to the National Academy of Sciences (1971) the minimum requirements per kilogram of feed, in grams are: for s t a r t i n g chicks (hatch to 8 weeks), 3.6; for growing chicks (8 to 18 weeks) 1.8; f o r laying hens, 2.2; and for breeding hens, 3.8. 24 The symptoms of r i b o f l a v i n deficiency in growing chicks are well known. The 2 most severely affected tissues are the epithelium and the myelin sheaths of the main nerve trunk. Dermatitis, diarrhea, and marked enlargement of the s c i a t i c and branchial nerve sheaths, with h i s t o l o g i c a l l y degenerative changes in the myelin sheath and axis cylinder as the r e s u l t of apparent accumulation of metabolic products within the myelin sheath, are observed, the l a t t e r r e s u l t -ing in c h a r a c t e r i s t i c "curled toe paralysis." These changes were f i r s t reported by P h i l l i p s and Engel (1938), and Engel et al_. (1940), and were restated by Scott et aj_. (1969) and the National Academy of Sciences (1971). Recently, Wyatt et al_. (1973) concluded that curled toe paralysis i s an infrequent aspect of r i b o f l a v i n deficiency i n the b r o i l e r chick, while reduced growth rate and posture changes are the most sensitive i n d i c a t o r s . The symptoms of r i b o f l a v i n deficiency in the hen are: decreased levels of r i b o f l a v i n in the muscles and l i v e r ; decreased egg production; increased s i z e and f a t content of the l i v e r ; and increased embryonic mortality (Lepkovsky et aj_. , 1938; Stamberg et a l . , 1947; Petersen et aj_., 1947 a and b; Scott et al_., 1969; and Scott and Krook, 1972). The effect of the r i b o f l a v i n content of the diet fed to laying hens on the ha t c h a b i l i t y of t h e i r eggs has been studied extensively. Early researchers reported the beneficial effect on hat c h a b i l i t y of the "vitamin G complex" found i n dried l i v e r , autoclaved yeast, dried whey, and some fishmeals (Bethke et a l . , 1933; Record and Bethke, 1933; Heiman and N o r r i s , 1933). Heiman and Norris (1933) and Heiman (1935) presented evidence that the "vitamin G complex" provided by dried skim milk and dried whey, was required for hatchabi1ity, and that hatchability apparently was d i r e c t l y related to the "vitamin G complex" content of the d i e t . Later workers found that r i b o f l a v i n was one of the components of the "vitamin G complex" and v e r i f i e d i t s necessity i n the diet for hatch a b i l i t y of eggs (Davis et al_., 1938 a and b; Lepkovsky et a l . , 1938; Schumacher and Heuser, 1939; Engel e_t aj^. , 1940; Romanoff and Bauernfeind, 1942; Petersen et al_., 1947 a and b). Typical results showed that when hens were placed on r i b o f l a v i n d e f i c i e n t d i e t s , a period of apparent r i b o f l a v i n depletion occurred, l a s t i n g 2 to 4 weeks, after which hatchability plunged from good (100 to 65%) to very low (12% to 0%) levels (Davis et al_., 1938 a and b; Schumacher and Heuser, 1939; Romanoff and Bauernfeind, 1942). Davis et_ al_. (1938b) reported that restoration of r i b o f l a v i to the diet of "vitamin G complex"--deficient hens resulted in attainment of maximum hatc h a b i l i t y during the second week after the s t a r t of supplementation. Lepkovsky et aj_. (1938) found that hatchability could be restored to r i b o f l a v i n d e f i c i e n t hens eggs by treatment of the hen with oral doses or subcutaneous i n j e c t i o n of aqueous r i b o f l a v i n s o l u t i o n . Injection of aqueous r i b o f l a v i n into the a i r c e l l of eggs from r i b o f l a v i n d e f i c i e n t hens was found to improve hatchability (Lepkovsky et al_., 1938); Bernier and Cooney, 1954). Engel et a l . (1940) found that the myelin sheath degeneration of the s c i a t i c nerve and spinal cord observed in developing embryos from r i b o f l a v i n d e f i c i e n t hens occurred less frequently i n eggs injected with aqueous r i b o f l a v i n s o l u t i o n , or eggs from r i b o f l a v i n d e f i c i e n t hens to which oral doses of r i b o f l a v i n had been given. The amount of r i b o f l a v i n present i n the egg has been shown to be a r e f l e c t i o n of the dietary intake of r i b o f l a v i n of the hen (Norris and Bauernfeind, 1940; Stamberg et al_., 1946a; Jackson et a l . , 1946; Petersen et a l . , 1947a; Mayf i e l d et a l _ . , 1953). There seem to be upper and lower l i m i t s of this response, however. Norris and Bauernfeind (1940) reported that White Leghorn hens maintained on a low r i b o f l a v i n diet (125 micrograms per 100 grams)(below the minimum requirement for hens), after 2 weeks leveled out at 1.39 micrograms per gram of egg, compared to a more normal 2.26 micrograms per gram. Whether chick embryos would survive such a low l e v e l , or whether there was an end to the plateau were not determined, nor the length of time the plateau maintained, stated. Norris and Bauernfeind (1940) and Jackson e_t aj_. (1946) reported that upper l i m i t s of u t i l i z a t i o n of dietary r i b o f l a v i n were found, beyond which no further amount was deposited in the egg. Norris and Bauernfeind (1940) found that the maximum level was 800-1000 micrograms of r i b o f l a v i n per 100 grams of d i e t , which gave about 32% more r i b o f l a v i n in eggs than those obtained from farm eggs from hens fed diets designed to promote good h a t c h a b i l i t y . Jackson e_t aj_. (1946) found that the highest level of r i b o f l a v i n to affect the concentration of r i b o f l a v i n in the egg was 1400-1600 micrograms per pound of feed. 27 Studies were conducted to locate where r i b o f l a v i n was depo-si t e d i n the egg. Norris and Bauernfeind (19'40) found that the concentration of r i b o f l a v i n was greater in the y o l k , than in the albumen on both normal and high r i b o f l a v i n d i e t s , and that the response to an increased level of r i b o f l a v i n in the diet gave a more rapid increase in the level in the albumen, which then plateaued sooner. Stamberg et al_. (1946b) reported that while in most (74%) eggs the r i b o f l a v i n concentration was higher in y o l k , in t h e i r study of White Leghorn, New Hampshire, Rhode Island Red, and Barred Plymouth Rock f l o c k s , an average of 58% of the total amount of r i b o f l a v i n i n the egg was found in the albumen, due to i t s greater weight in the egg. Most researchers studying r i b o f l a v i n n u t r i t i o n and egg hat c h a b i l i t y noted that there were marked bird to bird and breed to breed variations in response to the level of dietary r i b o f l a v i n r e f l e c -ted by amount of r i b o f l a v i n deposited i n the egg, and subsequent hatchability of eggs. Stamberg et al_. (1946a) noted that hens which produced eggs of lowest r i b o f l a v i n content on a low intake of r i b o f l a v i n responded to higher levels of r i b o f l a v i n by increased egg r i b o f l a v i n content, but s t i l l ranked lower than the other birds on s i m i l a r diet regimes. Jackson et al_. (1946) noted some variation i n the con-centration of r i b o f l a v i n in the eggs from various breeds, va r i e t i e s and individual hens on the same r a t i o n . Mayfield et a l . (1953) found that d i f f e r e n t hens l a i d eggs of d i f f e r e n t r i b o f l a v i n content, that the content was not related to rate of egg pro-duction, and that the sequence of the egg in the laying cycle did not influence the r i b o f l a v i n content of the egg. They also found 28 that White Leghorn hens put more r i b o f l a v i n and thiamine into t h e i r eggs than New Hampshire hens d i d . Conversely Stamberg et a l . (1946a) reported that the f i r s t egg of a clutch was generally lower in r i b o f l a v i n than other eggs, in a study using White Leghorns. Davis et_ al_. 1938(b) reported wide variation between individual hens in the h a t c h a b i l i t y of eggs during the depletion period on a r i b o f l a v i n d e f i c i e n t d i e t . In a d d i t i o n , in some families there was an excellent degree of consistency in t h e i r hatchability response to a d i e t d e f i c i e n t in r i b o f l a v i n . These observations would lead to the' view that there i s genetic control over the e f f i c i e n c y of u t i l i z a t i o n of r i b o f l a v i n and/or deposition in the egg and/or embryonic e f f i c i e n c y or requirement for r i b o f l a v i n in the egg. Maw (1954) reported the existencevof a single-gene-controlled, recessive t r a i t in which the a b i l i t y of the hen to put r i b o f l a v i n into the egg was markedly reduced. Lamoreux and Hutt (1948) found selection on the basis of hatchability on high or low levels of r i b o f l a v i n to be i n e f f e c t i v e . In further work they practiced 6 generations of selection for and against resistance and s u s c e p t i b i l i t y of growing chicks to r i b o -f l a v i n deficiency to 5 weeks of age. The selection c r i t e r i a were survival and body weight. They succeeded i n creating divergent lines having d i f f e r e n t i a l mortality and growth on a r i b o f l a v i n d e f i c i e n t diet and equal growth and s u r v i v a b i l i t y on a r i b o f l a v i n adequate d i e t . The response to the adequate diet indicated that the genetic differences shown were concerned with u t i l i z a t i o n of 29 r i b o f l a v i n . I t would be interesting to observe the s u r v i v a b i l i t y of embryos of the resistant s t r a i n in r i b o f l a v i n d e f i c i e n t eggs. Lerner and Bird (1948) in a smaller, but s i m i l a r study showed less consistent results than Lamoreux and Hutt, but indicated that t h e i r results were in general agreement, a t t r i b u t i n g the differences to less discriminating selection c r i t e r i a . ( d i e t s not so deficient) and the thereby possible confounding of selection for growth rate with selection for resistance to deficiency. A further complication arose in the "adequate" diets being not adequate on the t e s t . Bernier and Cooney (1954) found a relationship between melanin pigmentation and r i b o f l a v i n requirement during embryonic development of chicks. Embryos having heavily pigmented down were found to have higher r i b o f l a v i n requirements, and, conversely, i t was found that modifiers which tended to reduce the i n t e n s i t y of colouring were found to reduce the r i b o f l a v i n requirement. These observations led to a hypothesis that r i b o f l a v i n i s involved i n the process of melanin formation. The work of Landauer (1952) indicated that embryos of the Black Minorca Breed were more susceptible than White Leghorn embryos to the teratogenic effects of boric acid whose effects were thought to be mediated through effects on r i b o f l a v i n -containing enzymes. In addition to the overall hatchability of r i b o f l a v i n d e f i c i e n t hen's eggs, the stages at which embryonic mortality occurs, and the embryonic defects observed have been studied in such eggs. Chick embryos have 2 "natural" peaks of embryonic mor t a l i t y , presumably due to non-specific causes. These occur at the 30 (approximate) fourth and nineteenth days of incubation (Romanoff, 1972). Lepkovsky et al_. (1938) and Davis et_ a]_. (1938a) reported the appearance of a th i r d peak in embryonic mortality at the tenth to twelfth day of incubation of eggs from r i b o f l a v i n - d e f i c i e n t hens. This peak i s not considered to be r i b o f l a v i n s p e c i f i c , but can be a r e f l e c t i o n of the effects of other s p e c i f i c factors (Romanoff and Bauernfeind, 1942; Romanoff, 1972). The observations of Romanoff and Bauernfeind (1942) showed that the variation in the rate of depletion of r i b o f l a v i n reserves was very high and that i t was more rapid with some individual hens than with others. In the course of this depletion there was not only an increase in total embryonic mo r t a l i t y , but also a progressive s h i f t i n g in the major peak of mortality from the l a s t c r i t i c a l period (nineteenth day) to the second,(tenth to twelfth day), and then to the f i r s t (fourth day). In addition to the time of embryonic m o r t a l i t y , some studies have reported on the types of embryonic defects accompanying r i b o f l a v i n deficiency. The c h a r a c t e r i s t i c curled toe paralysis observed i n r i b o f l a v i n - d e f i c i e n t growing birds ( P h i l l i p s and Engel, 1938) and accompanying h i s t o l o g i c a l nerve changes were reported by Engel et al_. (1940) to be observed in dead embryos from r i b o f l a v i n d e f i c i e n t hens. These degenerative changes of the myelin sheath of the s c i a t i c nerve, often the myelin sheath of the spinal cord, and in severe cases, the axis of the s c i a t i c nerve^were also found to be reversible by in j e c t i o n of r i b o f l a v i n into the egg. They were thought to be the cause of embryonic mortality. 31 Romanoff and Bauernfeind (1942) reported curled toe paralysis in the mortality of the nineteenth incubation day in embryos from r i b o f l a v i n deficient hens. According to Landauer (1952) there i s no known cause of curled toe paralysis except developmental r i b o f l a v i n deficiency. A consistently reported embryonic defect in r i b o f l a v i n deficiency i s the dwarfing of embryos (Lepkovsky et a]_. , 1938; Engel et_ al_. , 1940; Romanoff and Bauernfeind, 1942). Romanoff and Bauernfeind (1942) found that these dwarfed embryos made up about one t h i r d of the mortality at 19 days, and they were less than 40% of the normal weight at that age. Edema of dead embryos during r i b o f l a v i n deficiency was reported by Lepkovsky et al_. (1938) and by Romanoff and Bauernfeind (1942). Lepkovsky et_ al_. (1938) described a defect of down formation occurring in r i b o f l a v i n d eficient embryos as " f a i l u r e of the down feather to rupture the surrounding sheath. As a result the feather i s c o i l e d and takes the shape of a french knot." Romanoff and Bauernfeind (1942) reported undeveloped feather growth in r i b o f l a v i n d eficient embryos. Clubbed down may not be s p e c i f i c to r i b o f l a v i n deficiency in that i t has been reported to occur in embryos in eggs injected with 1-B-D-arabinofuranosylcytosine (Karnofsky and Lacon, 1966; 3,3-dimethyl-l-phenyltriazene (Dagg et a l . , 1955); azaserine, (Dagg and Karnofsky, 1955); s u l f a n i -lamide (Landauer, 1954; eserine (Landauer, 1954) and pilocarpine (Landauer, 1953 and 1956) and in eggs from hens deficient in b i o t i n (Cravens et al_., 1944). Degeneration of the Wolfian bodies of the kidneys was r e -ported i n dead r i b o f l a v i n d e f i c i e n t embryos of 17 to 21 days' development by Lepkovsky et_ al_. (1938). This degeneration took the form of edematous swelling or caseous f i l l i n g of the mesonephros. Romanoff and Bauernfeind (1942) reported changes in l i v e r color of 14 day deficient embryos from normal light"yellow to brown or l i g h t green with areas of atrophy. They also reported micromelia (shortened limbs) and prognathism (lower mandible shorter than upper) in the d e f i c i e n t embryos. McFarlane et al_. (1930), using a diet that would now be considered r i b o f l a v i n d e f i c i e n t , and Lepkovsky et_ al_. (1938) reported the possible relationship between "anemia" (lack of haemoglobin) in unhatched chicks, and r i b o f l a v i n deficiency. I n a b i l i t y to reduce the incidence consistently with r i b o f l a v i n supplements to the hen led Lepkovsky et al_. (1938) to suggest that more than one factor was involved. It seems that chicks which hatch and are apparently normal from eggs that are p a r t i a l l y r i b o f l a v i n deficient may be affected for at least the early parts of t h e i r l i v e s . Engel et_ al_. (1940) found that the r i b o f l a v i n content of the l i v e r s of such chicks i s lower than normal, while Davis et al_. (1938a) found that chicks from r i b o f l a v i n low hens grew slower and had higher mortality than those from r i b o f l a v i n s u f f i c i e n t hens. It i s interesting to speculate whether a long term effect i s present in such chicks, in view of the work of Roberts et a l . (1973). 33 MATERIALS AND METHODS Two lines of chickens were used i n this study: an egg laying New Hampshire Line (NH) and a l i n e of Single Comb White Leghorn birds (CP) under selection f o r incidence of the c l e f t palate t r a i t . A. The New Hampshire Line The Univeristy of B r i t i s h Columbia (U.B.C.) population of NH birds was created by the intermating of several NH lines by Dr. S.S. Munro c i r c a 1940. The population has been maintained without intentional selection with a yearly population size that has.varied from 300 to 600 females and 100 to 200 males. C l e f t palate has not been found in the U.B.C. NH l i n e , nor has i t been reported in the NH breed. No cases of c l e f t palate were found i n a tot a l of 1415 observations (749 females,609 males, and 57 embryos which had died between the thirteenth and twentieth days of incubation). Considering the p o s s i b i l i t y that the c l e f t palate t r a i t might be lethal i n the NH l i n e , t h is does not prove that the t r a i t does not occur in the NH l i n e , but i t does suggest that the incidence i s low i f not zero. The adult NH birds used in th i s study were chosen at random from the U.B.C. population, and were a l l approximately 14 months o l d . 34 B. The Cl e f t Palate Line The CP l i n e of birds was a li n e under selection for the c l e f t palate condition. A l l CP l i n e birds were derived by selection of c l e f t palated individuals from the progeny of the "Minnesota 420 l i n e " of highly inbred White Leghorns maintained at U.B.C. This "420 l i n e " was developed by Shoffner et a l . (1953). I t has been maintained at U.B.C. since 1963 without intentional selection and with a yearly population size of approximately 100 females and 30 males. The derivation of the CP l i n e from the "420 l i n e " has been described by Roberts et al_. (1973), and involved the use of a maternal dietary r i b o f l a v i n deficiency which was shown to increase the frequency of the c l e f t palate anomaly. In this study 2 generations of CP birds were used. They were i d e n t i f i e d as 62 and G3. G2 was the result of 1 generation of selection from the "420 l i n e " and had a 30% frequency of c l e f t palate. G3 was the progeny of selected G2 and had a frequency of 50% c l e f t palate. Only phenotypically c l e f t palate birds from G2 and G3 were used in this study. The G2 birds used were of variable age, having been produced over a series of 16 consecutive weekly hatches on a low r i b o f l a v i n d i e t . At the time of use they were approximately 16 to 20 months of age. The hens had been used i n a p r i o r experiment involving one of the following r i b o f l a v i n diets: 16 weeks of low r i b o f l a v i n ; 16 weeks of normal r a t i o n ; or 4 normal, 8 low, and 4 normal ration weeks. Ten days elapsed between the end of the prior experiment and the use of the hens in t h i s study. The diet was normal during that period and previous experience indicated that the effect of the low d i e t , as measured by h a t c h a b i l i t y , would have been l o s t during that time. The G3 birds were of variable age, having been produced over the 16 week period of weekly hatches for the diets just described. Subsequent analysis (Roberts et_ al_. , 1973) indicated that there was no diet effect on the incidence of c l e f t palate produced for those d i e t s . At the time of use, the G3 were approximately 17 to 21 months of age. In both the G2 and the G3 b i r d s , considerable mortality (up to 50%) had taken place between hatch and the time of use. The vast majority of mortality was d i r e c t l y attributable to Marek's disease and lymphomatosis. (The "420 l i n e " had been selected for s u s c e p t i b i l i t y to avian leukosis and t h i s l i a b i l i t y was apparently s t i l l a character of the derived CP l i n e . How t h i s mortality affected birds of di f f e r e n t n u t r i t i o n a l o r i g i n i s not known.) 36 C. The Breeding Design A l l matings were carried out using a r t i f i c i a l insemination. Feed and water was provided ad libitum for a l l b i r d s , for the duration of the study. An F-| was obtained from reciprocal crosses between the NH and CP (G2) l i n e s . The F-j was f u l l - s i b mated to produce an F^. The F-| was also backcrossed to the CP l i n e (G3) in reciprocal crosses, creating a backcross generation (BC). 1. Production of the F-j Six NH males were each mated to 3 CP hens of G2, and 18 CP males of G2 were each mated to 2 NH hens. Hens were housed in standard 20.3 cm. laying cages and males in 30.5 cm. cages, i n one house. After 8 weeks a l l birds were moved to a second house containing 25.4 cm. laying--and 35.6 cm. male-cages. Eggs were collected d a i l y , i n d i v i d u a l l y i d e n t i f i e d , and stored at approximately 12.8°C. and r e l a t i v e humidity of approximately 70%. For 10 weeks eggs were set weekly in a Jamesway Model #252 incubator under standard incubation conditions, and were a l l transferred to pedigree hatching baskets on the eighteenth day of incubation. On the twenty-first day of incubation, a l l unhatched eggs and hatched chicks were removed from the incubator for examination. The hatched chicks from the f i r s t 2 hatches were i n d i v i d u a l l y i d e n t i f i e d at hatch, and reared using standard brooding and rearing procedures. 37 The CP hens were fed a standard, "normal" diet which was U.B.C. formulated and commercially mixed (Number 8 r a t i o n , Table 1, Appendix). The NH hens were fed the normal diet for the f i r s t 2 weeks, then 8 weeks of a r i b o f l a v i n d e f i c i e n t ration (formulated by Dr. J . B i e l y , Appendix, Table 2) followed by 2 weeks of normal r a t i o n . The males were fed the normal ration throughout the study. 2. Selection C r i t e r i a for Parents of the F-| A l l NH birds used were selected at random from the NH population. The CP l i n e males and females were not selected at random from the G2. Males were selected by 2 c r i t e r i a . The f i r s t was potential f e r t i l i t y and the second was the type of c l e f t possessed. Six males of each of the phenotypes "rig h t , " " l e f t " and " b i l a t e r a l " c l e f t s were chosen. This was not representative of the group as a whole, where " r i g h t , " " l e f t " and " b i l a t e r a l " occurred i n the approxi-mate ratios (after mortality) of 5:6:9 respectively. The female selection was based on the previous treatment of the hen and type of palate c l e f t . As described e a r l i e r there had been 3 previous dietary regimes, 2 of which had low r i b o f l a v i n content. Equal numbers of hens from each of the p r i o r treatments were used. Equal numbers of " r i g h t , " " l e f t " and " b i l a t e r a l " c l e f t s were used. The females were assigned to NH males at random with the r e s t r i c t i o n s that one of each type of c l e f t was represented for each male, and t h a t , i n ad d i t i o n , for most matings a l l 3 prior diet treatments were also represented. 3. Production of the and the BC For each family ( i . e . , the progeny of 1 or i g i n a l sire-dam combination), 1 F-| male was mated to 2 f u l l - s i b females, and to 2 CP l i n e G3 females. In addi t i o n , 1 or 2 F-j females were mated to 1 CP l i n e G3 male each. That i s , reciprocal backcross matings to the CP li n e were made. Hens were housed in standard 25.4 cm. laying cages and males in 35.5 cm. male cages in one house. Eggs were collected d a i l y , i n d i v i d u a l l y i d e n t i f i e d , and stored as described previously. For 16 weeks eggs were set weekly under the conditions described previously. The eggs were set i n order of hen cage number,incubation trays were assigned levels within the incubator for each hatch at random. At 18 days of incubation, a l l eggs were transferred to individual p l a s t i c hatching baskets (approximately 8 x 8 x 6 cm.) with wire l i d s affixed with masking tape, and trayed in order. Hatching trays were assigned levels at random within the "hatcher." A l l unhatched eggs and chicks were removed on the twenty-first day of incubation for data c o l l e c t i o n . A l l males were maintained on the normal ration for the duration of the study. The females were fed, for hatches 1 to 6 (H 1-6), the normal diet (Appendix Table 1). For hatches-7 to 10 (H 7-10), they received the r i b o f l a v i n d e f i c i e n t diet (Appendix Table 2 ) , and for the l a s t 6 weeks (H 11-16) they were fed the normal d i e t . I t was standard practice during any change from the normal crumbled diet to the de f i c i e n t mash diet to provide for the f i r s t 3 days of change a small amount 39 of crumbled normal diet on top of the mash in order to provide an adjustment period for the birds. 4. Selection C r i t e r i a f o r Parents of the Fg and BC It was planned for each mating pair of the parent cross that a minimum of 1 male and 3 females and a maximum of 1 male and 4 female progeny be used as parents of the Fg and BC. When there was more than 1 male cr 4 females a v a i l a b l e , those used were selected at random. Only 4 of the ori g i n a l 6 NH males were represented as parents producing the F^ and BC generations. Three of the ori g i n a l NH males had 2 families represented. The fourth male had only 1 family represented. Twelve of the or i g i n a l 18 CP males were represented as parents producing the Fg and BC generations. Eleven of the original CP males had one family represented; the twelfth male had both families represented. Since some of the G2 males and females of the CP l i n e which produced the F-j were the same as those which produced the G3, the following mating c r i t e r i a were used in this study: In the backcross for each family mating the F-j male could not be a ha l f - s i b to the 2 CP, G3 females. The same r e s t r i c t i o n was applied to the reciprocal cross for the 2 CP, G3 males. In addition, the 2 CP, G3 females could be neither f u l l - n o r h a l f - s i b s . Again the same r e s t r i c t i o n was applied to the male CP, G3 birds of the reciprocal backcross. However, whenever possible, 1 of the CP males and 1 of the CP females used 40 in t h e i r respective crosses to the F-| were full-sibs. The others, when a v a i l a b l e , v/ere not. D. The Data Collected for the CP Line, F 1, F^ and BC 1. The C l e f t Palate Line: G2 and G3 The CP l i n e (G2) was examined for the type of c l e f t palate contained ( i . e . , r i g h t , l e f t , or b i l a t e r a l ) , and scored according to a f i v e point scale where 0 = no c l e f t ; 1 = the mildest c l e f t s ; and 4 = the most severe c l e f t s . The CP l i n e (G3) was s i m i l a r l y examined and scored. The G3 females used in the backcross were weighed to the nearest gram at the end of the s i x t h week of the study, immediately pr i o r to being placed on the r i b o f l a v i n d e f i c i e n t d i e t . 2. The F-| Generation The hatched F-j progeny were examined for c l e f t palate and scored in the same manner as described previously. The unhatched eggs were broken open and mortality categorized into 5 periods: 1. Pips. 2. Dead-in-Shell (DIS) - birds which had absorbed most of the yolk and were 19 or 20 days of development. 3. Late Dead (LD+) - embryos that died between approximately 13 and 19 days of development and whose palate was observable. 4. Late Dead (LD-) - embryos that died between approximately 8 and 13 days of development and whose palate was not observable. 41 5. Early Dead (ED) - embryos that died between 1 and 8 days of development. I n f e r t i l e eggs were also recorded. A l l embryos attaining the LD+ stage or greater were checked for c l e f t palate and scored. Any external obvious malformations such as missing eyes or club down were noted. The F-| females used in the formation of the F^ a n d BC were weighed to the nearest gram at the time that the G3 females were weighed ( i . e . , immediately preceding the r i b o f l a v i n l e s s dietary period). 3. The ?2 ar|d BC Generation The hatched F^ a n d BC were examined for c l e f t palate and were scored in the following manner for each side: 0 = no c l e f t ; 1 = the mildest c l e f t s ; to 6 = the most severe c l e f t s . Throughout the study, the scores were determined by comparison to a set of drawings based on photographs of the t r a i t (Appendix, Figure 1). ( A l l scores in the study were evaluated by the author). The unhatched eggs were broken open and mortality categorized into 4 periods: 1. Pips. 2. Dead-in-Shell (DIS). 3. Late Dead (LD) - embryos that died between approximately 8 and 19 days of development. For some data this was further subdivided into LD- (8-12 days) and LD+ (13-19 days). 4. Early dead (ED). Embryos attaining 13 days of development or more were scored for c l e f t palate at that time. Embryos that had reached 8 to 12 days of develop-ment were preserved in Bouin's Fluid and the palates scored using a dissecting microscope. C l e f t palated embryos that had attained 13 days of development or more and the hatched c l e f t palated chicks were sexed by gonadal examination. Both embryos and hatched chicks were checked f o r club down, curled toe p a r a l y s i s , head and beak abnormalities and malpositions. As a matter of i n t e r e s t , since i t had been noted i n the G2 that an unusual t r a i t was present in possible association with c l e f t palate, that t r a i t , "palatal p i t s , " was also assessed. A l l hatched chicks were weighed to the nearest gram at hatch. E. Special Studies 1. Crosses of Phenotypically Normal CP Line Individuals This study used normal progeny from the CP l i n e (63). Five males were each mated at random to 2 females for 15 weeks. Eggs were collected as described previously. Eggs were set weekly, transferred at 18 days of incubation, and a l l eggs and hatched chicks removed for data c o l l e c t i o n at 21 days of incubation. For the f i r s t 3 and f i n a l 3 hatches, the diet consisted of a d i f f e r e n t r i b o f l a v i n s u f f i c i e n t ration (2.2 mg/kilo added to the r i b o f l a v i n d e f i c i e n t d i e t ) . 43 A r i b o f l a v i n low diet (1.1 mg/kilo added to the r i b o f l a v i n d e f i c i e n t diet) used for hatches 4 through 6, while hatches 7 through 12 used the r i b o f l a v i n d e f i c i e n t diet i t s e l f . Hatched chicks and embryos of 8 days development or more were assessed for the presence or absence of c l e f t palate. 2. The New Hampshire x C l e f t Palate Cross Tested  on the Riboflavin Deficient Diet The NH males and the surviving hens from the CP l i n e (G2) used to produce the F-j were remated. The birds continued to be housed in cages, the males in the same location as before, the females i n s i m i l a r cages in a d i f f e r e n t location having limited external l i g h t access, but incandescent l i g h t provided by time clock, and a trough-type water system. The eggs were collected and weekly hatches were set as previously described. At 18 days of incubation, the eggs were transferred to a small forced draft incubator for hatching. At 21 days a l l eggs and chicks were removed. Embryos attaining 13 days' develop-ment or more and hatched chicks were scored for presence or absence of c l e f t palate. The diet regime consisted of 9 weeks of normal ration followed by 6 weeks of the r i b o f l a v i n d e f i c i e n t r a t i o n , followed by a further 10 weeks of the normal r a t i o n . This experiment took place approximately 8 months after the production of the o r i g i n a l F^ i n d i v i d u a l s . 3. The Selected New Hampshire Male Study This study was run concurrently with and used the same f a c i l i t i e s as special study number 2 previously described. Three NH 44 males which had been selected based on the presence of grooves, which the author observed in the palatine ridge in the area where c l e f t s normally occurred, were each mated to 2 randomly selected CP (G3) females. Eggs were c o l l e c t e d , s e t , and hatched as described previously. Fourteen weekly hatches were set. The f i r s t 2 weeks on a normal diet were followed by 6 weeks of r i b o f l a v i n d e f i c i e n t r a t i o n , followed by 6 weeks of normal r a t i o n . Embryos attaining 8 days of development or more and hatched chicks were assessed for the presence or absence of c l e f t palate. F. General Considerations for Data Analysis Progeny from hens producing fewer than 6 eggs were not included in the analysis. Hens that died during the course of any study (6 birds in t o t a l ) were considered to have gone out of production except when production was the variable being considered for analysis. In general for the t r a i t under study the data were analyzed based on the hen frequency per week and comparisons of averages were made for dietary periods. Chi-square analyses were used for several comparisons (after Snedecor and Cochran, 1967). The test for "goodness of f i t " or "tests of independence" with 1 degree of freedom incorporated the Yates correction term (after Strickberger, 1968). The c l e f t palate frequency data of the F^ and BC generations were tested against a multiple recessive l o c i model. 45 Conversely, an additive model was assumed. The same data was also tested (including the F-j) using Wright's 1934 formulae for determining the number of genes d i f f e r i n g between inbred l i n e s . Table 1 l i s t s the abbreviations used to i d e n t i f y matings and progeny throughout this study. Table 1 - Breeding I d e n t i f i c a t i o n Used Throughout the Study of C l e f t Palate (CP) and New Hampshire (NH) Matings Producing the F,, F 0 and Backcross Generations. Symbol Matings* CP x NH F i NH x CP F2 (CP x NH) x (CP x NH) F2 (NH x CP) x (NH x CP) Fl X CP (CP x NH) x CP F i X CP (NH x CP) x CP CP X Fl CP x (CP x NH) CP X F i CP x (NH x CP) Male i d e n t i f i c a t i o n appears f i r s t in a l l matings. 47 RESULTS AND DISCUSSION A. Effect of Riboflavin Deficient Diet on Hatchability The response of hatchability of f e r t i l e eggs from hens pro-ducing the F-j, and BC progeny to the dietary regimes i s summarized in Table 2 and shown in d e t a i l i n Appendix Tables 3 to 10. A consistent diet effect was noted. For the normal diet the hatchability of f e r t i l e eggs from hens producing the F^ was 82.4%, followed by 64.8% hatchability on the r i b o f l a v i n deficient d i e t , which increased to a 79.1% hatchability when they were returned to the normal d i e t . The same respective values when the F-j hens were used to form the BC were 75.6, 67.4 and 73.6% h a t c h a b i l i t y . When the CP females (G3) were used to form the BC values were lower, but followed the same pattern: 62.4, 34.4, and 65.9% h a t c h a b i l i t y . Hens producing the F^ also showed the same pattern, in that the r i b o f l a v i n deficiency period gave 44.2% h a t c h a b i l i t y , which was preceded by and followed by 83.8 and 91.6% respectively. The comparable data was not available for hens producing the F-j, since i t was deemed advisable not to subject the CP hens (62) to a possible confounding with prior treatment e f f e c t s ; however, t h e i r average hatc h a b i l i t y over 10 weeks of normal diet was 81.3%. It should be recalled that the data for the F-j i s not comparable to that of the and BC. Table 2 - Mean Percent Hatchability for Riboflavin Normal (+) and Deficient (-) Dietary Periods for the FT, F 2 and Backcross Generations. Type of Cross H a t c h N u m b e r l-6(+) 7-10(-) . 11-16(+) 10(-) F, x F, 80.3 61.6 82.3 26.3 F; XF; 84.4 67.9 75.9 28.8 Mean 82.4 64.8 79.1 27.6 CP x F 1 80.1 63.2 77.5 41.8 CP x F^  71.1 71.5 69.8 45.8 Mean 75.6 67.4 73.6 43.8 F ] x CP 68.2 43.6 67.5 0.0 F\ x CP 56.7 25.2 64.3 0.0 Mean 62.4 34.4 65.9 0.0 CP x NH* 83.8 44.2 91.6 18.9 NH x CP** 81.3 * Cross was tested on the +, - and + diets for 2, 6 and 2 weeks respectively. ** Cross was made on + diet for 10 weeks. 49 Figure 1 shows the average hatchability response for the seventh to twelfth week of the study, for the hens producing Fg generation, and for the CP and F-j hens producing backcross progeny. Hatchability began to decrease after 1 to 3 weeks of feeding the r i b o f l a v i n d eficient r a t i o n , and continued to decrease u n t i l i t reached zero, or u n t i l the hens were returned to a "normal" r a t i o n , at week 10, at which time hatchability improved to reach nearly normal levels at the twelfth week. These results were e n t i r e l y consistent with the l i t e r a t u r e . The level to which hatchability had dropped by hatch 10 appeared to depend on two factors: the i n i t i a l "normal" hatchability level of the hens and the number of weeks that level could be sustained during r i b o f l a v i n deficiency. Thus the CP hens (G3), which had a lower i n i t i a l h a t c hability level (62.4%) and which showed e a r l i e r response to the de f i c i e n t diet (1 or 2 weeks) had reached zero hatch-a b i l i t y by hatch 10, while the F^ hens, with higher i n i t i a l hatchabil-i t i e s (82.4 and 75.6%) and slower response to diet (2 or 3 weeks) had reached 27.6 and 43.8% by hatch 10 (Table 2). The rate of loss of h a t c h a b i l i t y , once the process had begun, did not appear to be a major f a c t o r , as the slopes of the plotted decrease were s i m i l a r (Figure 1). The reciprocal F^ hens showed differences of less than 10% in the ha t c h a b i l i t y values attained within the productions of the F^ and BC generations respectively. Therefore i t can be concluded that there were minimal differences between F^ hens from reciprocal crosses with respect to h a t c h a b i l i t y . Figure 1 Percent hatchability across the r i b o f l a v i n d e f i c i e n t period (weeks 7 to 10) f o r F] generation hens producing F 2(a) or backcross chicks (b) and c l e f t palate l i n e hens producing backcross chicks ( c ) . 51 The level to which hatchability had decreased at hatch 10 (Table 2) f e l l into 3 d i s t i n c t groups: the eggs from CP hens, which reached zero h a t c h a b i l i t y ; the eggs from F-j hens containing Fg embryos, which averaged 27.6% h a t c h a b i l i t y ; and the eggs from F^ hens containing BC embryos, which averaged 43.8% h a t c h a b i l i t y . Assuredly a l l would have reached zero hatchability had the deficiency been continued further. The "normal" ha t c h a b i l i t y of the CP hens (G3) was low, being an average of 62.4% before the de f i c i e n t diet period. This less-than-optimum value was probably due, in part, to the r e l a t i v e l y advanced age of the hens (over 1 laying year), and i n part to inbreeding depression (Roberts et al_., 1973), and, perhaps, in part to the presence i n the hens themselves, of the c l e f t palate t r a i t . On the normal d i e t , the F^ hens used to produce the Fg averaged 6% greater h a t c h a b i l i t y than the F-| hens used to produce the BC. This difference did not appear to be meaningful. Two factors would seem to account for the groupings noted for hatch 10: the genotype of the dam and the genotype of the embryo within the r i b o f l a v i n d e f i c i e n t egg. I f one considers the CP hens and the F-j hens producing the BC chicks, the offspring are t h e o r e t i c a l l y genetically s i m i l a r . Therefore, with the exception of sex-linked genes, the differences in hatchability of progeny can be attributed to the effects of the dams. (Factors such as age of dam, egg s i z e , egg q u a l i t y , and, in addition, in this case, the r i b o f l a v i n content of the egg.) The CP hens at the s t a r t of hatch 7 were approximately 450 grams l i g h t e r than F, hens. Since r i b o f l a v i n i s contained i n muscles and l i v e r in small quantities i t seems l i k e l y that the F-j hens were slower to deplete t h e i r body content of r i b o f l a v i n , and reflected this difference i n continued input of r i b o f l a v i n into the eggs. It should be pointed out that further differences existed between the CP l i n e and the F-j producing the BC. The hatchability of the "420 l i n e " from which the CP l i n e had been derived, was markedly susceptible to r i b o f l a v i n deficiency (Shoffner ejt al_., 1953). The CP l i n e birds also strongly tended to go out of production u n t i l returned to the normal ration (Tables 9 and 10, Appendix), and this effect was only minor in the F-j hens (Tables 7 and 8, Appendix). Perhaps the l a t t e r response r e f l e c t s a basic genetic difference introduced by the NH a l l e l e s in the F-j. The difference between hatchability of progeny from F-j hens producing F^ and F-j hens producing BC at hatch 10 can be s i m i l a r l y examined. Consideration of the 2 groups of F-j hens shows that the hens may be considered to be genetically i d e n t i c a l and that differences i n h a t chability between them at hatch 10 should be r e f l e c t i o n s of the genotypes of the progeny. I t was noted in embryos, hatched chicks, and growing chicks of the that they contained about one-third to one-quarter of t h e i r number as segregates for feather pigment genes including extended black ("E") and recessive s i l v e r ("s"). A l l pigment a l l e l e s except "s" (recessive s i l v e r ) and "e" (recessive extended black) would have come from the CP l i n e which carries homozygous dominant e p i s t a t i c white ("I"). The NH l i n e i s recessive for a l l feather pigment a l l e l e s and i s homozygous for "s" and "e." 53 Considering that the BC chicks were white (either I i or II i n geno-type) and the F^ chicks pigmented, this pigmentation constitutes a part of the genetic difference between F^ a n c' BC chicks. Bernier and Cooney (1954) showed that pigmented embryos required more r i b o f l a v i n to survive to hatch than non-pigmented embryos of otherwise s i m i l a r genotype. Since the hatchability on a normal diet was not too d i s s i m i l a r for BC and F^ embryos from F-j hens (the rank, in f a c t , i s reversed), i t would seem very l i k e l y that the mortality of p i g -mented embryos of the F^ could account for the much lowered hatchabil-i t y of t h i s group compared to BC during r i b o f l a v i n deficiency of the hens. B. Effect of Riboflavin Deficient Diet on Embryonic Mortality Based on the l i t e r a t u r e , i t would be expected that as r i b o -f l a v i n deficiency of the hens continued, the time of embryonic mortality would s h i f t to progressively e a r l i e r stages. Data represent-ing the stages of embryonic mortality for the BC and F^ progeny produced as the r i b o f l a v i n deficiency progressed i s presented in Table 3. For ease of seeing the extent of change i n embryonic mo r t a l i t y , the values have been presented as the residual after the mean and 2 standard deviations from the f i r s t normal diet period have been removed. The dams and sires from the o r i g i n a l reciprocal crosses were pooled, since no very great differences were seen between them, and since both are equally represented i n the means contributing to the resulting categories. 54 Table 3 - Mean Percent Embryonic Mortality of Hatches 7 Through 16 that Exceeded that of the Mean of Hatches 1-6 and Twice i t s Standard Deviation (SD) for Riboflavin (R) Normal (+) and Deficient (-) Diets. Mating Hatch Number Early Dead Late Dead DIS PIP Hatch R F^CP and FjxCP CPxF and CPxF 1 1 Flx Fl and F1 xF' 1-6 24.7 6.6 2.8 3.4 62.4 + (SD) (6.8) (2.8) (1.5) .(3.6) (4.9) 7 a -0.1 - 3.2 - - -8 - 2.0 2.5 - -3.0 -9 - b 39.9 - - -33.0 -10 19.8 28.3 - - -52.6 -11 - 8.6 . - - - + 12 - - 0.6 - - + 13 - - 1.4 - - + 14 - - - - 3.8 + 15 - - 3.6 - - + -16 - - 2.0 - 4.0 + 1-6 14.2 4.0 1.3 4.1 75.6 + (SD) (5.8) (4.0) (1.2) (2.5) (6.1) 7 - - - - - -8 - - - - - -9 - 0.8 - - - -10 - 15.0 1.2 2.1 -19.6 -11 - 1.0 0.1 - -5.6 + 12 - - - - - + 13 - - - 0.7 - + 14 - - - - - + 15 - - - - - + 16 - - - - - + 1-6 8.4 4.0 2.8 2.5 82.3 + (SD) (3.0) (2.3) (2.4) (1.9) (5.9) 7 - - - - - -8 - - - - - -9 - 5.4 - 0.9 -7.1 -10 - 28.6 6.1 2.9 -42.9 -11 - 23.3 - - -9.5 + 12 - - - - - + 13 - - - - - + 14 - - - 0.4 - + 15 - - - - - + 16 - - - - - + For example, actual value was 11.0. bFor example, actual value was 58.1. 55 The largest portion of mortality contributing to reduction i n h a t c h a b i l i t y was found i n the LD period of embryonic mortality (8-18 days' incubation). This observation was in good agreement with the l i t e r a t u r e ' reports of a t h i r d peak appearing at 10 to 12 days' incubation during r i b o f l a v i n deficiency, in addition to the "normal" peaks at days 4 and 19. Based on the results of previous reports, i t might be expected, although the embryonic mortality had been shown to s h i f t to progressively e a r l i e r stages as the hens became more depleted, that the individual variations between hens (also documented) would tend to mask somewhat this process. Further, since the data reported i n t his study represented d i f f e r e n t numbers of days in the 4 mortality periods ( i . e . , ED = 8 days; LD = 10 days-; DIS = 1 day), no d e f i n i t e measure of this s h i f t was possible. However, some evidence that such a change was taking place was provided by the results i n the CP hens. The DIS category showed an i n i t i a l increase followed by a decrease while the LD category simultaniously swelled to a maximum of 39.9% at hatch 9. I t in turn then decreased while the ED category increased to 19.8% at hatch 10. No such clear-cut pattern was seen i n the F-j hens, but i t i s possible that since they showed a l a t e r response to the d i e t , the deficiency effect may not have been of long enough duration to show the progressively e a r l i e r pattern of mortality. The effect of r i b o f l a v i n deficiency on mortality continued for a l l 3 hen groups (Table 3) into hatch 11 but was no longer present by hatch 12. This fact may be of importance in the a l l o c a t i o n of the c l e f t palate chicks produced i n hatch 11. I t i s arguable that t h e i r frequency should be considered representative of r i b o f l a v i n 56 deficiency, despite the fact that t h e i r hens were being fed a "normal" ration again. C. Incidence of C l e f t Palate on Normal and Riboflavin Deficient  Diets 1. The F.j Data There was a d e f i n i t e low incidence of c l e f t palate in the F-j progeny (Table 4). Each reciprocal cross produced 1, non-hatching, chick having a severe b i l a t e r a l c l e f t . If these d e f i n i t e cases are taken to be the frequency of c l e f t palate in the , the values were 0.11% (1 out of 870) and 0.20% (1 out of 491) for NH and CP (G2) hens respectively. Whereas there were 2 d e f i n i t e CP chicks in the F-j, i n addition there were several individuals whose palatal structure was somewhat d i f f e r e n t than "normal." These were divided into 2 categories. The "probable" category (1 in the progeny from NH hens, and 2 in the progeny from CP hens) were highly suspicious of having c l e f t palate but i t was not c l e a r l y evident as being an expression of the t r a i t . The second group, the "possible" category, were even less well defined, but i t was evident to the scorer that some difference could be discerned from the probable group. Therefore the 4 individuals from the NH hens which appeared thus were recorded as "possible" c l e f t palates. I f the "possibles" and "probables" are included in the frequency of c l e f t palate in the F^, the frequencies were 0.85% and 0.66% for the NH and CP hens respectively. Table 4 - Incidence of Cle f t Palate (CP) in the F-| and F-j Progeny Over 10 Weekly Hatches of the CPxNH Cross and i t s Reciprocal on a Riboflavin (R) Normal (+) or Deficient (-) Diet-Mating (Progeny) Hatch No. R Number* Sires Dams Definite Number of CP Probable Possible *** Observations Maximum Percent CP CPxNH 1 + 15 29 0 0 0 111 0.00 2 + 17(1) 33(1) 0 1 0 130 0.77 (FT) 3 - 17(2) 33(2) 0 0 2 121 1.65 4 - 16(1) 27(1) 1 0 0 84 1.19 5 - 16 25 0 0 0 79 0.00 6 - 16 24 0 0 0 68 0.00 7 - 11(2) 15(2) 0 0 2 41 4.88 8 - 15 18 0 0 0 46 0.00 9 + 17 28 0 0 0 99 0.00 10 + 17 26 0 0 0 91 0.00 Mean • 0.85 NHxCP 1 + 6(1) 14(1) 0 ' • 1 0 45 2.22 2 + 5 14 0 0 0 52 0.00 (Fl) 3 + 6 17 0 0 0 66 0.00 4 + 6(1) 15(1) 1 0 0 46 2.17 5 + 6(1) 14(1) 0 0 45 2.22 6 + 6 14 0 0 0 51 0.00 7 + 6 15 0 0 0 47 0.00 8 + 6 13 0 0 0 42 0.00 9 + 6 15 0 0 0 44 0.00 10 + 6 15 0 0 0 53 0.00 Mean - 0.66 Bracket number sires and dams producing CP progeny. Also had exencephaly shortened upper beak, monophthalmia. Did not include late dead embryos or e a r l i e r stages that could not be evaluated. 58 There was much variation i n the structure of the palate of chickens in general, and no clear-cut l i n e of demarcation was found between the phenotypes "normal" and " c l e f t palate." The "possible" and "probable" types may represent the bottom of the range of expression of c l e f t palate. The s i m i l a r i t y i n incidence between the 2 types of hen suggests that l i t t l e , i f any, maternal eff e c t was i n evidence in the production of the t r a i t , i f dietary regimes are ignored. However the use of the r i b o f l a v i n d e f i c i e n t diet for 6 of 10 hatches from NH hens may be confounded with any maternal effect present. Although a cautious interpretation i s needed because of the low numbers of observations, i t i s inter e s t i n g that, for hatches 1 and 2, wherein the 2 lines received the same dietary treatment and can be compared, the frequencies of c l e f t palate were 0.38% and 1.11% for NH and CP hens respectively, indicating a p o s s i b i l i t y that on the normal diet CP hens tended to produce a higher frequency of c l e f t palate in thei r progeny than NH hens d i d . The comparison of occurrence of c l e f t palate between normal and r i b o f l a v i n d e f i c i e n t periods within NH hens shows an increase from 0.18% on the normal diet to 1.29% on the r i b o f l a v i n d e f i c i e n t d i e t . This trend i s consistent with the increase in incidence reported for normal and r i b o f l a v i n d e f i c i e n t diets for "420 l i n e " hens (Roberts et a]_., 1973). Consideration of the pedigrees producing c l e f t palate i n the F-j generation showed that a l l of the maximum of 6 c l e f t palates from NH hens were produced by di f f e r e n t hens while 2 of the s i x , but not the " d e f i n i t e , " were produced by 1 s i r e . Of the 3 cases produced 59 by CP hens, a l l were from d i f f e r e n t hens, while the 2 "non-definite" cases v/ere produced by 1 s i r e . Further evidence that the c l e f t palate t r a i t occurred in the F-| generation at frequencies that must be taken into account in any genetic explanation of the t r a i t was provided by 2 small a n c i l l a r y studies. A summary i s presented in Tables 5 and 6. The f i r s t of these studies involved 3 NH males selected because of the presence of "grooves" in the palatine ridge, each mated to 3 CP l i n e (G3) hens. The production of these hens was poor, and the data limited (Table 5). The effects of selection on the CP l i n e and of the r i b o f l a v i n d e f i c i e n t dietary regime were confounded, for comparison to the o r i g i n a l crosses producing the F^. The 2 c l e f t palated individuals produced in a total of 57 observations (3.5%) was much more than would be expected from unselected NH males mated to CP l i n e (G2) females during a normal diet (1.66%, Table 6). The second study involved the surviving NH sires and CP l i n e (G2) hens used to form the F-j 8 months e a r l i e r . As stated before, the CP hens at that time were not challenged with r i b o f l a v i n deficiency, since some of the CP hens had been involved i n previous r i b o f l a v i n low d i e t s , and i t was desired to avoid any confounding of residual effects with further treatments. This second small study subjected the hens to r i b o f l a v i n deficiency in the same manner as e a r l i e r * studies. As shown i n Table 6, the response i n incidence of c l e f t palate was marked, changing from approximately 1% during normal diet periods to approximately 12% during the maternal r i b o f l a v i n deficiency 60 Table 5 - Incidence of C l e f t Palate Progeny and Total Observations ( ) from New Hampshire Males (With Palate Grooves) Mated to C l e f t Palate Females of G3, On Riboflavin Normal (+) and Deficient (-) Diets. NHxCP Matings* H a t c h e s 1-2(+) 3-8(-) 9-10(-) Total. 1 0(4)** 0(2) 0(12) 2 0(2) 1(2) 0(14) 3 0(4) K6) 0(10) Totals 0(10) 2(10) 0(37) 2(57) Percent 0.0 20.0 0.0 3.5 Original mating was 1 NH to 3CP for each male, only 1 female from each mating continued in production. ** Total observations did not include embryos dying prior to 8 days of incubation. 61 Table 6 - Incidence of C l e f t Palate Progeny and Total Observations ( ) from the Remated NHxCP Cross On Riboflavin Normal (+) and Deficient (-) Diets Sire Dam H a t c h e s l-9(+) 10-15(-) 16-25(+) 1 1 0(26) 1(13) 0(15) 2 1(10) 2(11) 0(10) 3 0(17) 1(5) 1(9) 2 1 0(7) 1(11) 3 1 0(9) 2(9) 0(9) 2 0(3) 0(5) 4 1 0(14) 1(8) 0(18) 2 0(1) 1(6) 0(11) 5 " 1 0(4) 0(8) 2 0(9) 0(2) 0(18) Totals 1(96) 9(74) 1(108) Percent 1.0 12.2 0.9 Total observations did not include embryos dying prior to 12 days of incubation. period. Due to the age of the b i r d s , this study i s not d i r e c t l y comparable to the e a r l i e r study, but i t i s encouraging to note that on the normal diet the incidence of c l e f t palate from these hens during the e a r l i e r study (0.66%) was not very di f f e r e n t from the 1% observed in th i s second study. Considering the much less marked response of the NH hens of the reciprocal cross to r i b o f l a v i n d e f i c -iency (from 0.4% to 1.1%) in the e a r l i e r study, some importance of maternal genotype in the response to diet i s indicated. In general the c l e f t s counted were of much severity and l e f t no doubt as to the presence of the t r a i t , so that the data may be viewed as erring in the dire c t i o n of underestimation of frequencies, when the previously discussed problem i n determination i s considered. The outstanding resu l t of both of the a n c i l l a r y studies was the reinforcement of the observation of the presence of c l e f t palate in the F-j generation, and the knowledge that a l l explanations of results in the ¥^ and BC must provide f o r this f a c t . Perhaps the simplest explanation would be the presence of the c l e f t palate t r a i t i n the NH l i n e at undetected frequencies. 2. The F 2 and BC Data The percent c l e f t palate per hen for the 3 dietary periods for the reciprocal crosses producing Fg and and for the 4 reciprocal backcrosses i s presented i n Appendix, Tables 11, 12, 13, 14, 15 and 16, and i s summarized in Table 7. For both crosses producing Fg progeny and for 3 of the 4 crosses producing BC progeny, a clear increase in incidence of c l e f t 63 Table 7 - Mean Percent C l e f t Palate Progeny and Total Observations ( ) from Matings Producing the F 2 and 4 Types of Backcross Generations on Riboflavin Normal (+) and Deficient (-) Diets. H a t c h e s Mating l-6(+) 7-10(-) 11-16(+) F, x F,* . 1.6(390) 2.5(212) 0.8(326) F; x F; 0.0(227) 6.4(155) 0.4(221) Mean 0.8 4.4 0.6 CP xF1 7.8(361) 11.2(194) 5.5(298) CP x F^ 5.9(181) 12.3(122) 6.7(161) F1 x CP 6.5(358) 19.2(98) 4.6(171) F^ x CP 13.5(174) 6.7(39) 13.2(118) Mean 8.4 12.4 7.5 Hen 549 deleted. 64 palate was observed during the deficient diet period (hatches 7 to 10) compared to the normal f i r s t and t h i r d dietary periods (hatches 1 to 6 and 11 to 16). The change v/as to 4.4% from 0.8 and 0.6% for the F 2 and to 12.4% from 8.4 and 7.5% for the BC (Table 7). The f a i l u r e of one cross, Fj x CP to show a s i m i l a r pattern may be due to sampling er r o r . The CP hens were poor in egg production, especially during the r i b o f l a v i n deficiency period. For the cross under discussion, the number of hens used was also com-paratively small, so that the chance for sampling error was great. Of the 12 hens i n production during the f i r s t and l a s t hatch periods, 4 and 7 produced fewer than 10 eggs (after i n f e r t i l e and ED removed) respectively. Of the 9 hens in production during the r i b o f l a v i n d e f i c i e n t period, 7 produced fewer than 5 eggs (after i n f e r t i l e and ED removed). Although production was s i m i l a r l y poor for the other group of CP hens used in the reciprocal backcross, the number of hens involved (24) tended to offset the sampling problem. The response of increased production of c l e f t palate progeny during maternal r i b o f l a v i n deficiency i s i n agreement with the observations of Roberts et a]_. (1973) involving e a r l i e r generations of the CP l i n e . For the backcross, only 1 hen (number 470, Appendix Table 13) produced c l e f t palate progeny during the deficiency period that did not also produce c l e f t palate progeny during the normal diet period. Yet there were several hens which f a i l e d to produce any affected progeny at a l l . This would suggest that the increased frequency of c l e f t palate observed during r i b o f l a v i n deficiency was due to increased frequencies within dams producing c l e f t palate on the normal d i e t , and not to increased numbers of dams producing the anomaly. For the F 2 generation the 6 hens producing the t r a i t only on the def i c i e n t diet i s reasonable considering the sample size per dam and the very low frequency of the t r a i t on the normal d i e t . For an increased frequency to take place the number of hens had to increase. There was evidence of variation within the NH l i n e i n the frequency of c l e f t palate i n descendants. When the frequency of c l e f t palate in a l l descendants (F^ and BC combined) of the 4 NH males was computed, i t was found that the rank from most to least frequent was unchanged for normal and deficient types of diet respectively: s i r e 127, 11/38 and 20/104; s i r e 124, 9/71 and 11/104-; s i r e 123, 4/85 and 5/115; 'and s i r e 125, 1/88 and 0/152. The NH s i r e 125 apparently carried a l l e l e s which in h i b i t e d the appearance of c l e f t palate. The breeding design made possible the demonstration of family e f f e c t s , and showed that there was genetic v a r i a t i o n for the penetrance or incidence of c l e f t palate present i n the NH l i n e . Hen 549 Hen 549 was an F-j from a CP x NH cross, and was mated to a f u l l - s i b male to produce the F^. While the progeny of hen 549 were i n several ways unusual, those of a f u l l sib hen mated to the same male were e n t i r e l y unremarkable. 66 The progeny of hen 549 were notable in 4 respects: the incidence of "club down" on the normal d i e t ; the unusually high incidence of c l e f t palate on the normal d i e t ; the extreme severity of most of the c l e f t s produced, and the presence of an apparent syn-drome which appeared during hatches 12 and 13. Out of a to t a l of 18 LD and DIS produced, 10 had club down and c l e f t palate, 3 had c l e f t palate only, and 1 had club down only (Table 8). No other developmental stage reached showed either t r a i t . Compared to the overall incidence of c l e f t palate for other hens producing the F 2 (1.1%, 0.8%, and 2.5% for hatches 1 to 6, 11 to 16, and 7 to 10 r e s p e c t i v e l y ) , the frequencies of 20.7%, 25.0% and 5.6% for the same periods are e n t i r e l y a t y p i c a l . The occurrence of club down prior to deficiency was also unusual, the chicks from this hen being the only ones observed. Of the LD, club-downed individuals produced during hatches 11 to 16, 3 showed further abnormalities: a l l had abnormal spinal curvatures ( l a t e r a l l y ) , withered-appearing legs, and a f l u i d - f i l l e d sac in the mid region of the spinal column (r a c h s c h i s i s ) . The down colors were noted. Two were white or l i g h t in color; 1 was black. If the char a c t e r i s t i c s of this hen (which physically was in good health) were genetic in o r i g i n , i t would be interesting to speculate how the char a c t e r i s t i c s of the progeny would be related to the c l e f t palate t r a i t . Since club down and r i b o f l a v i n deficiency and c l e f t palate appear to be related (see review of Landauer, 1952 and Lepkovsky e_t al_ . , 1938) i t was possible that this hen sporadically 67 T a b l e 8 - I n c i d e n c e of Club Down (CD), C l e f t P a l a t e (CP) and the Combination of Both i n the Progeny of Hen 549 on a R i b o f l a v i n (R) Normal ( + ) and D e f i c i e n t (-) D i e t . Hatches R Stage CD CD and CP CP Normal T o t a l Hatched 0 0 0 20 20 P i p 0 0 0 1 1 DIS 0 0 0 0 0 LD 0 3 3 2 8 ED 0 0 0 1 1 T o t a l 0 3 3 24 30 Hatched 0 0 0 15 15 P i p 0 0 0 0 0 DIS 0 0 0 0 0 LD 0 1 0 2 . 3 ED 0 0 0 1 1 T o t a l 0 1 0 18 19 Hatched 0 0 0 17 17 P i p 0 0 0 0 0 DIS 0 2 0 0 2 LD 1 4 0 0 5 ED 0 0 0 4 4 T o t a l 1 6 0 21 28 68 (the abnormal chicks were interspersed among the normals) l a i d eggs which were def i c i e n t i n r i b o f l a v i n . I t i s also possible that a l l the eggs from this hen were unusually low in r i b o f l a v i n and that the genetics of the chick determined the adequancy or inadequacy of the amount of r i b o f l a v i n present. The observations of previous workers of the v a r i a b i l i t y of r i b o f l a v i n content of eggs between hens and the observations of higher r i b o f l a v i n requirements of pigmented embryos would support t h i s hypothesis. The severity of the c l e f t s and the additional abnormalities in 3 embryos suggests that the c l e f t palate t r a i t may usually have a modifier muting i t s effect which has been lo s t in hen 549 (mutation?). It i s also possible that this i s Asmundsen's "Abnormal Upper Mandible" t r a i t which could have been present in either l i n e , and segregating out i n the F^. The frequencies of c l e f t palate on the normal diet periods, 20.7% and 25% were compatible with a single locus recessive lethal t r a i t , as i s the 100% mortality observed in these embryos. I f so, these c l e f t palates may have nothing in common with the c l e f t palate t r a i t under study. Accordingly, since i t was believed that the progeny of this bird were unusual, they were deleted from the Fg data. 3. Facial Coloboma Although some researchers have previously dealt with " c l e f t palate" and " f a c i a l coloboma" as separate e n t i t i e s , i n this study no data pertaining to f a c i a l coloboma was taken. This was based on the author's early observations of c l e f t palate wherein the f a c i a l coloboma was present only in very severe cases of c l e f t palate, as shown in Figure 2. In a l l examinations of the "420 l i n e , " NH, CP, and crosses, no case of f a c i a l coloboma was found without the presence of a very severe c l e f t palate at that side. Therefore for the c l e f t palate t r a i t there has been no evidence in t h i s study that the f a c i a l coloboma i s anything but the resu l t of an extremely severe c l e f t palate, and an inspection of the l i t e r a t u r e has given no evidence to the contrary. 4.' Palatine Grooves The character which has been named "grooves" in this study i s shown i n Figure 2. During the observation of the palates of the F-j, Fg, and BC generations, the author became increasingly aware of small d e t a i l s i n the palate morphology. An interesting character, small grooves i n the palatine ridge i n the area where c l e f t s would appear, became notable. These grooves did not extend into the surround-ing t i s s u e , and were variable i n depth to the extent that the s l i g h t e s t ones could not be d i f f e r e n t i a t e d consistently from normal. Accordingly, in order to get some idea i f this " d i f f e r e n t " t r a i t could be related to the c l e f t palate manifestation, a small study using 3 NH males with d e f i n i t e "grooves" were each mated to 3 CP (G3) females for study, as previously described. Unfortunately only 1 female from each mating produced eggs, but the resulting data showed zero cases of c l e f t palate out of 10 and 37 observations during normal diet periods, and 2 cases of c l e f t palate out of 10 observations (20%) during a r i b o f l a v i n deficiency period (Table 5). 70 Figure 2 A, normal adult male; B, embryo with f a c i a l coloboma; C, D, adult male with b i l a t e r a l severe c l e f t s and b i l a t e r a l f a c i a l coloboma; E, adult with r i g h t strong groove and l e f t c l e f t ; F, embryo with b i l a t e r a l grooves. 71 This did not demonstrate a relationship between c l e f t palate and grooves in that the most comparable study, matings of NH males with CP (G2) females, tested on r i b o f l a v i n deficiency (previously described, Table 6) produced approximately 12% c l e f t palate during r i b o f l a v i n deficiency. If the grooves were in fact a mild manifestation of the c l e f t palate t r a i t , then the frequencies of c l e f t palate reported herein would be underestimates of the phenotypic frequencies of the t r a i t . D. Abnormalities Other than C l e f t Palate 1. Curled Toe Paralysis and Club Down Curled toe paralysis was seen only twice i n a l l birds hatched in t h is experiment. Both were i n hatched chicks during a r i b o f l a v i n deficiency period: an F-j chick and an chick. This low incidence may be p a r t i a l l y due to the limited observation of hatched, standing chicks, so that only cases severe enough to be noticed when chicks were handled for palate scoring would be recorded. Club down was noted during the r i b o f l a v i n deficiency periods. The frequencies are shown in Tables 9 and 10. Club down appeared in 4 Fg generation and 6 BC generation hatched chicks, and i n 44 Fg generation, 47 BC generation, and 12 F-j LD or older embryos. About 2 weeks of maternal r i b o f l a v i n deficiency preceded the appearance of the club down condition i n the progeny, and i t persisted i n low frequencies during the f i r s t hatch a f t e r the hens were returned to the Table 9 - Incidence of Club Down (CD) in F-j, Fg and Fg by Developmental Stages and by Sires and Dams (Producing CD) on Riboflavin Normal (+) and Deficient (-) Diets. S t a 9 e Total Percent Cross Hatches Sires Dams LD DIS Pip Hatch Total Sample CD Fl x Fl * * l-6(+) 11(1) 19(1) 0 0 0 0 0 390 0.0 1 1 7 (-) 9(0) 17(0) 0 0 0 0 0 53 0.0 8 (-) 7(0) 15(0) 0 0 0 0 0 59 0.0 9 (-) 8(4) 16(7) 3 3 5 0 11 56 19.6 10 (-) 8(6) 16(11) 5* 4 7 4 20 44 45.4 11-16(+) 11(4) 19(4) 3 1 1 0 5 326 1.5 F i x F i l-6(+) 7(0) 11(0) 0 0 0 0 0 227 0.0 i I 7 (-) 7(0) 9(0) 0 0 0 0 0 46 0.0 8 (-) 5(0) 9(0) 0 0 0 0 0 38 0.0 9 (-) 5(1) 9(2) 1* 2 1 0 4 38 10.5 10 (-) 4(3) 8(4) 3 4 1 0 8 33 24.2 n-i6(+) 6(0) 10(0) 0 0 0 0 0 221 0.0 CP x NH l-2(+) 17(0) 33(0) 0 0 0 0 0 241 0.0 3 (-) 17(0) 33(0) 0 0 0 0 0 121 0.0 4 (-) 16(0) 27(0) 0 0 0 0 0 84 0.0 5 (-) 16(8) 25(10) 0 n 0 0 11 79 13.9 6 (-) 16(1) v 24(1) 0 i 0 0 1 68 1.5 7 (-) 11(0) 15(0) 0 0 0 0 0 41 0.0 8 (-) 15(0) 18(0) 0 0 0 0 0 46 0.0 9-10(+) 17(0) 28(0) 0 0 0 0 0 190 0.0 * Also had c l e f t palate. * Hen 549 removed. Table 10 - Incidence of Club Down (CD) in the Backcross Progeny by Developmental Stages and by Sires and Dams (Producing CD) on Riboflavin Normal (+) and Deficient (-) Diets. S t a g e Total Percent Cross Hatches Sires Dams LD DIS Pip Hatch Total Sample CD CP x F 1 CP x F 1 F-, x CP Fj x CP l-6(+) 19 ,0) 19 ,0) 0 0 0 0 0 361 0.0 7 (-) 161 0) 16( 0) 0 0 0 0 0 55 0.0 8 (-) 12 2) 12< 2) 0 0 0 2** 2 49 4.1 9 (-) 12 ,3) 12( 3) 7 1 0 0 8 41 19.5 10 (-) 13 [8) 13 8) 7* 3 6 1* 17 49 34.7 11-16(+) 18 '3) 18( 3) 2* 0 0 1 3 298 1.0 l-6(+) 9 0) 91 0) 0 0 0 0 0 181 0.0 7 (-) 8 [0) 8 0) 0 0 0 0 0 27 0.0 8 (-) 9 ro) 91 o) 0 0 0 0 0 37 0.0 9 (-) 8 1) 8 1) 1 0 0 0 1 31 3.2 10 (-) 8 r5) 8 5) 4** 2* • 4* 2 12 27 44.4 n-i6(+) 9 r3) 9( 3) 2* 1 0 0 3 161 1.8 l-6(+) 12 0) , 24( 0) 0 0 0 0 0 358 0.0 7 (-) 10 0) 131 0) 0 0 0 0 0 33 0.0 8 (-) 9 0) 10( 0) 0 0 0 0 0 32 0.0 9 (-) 9 '3) 10( 3) 4 1* 0 0 5 21 23.8 10 (-) 7 [2) 7 2) 2 1 0 0 3 12 25.0 11-16(+) 11 '1) 17 1) 1 1 0 0 2 171 1.2 l-6(+) 7 '0) 12( o) 0 0 0 0 0 174 0.0 7 (-) 7 [0) 9 o) 0 0 0 0 0 21 0.0 8 (-) 4 [1) 41 1) 0 1 0 0 1 11 9.1 9 (-) 2 [0) 2 o) 0 0 0 0 0 5 0.0 10 (-) 3 [0) 4 o) 0 0 0 0 0 2 0.0 11-16(+) 7 '0) 12 ro) 0 0 0 0 0 118 0.0 Also had c l e f t palate. 2 birds had c l e f t palate, normal d i e t . In this respect the incidence of club down paralleled the effect of r i b o f l a v i n deficiency on hatchability (Tables 3 to 8, Appendix). Not a l l hens produced club down. As the frequency of club down increased, the number of hens producing the t r a i t also increased. For example, in the F^, as the incidence of club down rose from 19.6% to 45.4%, the number of hens producing i t increased from 7 to 11. As f o r hatchability (Table 2) the F-j-hens differed from CP (G3) hens i n the production of club down. The F-j hens produced an average maximum of 39.6% club down; the CP b i r d s , 17.5%. This apparent difference, however, may be a r e f l e c t i o n of sample sizes for the CP b i r d s . Among the 101 club-downed F^ and BC generation embryos and chicks produced, 3 males, 8 females, and 2 unsexed, were found which also had c l e f t palate. This coincidence of the t r a i t s club down and c l e f t palate was not s t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t (p>0.05) from the expectations for independence between the t r a i t s , the Chi-square values for the F^ generation and BC generation (1 d.f.) being 0.14 and 0.19 respectively. 2. Malpositioned and Strangled Embryos Table 11 shows the frequency of malpositions in the Fg and BC generations. Although malpositions were an infrequent occurrence, a clear increase in frequency during the r i b o f l a v i n d e f i c i e n t period, from 0.4% to 2.4%, was shown by the F^ progeny, which then dropped to 0.1% when the normal ration was resumed. The BC progeny did not show this increase during r i b o f l a v i n deficiency, having a frequency of Table 11 - Mai positioned Embryos (M) of the Fg and Backcross Progeny f o r 3 Hatch Periods on Riboflavin Normal (+) and Deficient (-) Diets. Hatches l-6(+) Hatches 7-10(-) Hatches 11-16(+) Dam Dam Dam M Percent M Percent M Percent Matings Dams Embryos Mean Dams Embryos Mean Dams Embryos Mean F xF ** 18(2)* 3 0.9 17(5) 5 2.7 18(1) 1 0.2 F' xF' 11(0) 0 0.0 9(2) 4 2.1 10(0) 0 0.0 CPxF] 19(1) 1 0.2 18(3) 3 1.1 18(1) 1 0.3 CPxF] 24(0) 0 0.0 15(1) 1 0.5 17(1) 1 0.4 F^CP 9(1) 1 0.6 9(0) 0 0.0 9(0) 0 0.0 F]xCP 12(1) 2 1.1 9(1) 1 0.9 12(1) 2 1.3 Summary F2 29(2) 3 0.4 26(7) 9 2.4 28(1) 1 0.1 Backcross 64(3) 4 0.5 51(5) 4 0.6 56(3) 4 0.5 * Number of dams whoseprogeny exibited t r a i t . ** Hen 549 removed. 0.5% malpositions during both normal diet periods, and 0.6% during the r i b o f l a v i n d e f i c i e n t period. There did not appear to be any maternal effects involved. The reason for the di f f e r e n t response of and BC generations to r i b o f l a v i n deficiency in incidence of malpositions i s not known. Malpositions were considered to be any noticeably "strange" position of dead embryos of approximately 16 to 19 days' incubation. Whereas the normal position of the chick i s to be lo n g i t u d i n a l l y oriented along the long axis of the egg with the head under the right wing and the beak in the area of the a i r c e l l , "malpositioned" embryos had the head between the thighs, under one l e g , or bent back to the l e f t . The malposition frequencies during the normal diet periods was consistent with a spontaneous level reported by many researchers, the f i r s t being Sanctuary (1925). Increases i n incidence of malpositions have been reported during maternal selenium poisoning (Poley et a l . , 1937), vitamin B ^ deficiency (Olcese e_t aj_., 1950), and high levels of carbon dioxide during incubation (Romanoff, 1972). Landauer (1961) suggested that some malpositions were merely symptoms of developmental retardation or expression of the fact that the embryo died at a time when the p a r t i c u l a r position was normal. On the other hand, Romanoff (1972) considered malpositions factual and a possible cause of mortality. I t i s clear that more work i s required before the suggested response to r i b o f l a v i n deficiency can be v e r i f i e d . I f the results for the Fg generation can be v e r i f i e d , i t would apparently be the f i r s t report of a malposition response to r i b o f l a v i n deficiency. The frequencies of the "strangled" character are shown in Table 12. The frequencies of this anomaly were very low, being, Table 12 - Strangled Embryos (S) of the F2 and Backcross Progeny for 3 Hatch Periods on Riboflavin Normal (+) and Deficient (-) Diets. Hatches l-6(+) Hatches 7-10(-) Hatches 11-16(+) Dam Dam Dam M Percent M Percent M . Percent Matings Dams Embryos Mean Dams Embryos Mean Dams Embryos Mean V i 18(2)* 2 0.4 17(0) 0 0.0 18(3) 3 0.8 F' xF' 11(0) 0 0.0 9(0) 0 0.0 10(0) 0 0.0 CPxF1 19(0) 0 0.0 18(3) 4 1.4 18(0) 0 0.0 CPxFj 24(1) 1 0.2 15(0) 0 0.0 17(0) 0 0.0 F^CP 9(0) 0 0.0 9(0) 0 0.0 9(0) 0 0.0 F^xCP 12(0) 0 0.0 9(0) 0 0.0 12(0) 0 0.0 Summary F2 29(2) 2 0.2 26(0) 0 0.0 28(3) 3 0.4 Backcross 64(1) 1 <0.1 51(3) 4 0.4 56(0) 0 0.0 * Number of dams whose progeny exibited t r a i t . ** Hen 549 removed. 78 across the f i r s t normal, r i b o f l a v i n d e f i c i e n t , and second normal, dietary periods, 0.2%, 0.0%, and 0.4% f o r the generation, and less than 0.1%, 0.4%, and 0.0% for the BC generation. No dietary effect on incidence was evident. Several hens were involved: 3 producing F^ generation chicks, and 4 producing BC generation chicks. No association between the strangled condition and the malposition eff e c t was seen i n that out of 22 hens which produced either of the t r a i t s , only 2 produced both. "Strangled" embryos were usually of about 14 days' incubation, and had died because of a cons t r i c t i o n in the neck area caused by i t s entanglement in an extra-embryonic membrane. They were d i f f i c u l t to assess as to position within the egg, but i t i s possible that some form of early malposition resulted i n the positioning of the neck between membranes. No F^ generation strangled (5) or malpositioned (13) embryos showed the c l e f t palate condition. Of the BC malpositioned embryos (12), 3 of the 4 produced during the r i b o f l a v i n d e f i c i e n t period also had c l e f t palate. Of the 5 strangled BC embryos, 1, produced during the f i r s t normal diet period, also had a c l e f t palate. A Chi-square test for independence could not be run f o r the c o i n c i -dence of strangled and c l e f t palate because a zero value was obtained for the isolated strangled condition during the normal diet period. A s i g n i f i c a n t (p<0.05) Chi-square value was found (6.87, 1 d.f.) for the test of independence between malpositions and c l e f t palate during the r i b o f l a v i n d e f i c i e n t period f o r the BC generation. The dependence of the t r a i t s f o r the BC during r i b o f l a v i n deficiency i s not readily explainable, but may lend support to Landauer's view that mal-positions reflected normal positions of development, preserved by the death of the embryo. 3. The Exencephaly Syndrome During the recording of incidence of gross abnormalities in embryos and chicks examined for c l e f t palate, i t was noticed that the following group of char a c t e r i s t i c s tended to occur together in various combinations: exposure of a l l or part of the brain as a "herniated" mass on the exterior of the s k u l l (exencephaly); absence of one or both eyes (monophthalmia, anophthalmia); shortening or absence of upper beak; crossed beak. That this was indeed a syndrome was shown by comparisons of expected and observed frequencies of the t r a i t s i n combination, based on the incidence of each in the total population (Table 13). A l l possible combinations exceeded t h e i r expected frequencies by between 12 times (cross beak with shortened upper beak) and 1 b i l l i o n times ( a l l four t r a i t s together) the expected number. With such differences, tests of s t a t i s t i c a l significance seemed to be redundant. The t r a i t appeared to be the same as that reported by Ancel (1955) i n White Leghorns exposed to colchicine treatments. No mention was made i n that paper of the t r a i t s being part of a syndrome, though photographs included showed a l l of the t r a i t s except crossed beak occurring in a single embryo. As would be expected from the types of deformity, the syndrome was l e t h a l . The majority died in the LD or DIS stages with only 1 out of 54 hatching. The eventual survival of this 1 was doubtful, because the brain was exposed. 80 Table 13 - Observed and Expected Frequencies of the "Exencephaly Syndrome Tr a i t " Based on a Total Sample 'of 5169 Observations. Calculated Expected Number T r a i t * Frequency Frequency Expected Observed a 61(10"4) -32 b 67 35 c 38 20 d 25 13 ab 4(10"5) 0.3 15 ac 2 0.1 17 ad 2 0.1 9 be 3 0.2 13 bd 2 0.1' 7 cd 10 0.5 6 abc 2(10"7) 1.0(10"3) 10 abd 1 0.5 4 acd 1 0.5 6 bed 1 0.5 1 abed 2(10"1 4) 1.0(10"10) 1 a: exencephaly, b: ano (mono-) phthalmia, c: cross beak, d: shortened upper beak. The incidence of the syndrome was low, ranging between zero and approximately 4 percent (Table 14). An effect of the type of hen involved was indicated when reciprocal crosses were compared. In the o r i g i n a l l i n e crosses, the incidence of the syndrome i n the Fj from CP l i n e (G2) hens (4.08%) f a r exceeded that of the F-, from NH l i n e hens (maximum 0.23%). The frequency of the syndrome i n the F,, generation was very low, i n the F,, being 0.45%, and in the F£ being zero. However, the data were not large enough to show convincing differences, since only 2 or 3 individuals could be expected in the l a t t e r cross. Comparison of incidence among progeny from the two types of backcross hens (F-j generation or CP (G3)) showed a generally higher incidence of the syndrome in the BC from CP hens (zero to 4.08%)than from F-| generation hens (zero to 1.15%). Consideration of the frequencies from the F-j and F.J hens used in the backcross, did not indicate any differences to e x i s t between them. The higher i n c i -dence of the syndrome i n the F^ and BC generations from the CP l i n e hens than from NH or F-j type hens suggests that some dominance (with incomplete penetrance) i s associated with the syndrome and that the t r a i t i s carried at a higher frequency in the CP l i n e than in the NH l i n e . I f the "420 l i n e " can be used as an indicator of gene frequencies in the CP l i n e , the l a t t e r point has been supported by the author's observations of frequencies of 1.84% (n = 326) and 0.25% (n = 388) for the exencephaly syndrome i n the "420 l i n e " and NH l i n e respectively. There was no consistent response in the frequency of the exencephaly syndrome to the r i b o f l a v i n d e f i c i e n t diet period (Table 14). Table 14 - Population Incidence of the Exencephaly Synchrome (En) and Total Observations (0) for Each Type of Cross on a Riboflavin Normal (+) and Deficient (-) Diet. Hatches l-6(+) Hatches 7-10(-) Hatches 11-16(+) Cross En 0 (%) En 0 {%) En 0 {%) CP x NH* 0 241 (0.00) NH x CP** 20 493 (4.08) Fl X Fl 2 390 (0.51) Fi X Fl 0 227 (0.00) CP X Fl 5 361 (0.14) CP X Fl 2 181 (1.15) Fl X CP 8 358 (2.24) Fi X CP 0 174 (0.00) 1 429 (0.23) 0 190 (0.00) 1 212 (0.47) 1 326 (0.31) 0 155 (0.00) 0 221 (0.00) 1 194 (0.52) 2 298 (0.67) 0 122 (0.00) 0 161 (0.00) 2 98 (2.04) 5 171 (2.92) 2 49 (4.08) 2 118 (1.68) *Hatches l-2(+); 3-8(-); 9-10(+). Hatches 1-10(+). CO r o 83 Only 4 chicks showed c l e f t palate and the exencephaly syndrome in combination. They were distributed across a l l diet periods. One was a progeny of a NH x CP cross, and was not a d e f i n i t e c l e f t palate. The remainder were backcross progeny: 1 was a progeny of a CP x F.j cross, and 2 were progeny of x CP crosses. The low frequency of the t r a i t s in combination indicated that there was no positive relationship between the t r a i t s . A negative relationship i s possible i n that extreme cases of shortened upper beak would be e p i s t a t i c to the c l e f t palate condition. 4. Palatal P i t s During the observation of palates f o r c l e f t s , another anomaly was noted and recorded. This c h a r a c t e r i s t i c consisted of a s i n g l e , round, r e l a t i v e l y deep pock-mark-like depression on either the right or l e f t or both sides of the surface of the palate, in the area where c l e f t s were commonly observed. The anomaly sometimes occurred in the presence of a c l e f t , either on the same side or on the other side. Photographs of the character, here named "palatal p i t s , " are presented i n Figure 3. No p r i o r report of any s i m i l a r t r a i t was found in any species. However, the pits of the lower l i p in humans exhibiting Van der Woude's Syndrome are associated with c l e f t palate. This syndrome has been reported to be controlled by a single dominant gene with variable penetrance and expressivity (Ross and Johnson, 1972), and must therefore be one of the 3% "explained" frequency of c l e f t palate i n humans. Thus, the question arose as to a possible Figure 3 Palatal p i t s : A and B adults: A, r i g h t p i t with l e f t c l e f t palate; B, l e f t p i t ; and C, D, and E embryos: C and D, b i l a t e r a l p i t s ; E, l e f t p i t . 85 relationship between the clef t palate t r a i t and the palatal pi t s . Table 15 presents the incidence of "pits" in the BC. No pits were observed in the F-j generation, nor in the F^ generation. In addition, the BC from F.J females failed to show occurrence of "pits." Of the 9 hens producing BC progeny with "pits," 1 was a CP line (G3) hen mated to an F^J male; 4 were CP line (G3) hens mated to F-j males; and 4 were F-j hens mated to CP line (G3) males. A total of 20 embryos and chicks having palatal pits were observed out of a total of 2275 BC observations (0.88%). There was no evidence of any effect of the riboflavin deficient diet on the incidence of "pits" (Table 15), with 0.9%, 0.8%, and 0.7% palatal pits averaged across the f i r s t normal, riboflavin deficient, and second normal diet periods, respectively. No evidence of any effect on hatchability by pits was found. Eighteen of 20 hatched, 2 died between 8 and 12 days of incubation. Mortality could be attributed to the riboflavin deficient diet in 1 of these cases. In order to detect any possible relationship between cl e f t palate and palatal p i t s , the frequencies of c l e f t palate produced by pit-producing and non-pit producing hens were compared (Table 16). Much variation in the frequency of c l e f t palate produced was present, but no pattern indicating any relationship was discernable. A second approach to examine the possibility of a relation-ship between these traits was taken. Two of the 20 "pitted" embryos and chicks also had c l e f t palates, 1 each being produced during the f i r s t and last hatch periods. Taking the approximate average Table 15 - Incidence of Palatal Pits in the Backcross Populations for Riboflavin Normal (+) and Deficient (-) Diets. H a t c h e s Hen Number l-6( + ) •7-10(-_ \ 11-16(+) Total Cross P i t 0 P i t 0 P i t 0 P i t 0 Percent CP x F1 1 2 3 4 1 0 1 0 20 22 2 26 1 6 0 0 19 18 2 5 1 4 0 1 23 28 3 24 3 10 1 1 62 68 7 55 4.83 14.70 14.28 1.81 Mean % Per(Hen) 3.1(18) 2.4(16) 1.4(16) • CP x 0 0 0 0 0 0 0 0 0.00 Mean % Per(Hen) 0.0(8) 0.0(9) ' 0.0(9) F1 x CP 1 2 3 4 1 1 0 0 22 17 17 24 0 . 0 0 6 10 12 0 2 1 3 13 23 1 1 2 1 31 17 40 59 3.22 5.88 5.00 1.69 Mean % Per(Hen) 0.4(24) 0.0(13) 1.4(14) F^ x CP 1 0 15 1 12 0 13 1 40 2.50 Mean % Per(Hen) 0.0(12) 0.9(9) 0.0(9) Cross Mean 0.9 0.8 0.7 CO cn Table 16 - Percent Cl e f t Palate for Hens that Did and Did Not Produce Palatal Pits for Riboflavin Normal (+) and Deficient (-) Diets. Hatches l-6(+) Hatches 7-10(-) Hatches n-i6(+) Cross P i t Non P i t P i t Non P i t P i t Non P i t CP x F] 14.8 5.4 2.7 11.4 3.0 5.8 F] x CP 2.5 7.3 16.1 20.0 2.6 5.4 Fj x CP 6.7 14.1 16.7 • 5.4 0.0 14.4 Mean 8.0 8.9 11.8 12.3 1.9 8.5 00 incidence of c l e f t palate as being 6% during the normal diet periods, and 15% during the r i b o f l a v i n deficient hatch period, and taking the incidence of pits to be approximately 1%, the expected frequencies of the 2 t r a i t s , occurring simultaneously can be calculated. The expected values for the combined f i r s t and l a s t hatch periods was 1.1 individuals out of 1822 observations (compared to 2 individuals observed), and for the r i b o f l a v i n d e f i c i e n t period was 0.7 individuals out of 453 observations (compared to zero individuals observed). The differences between the expected values for independent e n t i t i e s and the observed values do not support a relationship between c l e f t palate and palatal p i t . Furthermore, they do not support any s i m i l a r i t y to the human Van der Woude's Syndrome. In a further examination of the " p i t " data (Table 17) i t was found that there was no difference in the number of males and females showing the t r a i t (9 for each sex). The sex ratios within " r i g h t , " " l e f t , " and " b i l a t e r a l " palatal pits for males and females respectively were 7:3, 2:2, and 0:4. More data would need to be coll-ected before such results could be interpreted. The source of the palatal p i t t r a i t was the "420 l i n e " (Figure 4). No " p i t s " were observed in the NH l i n e (n = 1415), while 2 palatal p i t s (0.8%) were found i n 252 observations of the hatched and DIS "420 l i n e " replacement hatch of A p r i l , 1972. Further, i t i s known that " p i t s " occur in the CP l i n e , but the frequency has not been determined. A pedigree analysis showed that a l l matings producing " p i t s " in the BC had at least 1 l i n e of descent r e l a t i n g at least Table 17 - Number of Palatal Pits for Males (M) and Females (F) and Their Palate Location. L o c a t i o n Hatch Hen Number Right Left B i l a t e r a l Total M F Total M F Total M F Total M F l-6(+) 1 1 1 1 2 1 1 1 3 1* 1 1 4 1 1 1 7-10C-) 4 1 1 1 5 2 1 3 1 1 2 2 3 3 6 ]** 11-16(+) 4 1 1 1 5 2 1 3 1 1 2 2 7 1* 1 1 8 ] ** 9 1 1 1 Total 10 6 4 9 9 Also had c l e f t palate. * Did not hatch. CO Figure 4 Partial pedigree of right ( r ) , l e f t (1) and b i l a t e r a l (b) palatal pits from backcross generation, showing l i n e of descent from one mating of c l e f t palate l i n e . A l l birds ^ represented are c l e f t palate line except those marked "nh" (New Hampshire li n e ) and o their progeny. 91 1 parent to 1 o r i g i n a l mating in the "420 l i n e " generation that pro-duced the f i r s t CP generation 3 years prior to this study. I t seems l i k e l y that the gene or genes c o n t r o l l i n g the " p i t " t r a i t are recessive i n nature (no palatal p i t s appeared in the F-j), with variable penetrance (the family incidence ranged from 1.7% to 14.7%, Table 15). I f the t r a i t i s viewed as recessive, then i t can be considered that the penetrance or gene frequency might have been increased (due to random d r i f t ) i n that the frequency of occurrence in the BC (0.9%) was s i m i l a r to that in the "420 l i n e " (0.8%) when i t would be expected to be approximately 0.6%. It i s tempting to speculate that the " p i t " t r a i t i s related i n some way to the c l e f t palate t r a i t . The genetics and physiological development of palatal pits remain an interesting area yet to be investigated. E. Sex Ratios in the C l e f t Palate Progeny i n the F,, and BC  Generations The number of males and females with c l e f t palates found in the F^ and BC generations are presented in Table 18. For the F^ progeny 12 out of 26 observations were sexed, the remainder being unsexable. Of the 12 observations, 1 hatched male, 7 hatched females, and 2 males and 2 females dead before hatch were recorded. The numbers of hatched males and females were not s i g n i f i c a n t l y d i f f e r e n t from a 50:50 r a t i o (x2 = 3.12). Table 18 - Sex Differences in the Backcross and F 2 Generations for Hatched (H) and Dead (D) Progeny for Riboflavin Normal (+) and Deficient (-) Diets. BackcrossA F 2*B Males Females Males Females Hatch H D H D Non-Sexed H D H D Non-Sexed 1-6 (+) 30 8 24 4 4 1 0 3 1 0 7-10 (-) 15 4 10 15 25 0 1 3 0 13 11-16 (+) 13 5 15 4 4 0 1 1 1 1 Sub Total 58 17 49 23 1 2 7 2 Total 75 72 33 3 9 14 Backcross sex r a t i o : Hatched; x = 0.60 (p > 0.05) 1 d.f. Backcross sex r a t i o : Dead; x2 = 0.62 (p > 0.05) 1 d.f. F 2 sex r a t i o ; x2 = 3.12 (p > 0.05) 1 d.f. Hen 549 deleted. VO 93 S i m i l a r l y , for the BC data, 147 out of 180 c l e f t palate observations were sexed, and the 58 males and 49 females hatched (x2 = 0.60) and the 17 males and 23 females dead before hatch (x2 = 0.62) were not s i g n i f i c a n t l y d i f f e r e n t from a 50:50 r a t i o . Bearing in mind the l i m i t a t i o n s of the data, in that the sex of the embryos of very early mortality was not a v a i l a b l e , i t can be concluded that there was no evidence of any difference i n frequency of cleft"palates in males compared to females. This resul t was d i f f e r e n t from those reported for humans and mice. In man, males have been reported to have a higher frequency of c l e f t palate than females. For treated mice, the reverse has been reported. F. The Effect of Cle f t Palate, Diet, and Maternal Body Weight  on Hatch Weight of Chicks Reports in the l i t e r a t u r e indicate that the weight of r i b o -f l a v i n d eficient chick embryos i s lower than normal embryos u n t i l the sixteenth day of incubation, while those chicks that hatch from r i b o -f l a v i n d e f i c i e n t eggs have normal hatch weights (Davis et a]_., 1938a; Romanoff and Bauernfeind, 1942). Considering the c l e f t palate t r a i t might be associated with a slowed growth rate during a c r i t i c a l embryonic period, which might then be reflected in hatch weight, the mean hatch weights of the BC and F^ chicks were evaluated according to t h e i r genetic o r i g i n , palate type, and dietary period. These means are presented in Tables 19 and 20. There was l i t t l e i ndication of hatch weight differences between normal and c l e f t palated chicks, the differences Table 19 - Mean Hatch Body Weight (Gms.) of C l e f t Palate (CP) and Non-Cleft Palate (N) Chicks for the F? Generation for Riboflavin Normal (+) and Deficient (-) Diets.** H a t c h e s l-6(+) 7-10(-) 11-16(-) Mean Cross CP N CP N CP N CP N CPxNH 40.3 (40.1) 38.8 NHxCP* - (42.3) 43.4 Mean (41.2) 41.1 Hatch Mean 41 (41.1) 40.4 (41.8) 39.8 (41.0) (43.5) - (44.2) - (43.3) (42.3) (43.0) (42.2) 7 No c l e f t palate chicks in hatches 1-6 or 11-16. Hen 549 not deleted. Table 20 Mean Hatch Body Weight (Gms.) of Cleft Palate (CP) and Non-Cleft Palate (N) Chicks for the Backcross Generation for Riboflavin Normal (+) and Deficient (-) Diets. H a t c h e s l-6(+) 7-10(-) 11-16(+) Cross CP N CP N CP N CPxF] 39.4 (39.8) 40.8 (40.6) 42.5 (41.3) 40.9 (40.5) 40.7 CPxF^ 40.0 (40.0) 42.9 (41.2) 40.8 (41.4) 41.2 (40.9) 41.0 F^CP 33.5 (35.3) 35.3 (35.8) 39.7 (36.6) 36.1 (35.9) 36.0 F^xCP 36.8 (35.4) 32.8 (35.0) 39.2 (37.1) 36.3 (35.8) 36.0 Mean 37.4 (37.6) 38.0 (38.1) 40.5 (39.1) 38.6 (38.3) Hatch Mean 37. .5 38. .0 39. .8 Mean CP N Line Mean 96 between normal and c l e f t palated chicks, the differences of 0.3 grams for BC chicks being non-significant (p>0.05). Small sample sizes for c l e f t palated chicks voided s t a t i s t i c a l comparisons, but compari-sons between normal and c l e f t palated chicks from each cross suggested no differences (for example, 41.0 and 39.8 grams for normal and c l e f t palated F 2 chicks r e s p e c t i v e l y ) . Mean hatch weights for BC and normal F^ chicks (Tables 19 and 20) indicated a difference of approximately 1 gram between each period in order of time. For the F^ chicks (41.2, 42.3, and 43.0 grams) the differences were s i g n i f i c a n t (p<0.05) (Table 21). BC chicks using a l l data (37.6, 38.1, and 39.1 grams) also showed s i g n i f i c a n t (p<0.05) differences. The difference was found to l i e between the f i r s t and l a s t dietary period, but not between these 2 normal periods and the central r i b o f l a v i n d e f i c i e n t period. This indicated that the difference was a r e f l e c t i o n of time. The egg size of the study was l i k e l y to be increasing over time and the hatch weight would r e f l e c t that increase. The correlation between egg size and chick weight i s well known (Phalaraksh, 1972). Inasmuch as r i b o f l a v i n deficiency was not found to reduce the hatch weight of surviving chicks, these results were i n agreement with those of Davis et al_. (1938a), who explained the disappearance of the reduced r i b o f l a v i n d e f i c i e n t embryo weight by the death of those affected embryos before hatch. A difference of s t a t i s t i c a l significance (p<0.05) was found in the hatch weight means for the F 2 generation chicks from the reciprocal crosses (41.0 and 43.3 for F, and F, respectively. The 97 Table 21 - Analysis of Variance of Mean Hatch Body Weight (Gms.) of Non-Cleft Palate Chicks of the F 2 Generation for Riboflavin Normal (+) and Deficient (-) Diets. Source of Degrees of Sums of Var i a t i on Freedom Squares F Matings 1 8.16 816* Ration 2 3.29 165* l-6(+); 11-16(+) vs 1 7-10(-) 0. .05 5 l-6(+) vs 11-16(+) 1 3. .24 324* Error 2 0.02 Total 5 11.47 98 reasons for this difference are not apparent since they are the reverse of what might be expected based on hen body weight influences on chick body weight (Table 22). Means for hatch weights of the four types of BC progeny indicated that CP l i n e hens produced chicks approximately 4 grams l i g h t e r than those from F-j generation hens. As shown in Table 23, this difference was s i g n i f i c a n t (p<0.05). The difference i n BC chick hatch weight means from d i f f e r e n t dam types was probably a r e f l e c t i o n of the 300 to 600 gram difference between the r e l a t i v e l y small CP l i n e hens and the larger F-j generation hens. In general, smaller birds tend to lay smaller eggs, and this i s also true of the CP l i n e . When the mean body weights of the backcross hens were ranked and compared to the ranked mean body weights of the BC chicks (Table 22) a very good agreement between r e l a t i v e hen size and chick size was evident. It was inte r e s t i n g to note that for the BC- and Fg- produc-ing crosses within the 16 CP l i n e (G3) and 9F^ hens that went out of production during the r i b o f l a v i n d e f i c i e n t diet period, there was no s i g n i f i c a n t correlation between the number of days (average 5.6 and 6.0) from the s t a r t of the diet to the l a s t day of production and the body weight of the hens at the s t a r t of the deficient diet period (r = 0.05; r = -0.61). S i m i l a r l y the body weight and number of eggs to the l a s t egg l a i d were not s i g n i f i c a n t l y related, the average f o r the CP and F1 hens being 3.4 and 3.9 respectively (r = 0.04; r = -0.53). Of the 10 CP hens and 31 F-j hens which showed a marked drop i n hatchability during the r i b o f l a v i n d e f i c i e n t diet period, 99 Table 22 - Mean Body Weight (Gms.) of the F-|, ?\ and C l e f t Palate (CP) Dams and the Mean Hatch Weight of Their Respective F 0, F i and Backcross Progeny. Si re Dam Dam Chick Weight* Rank Weight Rank Fl 2262 1 40.4 2 F i F i 2244 2 43.3 1 CP Fl 2313 1 40.7 2 CP Fl 2078 2 41.0 1 F l CP 1790 3 36.0 3.5 F l CP 1737 4 36.0 3.5 * Dam weight taken at hatch 7. 100 Table 23 - Analysis of Variance of Mean Hatch Body Weight (Gms.) of C l e f t Palate and Non-Cleft Palate Chicks for the Backcross Generation for Riboflavin Normal (+) and Deficient (-) Diets. Source of Variation Degrees of Freedom Sums of Squares F Matings (M) 3 140.0 27.0* CPxFpF^CP vs CPxFj;F]xCP 1 0. .2 0.1 CPxF-j vs F^CP 1 66. ,0 37.9* CPxF] vs F^xCP 1 74. .8 43.0* Rations (R) 2 23.5 6.8* l-6(+);ll-16(+) vs 7-10(-) 1 2, .0 1.2 l-6( + ) vs. 11-16( + ) 1 21. .4 12.3* Palate Type (P) 1 0.7 0.4 M x R 6 19.2 1.8 M x P 3 < 0.1 <0.1 R x P 2 3.6 1.0 Error 6 10.4 Total 23 198.4 * S i g n i f i c a n t (p < 0.05). there was no s i g n i f i c a n t relationship within groups between the number of days to the second to l a s t hatched egg (average 10.1 and 17.2) and the body weight of the hens at the s t a r t of the deficiency period (r = -0.11; r = 0.08). S i m i l a r l y , the body weight and number of eggs to the second l a s t egg hatched were not s i g n i f i c a n t l y r e l a t e d , the average number of eggs being 6.5 and 12.2 for CP and hens respectively (r = -0.27; -0.04). Comparison of the mean body weights of these hatchabi1ity--or production--losing hens to the mean body weights of a l l hens of th e i r type used in the study indicated no pattern for those which lo s t h a t c h a b i l i t y , the F-j generation hens (2181 grams) being 15 grams l i g h t e r than the to t a l F-j hen group (2196 grams) and the CP hens (1784 grams) being 20 grams heavier than the whole CP hen group (1764 grams). On the other hand, the hens which lost production showed more s t r i k i n g differences from other hens of th e i r type. The F-j generation hens which stopped production (2437 grams) were 241 grams heavier then the total F^ hen group mean (2196 grams) while the CP hens which stopped production (1712 grams were 54 grams l i g h t e r than the average CP hen (1764 grams) i n the study. These results suggest that the response i n loss of hatch-a b i l i t y during the r i b o f l a v i n d e f i c i e n t period was probably not related to body weight within dam types. The response of stopping production (often accompanied by a molt) did seem to be related to body weight, the heavier hens and the l i g h t e r CP hens being more l i k e l y to respond in this way. However, the response was apparently almost immediate and uniform, and, once i n i t i a t e d , unrelated to body 102 weight. One i s l e f t to wonder what l i g h t CP hens and heavy F-| genera-tion hens have i n common which causes them to respond to a new diet (mash replacing crumbles) or to r i b o f l a v i n deficiency in this s i m i l a r manner. G. L i v e a b i l i t y o f Embryos Related to the Severity of C l e f t  Palate in the F,, and BC Generations During the c o l l e c t i n g of data i t was observed that there was an apparent tendency for hatched chicks to have less severe c l e f t palates than dead embryos. The c l e f t palate scores were examined on 2 bases: using as data the sum of right and l e f t scores within i n d i v i d u a l s ; and using only the score of the most severe side for each i n d i v i d u a l . Using both sets of data for the c l e f t palated chicks, pooled with respect to the or i g i n a l reciprocal crosses, analyses were undertaken to f i n d i f the mean severity of c l e f t s differed between the hatch periods, between hatched and non-hatching embryos, and among the reciprocal backcrosses and Fg generation. The mean "sum side" scores for the three diet periods and the 3 groups of progeny are presented in Table 24. The "strong side" scores for these same diet periods and groups are presented i n Table 25. The pattern of means for both systems was generally s i m i l a r . The mean severity of c l e f t s was greater in the non-hatching progeny than in hatched; being, respectively, 5.3 and 2.2 for the "sum side" scores and 3.3 and 1.7 for the "strong side" scores. These differences were both s i g n i f i c a n t (p<0.05), as shown in Tables 26 and 27. For Table 24 - Mean Sum of Right and Left Palate Scores of Hatched and Non-Hatched ( ) Chicks for the Backcrosses Pooled for Hen Genotype (CP x F-j and F-j x CP) and for the Pooled F 2 for Riboflavin Normal (+) and Deficient (-) Diets. -H a t c h e s Pooled Groups l-6(+) 7-10(-) n-i6(+) Mean Line Mean CP x F] 2.4 (6.2) 2.7 (5.1) 2.4 (4.0) 2.5 (5.1) 3.8 F1 x CP 2.9 (5.5) 3.4 (5.0) 2.1 (5.0) 2.8 (5.2) 4.0 F2 2.0 (7.0) 1.3 (4.5) 1.0 (5.1) 1.4 (5.5) 3.4 Mean 2.4 (6.2) 2.5 (4.9) 1.8 (4.7) 2.2 (5.3) Hatch Mean 4.3 3.7 3.2 o co Table 25 - Mean Strong Side Palate Scores for Hatched and Non-Hatched ( ) Chicks for the Backcrosses Pooled for Hen Genotype (CP x F-| and FT x CP) and for the Pooled F 2 for Riboflavin Normal (+) and Deficient (-) Diets. Pooled Groups H a t c h e s l-6(+) 7-10(-) 11-16(+) Mean Line Mean CP x F 1 1.9 ( 4 . 6 ) 2.1 (3.1) 2.0 (2.7) 2.0 ( 3 . 5 ) 2 . 8 F 1 x CP 2.1 (3.0) 2 . 3 ( 2 . 8 ) 1 .8 ( 3 . 3 ) 2.7 (3.0) 2 . 8 F 2 1 .2 (3.7) 1 .3 ( 2 . 5 ) 1.0 ( 3 . 6 ) 1 .2 ( 3 . 3 ) 2 . 2 Mean 1.7 ( 3 . 8 ) 1.9 ( 2 . 8 ) 1.6 ( 3 . 2 ) 1.7 ( 3 . 3 ) Hatch Mean 2 . 8 2 . 4 2 . 4 105 Table 26 - Analysis of Variance of Mean Sum of Right and Left Palate Scores of Hatched and Non-Hatched Chicks for the Backcrosses Pooled for Hen Genotype (CP x F] and F-j x CP) and for the Pooled F 2 for Riboflavin Normal and Deficient Diets. Source of Variation Degrees of Freedom Sums of Squares F Hatch (H) 1 41.10 166.42* Genotype (G) 2 0.77 1.56 Ration (R) 2 3.48 7.05* G x R 4 1.44 1.46 G x H 2 2.65 5.38 R x H 2 1.52 3.09 Error 4 0.99 Total 17 51.96 Si g n i f i c a n t (p <_ 0.05). 106 Table 27 - Analysis of Variance of Mean "Strong Side" Palate Scores of Hatched and Non-Hatched Chicks for the Backcrosses Pooled for Hen Genotype, (CP x F-j and F-| x CP) and for the Pooled F 2 for Riboflavin Normal and Deficient Diets. Source of Variation Degrees of Freedom Sums of Squares F Hatch (H) 1 10.28 39.37* Genotype (G) 2 0.823 1.58 Ration (R) 2 0.570 1.09 G x R 4 0.617 0.59 G x H 2 0.968 1.85 R x H 2 0.981 s 1.88 Error 4 1.045 Total 17 15.280 S i g n i f i c a n t (p < 0.05). 107 the "sum side" scores no s i g n i f i c a n t differences were found between the means of the BC from generation hens (3.8), BC from CP hens (4.0) and the F^ generation (3.5). The s i m i l a r means for "strong side" scores (2.8, 2.6 and 2.2) also showed no s i g n i f i c a n t differences. The 2 scoring systems did d i f f e r in the measurement of effects of dietary periods on mean scores. The "strong side" data showed no s i g n i f i c a n t differences between the means for the f i r s t normal (2.8), r i b o f l a v i n d e f i c i e n t (2.4), and second normal (2.4) diet periods. The "sum side" data did show s i g n i f i c a n t differences between these three diet periods (4.3, 3.7 and 3.2 respectively). . Neither scoring system gave s i g n i f i c a n t interactions between the main e f f e c t s . The s i g n i f i c a n t e f f e c t of dietary period on "sum score" means did not appear to be an effect of r i b o f l a v i n deficiency, since the score values steadily declined from the f i r s t to l a s t dietary period. I t would rather seem to be an effect of time ( i . e . age of b i r d ; increased egg size) or a gradual change i n scoring, despite the attempted standarization of the system. The difference between the "sum side" and "strong side" systems in the finding of this change in score across time i s not readily explainable, but does show that the 2 systems are not equivalent. The s i g n i f i c a n t l y higher means for either "sum side" or "strong side" score data in non-hatching embryos compared to hatched chicks indicates that the more severe c l e f t palated individuals tend to die before hatch. In other words, the c l e f t palate condition appears to be semi-lethal t r a i t . There i s natural selection against 108 higher levels of e x p r e s s i v i t y . The lack of s i g n i f i c a n t difference between mean scores embryos of the F^ generation and BC i s interesting because i t suggests that the severity of the c l e f t was not related to i t s presence or absence. I f the t r a i t were s t r i c t l y a d d itive, the mean severity of the scores would be expected to be less than that of the BC. This adds weight to the p o s s i b i l i t y that the expressivity of c l e f t palate i s under the control of modifiers independent of the penetrance of the t r a i t . H. Embryonic Mortality of the F,, and BC Generations Associated  with C l e f t Palate It has been shown that embryos of the F^ generation and BC having severe c l e f t palates tended to die (Tables 24 and 25). This implied that c l e f t palate per se might be viewed as being p a r t i a l l y l e t h a l . Another approach was taken to demonstrate t h i s l e t h a l i t y : an estimate of the percent c l e f t palate for each mortality period and for hatched chicks was calculated (Tables 28 and 29). This consisted of the average percent c l e f t palate within a mortality period averaged by hatches within a diet period for each genotype. Since the values of percent c l e f t palate for each mortality period and hatch would be expected to be the same i f c l e f t palate and embryonic mortality were unrelated, the values calculated were an index of the relationship between c l e f t - p a l a t e and embryonic mortality. The number of c l e f t palate observations for the F^ generation on the normal diet was limited (9). For the l a s t diet Table 28 - Cl e f t Palate as Percent of Hatched and Dead F ? Generation Progeny, Across Maternal Riboflavin (R) Normal (+) and Deficient (-) Diets. S t a g e Hatch (R) Mating LD" LD+ DIS Pip Hatch 1-6 (+) F l x F l 3.3 0.0 0.0 0.0 1.1 Flx Fl 0.0 0.0 0.0 0.0 0.0 Mean 1.6 0.0 0.0 0.0 0.6 7-10 (-) F lx Fl 5.7 0.0 0.0 0.0 1.2 Flx Fl 19.7 14.7 0.0 0.0 0.6 Mean 12.7 7.4 0.0 0.0 0.9 11-16 (+) F lx Fl 0.0 2.2 0.0 8.3 0.3 Flx Fl 2.8 0.0 0.0 0.0 0.0 Mean 1.4 1.1 0.0 4.2 0.2 o vo Table 29 - C l e f t Palate as Percent of Hatched and Dead Backcross Progeny Across Maternal Riboflavin (R) Normal (+) and Deficient (-) Diets. S t a g e  Hatch (R) Mating LD" LD+ DIS Pip Hatch 1-6 (+) CPxF1 CPxF] F^CP F]xCP Mean 0.0 0.0 11.7 11.1 5.7 38.9 0.0 22.8 5.6 16.8 25.0 0.0 0.0 12.5 9.6 0.0 0.0 12.5 0.0 3.1 5.4 6.5 3.9 8.3 6.2 7-10 (-) CPxF1 CPxF] F^CP F]xCP Mean 11.1 26.2 22.0 15.0 18.6 20.0 21.5 2.8 8.3 13.2 18.8 50.0 16.7 0.0 21.4 12.5 31.3 25.0 0.0 17.2 7.7 4.7 10.0 3.4 6.4 11-16 (+) CPxF] CPxF] F^CP F]xCP Mean 3.1 16.7 8.4 6.7 8.6 3.1 16.7 0.0 0.0 5.0 10.0 33.3 26.7 37.5 26.9 0.0 0.0 0.0 0.0 0.0 4.9 5.1 2.3 5.8 4.5 period (Table 28) the percent c l e f t palate during a l l mortality periods except DIS was greater than that for hatch. The difference was accentuated during r i b o f l a v i n deficiency where on the average 12.7% c l e f t palate was found i n the LD- and 7.4% in the LD+, compared to 0.9% in hatched chicks. S i m i l a r l y , for the BC generation, during the f i r s t normal diet period averages of 16.8% and 9.6% c l e f t palate were found in the LD+ and DIS embryos compared to 6.2% in hatched chicks. During the second normal diet period, where some residual effects of r i b o f l a v i n deficiency might be considered to have been present, based on h a t c h a b i l i t y data, (Table 29) 8.6% and 26.9% c l e f t palate were observed i n the LD- and DIS embryos compared to 4.5% in hatched chicks. During the r i b o f l a v i n d e f i c i e n t period, average values of 18.6%, 13.2%, 21.4%, and 17.2% c l e f t palate were found in the LD-, LD+, DIS, and Pip categories compared to 6.4% in the hatched category. During normal diet periods, the majority of chicks hatch, therefore the sample sizes from which the percent c l e f t palates were calculated for mortality periods were generally small, and that the large v a r i a b i l i t y seen in the percent c l e f t palate values calculated for each mating type (Tables 28 and 29) would be expected. Despite this v a r i a b i l i t y , there was a tendency in both the BC and F,, generations for the percent c l e f t palate to be greater in dead embryos than hatched ones, although no p a r t i c u l a r mortality period seemed to be consistently responsible. While the average percent c l e f t palate i n hatched BC and F 9 generations changed very l i t t l e during r i b o f l a v i n deficiency (from 112 6.2% and 4.5% to 6.4%, and from 0.6% and 0.2% t o 0.9%) the percent c l e f t palate in mortality periods increased greatly. In the generation the increase was seen i n the LD- and LD+ periods which rose from less than 2% to greater than 7%. In the BC, a l l embryonic mortality periods exceeded 13% c l e f t palate, and a l l were greater than the averaged respective values for the normal diet periods. The following conclusions can be drawn from these r e s u l t s : there was a lethal effect of the c l e f t palate condition during both normal and r i b o f l a v i n d e f i c i e n t diet periods; clefted progeny tended to be more affected ( i . e . died) by the r i b o f l a v i n d e f i c i e n t diet than normals, as evidenced by the increased percent c l e f t palate i n mortality categories. However, i t must be noted that the lethal effects of r i b o f l a v i n deficiency, the let h a l effects of the c l e f t palate condition, and the increased incidence of c l e f t palate during r i b o f l a v i n deficiency are confounded. A further demonstration of the lethal effect of the c l e f t palate condition i s the comparison of mean hen frequencies of c l e f t palate (Table 7) and mean frequencies of c l e f t palate i n hatched chicks (Tables 28 and 29). For a l l 3 diet periods, for both the F^ generation and the BC, the mean frequencies of c l e f t palate i n hatched chicks was less than that calculated for a l l progeny (0.6%, 0.9% and 0.2% versus 0.8%, 4.4% and 0.6%; 6.2%, 6.4% and 4.5% versus 8.4%, 12.4% and 7.5%). 113 I. The Frequency of Different Types of Clefts Observed in the Fg Generation and BC and the Response of the Frequency of  Each Type to Riboflavin Deficiency In addition to the parameter of "severity" as measured by "score," ( i . e . , the width of the c l e f t ) , the c l e f t palate anomaly was observed to show variation i n l a t e r a l i t y . That i s , c l e f t s could be found only on the r i g h t , only on the l e f t , on both sides with equal s e v e r i t y , or on both sides with either the right or l e f t more severe than the remaining side. The entire spectrum of "severity" was found within each of these types (Figures 5, 6 and 7). Since i t was demonstrated that the incidence of c l e f t palate increased during the r i b o f l a v i n deficiency period, and that the degree of severity was not influenced by the deficiency, the question arose as to whether the response in c l e f t palate frequency to the r i b o f l a v i n d e f i c i e n t diet was due to an increase of a l l types or only some s p e c i f i c l a t e r a l i t y types of c l e f t . I t was further questioned that the higher mortality of more severelyclefted individuals shown by either scoring method could be p a r t i a l l y due to d i f f e r e n t i a l mortality and severity of p a r t i c u l a r l a t e r a l i t y types. Table 30 presents for the pooled F^ generation and the BC pooled by dam genotype for the 3 diet periods, the percent hatched and non-hatched c l e f t palate types. Table 31 presents the averaged values from the 2 pooled BC. The averaged values for the normal diet periods (Table 30) summed within the b i l a t e r a l categories and within the u n i l a t e r a l categories were calculated for the generation, and showed that the b i l a t e r a l types (0.66%) were s l i g h t l y more frequent than the Figure 5 Palates of 8 to 12 day dead embryos: A, normal; B, mild l e f t c l e f t ; C, D, E, F, b i l a t e r a l c l e f t s in order of increasing severity. Figure 6 Palates of 8 to 12 day dead embryos: A, normal; B, severe l e f t c l e f t ; C, D, severe b i l a t e r a l c l e f t s ; E, very severe b i l a t e r a l c l e f t with olfactory lobes (?) showing. (U) Figure 7 Adult birds with c l e f t palate: A, normal; B, s l i g h t l e f t c l e f t ; C, b i l a t e r a l c l e f t stronger on r i g h t ; D, E, b i l a t e r a l c l e f t very s l i g h t on one side; F, u n i l a t e r a l l e f t c l e f t ; G, equal b i l a t e r a l c l e f t . Table 30 - Percent Hatched and Non-Hatched Cl e f t Palate Types for the Pooled F 2 and Backcross Pooled by Dam Genotype for Riboflavin Normal (+) and Deficient (-) Diets. P a l a t e T y p e Matings Equal Strong Strong (Pooled) Hatches Progeny Right Left B i l a t e r a l Right Left CPx(F1) 1-6 (+) 543 1 .29(0.00)* 2.21(0.37) 0.74(0.00 0.74(0.37) 0.37(0.18) 7-10 (-) 316 1.27(0.63) 3.16(2.22) 0.63(3.48) 0.00(0.95) 0.63(1.27) 11-16 (+) 459 1.31(0.44) 1.53(0.22) 0.65(0.65) 0.22(0.00) 0.44(0.00) (F-,)xCP 1-6 (+) 532 1.13(0.19) 1.88(0.19) 0.75(1.13) 0.19(0.56) 0.75(0.00) 7-10 (-) 137 0.73(2.19) 3.65(1.46) 0.73(5.84) 0.00(0.73) 0.00(2.19) 11-16 (+) 289 1.38(0.35) 1.04(1.04) 0.35(0.69) 0.00(0.35) 0.35(0.00) (F 1)x(F ]) 1-6 (+) 646 0.15(0.00) 0.00(0.00) 0.31(0.15) 0.00(0.00) 0.15(0.00) 7-10 (-) 386 0.78(0.00) 0.00(0.52) 0.00(2.33) 0.00(0.00) 0.00(0.78) 11-16 (+) 571 0.18(0.18) 0.00(0.70) 0.00(0.35) 0.00(0.18) 0.00(0.18) Hatched (Non-Hatched). Table 31 - Average Percent Hatch and Non-Hatched C l e f t Palate Types for the Backcross Data of Table 30. P a l a t e T y p e  Equal Strong Strong Hatch Right Left B i l a t e r a l Right Left 1 - 6 (+) 1.21(0.10) 2.04(0.28) 0.74(0.56) 0.46(0.46) 0.56(0.09) 7 - 10 (-) 1.00(1.41) 2.40(1.84) 0.68(4.66) 0.00(0.84) 0.32(1.73) 11 - 16 (+) 1.34(0.40) 1.28(0.63) 0.50(0.67) 0.11(0.18) 0.40(0.00) 119 u n i l a t e r a l types (0.60%). During the r i b o f l a v i n d e f i c i e n t period these values were 3.11% and 1.30%, respectively. The s i m i l a r calcu-cations f o r the normal diet periods for the BC (Table 31) showed a predominance of u n i l a t e r a l types (3.64%) compared with b i l a t e r a l types (2.36%). During the r i b o f l a v i n d e f i c i e n t diet period these values were 7.65% and 8.23%, respectively. While b i l a t e r a l types predominated in both diet regimes in the generation, the same trend was not found in the BC where un i l a t e r a l types predominated during the pooled normal diet periods, and b i l a t e r a l s i n the r i b o f l a v i n d e f i c i e n t period. These differences do not appear to be meaningful when viewed within diet periods, but i f r e l a t i v e change of frequency of types i s considered, the 2 f o l d increase of u n i l a t e r a l types' compared with the 3.5 f o l d increase of b i l a t e r a l types in the BC during r i b o f l a v i n deficiency would seem to warrant further i n v e s t i g a t i o n . Based oh the r a t i o of the frequency of each palate type during the deficient period to i t s frequency during the f i r s t normal period, i t was observed (Table 32) that for the BC, the equal b i l a t e r a l type showed the greatest change (4.11). The second greatest change was shown by the strong l e f t b i l a t e r a l type (3.15). The highest u n i l a t e r a l value was that of the l e f t (2.26). Thus i t can be inferred that there was a change in the r e l a t i v e proportions of types, due to diet (the frequencies having dropped to near t h e i r former levels when replaced on the normal d i e t ) . Due to limited sample size the comparable figures f o r the are d i f f i c u l t to i n t e r p r e t , but the values for equal b i l a t e r a l and strong l e f t b i l a t e r a l were 5.06 and 5.20 respectively, indicating a high rate of change. The right u n i l a t e r a l also had a value of 5.20, which did not seem to be comparable to the BC data. Table 32 Relative Change in Percent Cleft Palate Frequencies of .Hatches 7-10 and 11-16 Compared to Hatches 1-6 for Each Palate Type for the Average Backcross and F 0 Data of Tables 30 and 31. P a l a t e T y p e Pooled Matings Hatches Total Progeny Right Left Equal . Bi l a t e r a l Strong Right Strong Left Backcross 1-6 (+)* 1075 1.31 2.32 1.30 7-10 (-) 453 2.41(1.84)** 5.24(2.26) 5.34(4.11) 11-16 (+) 748 1.74(1.33) 1.91(0.82) 1.17(0.90) 0.92 0.84(0.91) 0.29(0.32) 0.65 2.05(3.15) 0.40(0.62) 1-6 (+) 7-10 (-) 11-16 (+) 646 386 571 0.15 0.78(5.20) 0.36(2.40) 0.00 0.52(-) 0.70(-) 0.46 2.33(5.06) 0.35(0.76) 0.00 O.OO(-) O.OO(-) 0.15 0.78(5.20) 0.18(1.20) ** Riboflavin Normal (+) and Deficient (-) Diets, Relative change. o 121 I t has been previously reported (Tables 24 and 25) that the tendency for embryos to die before hatch was increased with increased severity of the c l e f t , measured by either of the "strong side" or "sum side" systems. From the data showing d i s t r i b u t i o n of mortality among types of c l e f t (Table 33) i t can be seen that for the BC there was higher embryonic mortality during both diet regimes in the b i l a t e r a l types. With the exception of the strong l e f t during the second normal diet period, the mortality ranged from 14 to 100%. The s i m i l a r range for the u n i l a t e r a l types was 8 to 59%. Again, the l i m i t a t i o n of the Fg generation data gave no consistent i n t e r p r e t a t i o n . I t was interesting to note the r e l a t i v e simultaneous changes, due to d i e t , of frequency and mortality within a c l e f t palate type of the BC (Table 30). It was seen that for a l l categories except unil a t e r a l l e f t , there was no change i n the frequency of the hatched category, leaving the entire observed increase in c l e f t palate to increased mortality for each category. I t i s interesting to speculate that the c l e f t palated chicks hatching during normal and d e f i c i e n t diet regimes are the same phenomenon, while the increased frequency of the c l e f t palate anomaly i s due to the appearance of a new class of observations d i f f e r e n t from the former i n some way which causes t h e i r mortality (in addition to the r i b o f l a v i n deficiency). J . Multiple Regression Models An attempt to relate the "severity" of the parent palate c l e f t s to the frequency of c l e f t palate i n t h e i r progeny was performed using multiple regression techniques. Table 33 - Percent Mortality of Total Percent of C l e f t Palate Type for Combined Backcross Means of Table 31 and the F? of Table 30 with Riboflavin Normal (+) and Deficient (-) Diets. Palate Type Combined Equal Strong Strong Progeny Hatch Right Left B i l a t e r a l Right Left Backcross 1-6 (+) 7.63 12.07 40.00 50.00 13.85 7-10 (-) 58.51 35.11 87.26 1.00.00 84.39 11-16 (+) 29.85 32.98 57.26 62.07 0.00 F 2 1-6 (+) 0.00 - 32.61 , - 0.00 7-10 (-) 0.00 100.00 100.00 - 100.00 11-16 (+) 50.00 100.00 100.00 100.00 100.00 123 The "severity" of the c l e f t s was measured by either the sum of the scores of each side or the score of the most severe side. A l l models were run for each type of data. The multiple regression models assumed were: Y = a + b ]X 1 + b 2X 2 , Y = a + b ^2 + b 2X 22 , and Y = a + b-jlog X-j + b 2log X2 where Y = percent c l e f t palate progeny per hen per diet period, a, bj , and b 2 = constants, X-j = "severity" of o r i g i n a l parent c l e f t , and X2 = "severity" of backcross parent c l e f t . 2 Table 34 shows the R ( c o e f f i c i e n t s of determination) for the percent c l e f t palate per dam related to the "severity" of the c l e f t e d parent scores for the 3 diet periods. In general, the severity of the clefted parental scores as measured by either method were poor predictors of the incidence of 2 c l e f t palate i n t h e i r BC progeny. The R values for the 3 models were low and generally less than 30%. Significance was found occasionally, but i t was not consistent across the backcross matings. This may be due to sampling error (considering that sample sizes for this data ranged from 9 to 25 observations). Not enough data was available for Table 34 - Coefficients of Determination (R xlOO) for Percent C l e f t Palate Progeny Per Dam(Y) Related to Cle f t Palate Severity of the C l e f t Palate Line F] Parents (Xi) and the Cle f t Palate Line Backcross Parents (X 2) for Riboflavin Normal (+) and Deficient (-) Diets for 3 Multiple Regression Models (See Note Below). Hatches 1-6 ( + ) Hatches 7-10 ( -) ' Hatches 11-16 ( +) Mating N 1 2 3 N 1 2 3 N 1 2 3 CPxF] 19 6 6 7 18 12 11 12 18 4 2 6 CPxF' F]XCP 9 25 12 37* 12 43* 11 29* 9 16 20 32A 22 34A 17 30A 9 19 25 1 28 1 21 1 F^xCP 12 8 8 8 9 20 17 23 11 54* 56* 51A V_ 21 1!_ H 21 11 V_ 21 3_1 CPxF1 19 2 1 5 18 8 8 8 18 6 4 8 CPxF^ 9 6 7 6 9 16 17 15 9 16 18 15 F^CP 25 14 15 12 16 . 7 . 6 7 19 3 3 4 F^xCP 12 6 6 6 9 28 33 37 11 56* 58* 52* * , Sig n i f i c a n t (p < 0.05.) Approaching significance (0.10 > p palate scores. > 0.05) Models 1, 2 and 3 : X, and X2 = Sum of sides of c l e f t Note: Model 1, Y = a+b] x 1 +b 2 X2; Model 2, Y = a+b.,X f+bJ ; Model 3, Y = a+b, logX-j+bplogX 2' For Models T , 2' , and 3' : X1 and X2 = strong side score. 125 the Fg progeny to make possible s i m i l a r t e s t s . The second group of multiple regression models used to attempt to relate parental c l e f t severity to incidence of c l e f t palate in the progeny (Table 35) used the score of each side of each parent as the independent variables, and the percent c l e f t palate within hens and hatch periods as the dependent variable. That i s : Y = a + b]X1 + b 2X 2 + b 3X 3 + b 3X 3 + b 4X 4 , Y = a + b ^2 + b 2X 22 + b 3X 32 + b ^2 Y = a + bjlog X] + bglog X2 + b 3log X3 + b 4log X4 Y = percent c l e f t palate progeny per hen per diet period, a,b-|, b 2 > b 3 > b4. = constants, X-j = o r i g i n a l c l e f ted parent right side score, X2 = o r i g i n a l c l e f t e d parent l e f t side score, X3 = backcross c l e f t e d parent right side score, X4 = backcross c l e f t e d parent l e f t side score. This system of using a l l 4 c l e f t sides as predictors gave results i n -dicating that i t was a better predictor than the "sum side" or "strong side" systems, of incidence of c l e f t palate in the BC progeny. Although significance was rare, the R values were generally greater than 30%. No consistent advantage was found i n using the square of log-jQ or the independent variables. and where and Table 35 - Coefficients of Determination (R xlOO) for Percent Cleft-Palate Progeny Per Dam (Y) Related to Cleft Palate Scores of the C l e f t Palate Line F, Parents (Right Side: X], Left Side: X2) and the Cleft Palate Line Backcross Parents (Right Side: X3, Left Side: X4) for Riboflavin Normal (+) and Deficient (-) Diets for 3 Multiple Regression Models (See Note Below). Hatches 1-6 (+) Hatches 7-10 (-) Hatches 11-16 (+) Mating N 1 2 3 N 1 2 3 N 1 2 3 CPxF] 19 34 34 28 18 18 19 16 16 5 3 10 CPxF] 9 24 24 25 9 32 34 31 9 45 45 44 F^CP 25 44* 51* 39* 16 • 32 30 37 19 18 20 14 F]xCP 12 31 29 32 9 64 65 61 11 . 55 59 49 * S i g n i f i c a n t (p Note: Model 1, Y < 0.05). = a + b-j X l + b2x2 + b3x3 + B 4 X 4 2, Y 3, Y = a + = a + bl bl X2 + logX1 b2X2 + b3X3 + b 2logX 2 H + B 4 X 4 i- b3logX 3 + b 4logX 4 ro 127 Noting that the experiment being analyzed was not designed for t h is p a r t i c u l a r a n a l y s i s , and that score type data i s not e n t i r e l y 2 appropriate for regression a n a l y s i s , yet the R values being consistently greater than what would be expected i f the relationship were zero suggests that this i s an avenue of approach to c l e f t palate studies which should be further explored. Attempts to relate the severity of the parent palate c l e f t s to the severity of c l e f t palate in t h e i r progeny was performed using multiple regression techniques. The severity of the c l e f t s was measured as the score of the right s i d e , l e f t s i d e , or the sum of the sides. The multiple regression models assumed were: Y = a + b1X1 + b 2X 2 + b 3X 3 +. b 4X 4 , where Y = 1. right side score of the clefted progeny 2. l e f t side score of the clefte d progeny 3. sum of the scores for each side f o r the clefted progeny, X.j = o r i g i n a l c l e f t e d parent right side score, X2 = o r i g i n a l c l e f t e d parent l e f t side score, X^ = backcross clefte d parent right side score, X^ = backcross clefte d parent l e f t side score, a, b-j, b 2, bg. b^ = constants. 2 Table 36 shows the R values for c l e f t e d progeny scores related to o r i g i n a l parent and backcross parent scores. In general the parental scores were poor predictors of the clefted progeny scores i n the BC. Table 36 - Coefficients of Determination (R xlOO) f o r C l e f t Palate Progeny Scores f o r Right Side ( Y i j , Left Side (Y 2) and Sum of Sides (Y3) Related to Original Parent C l e f t Palate Line Scores (Right Side: X]; Left Side: X2) and C l e f t Palate Line Backcross Parent Palate Scores (Right Side: X3; Left Side: X4) for Riboflavin Normal (+) and Deficient (-) Diets Using the Model Y = a + b,X, + b~X„ + b,X, + b.X. . Hatches 1-6, 11-16(+) Hatches 7-10 (-) Mating CPxF, CPxF' F^CP FjxCP 38 21 26 26 4 9 13 1 31T 13 12 9 42* 4 13 9 37* 30 15 19 5 22 24 12 11 47 18 18 34 11 Sig n i f i c a n t (p <_ 0.05). ^ Approaching significance (0.10 > p > 0.05). Not enough data was available to make analysis f e a s i b l e . The R values were generally low (less than 30%) and showed no consistent pattern f o r r e l a t i v e values when the d i f f e r e n t Y's were compared. I t was notable that the models relat i n g parental severity of c l e f t to progeny incidence or progeny c l e f t s e v e r i t y , did give' values somewhat greater than would be expected i f the e n t i t i e s under consideration were in fact unrelated. This observation lends some weight to the view that with respect to genetic c o n t r o l , the c l e f t palate t r a i t i s under at least p a r t i a l l y additive c o n t r o l , wherein both incidence and severity i n progeny would be related to parental c l e f t s e v e r i t y . K. Incidence of C l e f t Palate in the F 0 Generation and BC  Compared to Expectations for Multiple Recessive Loci Table 37 shows the number of c l e f t palated and normal progeny f o r each type of BC and Fg f o r both diet regimes. The Chi-square tests for independence of type of BC and 2 type of palate (Table 38) showed for the normal diet (X = 5.46) and o r i b o f l a v i n d e f i c i e n t diet (X = 2.31) that these factors could be considered to be unrelated. Therefore the BC data was pooled for further analyses. The s i m i l a r tests for the F^ data (Table 39) also indicated o independence (X = 1.83 for normal diet and 1.36 for r i b o f l a v i n l e s s diet) and i t also was pooled for subsequent analyses. Using an assumption that the 2 lines used i n the study were homozygous at l o c i pertaining to the c l e f t palate t r a i t , Table 37 Number of C l e f t Palate and Normal Progeny i n the F~ and Backcross Generations f o r Riboflavin Normal (+) and Deficient (-) Diets. Matings Hatches 1-6 (+) Hatches 7-10 (-) Hatches 11-16 (+) Cle f t Normal Cleft Normal C l e f t Normal Fi * Fi 5 385 7 205 3 323 Fj x Fj 0 227 10 145 1 220 Total 5 612 17 '350 4 543 CPxF] 23 338 30 164 15 283 CPxFj 11 170 15 107 10 151 F^CP 19 339 19 79 7 164 FjxCP 17 157 5 34 9 109 Total 70 1004 69 384 41 707 Hen 549 deleted. Table 38 - Number of C l e f t Palate and Normal Progeny of the Backcross Generation f o r Riboflavin Normal (+) and Deficient (-) Diets. Hatches 1-6, 11-16(+)1 Hatches 7-10 (-) 2 Matings Cleft Normal Total C l e f t Normal Total CPxF1 38 621 659 30 164 194 CPxFj 21 321 342 15 107 122 F-jXCP 26 503 529 19 79 98 F]xCP 26 266 292 5- 34 39 TOTAL 111 1711 1822 69 384 453 5.46 (p > 0.05) 3 d.f. 2.31 (p > 0.05) 3 d.f. Chi-square, for independence: x = 2 Chi-square, for independence: x = Table 39 - Number of C l e f t Palate and Normal Progeny of the F* Generation for Riboflavin Normal (+) and Deficient (-) Diets. Hatches 1-6, 11-16 (+) 1 Hatches 7-10 (-)' Original Cross Cleft Normal Total C l e f t Normal Total CPxNH NHxCP Total 8 1 9 708 447 1155 716 448 1164 7 10 17 205 145 350 212 155 367 Chi-square, for independence: x = 1.83 (p > 0.05) 1 d.f. p Chi-square, for independence: x = 1.36 (p > 0.05) 1 d.f. Hen 549 deleted. CO ro 133 expected frequencies of segregation for 1 to 5 recessive " c l e f t palate" l o c i vn"th 100% penetrance were calculated for the BC and F^. Chi-square tests for goodness of f i t of the observed data to the expected numbers are shown i n Table 40. During the combined normal diet periods, the frequencies of c l e f t palate observed for the BC (111 in 1822 observations) did not d i f f e r s i g n i f i c a n t l y from the frequency expected for segregation of 4 recessive l o c i (6.250% or 114 c l e f t palates), and s i m i l a r l y that for the generation (9 in 1164 observations) also f i t 4 recessive l o c i (0.391% or 5 c l e f t palates). During the r i b o f l a v i n deficiency period the BC data (69 • c l e f t palates i n 522 observations) did not d i f f e r s i g n i f i c a n t l y from the frequency expected for segregation of 3 recessive l o c i (12.5% or 57 c l e f t palates), while the F^ generation frequency of c l e f t palates (17 in 367 observations) f i t that expected for 2 recessive l o c i (6.25% or 23 c l e f t palates). Data for the results of intermatings between phenotypically normal s i b l i n g s of the G3, CP l i n e (presented in Table 41) indicated that the penetrance of the c l e f t palate t r a i t in the CP l i n e was approximately 50%. No data was available on the incidence of c l e f t palate i n intermatings of t h e i r c l e f t palate s i b l i n g s (who were the CP parents i n the backcross study). Based on the observation that the incidence of c l e f t palate in the progeny of these phenotypically normal birds was very s i m i l a r to that of t h e i r own CP l i n e G2 parents, i t can be argued that the a l l e l e s f o r c l e f t palate i n the CP l i n e have been f i x e d , and that Table 40 - Chi-Square for Goodness of F i t to Expected Frequencies for Multiple Recessive Loci Assuming 100 Percent Penetrance for the F 2 and Backcross C l e f t Palate Progeny When Their Dams Were on Riboflavin Normal (+) and Deficient (-) Diets. Hatches !• -6, 11-16 ( + ) Hatches 7-10 (-) No. of Loci Expected Percent Cleft Palate Expected Expected Progeny Cle f t Palate Normal Chi-Square Cl e f t Palate Normal Chi-Square 2 1 25.000 _ _ 91.750 275.250 80.12 2 6.250 72.750 1091.250 58.66 22.938 334.062 1.38* 3 1.560 18.158 1145.842 4.19 5.725 361.275 20.60 4 0.391 4.547 1159.453 3.45* - - -5 0.098 1.137 1162.863 47.73 - - -Total Observed 9 1155 17 350 Backcross 2 25.000 - - - 113.250 339.750 22.53 3 12.500 227.875 1595.125 67.92 56.625 396.375 2.85* 4 6.250 113.938 1709.062 0.06* 28.312 424.698 60.85 5 3.125 56.969 1766.031 51.87 - - -Total Observed 111 1711 69 453 p > 0.05. Hen 549 deleted. Table 41 - Number and Percent of C l e f t Palate Progeny from Matings of Normal Progeny of Cl e f t Palate Parents of Generation 3, on Riboflavin S u f f i c i e n t (+) and Low (-) Diets. Hatches 1-3 (+) Hatches 4-12 (-) Hatches 13-15 (+) Sire Dam No. Eggs Set Cl e f t Palate No. No Eggs Set 0 Clef t Palate No. No. Eggs Set C l e f t Palate No. 1 1 14 13 8 61.5 9 9 5 55.6 2 13 11 4 36.3 11 8 5 45.5 2 1 11 10 6 60.0 29 23 15 65.2 10 8 8 100.0 3 1 13 11 4 36.3 19 12 5 41.7 13 8 5 62.5 4 1 15 15 10 66.7 41 37. 28 75.7 15 13 11 84.6 2 11 10 7 70.0 3 3 3 100.0 5 1 10 9 4 44.0 25 . 21 6 28.6 9 3 1 33.3 2 9 8 2 25.0 14 12 4 33.3 Mean 50.0 55.7 70.1 * Observed. 136 .selection (Roberts et aj_., 1973) was for a l l e l e s improving penetrance. There was no basis f o r any estimate of the potential penetrance of the NH l i n e . I t would seem that the penetrance f o r the BC, being under 3/4 control by CP l i n e a l l e l e s should be reasonably close to 50% on the average. Since the NH l i n e was unknown, the penetrance f o r the F,, generation remains a subject for speculation. With these l i m i t a t i o n s i n mind, the Chi-square tests for goodness of f i t were re-run for the BC and Fg data using the assumption of 50% pene-trance f o r the multiple recessive l o c i model. The results of these tests are presented in Table 42 and a summary comparing results for 100% and 50% penetrance models i s presented in Table 43. The assumption of 50% penetrance produced more consistency between the l o c i numbers estimated by the BC and Fg generation, being 3 for both on the normal diet and 2 for both during the r i b o f l a v i n d eficient diet period. In order to check that the effects of r i b o f l a v i n deficiency in r a i s i n g the incidence of c l e f t palate did not continue into hatch 11 to the extent of biasing the normal diet data in the manner i t was previously shown to affect h a t c h a b i l i t y , a l l of the Chi -quare tests described above were re-run for the normal diet with hatch 11 deleted. There was no change in the results for the F^ generation in any t e s t . The Chi-square value for the BC test of independence increased s l i g h t l y and became s i g n i f i c a n t , but this change was not reflected in any change i n Chi-square value for goodness of f i t . Therefore i t was concluded that there would be no great bias i n including hatch 11 in the data, a view that was further supported by a Table 42 - Chi-Squares for Goodness of F i t to Expected Frequencies for Multiple Recessive Loci Assuming 50 Percent Penetrance for the F^ and the Backcross C l e f t Palate Progeny When Their Dams Were on Riboflavin Normal (+) and Deficient (-) Diets. Hatches 1-6, , 11-16 (+ ) Hatches 7-10 (-) No. of Loci Expected Percent C l e f t Palate Expected Chi-Square Expected Chi -Square Progeny Cleft Palate Normal Cl e f t Palate Normal 2 1 12.500 _ 45.875 321.125 20.06 2 3.125 36.375 1127.625 20.50 11.469 355.531 2.28* 3 0.780 9.079 1154.921 0.02* 2.863 364.137 64.47 4 0.195 2.273 1161.727 17.09 - - -Total Observed 9 1115 17 350 Backcross 1 25.000 - - - 113.250 339.750 22.53 2 12.500 227.875 1595.125 67.92 56.625 396.375 2.85* 3 6.250 113.938 1709.062 0.06* 28.312 424.698 60.85 4 3.125 56.969 1766.031 51.87 - - -Total Observed 111 1711 69 453 * p > 0.05 . Hen 549 deleted. to 138 Table 43 - Number of Loci Controlling the C l e f t Palate T r a i t Based on the Goodness of F i t Chi-Square Tests for Riboflavin Normal (+) and Deficient (-) Diets. Penetrance Progeny 100 Percent 50 Percent Diet Diet F2 Backcross 139 lack of difference between the frequencies of c l e f t palate observed in the f i r s t and l a s t diet periods as previously shown (Table 7). Since the actual penetrance level i s unknown the results of the tests for goodness of f i t and for independence indicate the following: 1. the general independence in frequency of c l e f t palate i n both dietary periods indicates that there i s no maternal i n f l u -ence on frequency of c l e f t palate during either kind of d i e t . This result i s d i f f e r e n t than the report of maternal effe c t on mouse dietary response of c l e f t palate frequencies (Pollard and Fraser, 1968). 2. the results of goodness of f i t tests for multiple l o c i indicate that i f the c l e f t palate t r a i t i s controlled by a series of recessive a l l e l e s at several l o c i , then the number of l o c i i s probably among 2 to 4, i n c l u s i v e . 3. the increased frequency of c l e f t palate (which was associated with a decreased number of l o c i ) during maternal r i b o f l a v i n deficiency i s consistent with a masking of the genetic variation of 1 or 2 l o c i by the mimicking of the effect of the recessive a l l e l e s at those l o c i by the resultant condition due to r i b o f l a v i n deficiency. Thus those l o c i would no longer play a r o l e , being replaced by a physiological condition l i k e that produced by the recessive a l l e l e . Although based on the l i t e r a t u r e i t would be expected that there was genetic va r i a t i o n i n the control of egg r i b o f l a v i n content between hens used, the lack of clear-cut maternal effects between the F.j hens and the CP hens in the reciprocal backcrosses indicates that the r i b o f l a v i n related t r a i t was segre-gating in the progeny, not the hens. Thus, the suggestion by Roberts et aj_. (1973) that the locus involved was si m i l a r to Maw's (1954) r i b o f l a v i n l e s s t r a i t wherein i n -s u f f i c i e n t r i b o f l a v i n was deposited in the egg was not supported. Rather, a f a i l u r e of the embryo to use the r i b o -f l a v i n present in the egg was indicated. L. The Additive Model Applied to the Incidences of Cle f t Palate  in the Generation and BC Castle's o r i g i n a l formula (1921a) as modified by Wright (Castle, 1921b; Wright, 1934) for estimating the number of pairs of genes involved in the inheritance of a quantitative c h a r a c t e r i s t i c was applied.to the ¥^ generation and BC data. The conditions under which the formulae are v a l i d , as discussed by Lush (1948) are as follows: (a) the genes a l l have the same e f f e c t s , i . e . , A = B = C; a = b = c. (b) there i s no dominance. (c) the genes combine t h e i r effects in an additive fashion. (d) the parental lines are e n t i r e l y homozygous for the t r a i t involved. (e) one l i n e has a l l of the plus genes and the other has a l l of the minus genes. (f) the genes are independent of each other. (g) there i s a common environmental variance for the F-j, F^ and BC. (h) there i s a large number of individuals so that sampling errors are smal1. 141 The formula f o r the Fg i s D2 8(V F - VF ) r 2 r 1 and The formula for the BC i s D2 , 6< V8C " VF,> where D = difference between the means of the two parental l i n e s , VP = variance of the F,, rl ' VF = variance of the F 0, Vg^ = variance of the backcross and . n = number of l o c i affecting the t r a i t , that are di f f e r e n t between the 2 l i n e s . The data was transformed to Freeman-Tukey arc-sine values using the modified method f o r small samples of Mosteller and Youtz (1961) (Tables 17 to 25 Appendix). Table 44 shows the calculated values estimating the number of l o c i for the F 2 and BC data on the normal and def i c i e n t d i e t s . The number of l o c i affecting c l e f t palate estimated by the F 2 data was, fo r the normal diet 56.3, and for the de f i c i e n t d i e t , 5.7. For the BC 142 Table 44 - Estimation of Number of Loci (n) for an Additive Genetic Model Using Wright's Formulae for F 2 and Backcross Data Tested Generation Formula Components 1 Riboflavin Diet Normal Deficient D Mean V(F ]) Mean V(F 2) 33.02 3.239 5.658 56.34 33.02 6.469 30.286 5.72 Backcross Mean V(F.j) Mean V(BC) n 40.02 3.239 84.423 1.23 40.02 6.469 131.754 0.80 D = mean difference between parental l i n e s , Mean V = mean variance. Calculation of formula component values in Appendix Tables 17 to 25. 143 data the same 2 estimates respectively were 1.2 and 0.8. These values most d e f i n i t e l y did not agree with those obtained in the analysis based on the recessive model. However, i t was interesting to note that there was an agreement between the analyses i n that in both instances the number of l o c i estimated on the d e f i c i e n t diet was less than that on the normal. I t should be noted that the estimate for the recessive model was based on frequencies, while that for the additive model u t i l i z e d variances of frequencies. I t i s not surprising that the 2 models f a i l e d to agree in that they differ e d a major assumption: the recessive model considers that AA = Aa f aa; whereas the additive model considers that AA f Aa f aa with an equal unit effect between the 3 phenotypes. The special conditions necessary for Wright's formula to be v a l i d could not be met with t h i s data. For example, i t had been established that the NH l i n e was, in a l l l i k e l i h o o d , carrying some c l e f t palate genes. This meant that the F-j contained some genetic variance. In addition, the difference between the 2 l i n e means did not r e f l e c t a complete separation of the c l e f t palate and non-cleft palate genes. Furthermore, the CP l i n e was not homozygous for the genes influencing the c l e f t palate condition i n that the CP individuals used to produce the F^ showed response to selection when random mated among themselves (Roberts et a]_., 1973). A complete discussion of the problems associated with this type of estimate can be found i n Lush (1948). 144 M. Additional Considerations Obviously since this thesis i s only the second research e f f o r t investigating the genetics of the c l e f t palate t r a i t in chickens, many questions and problems arise that only additional studies can c l a r i f y . For example: "Is the c l e f t palate t r a i t in the chicken homologous (genetically or developmentally) to that i n mammals?" The posterior palate of the bird i s normally s l i t t e d (Figure 7 ) which suggests that the c l e f t s observed i n the bird may be analagous to c l e f t l i p in other species. While this study was not investigating the developmental aspects of c l e f t palate, i t was of int e r e s t to observe that based on the location of the c l e f t s , and the diagrams in Romanoff (1960) the formation of the c l e f t i n the chicken probably involves the junction of the following bones: the anterior end of the ju g a l ; the palative process of the maxillary, and the anterior end of the palatine bone. Since these bones are not "plates" but are rather elongated forms, improper fusion ( i . e . i n the wrong place) or misshapen bones would seem to be l i k e l y causes of the c l e f t s observed. Another question that arises from the data i s , "Were a l l the c l e f t s observed the same genetic t r a i t ? " C l e f t palate i s a descriptive term for an end point which could conceivably develop through many d i f f e r e n t routes. With the exception of the progeny of hen #549 i t has been assumed that a l l of the c l e f t palates observed were the same genetic t r a i t . This might not be true. However, based on this paper and the previous paper (Roberts et al_ . , 1973) the 145 c l e f t palate t r a i t seemed to be consistent within the population under study. A very pertinent question arises regarding the consequences of the r i b o f l a v i n d eficient d i e t s : "What other conditions could result from a maternal r i b o f l a v i n deficiency, and are they more d i r e c t l y associated with the c l e f t palate condition?" Although r i b o f l a v i n deficiency has been shown to increase the incidence of c l e f t palate in t h i s study, the effect may not be d i r e c t . The metabolism of other compounds, p a r t i c u l a r l y f o l i c and n i c o t i n i c acid would be expected to be disturbed i n the hen, and subsequent deposition in the egg altered. The d i f f e r e n t response between the CP and F-j hens in hatchability during r i b o f l a v i n deficiency, coupled with the s i m i l a r response in the incidence of c l e f t palate produced, may suggest that the c l e f t s were either a response to levels of r i b o f l a v i n not affecting h a t c h a b i l i t y , as indicated by Roberts et al_. (1973), or that other factors having less influence on hatchability than r i b o f l a v i n were also involved. This lack of difference between CP and F^ generation hens producing BC progeny i n c l e f t palate incidence compared to that of hatchability and the p o s s i b i l i t y that the hatchability differences may be a r e f l e c t i o n of body weight differences between F-j and CP hens suggests that there i s l i t t l e wrong with the CP hen's a b i l i t y to metabolize r i b o f l a v i n , and that the response to deficiency i s that of the embryo i t s e l f to the lowering of the levels of r i b o f l a v i n i n the egg. Another question, answerable only by further genetic studies i s "Can genetic models, other than those already discussed, be applied to the t r a i t and the v a r i a b i l i t y of the c l e f t palate observed?" 146 There was no doubt that the chicks had a low incidence of the c l e f t palate t r a i t . This could indicate a dominance factor associated with the character, although this appears unlikely i n view of the past history of the l i n e (Roberts et aJL , 1973; Shoffner et a l . , 1953). A simple additive model could also explain this occurrence, although the simple additive model was rejected by Roberts et a l . (1973) on the basis of t h e i r selection study forming the CP l i n e . Since none of- the F-j generation chicks with d e f i n i t e c l e f t palate hatched during any of the studies, i t could be speculated that a lethal factor associated with the NH l i n e was in evidence. In a d d i t i o n , when the lack of appearance of c l e f t palate i n the NH l i n e (zero in 1415 observations) i s coupled with the incidence in the F-j, some indi c a t i o n i s given that the t r a i t i s at very low frequencies in the NH l i n e and that i t s appearance i s also associated with an early mortality there (pr i o r to 13 days of development). A longer embryonic survival i n the F-| presumably conferred by the CP l i n e (or a heterotic effect between the 2 lines) may have allowed the detection of the c l e f t palate a l l e l e s in the NH l i n e . I t would be interesting i f this l a t t e r p o s s i b i l i t y were true, because i t would mean that despite the presence of c l e f t palate a l l e l e s in the NH l i n e , the l e t h a l i t y would have allowed only a l l e l e s from the CP l i n e to be transmitted to the and BC generations, and would provide the subsequent f i t to the multiple recessive l o c i model. Although Wright's formulae for determining the number of additive l o c i d i f f e r i n g between lines were applied to the data, there were indications that the t r a i t was probably not controlled i n an additive fashion: (1) The observation of Roberts ejt al_. (1973) that a l l matings of the CP l i n e produced c l e f t palate after 1 generation of s e l e c t i o n , while the incidence of c l e f t palate was approximately 30%, indicated that the data did not f i t an additive model. At a population frequency of 30%, some matings would be expected to have a zero incidence in t h e i r progeny; (2) The intermatings, discussed i n this t h e s i s , of phenotypically normal individuals of the c l e f t palate l i n e , showed a mean percent incidence of c l e f t palate i n th e i r progeny at least as high as that of t h e i r parent's progeny. The additive model was therefore again not supported, because the incidence would be expected to be less than i n t h e i r parent's progeny; (3) The 1% incidence of c l e f t palate in the , being much closer to that of the NH l i n e (zero) than to the CP l i n e (30%), also does not support an additive model. In the additive model the F^ mean would be expected to be midway between the parental l i n e s . In view of the manner i n which the CP l i n e was created (using an environmental factor to increase the incidence of the t r a i t i n an inbred l i n e ) and in view of the i n d i c a t i o n , using either the multiple recessive l o c i model or the additive model, that about one locus i s made irrel e v a n t to the t r a i t when r i b o f l a v i n deficiency i s imposed, the genetics of c l e f t palate should be examined b r i e f l y with reference to a group of models proposed f o r t r a i t s such as c l e f t palate which do not consistently f i t simple Mendelian segregations. 148 Genetic assimilation,(the appearance of r e l a t i v e l y high spontaneous incidence of a character, which prior to selection f o r the same character produced by a treatment, did not appear spontaneously in a population),has been described by Falconer (1960) on the basis of an underlying additive genetic control with 2 thresholds: 1 for spontaneous occurrence and 1 for "induced1'occurrence. The response to selection of induced incidence reflected by the spontaneous group i s described as being due to the movement of the entire d i s t r i b u t i o n r e l a t i v e to the thresholds. With the exception that there was a low spontaneous incidence of c l e f t palate i n the "420 l i n e , " the production of the CP l i n e by selection could be described by this model, and the frequencies of c l e f t palate i n the ¥^ and BC viewed as the product of the position of t h e i r d i s t r i b u t i o n s r e l a t i v e to the 2 thresholds. However the f a i l u r e of the observations to date to support the additive model make i t l i k e l y that the resemblance of the creation of the CP l i n e to genetic assimilation based on an additive model was a s u p e r f i c i a l one only. In considering "phenocopies" produced by various chemicals injected into chicken eggs containing developing embryos, Landauer (1957) stated that "the residual genetic factors which set the stage for sporadic developmental 'accidents' also predispose, and in a correlated manner, toward the phenocopy response. The fact that both situations are s i m i l a r l y affected by plus or minus modifiers strengthens the conviction of t h e i r being closely related." This view d i f f e r s from that of Falconer discussed e a r l i e r , i n that i t no longer assumes an underlying additive model of inheritance. From this point i t could be viewed that the spontan-eously induced c l e f t palate t r a i t depended on both major l o c i and modifying penetrance a l l e l e s (as proposed i n the 3 recessive l o c i , 50% penetrance model), and that the production of "phenocopy" c l e f t palates during r i b o f l a v i n deficiency depended on only 2 major l o c i with 50% penetrance. The phenocopy, by d e f i n i t i o n would be of the 1 locus made i r r e l e v a n t . Further, i t could be viewed that selection i n the CP l i n e was f o r the penetrance a l l e l e s , the major l o c i being f i x e d , but undetectable in the absence of favorable 9 modifiers. The response to selection being quite rapid (h~ = approximately 30%; Roberts, personal communication), the number of penetrance a l l e l e s present could be quite small ( i . e . , 3 or 4). The l a t t e r 2 points have been suggested previously by Roberts et a i . (1973). A further model, describing both "phenodeviants" and "canalization," was discussed by Lerner (1954) within the context of genetic homeostasis. Because of i t s generality, this concept can include major control of t r a i t s by both additive or non-additive genetic mechanisms. Lerner's thesis was, b r i e f l y , that i t i s the general level of heterozygosity in an organism which allows i t to be buffered from environmental forces or to respond in a manner which confers " f i t n e s s . " Inbreeding, by reducing heterozygosity reduces the general buffering capacity and allows the environment to influence t r a i t s to more extreme forms, which have less " f i t n e s s . " The"canalization" concept considered that developmental reactions as they occur in organisms submitted to natural selection are adjusted to bring about one d e f i n i t e end res u l t regardless of minor variations i n conditions during the course of the reaction. This canalization would therefore be dependent on buffering by heterozygosity. Thus, "phenodeviants" (abnormal morphological deviants) were seen as the result of the l o s s , due to inbreeding, of the canalization dependent on heterozygosity. It was also suggested that some major genes were involved in the production of phenodeviants, but that the expression was dependent on the accumulation of modifiers so widely spread in populations as to make the p o t e n t i a l i t y for the t r a i t ubiquitous. I t was further suggested that the p a r t i c u l a r direction that the organism may take outside i t s normal channel of development depends to a great extent on i t s s p e c i f i c gene contents. The multiple recessive l o c i model with penetrance a l l e l e s can cer t a i n l y be found to be compatible with this view of genetic homeostasis. The effect of the r i b o f l a v i n d e f i c i e n t diet i n producing additional phenodeviants normally controlled by a l l e l e s at 1 locus i n the presence of the modifiers i s also compatible with the concept of ca n a l i z a t i o n . A s l i g h t change in point of view of the development of the CP li n e would be needed, in that i f the major a l l e l e s were considered to be f i x e d , i t would not be e n t i r e l y selection per se, but the attenuating inbreeding which ad d i t i o n a l l y increased the penetrance of the c l e f t palate condition. Conditions which may not t o t a l l y f i t the canalization model are the questions whether the F 9 contained s u f f i c i e n t inbreeding to produce the 151 level of c l e f t palate attained, and whether the heterozygosity of the F-] could allow the level of c l e f t palate observed. While not refuting Gruneberg's model of quasi-continuous variation or the l a t e r refinement of Falconer's double threshold model, i t i s possible that f o r c l e f t palate in poultry i t i s not necessary to use such additive genetic models to explain the genetic observations to date. With regard to the multiple recessive l o c i model with penetrance a l l e l e s and ca n a l i z a t i o n , the corollary of ubiquitous d i s t r i b u t i o n of penetrance a l l e l e s associated with the c l e f t palate t r a i t was supported by the l i t e r a t u r e reporting spontaneous and induced c l e f t palates i n many lines and up to about 50% incidence, though generally much lower. I t i s possible that each of the major l o c i mimic, in e f f e c t , the condition of one s p e c i f i c environmental deficiency or metabolic upset. I t would be inter e s t i n g to determine i f n i c o t i n i c acid antagonists or f o l i c acid antagonists which would disturb at least one of the same metabolic pathways as r i b o f l a v i n deficiency could also produce increased incidence of c l e f t palate due to elimination of effects of the same or di f f e r e n t l o c i i n the group. It would also be inter e s t i n g to determine i f the inferred role of disturbed mucopolysaccharide biosynthesis in the production of c l e f t s i s in fact true. Previous work (Roberts et_ al _ . , 1973) suggested the following model to explain the variation observed associated with the c l e f t palate t r a i t : 152 1. One or 2 l o c i , recessive in nature, associated with the t r a i t per se. 2. A few l o c i , probably no more than 4,acting in an additive fashion associated with the penetrance of the t r a i t . 3. A maternal sex-limited genetic f a c t o r , and/or an imposed environmental factor associated with the level of r i b o -f l a v i n deposited in the egg by the dam, which resulted in a threshold affecting the developing embryo. The data of this thesis did not disagree with the f i r s t 2 proposals. However, no evidence of a sex-limited genetic factor was found, since reciprocal backcrosses gave e s s e n t i a l l y the same response in c l e f t palate incidence. In addition the response to r i b o f l a v i n deficiency of increased' incidence of c l e f t palate reported for deficient but not moderately low r i b o f l a v i n diets by Roberts et al_. (1973) was confirmed by the data for the and BC generations. The suggestion that there might be a genetic factor associated with r i b o f l a v i n level deposited in the egg was p a r t i a l l y borne out i n that i t appeared that segregation f o r a factor associated with r i b o f l a v i n on normal diets was necessary for production of c l e f t palate and no longer relevant in the absence of normal r i b o f l a v i n l e v e l s . However, this segregation must have taken place in the embryo from the fact' that the BC progeny of the F-j and CP hens were not e s s e n t i a l l y d i f f e r e n t i n c l e f t palate incidence. Roberts et aj_. also suggested that a possible l e t h a l 153 effect might be associated with the c l e f t palate t r a i t . The present data from the backcross and f i r s t segregating generation of a cross of the CP l i n e and the non-cleft New Hampshire l i n e c l e a r l y demon-strates that the phenotype, c l e f t palate, i s semi-lethal. Furthermore, i t shows that the l e t h a l i t y was associated with the expressivity of the t r a i t . 154 SUMMARY The genetic basis of, and the influence of maternal r i b o f l a v i n deficiency on the incidence of the c l e f t palate condition in chickens was investigated. For both maternal normal and r i b o f l a v i n d e f i c i e n t diet periods, the incidence of c l e f t palate was observed in the F-j of a cross between a inbred Leghorn l i n e having high c l e f t palate incidence (CP) and a non-cleft palate New Hampshire li n e (NH), in the F^ of that cross,, and i n the progeny of the f i r s t backcross to the CP l i n e . The CP l i n e was under selection f o r the c l e f t palate t r a i t concurrent with i t s use in this study. Only phenotypically c l e f t palated individuals.were used. The incidence of c l e f t palate was approximately 30% in the generation used to form the F-J and approximately 50% in the generation used to form the backcross progeny. A l l matings were reciprocal crosses. Eighteen CP males, 18 CP females, 6 NH males and 36 NH females produced the 1361 F-| embryos and chicks examined. Twenty-eight CP males, 36 CP females, 20 F-j males, and 28 F^ females were used to produce the 2275 backcross observations; 18 F-j males and 29 F-j females produced the 1531 F,> observations. The data collected indicated the following: 1. The response to r i b o f l a v i n deficiency as measured by loss of h a t c h a b i l i t y of eggs was found in a l l types of hens used, at from 1 to 3 weeks af t e r introduction of the r i b o f l a v i n d e ficient d i e t , which was consistent with the reports in the l i t e r a t u r e . No abnormalities in hatchability response were indicated f o r the CP l i n e hens, and a hypothesis of abnormal r i b o f l a v i n metabolism in that l i n e was rejected 2. The genotype of the embryo was indicated to play a role i n the hat c h a b i l i t y response to r i b o f l a v i n deficiency. The greater loss of hatchability of Fg chicks than of backcross chicks from F-| hens was assumed to be a result of the presence i n the F^ of expressed pigmentation a l l e l e s with a concomitant elevated r i b o f l a v i n requirement. 3. In agreement with the l i t e r a t u r e , there were indications that embryonic mortality took place progressively e a r l i e r as the r i b o f l a v i n deficiency continued, and approximately 1 week was required f o r h a t c h a b i l i t y levels to be recovered when the hens were returned to the normal d i e t . 4. There was a d e f i n i t e low incidence of c l e f t palate i n the F-j generation on the normal d i e t , and subsequent evidence that the incidence increased during maternal r i b o f l a v i n deficiency i n a l a t e r study. 5. There was some suggestion of a maternal influence of the CP l i n e on the incidence of c l e f t palate i n the F^ furing r i b o f l a v i n deficiency of th i s study although an age effe c t could have been responsible. 6. The severity of several of the c l e f t s observed i n the F^ chicks were at least as severe as any observed i n the or i g i n a l CP l i n e and the condition was apparently lethal i n the F, generation. 7. C l e f t palate was recovered i n the and backcross progeny during normal diet periods at incidences of approximately 1% and 8% respectively. 8. The incidence of c l e f t palate in the Fr, and backcross progeny showed a marked increase during the maternal r i b o f l a v i n deficiency period, being approximately 4% and 12% respectively. 9. L i t t l e evidence of maternal effects were found in the F^ or back-cross generations for c l e f t palate incidence. Comparison of hatch-a b i l i t y and c l e f t palate data f o r CP and F-j hens indicated that the level of r i b o f l a v i n deficiency which induced c l e f t palate i s not one which affected h a t c h a b i l i t y . 10. There was evidence that the response in incidence of c l e f t palate to r i b o f l a v i n deficiency depended upon a predisposition, in that the increase was found to be within hens, but not between them in the backcross. This predisposition was considered to be asso-ciated with penetrance a l l e l e s contributed through the F^ from the NH l i n e . 11. A condition of multiple abnormalities including c l e f t palate was recovered in the F^ progeny from 1 hen at frequencies suggesting segregation of 1 recessive a l l e l e . This condition was considered to be separate from, and those progeny excluded from, the c l e f t palate condition under study. 12. No dependence was found, in the F^ and backcross generation, between c l e f t palate and club down or an exencephaly syndrome, while the dependence shown between malpositions and c l e f t palate during r i b o -f l a v i n deficiency for backcross progeny requires further work to be explained. 13. An apparently new t r a i t , "palatal p i t s , " was observed. Although i t apparently originated from the CP l i n e , there was no evidence of i t s being related to c l e f t palate. 14. No evidence of d i f f e r e n t frequencies of c l e f t palate between sexes was found. 15. No effect on hatch weight was found f o r r i b o f l a v i n deficiency or the c l e f t palate condition, for the Fg and backcross progeny. The expected e f f e c t of maternal body weight was found in the reciprocal backcrosses. 16. There was no clear relationship within dam genotypes (F^ or CP) between body weight and loss of hatchability or production during r i b o f l a v i n deficiency. 17. A d e f i n i t e embryonic l e t h a l i t y of the more severe c l e f t palate condition during normal and r i b o f l a v i n deficient diet periods was shown for F,, and backcross progeny. 18. No effect on c l e f t palate severity was found for maternal genotype or progeny genotype for the F^ and backcross progeny. 19. An inconsistent effect of diet periods was found, which appeared under one scoring system of severity and not under another. Where i t was found, i t was not a r e f l e c t i o n of r i b o f l a v i n deficiency, but rather a decreasing severity over time; of unexplained o r i g i n . 20. The semi-lethality of the c l e f t palate condition was further demonstrated by the generally higher frequencies of the t r a i t among dead embryos compared to hatched chicks although no 158 p a r t i c u l a r period of mortality predominated. 21. The increase in c l e f t palate incidence during the r i b o f l a v i n d e f i c i e n t period was found only among the mortality periods. The diet l e t h a l i t y and l e t h a l i t y of the induced t r a i t were confounded. 22. For the backcross the u n i l a t e r a l types of c l e f t predominated during normal diet periods, and b i l a t e r a l s , owing to t h e i r greater rate of increase during r i b o f l a v i n deficiency. During both types of d i e t , b i l a t e r a l types showed greater mortality than u n i l a t e r a l type of c l e f t . The F 2 data did not agree, but was very l i m i t e d . 23. A low relationship (R < 30%) was found between c l e f t palate parent severity and incidence of c l e f t s i n the F 2 and BC progeny. 24. A low relationship (R < 30%) was found between c l e f t palate parent severity and c l e f t palate progeny s e v e r i t y . 25- With 100% penetrance, both the F^ and backcross frequencies f i t expectations of 4 segregating l o c i on the normal d i e t . During r i b o f l a v i n deficiency the F 2 frequencies f i t segregation of 3 l o c i while backcross frequencies f i t 2 l o c i . 26. With 50% penetrance, both groups f i t 3 l o c i during the normal diet and 2 l o c i during r i b o f l a v i n deficiency. 159 27. Wright's formulae for determining the number of l o c i d i f f e r i n g between lines on the basis of the F-j, Fg and backcross variances were applied. The results were inconsistent, which was a t t r i -buted to the f a i l u r e of the data to meet the conditions of the model. 28. 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P e r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 10 Weekly Hatches of the F] Progeny from CPxNH Matings on a R i b o f l a v i n (R) Normal ( + ) or D e f i c i e n t (-) D i e t 3 4. P e r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 10 Weekly Hatches of the F-j Progeny from the NHxCP Matings on a Normal R i b o f l a v i n D i e t . . . . . 4 5. P e r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 16 Weekly Hatches of the F 2 Progeny from the F -i x F -| Matings on a R i b o f l a v i n (R) Normal ( + ) or D e f i c i e n t (-) D i e t 5 6. P e r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 16 Weekly Hatches of the F 2 progeny from the F-jxF-j Matings on a R i b o f l a v i n (R) Normal ( + ) or D e f i c i e n t (-) D i e t 6 7. P e r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 16 Weekly Hatches of the B a c k c r o s s progeny from the CPxF-] Matings on a R i b o f l a v i n (R) Normal ( + ) or D e f i c i e n t (-) D i e t 7 8. P e r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 16 Weekly Hatches of the B a c k c r o s s Progeny from the CPxF-j Matings on a R i b o f l a v i n (R.) Normal ( + ) or D e f i c i e n t (-) D i e t 8 9. P e r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 16 Weekly Hatches of the B a c k c r o s s Progeny from the F-ixCP Matings on a R i b o f l a v i n (R) Normal (+) or D e f i c i e n t (-) D i e t . . . 9 /4ii Appendix Table Page 10. Percent Hatchability and Mortality for 16 Weekly Hatches of the Backcross Progeny from the FjxCP Matings on a Riboflavin (R) Normal (+) or Deficient (-) Diet 10 IT. Percent C l e f t Palate (CP) Progeny and Total Observations (0) for the F ? Progeny of F-jxFn Matings on a Riboflavin Normal (+) and Deficient (-) Diet 11 12. Percent C l e f t Palate (CP) Progeny and Total Observa-tions (0) for the VI Progeny of F-JxF-i Matings on a Riboflavin Normal ( + ) and Deficient (-) Diet 12 13. Percent C l e f t Palate (CP) Progeny and Total Observa-tions (0) for the Backcross Progeny of CPxF] Matings on a Riboflavin Normal (+) and Deficient (-) Diet 13 14. Percent C l e f t Palate (CP) Progeny and Total Observa-tions ()) for the Backcross Progeny of CPxFj Matings on a Riboflavin Normal ( + ) and Deficient. (-} Diet . . 14 15. Percent C l e f t Palate (CP) Progeny and Total Observa-tions (0) for the backcross Progeny of F-|xCP Matings on a Riboflavin Normal (+) and Deficient (-) Diet . . 15 16. Percent C l e f t Palate (CP) Progeny and Total Observa-tions (0) for the Backcross Progeny of F-jxCP Matings on a Riboflavin Normal (+) and Deficient (-) Diet . . 16 17. Arc-sine Values of Percent Cl e f t Palate Progeny Used for Calculations of Sire Variances of the F, from CPxNH Matings '. . . 17 18. Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculations of Sire Variances of the F, from NHxCP Matings 18 19. Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculations of Sire Variances of the F ? from F-jxF-j Matings 19 20. Arc-sine Values of Percent C l e f t Palate Progeny Used fo r Calculations of Sire Variances of the F« from F'xF-J Matings 20 4 i i i Appendix Table Page 21. Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculations of Sire Variances of the Backcross Progeny from CPxF-j Matings 21 22. Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculations of Sire Variances of the Backcross Progeny from CPxFj Matings 22 23. Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculations df Sire Variances of the Backcross Progeny from F^xCP Matings 23 24. Arc-sine Values of Percent C l e f t Palate Progeny •Used for Calculations of Sire Variances of the Backcross Progeny from F-jxCP Matings 24 25. Mean Sire Variances (Arc-sihe) Values and C l e f t Palate (CP) and New Hampshire (NH) Mean Line Differences Used for the Calculation of Number of C l e f t Palate l o c i • 25 /liv LIST OF APPENDIX FIGURES Appendix Figure Page 1. Sketches Used for Standard Scores of C l e f t Palates i n the F 2 and Backcross Generations . . . . 26 / l l Table 1 - University of B r i t i s h Columbia Number 3 Ration. Ingredient Percent No. 1 Ground Screenings 68.25 Ground Oats 10.00 Meatmeal 3.00 Soybean Meal (48.5%) 4.50 Fishmeal 3.00 Dehydrated Grass 5.00 Ground Limestone 1.00 Dicalcium Phosphate 0.35 Iodized Salt 0.05 Pryferm (Fermentation By-Product) 0.85 Tallow 1.00 UBC#8 Premix 0.85 UBC#8 Premix Mg/kilo Vitamin A 1683 I.U. • Vitamin D3 1122 I.C.U. B12 4.8 meg Riboflavin 4.4 mg Maganese Sulfate 190.0 mg Zinc Sulfate 80.0 mg Table 2 - Riboflavin Deficient Diet*. Ingredient Percent Ground Wheat 61 .00 Pulverized Oats 15 .00 Soybean Meal (48.5%) 15 .00 Bonemeal 1 .75 Ground Limestone 5 .40 Iodized Salt 0 .35 Feeding Oil (3000D:1500A) 0 .50 Premix 1 .00 Premix Mg / k i l o Manganese Sulfate 249.70 3.30 Niacin 11.00 Calcium pantothenate 3.34 Folacin 0.33 Choiine 1100.00 B i o t i n 1.32 Pyridoxine HC1 4.40 Vitamin K 0.55 * Formulated by Professor J . B i e l y , Department of Poultry Science, University of B r i t i s h Columbia. T a b l e 3 - P e r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 10 Weekly Hatches of the F-| Progeny from the CPxNH Matings on a R i b o f l a v i n (R) Normal (+) or D e f i c i e n t (-) D i e t . Mean Average P e r c e n t of F e r t i l e Dam Hatch Number* T o t a l T o t a l P e r c e n t E a r l y Late Late Dead R Number S i r e s Dams Eggs F e r t i l e I n f e r t i l e . Dead Dead(-) Dead(+) i n S h e l l P i p Hatch D i e t 1 15 29 146 113 22.3 0.7 0.8 1.5 0.8 16.2 79.9 + 2 17 33 145 137 5.3 3.6 2.1 0.0 0.6 6.1 87.7 + 3 17 33 140 128 8.0 4.8 0.7 0.0 1 .2 5.3 88.1 -4 16 28 96 91 5.0 6.0 4.2 0.6 1.8 8.3 79.1 -5 16 25 109 106 3.6 11 .2 15.2 ' 0.0 22.4 16.4 34.7 -6 17 28 122 117 4.4 11.4 27.0 13.2 8.8 10.9 28.7 -7 16 29 116 106 9.3 7.7 49.6 3.4 18.7 4.7 15.9 -8 17 29 116 111 3.7 17.4 36. 3 13.4 8.5 5.4 18.9 -9 17 28 n o 100 3.6 4.4 1 .7 0.0 0.0 0.6 94.0 + 10 17 26 102 96 4.3 6.4 0.0 0.7 0.0 3.2 89.2 + * Maximum number of s i r e s and dams was 18 and 36 r e s p e c t i v e l y . to Table 4 - P e r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 10 Weekly Hatches of the F-J Progeny from the NHxCP Matings on a Normal R i b o f l a v i n D i e t . Hatch Number Number* S i r e s Dams T o t a l Eggs T o t a l F e r t i 1 e Mean Dam Perc e n t I n f e r t i 1 e E a r l y Dead Average Late Dead(-) P e r c e n t Late Dead(+) of F e r t i l e Dead i n S h e l l P i p Hatch 1 6 15 75 51 33.7 16.7 1.7 0.0 8.3 0.0 73.3 2 5 14 65 55 14.0 6.1 0.0 0.0 8.4 0.0 85 .5 3 6 17 76 70 8.2 6.4 0.0 0.0 2.8 0.0 90 .8 4 6 15 51 45 12.0 3.9 0.0 6.3 0.0 0.0 89.8 5 6 14 56 50 11.4 4.6 2.8 0.0 2.8 0.0 89.8 6 6 14 59 58 1 .1 14.1 2.2 2.2 11 .8 0.0 69.7 7 6 16 60 55 8.5 10.2 5.7 0.0 0.0 1 .9 82.2 8 6 15a 57 47 23.6 13.0 1.1 4.2 1 .1 1 .1 79.5 9 6 15 56 54 6.9 20.1 2.8 0.0 2.8 1 .1 73.2 10 6 15 59 56 5.1 8.1 0.0 1 .9 8.5 2.8 78.8 Maximum number of s i r e s and dams was 6 and 18 r e s p e c t i v e l y . Number of dams reduced by 1 f o r f e r t i l e eggs. Table 5 - P e r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 16 Weekly Hatches of the F 2 Progeny from the F]xF-| Matings on a R i b o f l a v i n (R) Normal ( + ) or D e f i c i e n t (-) D i e t . Average P e r c e n t of F e r t i l e Number* Mean Dam Hatch T o t a l T o t a l P e r c e n t E a r l y Late Dead R Number S i r e s Dams Eggs F e r t i l e I n f e r t i l e Dead Dead i n S h e l l P i p Hatch D i e t b 1 10 18 81 75 7.8 16.3 9.4 6 .2 1 .1 67 .0 + 2 11 19 81 77 5.4 5.2 10.9 1 .8 6 .8 75 .4 + 3 11 19 84 84 0.0 5.4 4.3 1 .1 0 .9 88 .3 + 4 10 18 78 78 0.0 12.1 4.6 3 .3 2 .5 77 .4 + 5 10 18 74 74 0.0 3.9 2.2 0 .0 0 .0 93 .9 + 6 8 16 71 71 0.0 9.4 7.8 2 . 1 1 .3 79 .5 + 7 9 17 67 67 0.0 13.9 1 .5 2 .9 0 .0 81 .7 -8 7 15 71 71 0.0 9.3 3.3 1 .3 2 .4 83 .6 -9 8 16 73 71 6.3 17.7 14.2 5 .8 7 .6 54 .7 -10 ga 17a 60 54 9.8 15.5 41 .4 5 .9 10 .9 26 .3 -11 8a 15b 59 54 15.6 13.7 16.9 9 .6 1 .5 58 .2 + 12 8a 17a 67 63 10.3 6.8 2:8 1 .3 0 .0 89 .2 + 13 9 17 71 70 2.0 9.4 3.5 0 .0 0 .0 88 . 2 + 14 9 17 62 61 2.0 5.0 1 .5 1 .2 2 .9 89 .4 + 15 9 17 67 67 0.0 7.1 0.0 9 .4 1 .2 82 .4 + 16 8 16 62 62 0.0 1.6 2.8 6 .3 3 .1 86 .3 + Maxi mum number of s i r e s and dams was 11 and 19 r e s p e c t i v e l y . Number of s i r e s or dams reduced by 1 f o r f e r t i 1 e eggs Number of dams reduced by 2 f o r f e r t i l e eggs Table 6 - P e r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 16 Weekly Hatches of the F£ Progeny from the F^ x F i Matings on a R i b o f l a v i n (R) Normal (+) or D e f i c i e n t (-) D i e t . Average P e r c e n t of F e r t i l e Number* Mean Dam Hatch T o t a l T o t a l P e r c e n t E a r l y Late Dead R Number S i r e s Dams Eggs F e r t i l e I n f e r t i l e Dead Dead i n S h e l l P i p Hatch D i e t 1 7 11 46 46 0.0 2 6 9 41 38 6.7 3 7 11 52 52 0.0 4 7 11 49 49 0.0 5 7 11 50 50 0.0 6 7 11 44 43 1 .8 7 5 9 45 45 0.0 8 5 9 43 43 0.0 9 5 9 40 39 2.2 10 4 8 41 41 0.0 11 4 8 34 33 2.5 12 6 10 40 40 0.0 13 6 10 41 41 0.0 14 6 10 40 39 5.0 15 6 10 42 42 0.0 16 6 10 43 43 0.0 0.0 2.3 7.1 4.1 86.5 + 3.7 3.7 7.4 4.1 81 .1 + 13.5 0.0 0.0 0.0 86.5 + 14.5 2.3 2.3 5.5 75. 5 + 9.4 0.0 1 .8 2.3 86.5 + 8.2 0.0 0.0 1 .8 90.0 + 0.0 4.4 2.2 2.2 91 .1 -11.7 4.4 0.0 4.4 79.4 -2.2 13.9 5.0 6.7 72.2 -9.2 33.1 21 . 5 7.5 28.8 -3.1 26 .9 0.0 6.3 63.8 + 12.3- 5.0 0.0 5.0 77.7 + 12.8 6.7 4.5 7.0 69.0 + 3.3 3.3 6.5 12.5 74.3 + 6.0 0.0 2.5 4.0 87.5 + 7.8 0.0 2.5 6.5 83.2 + * Maximum number of s i r e s and dams was 7 and 11 r e s p e c t i v e l y . T a b l e 7 Pe r c e n t H a t c h a b i l i t y and M o r t a l i t y f o r 16 Weekly Hatches of the Backcross Progeny from the CPxFi Matings on a R i b o f l a v i n (R) Normal (+) or D e f i c i e n t (-) D i e t . Average P e r c e n t of F e r t i l e Number* Mean Dam Hatch T o t a l T o t a l P e r c e n t E a r l y L ate Dead R Number S i r e s Dams Eggs F e r t i l e I n f e r t i l e Dead Dead i n S h e l l P i p Hatch D i e t 1 18b 18b 74 57 26. 7 2 16 16 68 57 16.1 3 18 18 78 78 0.0 4 18 18 83 79 5.9 5 17 17 74 73 1 .5 6 17 17 68 66 2.4 7 16 16 65 61 6.3 8 12 12 56 52 6.7 9 12 12 53 44 18.3 10 13 13 57 53 6.2 11 143 14a 54 49 12.9 12 16 16 64 58 9.4 13 15a 15a 59 57 6.7 14 15 15 61 58 5.0 15 13 13 50 50 0.0 16 16 16 65 65 0.0 9.8 1.3 1.3 3.1 84.6 + 9.1 2.5 5.0 7.5 76 .0 + 12.5 1 .1 1 .1 5.5 79.8 + 8.1 6.9 3.6 1.1 80.2 + 11 .2 3.1 0.0 4.3 81 .4 + 10.9 7.3 0.0 3.4 78.4 + 12.5 4.9 7.3 4.2 71 .1 -15.3 0.0 6.1 5.6 73.1 -6.3 20. 6 3.3 3.5 66.4 -7.2 35.9 3.5 11.7 41 .8 -7.3 17.2 5.8 9.9 63.7 + 7.5 3.1 1 .0 3.8 84.6 + 6.7 0.0 0.0 4.4 88.9 + 19.8 6.0 1 .1 0.0 73.1 + 15.8 0.0 0.0 9.0 75.3 + 4.2 4.6 5.9 5.7 79.6 + Maximum number of s i r e s or dams was 19. Number o f s i r e s or dams reduced by 1 f o r f e r t i l e eggs. Number of s i r e s or dams reduced by 2 f o r f e r t i l e eggs. Table 8 - Percent Hatchability and Mortality for 16 Weekly Hatches of the Backcross Progeny from the CPxR Matings on a Riboflavin (R) Normal (+) or Deficient (-) Diet. 1 N u m b e r * Average Percent of F e r t i l e Mean Dam Hatch Total Total Percent Early Late Dead R Number Sires Dams Eggs F e r t i l e I n f e r t i l e Dead Dead in Shell Pip Hatch Diet 1 8 8 38 26 29.4 12.5 0.0 4.2 8.3 75.0 + 2 8 8 33 29 11.3 22.7 16.7 0.0 8.3 52.2 + 3 7 7 33 33 0.0 32.1 0.0 0.0 0.0 67.9 + 4 8 8 34 34 0.0 10.0 9.4 0.0 0.0 80.6 + 5 8 8 39 39 0.0 22.9 0.0 0.0 2.5 74.6 + 6 9 a ga 37 36 11.1 18.8 0.0 0.0 5.0 76.3 + 7 8 8 34 34 0.0 18.8 0.0 0.0 2.5 78.8 -8 9 9 44 43 2.2 14.0 1.6 0.0 4.1 80.3 -9 8 8 36 34 5.0 9.8 5.0 0.0 4.2 . 81.0 -10 8 8 34 33 2.5 19.2 18.1 6.3 10.6 45.8 -11 9 9 40 40 0.0 33.7 9.6 1.9 2.8 52.0 + 12 9 9 41 41 0.0 30.6 0.0 • 0.0 0.0 69.4 + 13 9 9 38 38 0.0 18.0 0.0 7.2 16.1 58.7 + 14 8 8 34 34 0.0 8.1 2.1 0.0 3.1 86.7 + 15 8 8 33 33 0.0 25.8 0.0 2.5 0.0 71.7 + 16 8 8 33 33 0.0 17.1 0.0 0.0 2.5 80.4 + Maximum number of sires or dams was 9. Number of sires or dams reduced by 1 for f e r t i l e eggs. Table 9 - Percent Hatchability and Mortality for 16 Weekly Hatches of the Backcross Progeny from the F,xCP Matings on a Riboflavin (R) Normal (+) or Deficient (-) Diet. ' Average Percent of F e r t i l e Number* Mean Dam Hatch Total Total Percent Early Late Dead R Number Sires Dams Eggs F e r t i l e I n f e r t i l e Dead Dead in Shell Pip Hatch Diet 1 13 22b 75 60 28.2 2 13 21a 79 72 11.8 3 13 24 98 97 1.4 4 13 24 91 90 0.8 5 12 21 77 73 4.8 6 13 19 59 58 2.6 7 10 13 42 41 3.3 8 9 10 38 36 5.0 9 9 10 27 26 2.0 10 7 7 24 24 0.0 11 8 8 23 22 3.1 12 69 1 4 a 42 37 14.3 13 10 14 39 36 7.7 14 10 12 37 37 0.0 15 11 13 43 43 0.0 16 10 11 39 38 2.3 27.6 2.8 1.8 0.0 67.8 + 12.2 9.2 8.9 2.5 67.2 + 17.2 4.8 3.5 3.3 71.3 + 17.3 8.0 1.4 2.4 70.9 + 25.0 4.8 0.0 1.2 69.0 + 30.0 3.1 3.9 0.0 63.0 + 14.6 9.6 5.0 3.8 66.9 -10.0 13.3 3.3 5.0 68.3 -13.3 37.5 10.0 0.0 39.2 -42.9 54.3 2.9 0.0 0.0 -12.5 22.7 8.8 6.3 49.8 + 28.3 0.0 12.8 4.5 54.4 + 32.0 1.8 7.1 0.0 59.0 + 19.7 0.0 0.0 0.0 80.3 + 15.4 0.0 6.4 3.8 74.4 + 6.1 2.3 4.5 0.0 87.1 + Maximum number of sires and dams was 13 and 24 respectively. a Number of sires or dams reduced by 1 for f e r t i l e eggs, k Number of sires or dams reduced by 3 for f e r t i l e eggs. Table 10 - Percent Hatchability and Mortality for 16 Weekly Hatches of the Backcross Progeny from the F^xCP Matings on a Riboflavin (R) Normal (+) or Deficient (-) Diet. 1 Average Percent of F e r t i l e Number* Mean Dam Hatch Total Total Percent Early Late Dead R Number Sires Dams Eggs F e r t i l e I n f e r t i l e Dead Dead in Shell Pip Hatch Diet 1 7 12 44 39 11.3 23.3 5.8 2.1 0.0 68.8 + 2 7 11 36 34 4.1 11.4 9.1 0.0 17.0 62.6 + 3 " 7 11 43 43 0.0 38.0 4.5 2.3 0.0 55.2 + 4 7 11 45 45 0.0 30.2 14.7 0.0 0.0 55.2 + 5 7 11 41 40 2,3 32.6 4.5 4.5 4.1 54.2 + 6 7 10 39 35 10.7 31.7 7.5 5.8 10.8 44.2 + 7 7 103 24 23 10.0 7.4 5.9 13.0 3.7 70.0 -8 4 4 17 15 10.0 30.8 15.0 13.3 10.0 30.8 -9 2 2 7 7 0.0 33.3 66.7 0.0 0.0 0.0 -10 2 2 8 8 0.0 73.3 26.7 0.0 0.0 0.0 -11 4 a 5a 12 11 20.0 18.8 18.8 0.0 6.3 56.3 + 12 6a gb 21 18 33.3 27.8 11.1 0.0 0.0 61.1 + 13 6a ga 27 23 21.3 7.3 12.5 7.3 4.2 68.8 + 14 7 9 33 31 5.6 14.1 2.8 9.3 2.2 71.7 + 15 7 8 27 26 4.2 12.5 12.5 12.5 0.0 62.5 + 16 7 9 31 31 0.0 23.5 0.0 11.1 0.0 65.4 + Maximum number of sires and dams was 7 and 12 respectively. Number of sires or dams reduced by 1 for f e r t i l e eggs. Number of sires of dams reduced by 3 for f e r t i l e eggs. Table 11 - Percent C l e f t Palate (CP) Progeny and Total Observations (0) for the F 2 Progeny of F-.XF-, Matings on a Riboflavin Normal (+) and Deficient (-) Diet. Mating Identification H a t c h e s Original Fl Fl 1 -6(+) 7-10( -) 11 -16(+) CP NH 0 CP 0 CP 0 CP 60 65 89 543 25 0.0 16 0.0 24 0.0 544 16 0.0 10 0.0 26 0.0 60 66 88 546 16 0.0 2 0.0 9 0.0 37 69 87 547 17 5.9(1)* 6 0.0 11 0.0 . 548 12 0.0 1 0.0 1 0.0 46 71 86 550 21 0.0 16 0.0 26 0.0 44 89 84 553 18 0.0 16 0.0 15 0.0 554 24 0.0 7 0.0 8 0.0 05 98 83 555 24 0.0 13 0.0 22 0.0 556 30 0.0 17 5.9(1). 22 4.5(1) 01 97 82 558 13 0.0 0 - 12 0.0 38 63 80 562 30 0.0 22 0.0 28 0.0 21 77 79 563 24 8.3(2) 17 11.8(2) 16 6.3(1) 564 27 3.7(1) 17 0.0 26 0.0 04 74 77 567 10 10.0(1) 2 0.0 11 0.0 568 26 0.0 14 14.3(2) 20 0.0 47 92 76 569 29 0.0 18 5.6(-l) 26 3.3(1) 570 28 0.0 18 5.6(1) 23 0.0 Total 390 (5) 212 (7) 326 (3) Mean 1.6 2.5 0.8 * Number of c l e f t palates. Table 12 - Percent C l e f t Palate (CP) Progeny and Total Observations (0) for the F' Progeny of F'xF' Matings on a Riboflavin Normal (+) and Deficient (-) 1 Diet. 1 1 Mating Ide n t i f i c a t i o n H a t c h e s Original l-6(+) 7-10(-) 11-16(+) NH CP Fl F l 0 CP 0 CP 0 CP 123 471 90 542 26 0.0 10 0.0 20 0.0 469 91 450 18 0.0 16 0.0 23 0.0 499 25 0.0 19 0.0 14 0.0 124 475 95 442 26 0.0 15 13.3(2)* 29 0.0 441 28 0.0 20 0.0 27 3.7(1) 452 96 440 25 0.0 18 22.2(4) 23 0.0 439 28 0.0 20 0.0 27 0.0 125 457 93 445 13 0.0 1 _ 0 _ 461 94 444 15 0.0 0 - 15 0.0 127 474 92 448 23 0.0 19 5.3(1) 22 0.0 447 . 0 - 18 16.7(3) 21 0.0 Total 227 (0) 155 (10) 221 (1) Mean 0.0 6.4 0.4 Number of c l e f t palates. Table 13 - Percent C l e f t Palate (CP) Progeny and Total Observations (0) for the Backcross Progeny of CPxF, Matings on a Riboflavin Normal (+) and Deficient (-) Diet. Mating Identification H a t c h e s Original l-6(+) 7-10(-) 11-16(+) CP NH CP Fl 0 CP 0 CP 0 CP 60 65 3 473 26 0.0 15 0.0 24 4.2(1) 4 472 13 0.0 2 0.0 13 0.0 66 5 471 4 0.0 9 0.0 6 16.7(1) 6 470 23 0.0 4 50.0(2) 9 0.0 37 69 7 469 24 4.2(1)* 0 - 15 6.7(1) 46 71 9 467 28 10.7(3) 20 30.0(6) 19 10.5(2) 44 89 11 465 2 50.0(1) 2 0.0 3 0.0 05 98 12 464 20 5.0(1) 19 5.3(1) 23 4.3(1) 01 97 15 461 23 0.0 5 0.0 0 -38 63 18 458 12 8.3(1) 1 0.0 8 25.0(2) 21 77 19 457 18 0.0 1 0.0 19 5.3(1) 20 456 23 8.7(2) 17 29.4(5) 22 4.5(1) 04 74 23 453 15 6.7(1) 13 0.0 18 0.0 24 452 22 27.3(6) 15 33.3(5) 21 9.5(2) 47 92 25 451 26 15.4(4) 21 38.1(8) 24 8.3(2) 23 80 21 455 22 9.1(2) 18 5.6(1) 28 3.6(1) 22 454 27 3.7(1) 22 9.1(2) 29 0.0 42 67 16 460 18 0.0 3 0.0 8 0.0 17 459 15 0.0 7 0.0 9 0.0 Total 361 (23) 194 (30) 298 (15) Mean 7.8 11.2 5.5 Number of c l e f t palate. CO Table 14 - Percent Cleft Palate (CP) Progeny and Total Observations (0) for the Backcross Progeny of CPxH Matings on a Riboflavin Normal (+) and Deficient (-) Diet. Mating Identification Original l-6(+) 7-10(-) 11-16(+) NH CP CP Fl 0 CP 0 CP 0 CP 123 471 2 474 23 8.7(2)* 20 5.0(1) 24 8.3(2) 469 1 475 12 0.0 8 0.0 18 0.0 124 475 31 438 28 7.1(2) 11 18.2(2) 15 6.7(1) 452 32 437 19 5.3(1) 19 21.1(4) 12 8.3(1) 125 457 28 478 9 0.0 8 0.0 16 0.0 29 479 23 0.0 13 0.0 20 0.0 461 30 480 19 0.0 18 0.0 25 0.0 127 474 26 476 19 31.6(6) 15 26.7(4) 25 20.0(5) 27 477 29 0.0 10 40.0(4) 6 16.7(1) Total 181 (11) 122 (15) 161 (10) Mean 5.9 12.3 6.7 Number of c l e f t palates. Table 15 - Percent C l e f t Palate (CP) Progeny and Total Observations (0) for the Backcross Progeny of F,xCP Matings on a Riboflavin Normal (+) and Deficient (-) Diet. Mating Ide n t i f i c a t i o n u . . n a t c n e s Original Fl CP l-6(+) 7-10C-) 11 -16(+) CP NH 0 CP 0 CP 0 CP 60 65 89 538 16 0.0 1 0.0 12 16.7(2) 537 3 0.0 0 - 0 -66 88 536 17 0.0 10 40.0(4) 13 7.7(1) 535 3 0.0 0 - 1 0.0 37 69 87 534 . 22 4.5(1)* 3 0.0 18 5.6(1) 533 19 10.5(2) 15 20.0(3) ' 15 0.0 46 71 86 532 21 0.0 13 15.4(2) 4 25.0(1) 531 21 9.5(2) 0 - 9 l l . K D 44 89 84 528 15 0.0 4 0.0 11 0.0 527 20 0.0 1 0.0 0 -05 98 83 526 9 11.1(1) 0 - 6 16.7(1) 525 24 4.2(1) 12 8.3(1) 23 0.0 01 97 82 524 14 0,0 0 - 0 -523 20 0.0 1 0.0 12 0.0 38 63 80 520 7 0.0 0 - 4 0.0 519 10 10.0(1) 0 - 0 -21 77 79 518 17 5.9(1) 0 - 0 -517 12 0.0 9 11.1(1) 3 0.0 04 74 77 514 17 5.9(1) 5 0.0 13 0.0 47 92 76 512 5 20.0(1) 6 66.7(4) 6 0.0 511 6 50.0(3) 1 100.0(1) 0 -23 80 78 516. 17 5.9(1) 11 27.3(3) 3 0.0 515 22 0.0 6 0.0 3 0.0 42 67 81 522 21 19.0(4) 0 - 10 -Total 358 (19) 98 (19) 171 (7) Mean 6.5 19.2 4.6 Number of c l e f t palates. Table 16 - Percent C l e f t Palate (CP) Progeny and Total Observations (0) for the Backcross Progeny of RxCP Matings on a Riboflavin Normal (+) and Deficient (-) Diet. Mating Ide n t i f i c a t i o n H a t c h e s Q r 1 g i n a l 1-6Q-) 7-10(-) 11-16(+) NH CP Fl CP 0 CP 0 CP 0 CP 123 471 90 539 15 6.7(1)* 12 16.7(2) 13 0.0 469 91 391 18 0.0 0 - 5 20.0(1) 392 21 4.8(1) 2 0.0 22 0.0 124 475 95 399 18 11.1(2) 11 18.2(2) 21 0.0 400 9 0.0 0 - 2 50.0(1) 452 96 401 3 66.7(2) 4 25.0(1) 2 0.0 402 5 0.0 1 0.0 6 16.7(1) 125 457 93 395 16 0.0 1 0.0 6 0.0 395 18 0.0 3 0.0 16 0.0 461 94 397 20 0.0 3 0.0 5 20.0(1) 127 474 92 393 23 34.8(8) 2 0.0 13 23.1(3) 394 8 37.5(3) 0 - 7 28.6(2) Total 174 (17) 39 (5) 118 (9) Mean 13.5 6.7 13.2 * Number of c l e f t palates. AM Table 17 - Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculation of Sire Variances of the CP x NH Matings Riboflavin Diet Normal Deficient Arc-sine* CP NH C l e f t C l e f t >ire Dam Palate Total Palate Total Normal Deficiei 1 1 0 10 0 16 8.12 9.54 2 0 14 0 5 2 1 0 16 0 12 6.92 14.24 2 0 17 1 11 3 1 0 17 0 8 7.80 8.02 2 0 10 0 20 4 1 0 18 0 8 6.82 8.18 2 0 16 0 18 5 1 0 8 0 21 8.02 6.22 2 0 20 0 20 6 1 0 20 1* 14 6.38 6.68 2 0 19 0 23 7 1 0 15 1** 21 7.24 8.25 2 0 15 ] ** 7 8 1 0 16 0 9 9.06 8.48 2 0 6 0 13 9 1 0 10 0 24 11.02 6.76 2 0 4 0 13 10 1 0 18 0 10 7.06 13.20 2 0 14 0 2 11 1 0 8 0 3 9.48 11.88 2 0 9 0 10 12 1 0 13 0 23 12.69 7.14 2 0 2 0 11 13 1 0 16 0 9 7.90 10.64 2 0 10 0 5 14 1 0 12 1** 21 7.64 7.46 2 0 15 0 10 15 1 0 12 0 7 9.58 11.20 2 0 6 0 5 16 1 0 9 0 23 11.25 5.89 2 0 4 0 0 17 1 1** 17 0 14 6.56 6.42 2 0 20 0 28 Sire Variance 3.316 6.469 Arc-sine transformations for small samples after Mosteller and Youtz, 1961. * Suspect c l e f t palate removed from calculations of s i r e variance. A18 Table 18 - Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculation of Sire Variance of the NH x CP Matings. Normal Diet NH CP C l e f t Sire Dam Palate Total Arc-sine* 1 1 0 19 5.18 2 0 35 3 0 44 2 1 0 30 8.72 2 1 24 3 0 16 3 1 ]** 32 5.18 2 ]** 28 3 0 30 4 1 0 12 8.34 2 0 9 3 0 13 5 1 0 21 5.29 2 0 30 3 0 39 6 1 0 38 4.79 2 0 37 3 0 31 Sire 3.162 Variance * See Appendix Table 17. ** Suspect c l e f t palate removed from ca l c u l a t i o n of s i r e variance. A19 Table 19 - Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculations of Sire Variances of the F 0 from F-, x F-, Matings. L 1 1 Riboflavin Diet Normal Deficient Arc-sine* >ire Fl Dam C l e f t Palate Total C l e f t Palate Total Normal Deficient 1 1 0 33 0 16 5.02 8.68 2 0 31 0 7 2 1 0 47 0 16 4.15 7.02 3 1 0 51 1 18 6.68 16.10 2 1 55 1 18 4 1 0 25 0 2 5.65 17.63 5 1 0 49 0 16 4.20 7.90 2 0 43 0 10 6 1 1 52 1 17 6.88 12.15 2 0 46 0 13 7 1 3 40 2 17 13.26 . 14.30 2 1 52 0 17 8 1 0 58 0 22 3.62 6.02 9 1 0 25 0 0 5.65 -10 1 1 21 0 2 9.56 20.81 2 0 46 2 14 11 1 0 13 0 1 10.36 16.80 2 1 28 0 6 Sire Variance 9.175 26.375 * See Appendix Table 17. A 20 Table 20 - Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculation of Sire Variances of the FX from F-! x F-j Matings. c 1 1 Riboflavin Diet Normal Deficient Arc-sine* Fl Fl C l e f t . C l e f t Sire Dam Palate Total Palate Total Normal Deficient 1 1 0 46 0 10 • 4.19 8.77 2 1 0 41 0 16 4.50 6.74 2 0 39 0 19 3 1 0 45 1 19 5.20 20.52 2 0 21 3 18 4 1 0 13 0 0 7.75 -5 1 0 30 0 0 5.17 -6 1 0 55 2 15 7.08 14.74 2 1 55 0 20 7 1 0 48 4 18 3.94 17.80 2 0 55 0 20 Sire Variance 2.141 34.196 See Appendix Table 17. /421 Table 21 - Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculation of the Sire Variance of the Backcross Progeny from CP x F-, Matings. Riboflavin Diet Normal Deficient Arc-sine* CP F, C l e f t C l e f t Sire Dam Palate. Total Palate Total Normal Deficient 1 1 0 23 0 5 5.89 12.05 2 1 8 43 5 15 26.06 35.87 3 1 1 33 0 13 11.96 7.75 4 1 3 20 0 1 24.04 22.50 5 1 0 26 0 3 5.55 15.00 6 1 0 24 0 7 5.77 10.35 7 1 2 42 1 19 13.89 15.68 8 1 1 39 0 1 11.01 22.50 9 1 3 45 5 16 15.97 34.64 10 1 6 50 8 21 20.90 38.42 11 1 1 10 0 9 21.39 9.22 12 -1 0 32 2 4 5.01 45.00 13 1 1 50 0 15 9.74 7.24 14 1 0 26 0 2 5.55 17.63 15 1 1 6 0 2 27.26 17.63 16 1 5 47 6 20 19.77 33.79 17 1 1 56 2 22 7.69 19.16 18 1 3 50 1 18 15.15 16.10 19 1 2 39 0 0 14.41 -Sire Variance 65.371 233.292 * See Appendix Table 17. All Table 22 - Arc-sine Values of Percent C l e f t Palate Progeny Used f o r Calculations of Sire Variances of the Backcross Progeny from CP x F-j Matings. Riboflavin Diet Normal Deficient Arc-sine* CP F^ C l e f t C l e f t Sire Dam Palate Total Palate Total Normal Deficient 1 1 3 43 2 11 16.34 27.05 2 1 2 32 4 19 15.90 28.28 3 1 0 25 0 8 5.65 9.74 4 1 0 43 0 13 4.34 7.75 5 1 0 45 0 18 4.34 6.63 6 1 11 44 4 15 31.99 31.99 7 1 1 35 4 10 11.61 39.74 8 1 4 47 1 21 14.93 14.93 9 1 0 31 0 8 5.09 9.74 Sire Variance 81.108 151.854 * See Appendix Table 17. A 23 Table 23 - Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculation of Sire Variances of the Backcross Progeny from F, x CP Matings. Riboflavin Diet * Normal Deficient Arc-sine F1 CP C l e f t C l e f t Sire Dam Palate Total Palate Total Normal Deficient 1 1 0 25 0 6 9.68 21.87 2 1 25 3 11 2 1 2 35 3 16 14.81 20.97 2 2 39 0 3 3 1 0 20 0 1 5.93 17.89 2 0 26 0 • 4 4 1 3 30 0 0 16.65 24.89 2 1 25 2 13 5 1 3 6 1 2 32.72 49.20 2 1 11 4 6 6 1 1 30 4 10 12.91 39.74 2 0 4 0 0 7 1 0 3 0 0 16.00 22.50 2 2 28 0 1 8 1 2 14 0 0 17.02 19.60 2 1 47 1 12 9 1 0 15 1 9 11.90 22.50 2 1 17 0 0 10 1 1 10 0 0 15.08 -2 0 10 0 0 11 1 4 31 0 0 21.99 -12 1 0 32 5 1 6.25 22.50 2 0 14 0 1 13 1 1 30 0 5 12.53 12.05 Sire Variance 48.144 109.49] * See Appendix Table 17. /424 Table 24 - Arc-sine Values of Percent C l e f t Palate Progeny Used for Calculation of Sire Variances of the Backcross Progeny from F\ x CP Matings. Riboflavin Diet Normal Deficient Arc-sine* F-J CP C l e f t C l e f t Sire Dam Palate Total Palate Total Normal Deficient 1 1 1 28 2 13 12.96 24.89 2 1 1 25 0 0 12:i0 17.63 2 1 43 0 2 3 1 11 36 0 2 34.88 17.63 2 5 15 0 0 4 1 0 22 0 1 5.41 18.75 2 0 35 0 3 5 1 1 25 0 3 13.71 15.00 6 1 2 40 2 11 16.92 27.05 2 1 12 0 0 7 1 2 3 1 3 36.47 30.00 2 1 11 0 1 Sire Variance 143.070 32.380 * See Appendix Table 17 . A 25 Table 25 - Mean Sire Variances (Arc-sine) and C l e f t Palate (CP) and New Hampshire (NH) Mean Line Differences Used for the Calculation of Number of C l e f t Palate Loci Different Between the CP and NH Line, After Wright (1921, 1934) Riboflavin Diet Matings Normal Deficient CPxNH(F1) NHxCP(Fj) Sire Mean 3.316 3.162 3.239 6.469 6.469 F-jxF* (Fp Sire Mean 9.175 2.141 5.658 26.375 34.196 30.286 CPxF1 CPxF^ F^CP F]xCP Sire Mean 65.371 81.108 48.144 143.070 84.423 233.292 151.854 109.497 32.380 131.754 C l e f t Palate Line Mean Percent Generation 1 29.7 Generation 2 47.4 Line Difference (D) F 2 = 33.02 - 0 • = 33.02 Backcross = [1/3(33.02)+2/3 (4351)]-0 = 40.02 Arc-sine 33.02 43.51 Figure 1 Sketches used for standard scores of c l e f t palates i n the F 9 and backcross generations. 

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