GENEHCIWUUWMJERS{M: LXSWEEWHmflRENENfilleHEtfificK Thesis for the Degree of PM). NHCHKUWlSIKTElfluVEmyTV EKfiNARDIEEEENOS 1971 WWW . 4' I 3"; 3;? «u‘ l. a- g a a) -—s i. . o_ ". ‘1- I '1 '1‘ v ‘ ' V’s . 1;, _. _‘ . . ‘ fig), "190 let-{is 36,51,33 SI 2 Dim; want}: t". This is to certify that the thesis entitled GENETIC PARAMETERS OF LYSINE REQUIREMENT BY THE CHICK presented by Howard L . Enos has been accepted towards fulfillment of the requirements for Ph.D. Jegreein Poultry Science- %%%w Major professor Date May 19, 1971 04639 . .. -3...» ABSTRACT GENETIC PARAMETERS OF LYSINE REQUIREMENT BY THE CHICK BY Howard Lee Enos Among the common plant protein sources of the world, only soybean meal is rich in lysine. When soybean oil meal is unavailable or it is not economically feasible to include it in the growth diet of monogastrics such as chicks, rats and man, lysine is the first- limiting amino acid for growth. This experiment was designed in an attempt to improve the efficiency and growth rate of chicks on low lysine diets. A bi-directional selection experiment was conducted to evaluate the magnitude of genetic mechanisms regulating lysine requirement. A heterogenic base population of egg-type chickens was randomly separated into four lines and each line was closed with regard to matings for each subsequent generation. Data from four generations plus the base population were evaluated for each line selected for either high or low growth rate in a lysine deficient dietary environ- ment. A third line identified as a natural selection line was main- tained by random matings among survivors of those fed the lysine deficient diet. The fourth line, a random control line, was reproduced by random matings among individuals fed only the control diet. The control diet utilized in this experiment was formulated to be adequately balanced with all known nutrients for optimum growth of chicks and it had a 1.0 percent lysine level. The deficient diet was identical in composition to the control except that it contained only 0.5 percent lysine. Full sibs of the same sex were randomly divided with regard to opportunity to express their growth potential in each of the two nutri- tional environments. Juvenile growth rate (measured as the gain from one day to three weeks of age) was analyzed for diet, sex, generation and line differences. Realized heritability computed as deviations from the random control line as measured in the lysine deficient environment was 1. 33 for the line under natural selection, .10 for the high growth rate selected line and -.19 for the low growth rate selected line. The component of variance procedure for estimating heritability provided slightly higher estimates than those computed as realized heritability. In the 0.5 percent lysine deficient environment average heritability estimates were .23 from the sire source of variance, .26 from the dam and .25 from the combined sire plus dam component of variance. Contemporary full-sibs were fed the 1.0 percent lysine control diet and heritability estimates from the component of variance method were .35, .39 and .37, respectively, for sire, dam and combined sire plus dam sources of variance. Reciprocal cross line progeny were analyzed in comparison to pure line progeny from the second, third and fourth generations. A highly significant diet by line interaction effect was shown and the cross line progeny always grew best in the 1.0 percent lysine control environment. For two of the three generations, pure line progeny grew better than the cross line progeny in the 0.5 percent lysine environment. Cross line progeny showed a positive heterosis and it was calculated to average 17 percent in the nutritionally adequate environment with (1.0% lysine) and a negative heterosis averaging five percent for cross line progeny in the deficient environment with 0. 5 percent lysine available from the ration. Results of this short-range experiment to alter the lysine re- quirement of chicks for growth indicate that mass selection as a technique was relatively inefficient. However, heritability estimates and the expression of heterosis among cross line progeny indicate the need for further research using large numbers per line, applying greater selection pressure and the utilization of a more rapidly grow- ing broiler type stock. In each generation, mortality was higher for chicks fed the 0.5 percent lysine diet than for those fed the 1.0 percent lysine diet. Males exhibited a higher mortality rate on the 0. 5 percent lysine diet while females had the highest mortality rate when grown in the 1.0 percent lysine environment. From an analysis of differences in fertility, hatchability, egg production and adult livability rates, it was apparent that the 0. 5 percent lysine diet fed during the early growth period, one day to three weeks of age, had no latent consequences or long - range manife stations . GENETIC PARAMETERS OF LYSINE REQUIREMENT BY THE CHICK By Howard Lee Enos A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Poultry Science 1971 "l 'x "'3 ”‘3 .' .' I - 1 "x v ./ .J ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to all those who assisted him with understanding, guidance, inspiration, and financial support during the tenure of his graduate program. The author is grateful to his graduate committee: Dr. Theo H. Coleman, major professor; Dr. William T. Magee, for his consul- tation on the statistical and genetic analysis; and to others of the committee, Dr. Howard C. Zindel, Dr. Mason E. Miller, and Dr. Cal Flegal, for their guidance in a program of study and the develop- ment of this dissertation. Special thanks are extended to Dr. Harry C. Muller and Dr. Robert E. Moreng of Colorado State University for their cooperation with project supervision and data collections during the author's absence from the laboratory. The author desires to recognize the cooperative attitude of both Colorado and Michigan State Universities' Experiment Stations, per- mitting the release of data by the former to be used by the author in this dissertation. In addition, thanks to Michigan State University for a graduate fellowship and to the Department of Poultry Science, Michigan State University, for office accommodations and a computer Science ii budget and to Colorado State University for significant computer science allocations. Invaluable assistance was rendered by Mr. Forest Nelson and Miss Patricia Biondini with computer program- ming. Acknowledgment and thanks also to the Ralston Purina Company, St. Louis, Mo. , who annually performed the routine amino acid analysis of the experimental diets. Appreciation is expressed to Hoffmann-La Roche, Inc. , Nutley, New Jersey, for many vitamins utilized in growth trial and laying hen diets and to Merck Company, Rahway, New Jersey, for methionine utilized in the diets. Also recognized are the generous supplies of corn-gluten meal for the growth diet from Clinton Corn Processing Co. , Clinton, Iowa, and Penick 8: Ford Limited, Cedar Rapids, Iowa. The author also expresses heartfelt appreciation to his family and to persons too numerous to name involved in duties of experi- mental trial work. A special thanks to Clark F. Overton, Avian Science Research Farm Superintendent, laboratory technicians, re- cording secretaries, and to others involved in key punching, typing, proofreading and tasks related to this study. Chapte r I II III TAB LE OF CONTENTS Introduction . . . . . ........... . . . . . . Review of Literature ...... . ....... . . . Materials and Methods . ............. . . A. Experimental Design. ........... . . B. Nutritional Environment. . . . . . . . . . . . C. Genetic Material. . . . ..... . . . . . . . D. Selection Procedure . . . . . . . . . . . . . . E. Other Measurements... . . . . . . . . . . . . F. Physical Environment . . . . . . . . . . . . . G. Statistical Procedure . . . . . . . . ..... Results and Discussion . ..... . ...... . . . Conclusions..... ........... Bibliography . . . ..... . . . ...... . . . . . iv Page l4 14 17 20 21 27 27 29 37 98 101 Table 10 LIST OF TAB LES Composition of 20 percent protein chick starter . Procedures for creating and maintaining four independentlines Hierarchial analysis of variance model and expected mean squares . . . . . . . . . . Computational formula for variance components, heritability estimates and standard error of heritability . . . . Protein and amino acid analysis of the control dietformulated to contain 1. 0% lysine and the deficient diet to have 0. 5% lysine O O O O O O 0 Least squares analysis of variance, fixed factorial design, for three week gain (grams) in weight for all four lines of birds in the experiment. . . . . . . Least squares analysis of variance, fixed 0 O I O O O factorial design, for three week gain (grams) for all four lines on the control diet ( 1.0% lysine) . . . Least squares analysis of variance, fixed factorial design, for three week gain (grams) lysine)....... ' for all four lines on the deficient diet (0. 5% Least squares analysis of variance, fixed factorial design, for three week deficient/ control (D/C) percent, and for all four lines in the experiment . . . Least squares analysis of variance, fixed factorial design, for three week gain (grams) for selected lines HS and LS on the control diet(l.0% lysine) . . . V Page 19 23 33 35 38 40 41 43 45 46 Table 11 12 13 14 15 16 l7 l8 19 LIST OF TABLES ( Cont. ) Least squares analysis of variance, fixed factorial design, for three week gain (grams) for selected lines HS and LS on the deficient diet (0.5% lysine) . . . . ..... . . . ........ Least squares analysis of variance, fixed factorial design, for three week deficient/ control (D/C) percent for selected HS and LS lines . . . . . ........... . ......... Correlation (r) between three week gain (grams) and deficient/control (D/C) percentage com- puted separately for sex of progeny of the HS andLSlines...... ..... . .. ......... Mean three week gain (grams) by diet, generation and line with sexes pooled .......... Percent change in mean (%A.) for three week gain (grams) by diet, generation and line with sexegpooled ........ ..... .... ...... Arginine : lysine ratio computed for each diet and generation . . . . ........ . ......... Computed realized heritability using deviations ' from the mean of the random control (RC) line by response/selection differential for trait one (t ) three week deficient/control (D/C) percent with sexes pooled . . . . . ............... Computed realized heritability using deviations from the mean of the random control (RC) line by response/secondary selection differential for trait two (t ) of gain (grams) with sexes pooled and fed The 0.5% lysine diet - ---------- Numeric example of analysis of variance for the hierarchal analysis design with its appropri- ate expected mean squares for generation four (G4) of the low selected line (LS) provided the O. 570 lysine diet ..... . . . . .......... Page 50 51 53' 57 LIST OF TABLES (Cont.) Table Page 20 Numeric example showing computations for component of variance, heritability and standard error of heritability from the sire variance component for the low selected line (LS) at the fourth generation interval (G4) on the 0.5% lysine diet .................... 69 21 Heritability estimates from component of variance for three week weight gain (grams), with sexes pooled on the 1.0% lysine diet ........ 70 22 Heritability estimates from component of variance for three week weight gain (grams), with sexes pooled on the 0.5% lysine diet ........ 72 23 Summary of calculated heritability estimates by line for diets separately and generations pooled for each level of the component of variance .......................... 74 24 Average malezfemale ratio by line during the experiment . . ...................... 76 25 Average male:female ratio by generation during the experiment .................. 77 26 Coefficient of inbreeding "F" by line and generation interval . . . . . . .............. 79 27 Least squares analysis of variance for three week gain (grams) for pure line HS and LS and for F cross line progeny with generations G2, G3 and G4 pooled .................. 82 28 Mean three week gain (grams) for pure line and cross line progeny by diet and generation interval with sexes pooled . ............... 83 29 Two-way contingency tables for cell means showing diet by line interaction effect for line of sire and line of dam by diet and generation ..................... . . . . 85 vii Table 30 31 32 33 34 35 36 LIST OF TABLES ( Cont.) The influence of nutritional environment on heterosis for three week gain (grams) of chicks from the high (HS) and low (LS) lines selected for growth on a 0.5 percent lysine diet OOOOOOOOOOOOOOOOOOO O ..... Percent mortality from one day to three weeks of age by diet, sex, generation and line ...... Average number of eggs produced for survivors during 40 weeks (280 days) on test ......... Percent egg production for survivors during 40 weeks (280 days) on test ............. Laying house mortality during 40 weeks (280 days) on test ....... . .......... Percent fertility and hatchability by generation for pure line 3 .................... Percent fertility and hatchability by generation for reciprocal line crosses ............. viii Page 87 89 91 92 93 95 96 Figure LIST OF FIGURES Pathways for biosynthesis of lysine . . . . . Theoretical model of a two—way selection for growth and a random control line . . . . Deviations by line (NS, HS and LS) in grams gained from mean of control (RC, line) on thel-O‘Volysinediet. . . .. Deviations by line (NS, HS and L8) in grams gained from mean of control (RC, line) on the0.5%lysinediet. . . . . . . . . . . . . . CHAPTER I INTRODUCTION In the face of an increasing world population, poultry raising must meet several challenges, and among these, the greatest is that of competing with man for food. Unquestionably, poultry maintains high status by its efficiency in converting feedstuffs to high quality meat and eggs needed to feed the human population. However, poultry daily consume large quantities of cereal crops, thereby threatening their own existence as they compete directly with man for foodstuffs. This dictates the necessity of increasing the conversion efficiencies of poultry and is one challenge to the poultry scientist of today. Differences in individual requirements based upon the physio- logical ability to utilize certain diets may be traced to the genetic constitution of an individual or a family. Many instances arise in nutritional studies where birds grow and lay at comparable rates under similar environments but react at varied rates to nutritional stresses. Why do certain birds show resistance to deficiencies in the ration, while others react quickly to them? Why do some birds perform well on a given nutrient level, while others show definite lags in growth and production performance? It seems important, then, that geneticists study these differences to elicit more efficient utilization of nutrients. A population of organisms with variable genetic backgrounds, as well as individuals within a species, have independent and dis- tinctive nutritional requirements which must be met for optimum well being. The importance of these differences depends clearly upon the degree and cause of the variability among individuals. Theoretically, additional knowledge of genetic variability for the utilization of specific amino acids would enhance breeding pro- gress and the development of strains of birds which may more efficiently utilize feed for growth and other performance character- istics. The individual and family traits identified under dietary stressing conditions would establish selection criteria to assist in the development of genetically more uniform stock according to its in— herent ability to utilize nutrients in the ration. Lysine (C N202), was selected as the amino acid for in- 6Hl4 vestigation because it is essential for growth of chicks (Almquist, 1957). The L-isomer is the only biologically active‘form utilized by the chick (N.R.C., 1960) and by man (Ryan & Wells, 1964), therefore, L-lysine is the nutrient being considered. L-lysine is unlike other amino acids and is a desirable nutrient for study because it is be- lieved to exist biochemically as a single component required to support metabolic activity, and this amino acid ( L-lysine) is not known to be "spared" by other nutrients in the diet. Among the common plant protein sources of the world, only soybean oil meal is rich in lysine (Almquist, 1957; Dean and Scott, 1965; Merck, 1961; Morton and Amoroso, 1967 and Nyhan, 1967). When soybean oil meal is unavailable or it is not economically feasible to include it in the growth diet of chicks, lysine is the first- limiting amino acid for growth. Lysine is also the first-limiting amino acid for growth of young children and some other monogasteric (single stomach) species when the daily protein intake is predomi- nately of the following cereal grain or legume: corn, wheat, rice, peanut meal or zein. A survey of physiology and biochemical texts as well as other sources of information failed to identify the biosynthesis of lysine with other nutrients, enzymes or hormonal systems. Recently, Ne sheim ( 1969) said "It appears that the chick may have two pathways of lysine degradation. " Fig. l is redrawn from Wagner and Mitchell (1964) for the reader's inspection; attention is called to the independ- ent nature of lysine or the need of lysine before the metabolic pro- cess may continue into the citric acid cycle. The impending consequence of the world's population and food supply situation requires scientific consideration of the genetic- nutrional interrelationship to avoid a crisis of disease and starva- tion. In addition, acknowledgment of the independent role of the «.me Succumzwuonwag 8050a Bow on ogmocmfiwmv inseam moow Eon 0:053 O “U .825an ~ moow :00w “.8 £96 roou I ...U o .w \\I.. «3.. . $00 . he its... 92-.., W W I s/ o _ . o“ o I z I u oh :OOw _\ U U . U . . $000 «:2 I o _ oHo I z I o u _ r «:2 I w :80 o «Essa $000 “w . o§< _ . mooo smolw E 32.10 /~=z w Iw U U . _ Essa»: ~=z I + w TIT I... w :8 _ Eon can“; 0 .11 U U C . - oamfi< u we \ w moow ~22 I «:2 I w «:2 I w «\ o l/ .o :08 Bus oneness \ \V w \ o «:2 I + \ \ . \ . .l 2/ \ \ “w \ \ w “w/ :08 “_V w \ \ 05 v :08 a I o I o 38 o/uxo Eon use-30 3:82 as}. no stasis 8“ $2.53 4 .3... amino acid lysine as a component of protein metabolism motivated the author to develop experiments with the chick as a laboratory tool. Consequently the following objectives were designed into an experi- ment: 1. To demonstrate that lysine requirement is inherited by selecting divergent lines through mass selection for growth rate. 2. To evaluate the genetic parameters for lysine re- quirement as expressed through growth and to estimate heritability of the trait. 3. To investigate the latent effects of a low lysine diet consumed during early growth and the consequence of imbalanced dietary protein intake during early growth on reproductive performance. CHAPTER II REVIEW OF LITERATURE As early as 1902 Sir Archibald Garrad recognized significant variability for metabolic function and he called these findings "inborn errors of metabolism". A few years later Mendel ( 1915) recognized lysine as the first-limiting amino acid of the diet for growth. In Mendel's ( 1915) experiments, the rats being fed zein as the sole source of protein lost weight and died. With the addition of the amino acid tryptophan, life was sustained for experiments six months in duration and the rats showed essentially no change in body weight. Gordon ( 1963) using the chick in a 200 day feeding trial confirmed Mendel's work. When the zein-trypotophan diet was supplemented with lysine in subsequent experiments, growth and body weight change occurred. From this experiment it was concluded that lysine was the first-limiting amino acid for growth, a finding also in agreement with Almquist, (1957) Schwartz 23.11: (1958), Dean and Scott (1965), and others. Numerous studies of genetic -nutritional interrelationships have been reported among a wide range of organisms. Beadle and Tatum (1941) found different nutritional requirements between strains, and within strains, of Neurospora. These researchers concluded that 6 different mutations block Specific chemical steps in the biochemical reactions which make up the patterns of metabolism. Mitchell and Houlahan(1946) demonstrated that a "riboflavinless mutant" strain of Neurospora had different and variable quantities of a particular enzyme, and when deficient of the enzyme it did not synthesize ade- quate riboflavin. Because it was found that between and within strain variation existed these workers proposed the theory of "leaky genes", a concept also described as ”partial genetic blocks '1. The text Genetics and Metabolism by Wagner and Mitchell ( 1955) is recognized as the initial attempt to organize the literature on this subject. Cited in their book, and from their own scientific papers, Wagner and Mitchell ( 1955) reported that in one group of five closely related species of Drosophila, three of the species were different from one another in respect to their nutritional requirement. In mammals, variations in nutrient requirement and utilization have also been demonstrated. Marked differences between strains of inbred rats have been shown for thiamine utilization, and a series of two-way selection experiments for body size in mice (Falconer and Latyszewski, 1952; and Falconer, 1960) showed significant vari- ation for feed utilization on high and low planes of nutrition. Their studies showed the differences in growth and feed utilization to repre- sent attainable genetic parameters . In man, Williams ( 1951) identified a genetic weakness in the metabolic system which tended to enhance chronic alcoholism. Vita- min B deficiencies have been determined as one of the main causes of the alcoholic problem. Williams (1956) demonstrated that laboratory rats had a high degree of individuality for alcohol consumption. Inbred lines were uniform among individuals of the same line but the lines were distinctly different in their consumption patterns. Phenylketonuria in man is genetically linked to a deficiency of the enzyme phenylalanine hydroxylase (Wallace eta” 1957). More re- cently, Colombo e_t_a_1. (1964) and Ghadimi 932.1. (1965) described a condition called "hyperlysinemia" in children, and these researchers found similar biochemical abnormalities among clinical patients. The cases reported to date of hereditary hyperlysinemia (Nyhan, 1967) have a high incidence of consanquinity of the parents. Children show- ing a mild case of lysine intolerance are mentally retarded, while those with a severe lysine intolerance usually die. Ryan and Wells ( 19 64) identified a hereditary block in a subsidiary pathway which inhibits lysine metabolism. These types of inherited metabolic variations are probably more prevalent in animals and man than has yet been reported. Perhaps the classic example of genetic differences for meta- bolic functions within the classes of a species is found in the Dalmatian dog. High volumes of uric acid are excreted in the urine of higher primates, such as man and chimpanzee, and also by the Dalmatian dog. The Dalmatian is unlike other carnivores which excrete low quantities of uric acid and a higher proportional volume of allantoin (Friedman and Byers, 1948). Among dogs, the difference was re- solved by determining that uric acid is not efficiently reabsorbed in the kidneys of the Dalmatian as it is for other dogs. It appears that this intra-species difference is genetically based and may or may not be directly involved with some enzyme process which influences the transport of uric acid through the cell membranes. The chicken is a superior laboratory animal and has been studied extensively. Results of many investigations show that its ability to withstand certain types of dietary deficiencies is inherited. These differences have been demonstrated between (and within) strains, breeds, and strain crosses. A specific example from the area of vitamin nutrition is the report of Lamoreux and Hutt ( 1948). They demonstrated, with five generations of selection, differences for the utilization of riboflavin within the Single Comb White Leghorn breed. In considering further the inherited differences for vitamin, mineral, protein and amino acid requirements in poultry, the reader is referred to comprehensive reviews by Lerner (1958) and Nesheim ( 19 66). The following citations deal with the variation of performance for the amino acids and are considered to have more than a casual relationship to lysine, the topic of this dissertation. 10 Griminger (1955) reported that the requirement for either DL- methionine, L-tryptophan, or L-lysine did not vary for rapid or slow-growing chicks. The study, however, did not compare the genetic influence on chicks for differences in their ability to utilize these nutrients. A later study (Griminger and Fisher, 1962) showed significant differences among dams in the growth potential of their offspring on arginine and lysine—deficient diets. Nesheim and Hutt ( 19 62) reported strain differences in the arginine requirement among Single Comb White Leghorn chickens. Subsequent selection and testing (Hutt and Nesheim, 1966) showed that the two lines were widely different in their arginine requirement but, when tested for biological efficiency, the lines were not signifi- cantly different. More recently, Hutt and Nesheim(1967) stated that, following four generations of selection for high and low arginine utilization, and by backcrossing to produce F individuals, they I acquired intermediates, "as in typical polygenic inheritance".‘ Hess 9311.. (1962) reported an experiment in which selection for growth rate differences on a methionine-deficient diet was con- sidered. The experimental results indicated widespread differences between the F-line (fast-growing) and the S-line (slow-growing) as measured by body weight at three weeks of age. In a more recent report, Wilson and Hess ( 1968) confirmed that ”selection was quite effective in separating high and low three-week body weight lines 11 on both normal and methionine deficient diets. " However, genetic differences for methionine requirement were not conclusively shown following a series of three-week growth trials. In their studies four selected lines and a control were tested in a factorial design over three generations on both methionine deficient and adequately sup- plemented rations. The conclusion, following a series of growth trials on graded levels (0.19, .29, .39, .49, .59, and .69 percent) of methionine, was that differences in growth rate were linked with appetite and not due to a change in methionine requirement. Williams and Grau (1956) reported better growth on any lysine level with a reduction of dietary energy simply through stimulation of feed intake. Lowering the energy concentration did not change the lysine requirement proportional to other nutrients but did influence the growth response on low level lysine diets. Although numerous workers have reported that dietary protein level influences the re- sponse of chicks at various ages to amino acid level, the requirement may vary in exact proportion of the amino acid content of the diet to the total protein available. Singsen gt 11. (1965) reported that a lysine deficient diet (0.59 percent lysine) fed to four weeks of age effectively retarded the onset of egg production among meat-type chickens. Mortality was not significantly affected by the lysine- deficient ration, while skeletal weight was slightly less but approached 12 "normal". Tissue weight was approximately one third that of (nor- mal) control birds. Evidence of an apparent inherited difference for lysine was provided by Enos and Moreng ( 1965). Individuals, representing different pedigreed sire families, exhibited significant differences in growth rate to four weeks of age when consuming a dietary lysine deficient ration. More recently, Godfrey (1968) reported low herit- ability for lysine utilization in Japanese quail (Coturnix coturnix japonica). At generations eight and ten in his experiment the D (deficient lysine) line consumed significantly more feed and grew significantly faster than either the F (full lysine) line or the C (con- trol) line. Even though continued selection did not change the mean performance on the deficient diet, he concluded that the lysine re- quirement must have changed, but that it required the better environ- ment for expression of the genetic change. After the present experiment was designed and particularly during 1966-1968, a host of scientific papers appeared on the rela- tionship between various amino acids and especially the antagonism between arginine and lysine. When the dietary imbalance among amino acids is great, or when the lysine level is excessive (more than 1.0 percent), chicks show marked increases in their arginine requirement. Authors of the following citations have considered the problem of arginine-lysine antagonism: Jones (1961 and 1964), Boorman and 13 Fisher (1966). O'Dell and Savage (1966), Smith and Lewis (1966), Jones 2311. (1967), Dean and Scott ( 1968), Hill and Shao (1968), Nesheim ( 1969), Smith ( 1968) and Squibb (1968); however, in these experiments, excess lysine was the problem. To this author's knowledge only Hill and Shao (1968) have considered the reverse situation of an antagonistic response when a lysine deficiency exists. Hill and Shao (1968) included in their study lysine as the first-limiting amino acid, and it resulted in a reduction of weight gain. They also reported that with high ar ginine levels in the diet and an existing lysine deficiency, the consequence of the imbalanced amino acids was augmented by further depressing growth rate. CHAPTER III MATERIALS AND METHODS A. Experimental Design Selection has been one of the most important tools of animal and plant breeders for many years. Centuries ago man realized that selection was a powerful tool for ”improving" a species when ”im- provement" means change favoring the breeder's ideal. Selection favoring the breeder's choice of a phenotype operates as a force to change the gene frequency, thus changing the genotypes within the offspring population. Consequently, the genotype is modi- fied in varying degrees depending upon the selection intensity for a phenotype (trait observed) performing in a particular environmental situation. This experiment was designedto examine the lysine requirement for optimum growth performance of the chick and to determine if the requirement is inherited (genetically controlled). If the assumption is made that the total variance among individuals for growth response in a poor nutritive environment is large, the individuals that deviate farthest in each direction from the mean could possibly have highly inherited different genetic make-ups and different nutritive require- ments . /‘/ 15 Since lysine is the first-limiting amino acid and biologically independent for growth of chicks and other species, it was theorized that a diet deficient in lysine would impose rigorous stress on a population. Thus it was hypothesized that two-way directional selec— tion pressure for growth response under dietary stressing conditions (low level lysine, 0. 5 percent) would separate genotypes due to inherited differences for lysine requirement. To test the hypothesis a bi -directional selection experiment was designed (Fig. 2). The performance of the selected lines was also compared with that of a random control population. If the Chick's genotype for lysine requirement as expressed by growth (phenotype) responds to directional selection in an additive genetic fashion, then two lines may be deve10ped, one for high growth rate and another for low growth rate under conditions of low dietary lysine availability. Numerous experiments have been reported of divergent directional selection: Falconer, (19 53), Martin and Bell, (1960), Siegel, (1962) and Maloney £11., (1963) to name a few. Two-way divergent selection was used as a method to eliminate envi- ronmental bias as related to selection response. To observe the bi-directional phenomenon the following conditions were assumed: (a) The trait (gain in weight) has moderate to high heritability. (b) Gene action is on an additive genetic scale. (c) Individual phenotype selection should be effective in changing the mean of the population. l6 nomad.“ 280 so mo No — _ so WNUUNOMJ 30 q HobsoO Eovsdm ueew ut 3811qu on: Houucoo Eocene .m was fiBOum new somuoofiom >d3103u a mo fiopocu Hdofiouoosfi .m .mrm 17 (d) The effect of appetite was considered to be random among lines and no attempt was made to study quantitative dietary ly sine consumption . B. Nutrient Environment It was hypothesized that differences of genetic potential for growth could be identified under poor environmental conditions, as opposed to otherwise adequate environments which tend to protect the less fit individuals of a population. A dietary stressing environment was considered suitable as an aid to selection; therefore, a dietary deficiency was designed into an experiment. Growth rate was selected as the criterion since it is the first characteristic influenced by lysine level above maintainance require- ment. Almquist ( 19 57) states that about 30 percent of the optimal amount of lysine is maintenance value and all other available lysine is utilized for growth. It was concluded that a diet calculated to pro- vide 50 percent of the L-lysine requirement would, in fact, make available only about 20 percent (SO-30:20) to promote growth. In addition, L-lysine was selected as the nutrient for study because it is not synthesized by the body and cannot be spared by other nutrients, therefore exhibiting an independent role as an essential element in protein metabolism. As cited previously the L-isomer is the only active form of lysine used by the chick and L—lysine‘is believed to be biologically the first-limiting amino acid for growth. The experiment 18 was designed with two specific diets to be fed to sets of sexed progeny as contemporaries within generation. The diet was mixed as a com- plete twenty percent protein chick starter (Table 1) annually as one lot of feed. The first mixing of ingredients was complete except for L-lysine. After the first mixing, the batch of feed was divided in half by weight. One half was labeled "deficient" lysine as it contained only lysine from the raw feedstuffs at a formulated level of 0. 5 per- cent lysine. To the second half of feed, 0.5 percent "L-lysine monohydrochloride" (N.B.C. 1964 - 68) was added, and the mixer was run briefly to blend the added 0. 5 percent lysine throughout the supply of feed. This lot was labeled ”control" lysine ration. Here- after in the text the nutrient environments containing L-lysine will be referred to as 1.0 percent lysine or 0.5 percent lysine. The chick starter diets for the growth trials were calculated to provide 2, 020 productive energy (Calories) per kilogram of feed and 20 percent protein. Formulation work related to the growth diets (Table l) was based on values in the NOPCO (1962) feed ingredient analysis chart. The two diets were: (a) Control nutrient environment with 1.0 percent lysine and adequate in all other known nutrients (Almquist 1957, Klain gia_l_., 1960, and N.R.C. 1960). (b) Deficient nutrient environment with 0. 5 percent lysine and adequate in all other known nutrients. 19 Table 1. Composition of 20 percent protein chick starter Ingredients Percent Ground Corn (yellow) 49 . 5 Corn Gluten (42 . 5%) 28. 0 Soybean Oil Meal (50. 0%) 3 . 5 Ground Oats 11. 0 Dehydrated Alfalfa Meal ( l7 . 0%) 4. 0 Limestone 1. 0 Dicalcium Phosphate 2 . 0 Iodized Salt 0. 5 Subtotal 99 . 5 Minerals added per kilogram of diet MnSO4 (70%) 154. 00 mg Zn Oxide 96.80 mg Vitamins addedmr kilogram of diet Vitamin A 5280. 00 IU Vitamin D3 1100. 00 IU Folic Acid 0.66 mg Pyridoxine (B6) 3. 30 mg Vitamin B12 11. 00 mcg Vitamin K 0. 55 mg Vitamin E 22. 00 IU Riboflavin 2 . 20 mg Pantothenic Acid 2 .20 mg Choline 2635 . 00 mg Niacin 56. 00 mg Amino Acids added per kilogram of diet L Arginine 0 . 41% L 'Lysinez 0. 50% DL Methionine 0. 10% DL Tryptophan 0. 10% 1 2, 020 productive energy (Cal.) per kilogram; Cal. /protein ratio = 101:1 2 . For the L-lysine deficient diet, omit 0.50 percent L-lysine 20 C. Genetic Material Two cases of hatching eggs (720 eggs) representing a hetero- genic germ plasma source of egg-type chickens, (Enos and Moreng, 1965) were obtained from a commercial poultry breeder in 1959 by the Colorado Agricultural Experiment Station. From 1959 to 1964 the Colorado State University Avian Science Research Center main- tained the stock under a closed flock breeding program. The original egg supply produced 508 potential breeders at 21 weeks of age, and, until 1964, this flock of egg—type chickens was reproduced by random matings among 17.25 sires and 85.75 dams on the average, annually. The consequence of the relatively small population before the initiation of the selection experiment was considered. The two necessary assumptions concerning the base population were: 1 . Randomness existed in the population structure and the effective population size was adequate to maintain heterogenic variance and approximately zero inbreeding. 2. Randomness did exist in the population structure, but the effective population size was inadequate, and while a heterogenic variance still existed, the inbreeding level was increasing. The coefficient of inbreeding was estimated for the base popu- lation and calculated from the actual number of males and females for each generation using the following approximation formula (Lush, 1948): 21 1 1 F=F'+ + (l+F"-2F') 8Nm 8Nf where: F is the expected inbreeding in the present generation; F' is the inbreeding of the previous generation; F" is the inbreeding of two generations ago; Nm is the actual number of breeding males; Nf is the actual number of breeding females . Using this formula the inbreeding was calculated to be about four percent at the time of initiation of this selection study. Selection experiments generally assume the inbreeding level to be zero in the base population. With an estimated four percent inbreeding level among individuals of the base population, inbreeding was considered to be of little consequence and will hereafter, as in most selection experiments, be considered zero. D. Selection Procedure The base population after random selection from the initial population consisted of 32 males and 192 females. Each male was randomly assigned to one of four lines and then mated to a minimum of six females respectively. Because of increasing concern over low numbers per line the number of females selected for mating with each male was increased from six to nine females per sire in producing the fourth generation. The 32 sire-dam groups used to launch the 22 study were subdivided into four lines and is presented in diagramatic form in Table 2. One line was designated as the restricted random control (RC) line. Restricted random, meaning that one male off- spring (son) each generation was retained at random from those available and this procedure was carried out respectively for each of the eight sires; thus, the sire pool was maintained as eight families, but each sire was mated to females on a random choice basis from within its own line. Throughout the experiment the restricted random mating line (RC) was tested in both nutritive environments. However, breeding stock for the restricted random control, hereafter to be identified as the RC, line was fed only the 1.0 percent lysine ade- quately balanced diet. In addition to the RC line another type of control line was main- tained, and it differed from the RC line only in that the breeding stock of the NS line had survived when fed the 0.5 percent lysine deficient diet from one day to three weeks of age. This line was influenced by lysine deficiency stress and will in the future be re- ferred to as line NS (Table 2), meaning natural selection. Males of the natural selection (NS) line were selected on the basis of one son per sire family by random choice and mated at random to females within its own line. However, only birds that were fed the specific deficient 0. 5 percent lysine diet could represent the closed line. The use of the notation "NS" in this paper should not be confused by the 23 Table 2. Procedures for creating and maintaining four independent lines. Random Base Population Distribution of "N" (Nd: 68, N9 = 457) Males 8 8 8 8 Females 6/d 6/d 6/d (3/6 5 Li” jg N54 H55 A Sex2 MF MF MF MF MF MF MF MF Diet3 D D C D C D C it at at “it “use it “as at :> ID '2 '2 '2 '2 :9 D U 2 :2 32 D :33 5252 5252 as 9930 an as Z Z Z Z 43 '50 Z 3 3 Z s s 3 3 l 2 M = Male, F = Female. 3 RC = Random Control; NS = Natural Selection; HS = High Selection; LS = Low Selection. D = Deficient (0. 5% lysine), C 2 Control ( l. 0% lysine). 4Restricted random control (RC); one restriction was imposed on the line in that one son was selected at random from each sire's off- spring and subsequently was mated to a random choice of females fed The same restriction applied to the reproduction phase of the NS line except breeders came from only birds that were reared, one day to three weeks of age, on the deficient diet. the control diet. 5Pedigreed lines (HS) and (LS) were restricted by the selection of the son for high or low growth performance of each sire's offspring and further restricted in that no close (full-sib) matings were permitted. 6 D/C%; means that the merit of an individual was determined by a mathematical procedure of dividing its own three week gain (grams) in weight by the average gain of its contemporary full-sibs of the same sex on the control diet. 24 reader with another common definition of "natural selection" meaning a non-controlled environmental situation, but is in fact a selection of the more fit (survivors) among the total number of the Species within the closed line being subjected to a poor nutritive regime of low level. 0. 5 percent available, lysine. Artificial selection was imposed by the investigator for highest growth rate (gain in grams) among pedigreed individuals of another line and this was termed the high selected line (HS) (Table 2). The low selected (LS) line (Table 2) was reproduced among pedigreed individuals with the poorest gain in weight on the experimental lysine deficient diet (0. 5 percent lysine). The use of the restriction of no full-sibs in the mating system for both lines HS and LS was practiced in order to suppress, for the short term (four generation) experiment, the increasing rate of inbreeding common in small populations. The unit of data for analysis was the gain in weight from one day to three weeks of age for chicks. This age period represented the accelerated portion of the growth curve (Almquist, 1957) and, in addition, the dietary requirement for lysine declines with age beyond four weeks. Gain data for progeny on the deficient diet, when com- pared with the average of their contemporary full-sibs of the same sex on the control diet were expressed as rations; deficient/control x 100 = percent (D/C%). This percentage was used as the criterion for selection. The following example should help clarify the pro— cedure: (a) (b) (C) (d) (e) (f) (g) 25 Assume sire No. 410 from the HS line was mated to six dams and produced 60 progeny. Sexed by vent method and assume equality of sexes, thus 30 males and 30 females. Randomly assign individuals, within sexed groups, to con- trol (1.0% lysine) or deficient (0.5% lysine) diet. For un- equal "N" offspring, assign the extra chick to the deficient environmental regime. Assume two mortality per sex and one each per dietary treatment combination; thus, 14 male control, and 14 male deficient, 14 female control, and 14 female deficient chicks yield data. Average the weights of full—sibs of the same sex on the control diet, and assume they gained 120 grams. Divide each individual gain value for chicks on the deficient diet by the average of their full-sib controls of the same sex. Example: progeny No. 620, weighed 80 grams; therefore, 80/120 (100) = 66.67%, and No. 630 weighed 30 grams so, 30/120 (100) = 25.00%. Selected from this example among the HS line would be progeny No. 620, because it had the highest percent gain relative to his controls. 26 (h) Similar computations were made for each individual in both the HS and LS lines at every generation interval. (i) If this example had assumed growth response of individuals among the LS line, then progeny No. 630 would be selected. In reality the following question must be answered. Which indi— vidual, No. 620 or No. 630 from the previous example, is the better? Number 620 with a 66.67% value was selected for highest growth rate (grams gained from 1 day to 3 weeks of age) on the deficient diet and apparently has the lowest lysine requirement. This concept of weighing individuals for merit or breeding value has been employed in this experiment since 1964. Wilson (1967) proposed a similar procedure for selecting desirable families in a discussion of alternative systems that may enhance selection effi- ciency. Individual selection was utilized in this experiment while Wilson’s recommendation was a family selection scheme; however, both designs use the same method of estimating breeding values. For each generation after the primary selection was made, in lines RC, NS, HS and LS, alternate males were chosen to replace any male lost because of an accident, normal mortality, infertility, or other cause. For the high and low growth rate lines, respectively, the first choice alternates were full-brothers, with half—brothers as further removed choices. 27 E. Other Measurements Throughout the growth studies, individual mortality was re- corded daily with regard to diet, sex and line. While the measure- ment for genetic change was restricted to gain in weight, several response characteristics were observed. Among them were the reproductive fitness traits, fertility and hatchability, which were measured as percent. Individual egg production was recorded daily for 280 days (ten, 28-day periods). F. Physical Environment Growth trials were conducted for sexes separately in groups of approximately 20 chicks. Chicks were assigned at random to growing battery locations. The electrically heated, wire floored batteries were thermostatically controlled for uniform brooding temperatures and adjusted to provide the following: (a) 1 day to 1 week of age, approximately 550 Centigrade. (b) 1 week to 2 weeks of age, approximately 52.50 Centigrade. (c) 2 weeks to 3 weeks of age, approximately 500 Centigrade. The battery room p_e_£ 2 was artificially illuminated with fluOrescent lighting fixtures to provide a constantly lighted environment. The room was equipped with ventilation fans adjusted to maintain the temperature range of 39° to 44° Centigrade. Feed and water were checked daily and provided on an ES. libitum basis throughout the studie s . l1 ll‘ll‘l‘ll. I- llll...’ I .l. 1 28 Following the experimental three week growth period, the chicks were provided a standard chick starter diet for one week in the battery room. During that week the electric heaters were shut off and the room temperature lowered to condition the chicks for cold room brooding on the research farm. At four weeks of age the chicks were moved to the farm where a floor rearing management program was utilized. The flock was vaccinated two days after delivery to the farm for Newcastle disease. During the rest of their lives, standard feeding programs, vaccination and management practices were followed for optimum growth, development and reproductive success. Since the chicks were hatched in April, annually, there was no particular physiological advantage in utilizing a special lighting pro- gram. Therefore, the birds grew and developed under natural day- light conditions at 400 latitude until housing time. When the pullets were twenty weeks old they were housed in individual wire cages. Feed and water were available 31 libitum throughout the 10 consecu- tive 28-day egg production periods. The environmental control features of the windowless poultry laying house had the following specifications: (a) Fourteen hours of artificial light per 24 hour day. (b) Four cfm (cubic feet per bird per minute) of outside fresh air supplied as forced air against 3/8 inch static pressure. (c) Direct drive, blade type, thermostatically controlled fans. 29 (d) Winter conditions - If the environmental temperature dropped below 220 Centigrade the fans were regulated by time clock for minimum air movement (1 cfm for 2 con- secutive minutes for every 10 minutes of total time). (e) Summer conditions - Evaporative pads were placed around the fans and flushed with water so that all incoming air was pre-cooled. With this system 110 Centigrade temperature differential was realized, even at high environmental temperatures of about 560 Centigrade. G. Statistical Procedure In dealing with the data, the first question to be answered was the presence of, or lack of, significant differences between main factors of diet, sex, generation and line effects. In addition, the existence of, or lack of, interactions involving lines was considered critically important. Least squares analyses of variance with un- equal numbers in computerized programs were used to statistically evaluate the data. Standard tests for homogeneity of variance were employed (Bartlett, 1937; and/or Pearson and Hartley, 1954). The Duncan's (19 55) Multiple Range F test for comparison of means was used to statistically compare different groups. The first statistical model employed in the general analysis was a four -way fixed factorial analysis of variance with unequal number of observations per treatment combination. 30 Yijkln = u + Di + Sj + Gk + L1+ DSij + DGik + DLil + Sij + Sle + GLkl + DSGijk + DSLijl + DGLikl + SGLjkl + DSGLijkl + eijkln where: . th . . th . Yijkln = the observation for the n chick in the 1 line of the kth generation for the jth sex on the itlrl diet; u = the overall common mean; .th . . Di = the effect of the 1 d1et, 1 = 1, 2; .th . S, = the effect of thej sex, j = 1, 2; J Gk = the effect of the kth generation, k = 1. . .5; th . L1 = the effect of the 1 line, 1 = l. . .4; DSij DGik’ DLil’ Sij, Sle and GLkl = the two-way interactions associated with the designated sub- classes; DSGijk' DSLijl' DGLikl and SGLjkl = the three -way inter- actions associated with the designated subclasses; DSGLijkl = the four -way interaction associated with the designated subclasses; e.. = the random error among observations. ijkln In addition to the four-way analysis of variance, several three- way, two-way and one ~way analysis of variance calculations were made. A special computerized program was written to perform the necessary calculations to obtain the appropriate deficient/control 31 (D/C) percent value. Each D/C percent was used as the best esti- mate of the individual merit or breeding value in the selection phase of the experiment. These analyses were computed for three week gain in weight of all chicks (without regard to sex) in the control nutritive environment (1.0% lysine) and for growth of their contemporary full-sibs in the deficient nutritive environment (0.5% lysine). In addition, the D/C percent estimates of breeding value were analyzed in the same man- ner. Least squares analysis of variance (Harvey, 1960) with unequal subclass frequencies of a hierarchial design for sires, dams within sires and offspring (progeny) nested within dam within sire was used for each set of data separated by diet, generation interval and by RC, NS, HS and LS line. The statistical model for this hierarchial analysis of variance design was: Yijk = u + Si + Dij + eijk where: . th . . . . .th Yijk 18 the record of the k 1nd1v1dual chick from the j dam mated to the ith sire; the overall common mean; C: II additive effect of the ith sire; (I) II 32 additive effect of the jth dam in the ith sire group; .9 ll — experimental random error among offspring. (D I The format for handling the data, identifying source of variation and the expected mean squares (EMS), appears in Table 3. The coefficients k1, k2 and k3 were calculated according to the method derived by Henderson ( 1953) and King and Henderson ( 1954) where: kl and k2 = effective number or coefficient of dams with unequal numbers per set; k3 = effective number or coefficient for sires with unequal numbers per set. The general formula for computing the coefficient, k, is: where: k = coefficient being computed; l a? = one /degrees freedom for the specific k; N = the total number of progeny; 2 .th . Zn = the sum of progeny for each 1 quantity squared. i The variance components for sires (oz) and dams ( o- :3) were computed by using the Henderson (1953) method. Formulas for these computations are in Table 4. Lush (1948), in his mimeographed notes, effectively described the use of the component of variance as a 33 3m 3 v acmuopcom was weak new $2: v “593933 you we wousmEoU mx pad mx Lx mucowommmooO H >~>b 3mg Q- a mofim\ made mnmhmmmwo Wbflx + >N>b sz mum mofim\ mEmQ meme + o ems + 3b mm: Tm mohm N N N 92H mg mp eon—mmnsr mo condom moudsvm smog 630098 was HopoE consist, mo maniacs ddweoumnoflm .m candfi 34 means of estimating heritability from data for sets of full-sibs and half-sibs, as well as a procedure to estimate heritability by combin- ing the sire and dam components of variance. Also shown in Table 4 are the formulas for computing standard error of heritability esti- mates, Dickerson (1960) and Becker (1967). Realized heritability, defined as the response to selection, was computed by using the following formula: N mIFU where: 2 h realized heritability; R response (gain); S = selection differential (intensity). Falconer (1960) says of this method, "it provides the most use- ful empirical description of the effectiveness of selection. " Magee (1965) proposed that the term selection differential be applied only in situations where mass selection is for only one trait; its computational formula is: 'U( I *Ul AG: where: AG genetic change "U( ll phenotypic average of selected animals 35 3.2.: Hoxoom was 309: GOmuoxomQ 81: V £3 32.: couucecem m No.30 pumped: was mouse—Sumo fiflwnmfinos N N a a. o m 3 o m b + b + b b + b + b N N N N N N . 3m 3 .. 3 a 3 o m - a mi I N b x 3 a me o m N A b + 3N N War 1 .6 .N1. N + b + b N N m2 N N x N N N Bb+ob+m Sb+ob+mb a N N N N N N u e: s u o b o o < .3 3% as we 71. N 3. N m2 - om: N + I 3 o m2 m2 N N N 3b+ob+m Bb+ob+mb N N N N N N N u m; x ... m b owe me mg a mi. N< nob Nx + ems: - mm: N + N N N n m 2 Nm ,Nmz N I mev A my «383mm 233388 Nahum pumvcmam ufimnmfinvm 005339? 323322 No .mucoeonrhoo oocmwndcy new desEHOH fidcoflmusmfioo .v QBNH. 36 P = phenotypic average of all the population in which they were born. With A G being equated to the response, meaning gain from the mean of one generation to the mean of the next generation, and (P - P) being equal to intensity or selection differential, then realized herit- ability could be calculated. In another phase of the experiment, pure line and reciprocal cross line matings between the HS and LS selected lines were made. This was done to find the mean of the F1 offspring populations. The mean of the cross line population was expected to lie at some mid- point between the selected lines. The F1 cross line progeny provided data for a measure of heterosis with respect to growth rate performance following selection in a low (0.5% lysine) nutritive environment. Mathematical proce- dures of Crow ( 1952) and Falconer (1960) were utilized in computing realized heterosis. Cross line Pure line Percent _ mean mean heterosis \ Pure line mean x 100 Percent heterosis is the degree of response; Pure line mean is the average of the parental types HS x HS plus LS x LS; Cross line mean is the average of the F1 progeny types HS x LS plus LS x HS. CHAPTER IV RESULTS AND DISCUSSION Annually, feed samples of each diet, control (1.0% lysine) or deficient (0.5% lysine), were taken for chemical analysis. As pre- sented in Table 5, one may observe that some variation existed among the feed supplies used during this experiment. Actual amino acid determinations were made from two random samples collected by the investigator and again randomly divided into three samples by the laboratory. This procedure was routinely followed except in 1967 when the samples were destroyed by weevil. The samples were pro- cessed in a commercial chemical laboratory on a "Spinco Amino Acid Analyzer", courtesy of the Ralston Purina Company. Each amino acid value shown in Table 5 represents the mean of six determinations while the D/C percent represents the ratio of available dietary lysine between the two diets calculated for the specific generation interval, along with the overall average for the duration of the experiment. Sufficient variation was present between the diets annually so that the percentages (D/C) ranged from a low of 48.42 percent to a high of 64. 37 percent. In theory, the actual difference annually in lysine should be . 50 percent, however, the actual differences as determined by chemical analysis were .49, . 31, . 31, --, and . 37 37 38 Table 5 . Protein and amino acid analysis of the control diet formu- lated to con ain l. 0% lysine and the deficient diet to have 0. 5% lysine Egg Protein Lysine Ar ginine Tryptophane Cy stine Methionine Bean. P rote in Lysine Ar ginine T ryptophane Cy stine Methionine D/C (Lysine) Control Diet (1.2% Lysine) .95 1.25 .18 .50 .49 a E; 2.3. a 23.4 25.4 --2 23.5 23.3 .83 .87 -- .87 .88 1.22 0.91 -- 1.01 1.10 .17 .23 -- .15 .18 .59 .64 -- .57 .57 .44 .42 -- .53 .47 Deficient Diet (0. 5% Lysine) .46 1.00 .17 .49 .36 48.42 it. Ea .63 214. Avg' 21.9 24.3 --2 22.3 21.9 .52 .56 -- .50 .51 1.25 1.33 -- .86 1.11 .17 .23 -- .13 .18 .57 .64 -- .59 .57 .46 .47 -- .51 .45 62.65 64.37 -- 57.47 57.95 ,7 Amino acid assay values, Spinco process, courtesy of Ralston Purina Co., Checkerboard Square, St. Louis, Missouri. (1964-68) Samples were destroyed by weevil 39 percent, respectively, for generations designated as P0 for the base generation and each subsequent generation denoted by the sub-gener- ation number G1, G2, G3 and G4. The overall average difference in the two diets for level of lysine was . 37 percent. Growth rate, as measured by grams of weight gained during the juvenile period from initial hatch weight at one day of age to three weeks of age, was analyzed for main effects of diet, sex, generation and line, each being considered as a fixed factor in the design. Table 6 shows the numerical values for this computation for the main effects as well as all two-way and three—way interactions. All of the categories showed statistically significant variability at the five per- cent or higher level of probability. By design it was intended that the 0. 5 percent lysine diet would, on the average, severely depress growth as compared with the 1. 0 percent lysine control diet; therefore, the data on punch cards were sorted and reanalyzed as though they represented two different and separate experiments. Displayed in Table 7 is the analysis of vari- ance result for chicks being fed on the control diet ( l .0% lysine). For the analysis with diet removed as a factor, the remaining main effects being analyzed in the three-way were sex, generation and line. For birds on the control diet (1.0% lysine), the effects of sex and generation were found to be statistically significant; however, no significant differences existed between the four experimental lines of birds (Table 7). 40 Table 6. Least squares analysis of variance, fixed factorial design, for three week gain (grams) in weight for all four lines of birds in the experiment Source of Variation df MS F Sig. Total 7362 Diet 1 6573375.97 15671.86 .005 Sex 1 6206.20 14.80 .005 Gen. 4 74354.79 177.27 .005 Line 3 1273.17 3.04 .05 DxS 1 9366.22 22.33 .005 DxG 4 45979 .94 109 . 62 . 005 DxL 3 1671.58 3.99 .01 SxG 4 12192.59 29.07 .005 SxL 3 1675.06 3.99 .01 GxL 12 2671.96 6.37 .005 DxSxG 4 10595.17 25.26 .005 DxSxL 3 3502.97 8.35 .005 DxGxL 12 744.96 1.78 .05 SxGxL 12 1853.17 4.12 .005 Error* 7295 419 .44 31‘ The 4-way interaction DxSxGxL (F = l. 50) was not significant (P > .05) and this source of variation was pooled in the overall error te rm 41 Table 7. Least squares analysis of variance, fixed factorial design, for three week gain (grams) for all four lines on the control diet (1. 0% lysine). Source of Variation df MS F Sig. *— Total 3610 Sex 1 14169.00 25.82 <.005 Gen. 4 103153.87 187.95 <.005 Line 3 200.83 0.37 N.S.* SxG 4 21465.82 39.11 <.005 SxL 3 3444.98 6.28 <.005 GxL 12 2243.91 4.09 <.005 SxGxL 12 2883.79 5.25 <.005 Error 3571 548.84 3|: N.S. means not significant here and throughout the text 42 Data for the birds being fed the 0. 5 percent lysine deficient diet were also analyzed by the least squares analysis of variance for un- equal numbers considering sex, generation and line as fixed factors. From this analysis, and as shown in Table 8, the effect of sex difference was no longer statistically significant while generation effects were significant as they were for the contemporary full-sibs on the control (1.0% lysine) diet (Table 7). Referring again to Table 8, there was a statistically significant difference among the four lines when they were fed the deficient diet with 0. 5 percent lysine. It is apparent that only one interaction was significant, that being the G x L (generation x line) effect. This interaction of generation x line was highly significant (P < .005). Theoretically, this interaction G x L (Table 8) should be significant if the selection experiment did indeed produce divergently separating lines as measured by the criterion, three week gain(grams) in weight. The importance of this interaction will be discussed later in a de- tailed study of means by line per generations of time. As described earlier in the experimental design, Chapter 111, Materials and Methods, Section A, a computed percentage value for each individual chick on the deficient (0. 5% lysine) diet was used to select breeders in each the HS (high selection) and LS (low selection) lines for growth, from one day to three weeks of age, rather than selecting for each individual gain (grams) in weight. 43 Table 8. Least squares analysis of variance, fixed factorial design, for three week gain (grams) for all four lines on the deficient diet (0. 5% lysine) Source of Variation df MS F Sig. Total 3751 Sex 1 104.75 0.36 N.S. Gen. 4 12770.03 43.65 <.005 Line 3 2694.62 9.21 <.005 SxG 4 493.94 1.69 N.S. SxL 3 139.10 0.48 N.S. GxL 12 1134.06 3.88 <.005 SxGxL 12 136.96 0.47 N.S. Error 3712 292.53 44 The preceding discussion in this chapter dealt with the actual gain (grams) in weight per bird from one day to three weeks of age. The next consideration was the statistical analysis for the deficient/ control (D/C) percent values which were the actual criteria for selection among the HS and LS, divergently selected lines. The least squares analysis of variance technique was used for the data with unequal numbers per subclass. A tabular diSplay of results for the three factor analysis, including sex, generation and line as main effects, appears in Table 9 . This analysis indicated that all main effects and each two-way interaction were highly signifi- cant (P < .005). Since all four lines of the experiment were included in the preceding analysis but only two of them, HS and LS, were artificially influenced by a high degree of selection pressure, the data were sorted to permit reanalyzation by analysis of variance for only the selected lines, HS and LS. The results of the analysis for those individuals within the selected lines fed the control (1.0% lysine) diet are presented in Table 10. The main effect of generation was highly significant(P < .005); however, the factors of sex and line were not significant (P > .05). In a similar manner, the data for the full-sibs on the deficient (0.5% lysine) diet were analyzed (Table 11). Of particular interest was the non-significant differences for sex and line. The comparison of the analysis for the 1.0 percent lysine and 0.5 percent lysine treatments is in exact agreement with the main 45 Table 9. Least squares analysis of variance, fixed factorial design, for three week deficient/control (D/C) percent and for all four lines in the experiment Source of Variation df MS F Sig. Total 3751 Sex 1 2436.39 8.61 <.005 Gen. 4 16379.70 57.85 <.005 Line 3 2981.41 10.53 <.005 SxG 4 4610.71 16.29 <.005 SxL 3 1680.23 5.94 <.005 GxL 12 683.96 2.42 <.005 SxGxL 12 403.90 1.43 N.S. Error 3712 283.13 Each D/ C percentage value was computed individually within dam of sire family and of the same sex Table 10. 46 Least squares analysis of variance, fixed factorial design, for three week gain (grams) for selected lines HS and LS on the control diet (1.0% lysine) Source of Variation df MS F Sig. Total 1750 Sex 1 595.21 1.01 N.S. Gen. 4 55912.37 94.71 <.005 Line 1 405.42 0. 69 N.S SxG 4 14276.10 24.18 <.005 SxL 1 2376.26 4.03 <.05 GxL 4 947.49 1.60 N.S SxGxL 4 1304.02 2.21 N.S. Error 1731 590.37 47 Table 11. Least squares analysis of variance, fixed factorial design, for the three week gain (grams) for selected lines HS and LS on the deficient diet (0.5% lysine) Error 1805 302.05 Source of Variation df MS F Sig. Total 1824 Sex 1 30.20 0.10 N.S Gen. 4 8036.25 26.61 <.005 Line 1 417.24 1.38 N.S SxG 4 599.55 1.98 N.S SxL 1 62.06 0.21 N.S GxL 4 278.76 0.92 N.S SxGxL 4 70.79 0.23 N.S 48 factors of sex, generation and line for the analysis of selected HS and LS lines (Tables 10 and 11 for 1.0% and 0.5% lysine dietary environ- ments, respectively). When dietary groups were analyzed separately, it became clear that pooling of some two-way and the higher order interaction of SxGxL could be carried out. However, this was not done in order to look at the relative effects of the possible interactions from one dietary environment to the other (1.0% lysine vs. 0. 5% lysine). A comparison of the various interactions points out that sex was involved in two of the two-way interaction levels for the 1.0 per- cent lysine environment but had no interaction effect relative to the analysis for the 0. 5 percent lysine environment. Sex, as a main factor, was not itself a significant source of variation among main effects for either dietary regime. The least squares analysis of variance result for deficient/ control (D/C) percent (Table 12) also points out that sex differences were not of significant concern (P > .05). The analysis of D/C infor- mation, since it is based on full-sibs of the same sex, reduces the model from two nutritional dimensions (1 . 0% vs. 0. 5% lysine) to one effect. The reduction of the model for analysis in this way can also be acceptable since sex differences were not significant in either dietary environment. Having compared the analysis for diets, separately, and from the D/C approach, one arrives at the same conclusion: sex as a main factor can be pooled for further analysis 49 Table 12. Least squares analysis of variance, fixed factorial design, for three week deficient/control (D/C) percent for selected HS and LS linesl Source of Variation df MS F Sig. Total 1824 Sex 1 140.36 0.47 N.S. Gen. 4 7047.96 23.85 <.005 Line 1 515.38 1.74 N.S. SxG 4 3064.64 10.37 <.005 SxL 1 53.45 0.18 N.S GxL 4 408.35 1.38 N.S SxGxL 4 193.93 0.66 N.S. Error 1805 295.53 1 Each D/ C percentage value was computed individually within dam of sire family and of the same sex 50 and generations as an effect are highly significant (P < .005); thus, future evaluation should be considered for each generation interval .G.G (Po' G1 2 3 , and G4) separately. Although the least squares analysis of variance result indicated that sex as an effect could be pooled for both dietary regimes, the correlation of three week gain (grams) with D/C for male, female and pooled sexes, was considered. A stepwise linear computerized program was employed to measure the degree of correlation between three week gain in grams with the three week deficient/control values for males, females and sexes pooled within line HS and LS. Presented in Table 13 are the appropri- ate correlation "r" values each being highly significant (P < .01). These high correlations between the two measurements, the com- puted D/C value and the empirical grams of gain value, add support to the previously drawn conclusion that sexes could be pooled and that either one, male or female, is as good as the other in making pre- dictions. In Table 13 one can also see that the correlations within sex and for sexes pooled are comparable between lines HS and LS; thus, any error committed by pooling sex effects within line would be no greater in the HS line than in the LS line. Comparisons between lines can be made, having previously pooled the data for within line effects. The average of all data in Table 13 is .798 with a range no greater or less than the mean by . 065 . 51 Table 13. Correlation (r) between three week gain (grams) and deficient/control (D/C) percentage computed separately for sex of progeny of the HS and LS lines HS LS 3101! #31! Sex N r N r Male (424) .733 (531) .753 Female (385) .863 (485) .838 Pooled (809) .798 (1016) .795 ** All computed r values are highly significant (P < .01), Snedecor and Cochran (1967) 52 The principal factors of this experiment were diet (two levels), two sexes, a base generation (PO) and four subsequent generations (G G.G 1, 2 3 and G4) for each of four separate lines. In the previous discussion, the effect of these main factors was considered for their probable effect on interpretations from the experiment as a whole. Both diet and generation effects were found to be highly significant (P < .005). Further, it was determined that sex as a main effect and as a factor of the overall experiment could be discounted; thus, the two sexes, male and female, could be pooled with regard to the parameter, grams gained from one day to three weeks of age, being investigated. The mean three week gain (grams) and its standard error (S.E.) for the specific number of observations per diet, generation and line with sexes pooled, are presented in Table 14. The Multiple Range F test for differences in means (Duncan, 1955) was applied to the data and. where the means were found to be significantly different (P < .05). they have been identified (Table 14). At the base generation interval (PO), there were no significant differences (P > .05)among lines within each nutritive environment. Consequently, one may conclude that the chance segregation of the base population, as genetic material, did fit the expectation of complete randomness of sampling as measured by mean three week gain (grams) of their progeny. There were approximately equal numbers of full-sibs of the same sex fed on each dietary regime (1.0% or 0. 5% lysine). 53 Table 14. Mean three week gain (grams) by diet, generation and line with sexes pooled 1. 0% Lysine 0 . 5% Lysine Gen. Line N 35 gms. P<.05 S.E. N ngs. P<.05 S.E. P0 RC 114 121 8.1 1.91 133 55 a1 1.56 NS 106 115 a 2.06 120 55 a 1.78 HS 115 115 a 2.03 123 58 a 1.66 LS 132 117 a. 1.85 159 56 a 1.50 CI RC 241 130 a 1.60 229 45 1.22 NS 253 129 a 1.40 286 47 a 1.07 HS 230 131 a 1.75 248 51 a 1.28 LS 240 131 a 1.76 266 49 a b 1.22 G2 RC 125 102 a 2.01 127 48 a b 1.39 NS 108 100 a. 2.40 139 49 a 1.30 HS 107 97 a 2.10 107 43 1.46 LS 129 96 a 2.45 147 45 a 1.25 G3 RC 109 95 b 3.13 114 38 b 1.46 NS 124 106 a 2.21 145 48 a 1.34 HS 109 109 a 2.58 116 47 a 1.30 LS 130 103 a b 2.40 140 43 a b 1.26 (:4 RC 322 121 a b 1.27 289 47 b 0.99 NS 358 117 b 1.20 345 55 a 0.78 HS 234 121 a b 1.66 215 52 a 1.16 LS 325 124 a 1.29 304 53 a 0.88 1Within each dietary and generation group of four lines, the means with the same subscript letter are not significantly different at the five percent level of probability (Duncan, 19 55) 54 To illustrate the effect of change from generation to generation, the means in Table 14 for each line on the control (1.0% lysine) diet have been graphed in Fig. 3 as deviations from the control (RC, line) mean. In a similar graph (Fig. 4), the deviations from the control (RC, line) for the 0.5 percent lysine dietary treatment effect are shown. In addition to the change for selected lines NS, HS and LS from RC line means, the actual grams deviation for the RC line itself has been plotted as the broken line in Figures 3 and 4 to illus- trate the generation to generation effect. These presentations make it very clear that there was some poorer growth performance for all four lines in the early generations but all lines showed considerable improvement in the later phase of the study. Due to the extreme variation, with reference to change in means from generation to generation, another approach for presentation of the data was considered. Table 15 shows the percent change from the RC line for each selected line (NS, HS and LS) by generation interval, along with the calculated percent change in mean on a within line basis from generation to generation. Each calculation of percent change (070A?) from line RC or change from generation to generation within the same line was made for both the control ( l . 0% lysine) and deficient (0. 5% lysine) nutrient environmental treatments. The percent change (70A,?) from previous generation and direc- tion, either upward or downward, was extreme (Table 15); the most 55 8:. out: so .2 s: so “on: .05 Nob—coo mo :noE Scum possum magnum 5 Amd new mm .96 on: can magnetron .m .mfim soflamuoaoO w 0 M0 N0 PU Cam 1 . _ _ on- |.. 1A0»: 988 finance 3 omnmsO O h u. l I. \ // m. // an \\ // \al \ l/ . 1 ON... d \ fl 2 \ / m \ x - ( / I. \ J N / . m N / I 3- \ z m. \ m mz ,\/ mu W mm ‘1 um o m D W a m 1 2+ - 2+ 56 «amp 0593 cam .o 05 no Ave: .Omv dosages m0 535 Esau pofidm madam 3 $4 was mm £73 on: .3 nowumgon— .v .mfm nowadaoaoO a. m N g o O O O 0 nm _ q q _ .I 1A0”: Geog deduce E 09530 \4’ \ / 1 \ ./ \ I \ / \\ // \ / 11$ 1 \ // l .111. / .\ l. / 4 4 / / 2 / / a / // om / .. _ ‘0 1 if (DU) om: 0N1 can o~+ m~+ news (33) paztuqezs mos; (sures?) 3811qu 57 msom>onm 50: Id ex: on N unomonm 80.5 14 axe «3.3 a... .o N osogoum Eoum Ides on unomonm 80.3 I 4 om. 928qu $0 4 N.NN+ N.N2+ «.oN+ 9N + No as. - N.2+ N; + TN + No N.N - N3 - fieN- a... .. 20 SN? as + 9N: N.o + o 3 v 8.2+ 92+ 9:- o.o - No N4. + N.NN+ TN: 5.3+ No v.2- +57 SN. 3. - ao 1N7 92+ 92.. N.o + 0 mm n2.3+ o.:+ To: win - Mo 1N - 98+ as + n22+ No N4. + 1N + m.NN- o.N - so 93. +9 + N.Nz+ N5 - 0 m2 58+ o.o 213+ o.o Mo N.S- ed as - 9o No as + ca 9:. as _o N.S- o.o a; + as o om ”GHQ 086m MO M .GGO 0m QGMH HO Magma 0am." ”ENG HO MM 3:00 0.“. ”am." HO MM ocmo eggo OGMNIH poaoom mouse at“? on: new somusnonom some .3 3:8va Saw #00? no.3» MOM AMdssv smog 5 09830 uncouonm .3 39mm. 58 extreme being +27.4 percent for the RC line on the 1.0 percent lysine diet. The least extreme case occurred for the NS line on the 0.5 percent lysine diet with -2.1 percent. In view of the extreme gener- ation to generation variation in growth performance, one becomes quite concerned with the probable causes and desires to learn if the source of variation is predominately genetic or environmental. Detailed analysis of the possibility of genetic change will be presented later after including data and information obtained for cross line progeny of the selected HS and LS lines and performance tested on each nutritional plane. Referring to the environmental source of variation, there are two possibilities that must be considered: first, an actual change in the nutritional environment or the diets fed annually; second, the possibility of differences of the physical environment, including daily care and management during the growth trials. When considering nutritional change from one generation (annually) to the next, it is important to recognize the possibility that, although the formulation procedure was not changed, the feedstuff ingredients and the micro nutrient supplies, including vitamins, minerals and amino acids (such as the "L-lysine monohydrochloride”), were acquired annually without specific quality control measures being employed. Some variation might arise at this level. The method used in this experiment to identify the possibility of meaningful S9 variation due to the diets, annually, was presented in Table 5. These results were from chemical laboratory analysis of the diets fed, analyzed especially for protein and amino acid levels. Some variation was noted and, in light of the numerous papers recently published concerning the problem of an arginine-lysine antagonism, the ratio of these two amino acids was considered within each diet fed at each generation interval (Table 5). The ratio of the amino acids, arginine: lysine, has been computed from the data in Table 5 and is presented in Table 16. To the author's knowledge, only Hill Bill.- (1961) Hill gal. ( 1966), and Hill and Shao (1968) have considered the effect of arginine to lysine where lysine was deficient. All other refer- ences cited in the review of literature dealt with the arginine-lysine antagonistic response where lysine was considered to be in excess of the requirement for the chick. A visual comparison of the growth performance graphs, Fig. 3 and Fig. 4, with the computed arginine: lysine ratios, Table 16, is interesting. In general, as the direction of the annual deviation for the A : L ratio (Table 16) shifted away from the base generation (Po), a shift to the opposite direction occurred for the mean three week gain of offspring on the l. 0 percent lysine diet (Fig. 3). A similar comparison for the full-sibs on the 0.5 percent lysine diet does not appear nearly as obvious with regard to the magnitude of directional change; however, the A : L ratios were only slightly variable (Table 16) for the deficient dietary treat- ment as compared to the control nutritive supply. 60 Table 16. Arginine: lysine ratio computed for each diet and genera- tion % Arginine % Lysine A:L Diet Gen. in diet in diet ratio Control Po 1.25 0.95 1:0.76 (1.0% lysine) G1 1.22 0.83 1:0.68 G2 0.91 0.87 1:0.96 G3 -- -- -- G4 1.01 0.87 130.86 Deficient P0 1.00 0.46 1:0.46 (0.5% lysine) Cl 1.25 0.52 1:0.42 G2 1.33 0.56 1:0.42 G3 -- -- -- G4 0.86 0.50 1:0.58 61 Annually, the wire floored brooding batteries were located in the same fan ventilated, light controlled facility; however, the animal caretaker was not the same from year to year. Responsibility and management instructions for the caretaker were identical, but the performance in relation to the execution of work assignments intro- duces a potential for variation. For example, change from genera- tion to generation could be attributable to a lack of precision in the adjustment of heating elements, for optimum brooding temperature relative to age of chicks, or perhaps to the maintenance of feed and water level in the troughs throughout the experiment. Since there was a high degree of correlation (Table 13) between the actual empirical three week gain in grams with the individuals' computed D/C percent, it appears unnecessary to evaluate realized heritability on but one of these measurements. Magee (1965) has described the effective relationship of heritability to genetic change (GA) and concluded that genetic change should be estimated only for the trait under direct influence through selection intensity. In this two—way directional selection experiment, the BIG per- cent value was the criteria for choosing breeding stock. Consequently, realized heritability estimates, as shown in Table 17, were calcu- lated for each selected line as a deviation from the control (RC) line for the D/C parameter. The ratio for each deviation response (gain) from one generation to the next was divided by the selection 62 00.0- 0.07 0.0+ 04 3.0+ 0.00+ 0.0+ mm 3.0+ +6? 0.0+ mz 38030 0300355000. 00.0- 0.0+ 0.00 0.0 - 0.0+ 0.0+ 0.0¢ m4 2.0+ 0.0+ 0.00 0.00+ 0.0+ 0.0+ 0.0¢ mm 00.0+ 0.0+ 0.00 0.0 + 0.0+ 0.0+ 0.0¢ 02 ¢ - - 0.00 - - - 0.00 0% O 00.0- 0.0+ 0.0¢ 0.0 - 0.0- 0.0+ 0.0¢ 04 00.0- 0.0+ 0.0¢ 700+ 0.0- ¢.0+ 0.00 03 000+ 0.0+ 0.0¢ 0.2+ 0.0- 0.0+ 0.¢¢ 02 0 -- -- is -- -- -- 1mm om o ~¢.~+ 0.0+ 0.0¢ 0.0 - 0.0+ 0.0+ 0.00 mA 00.¢- ¢.0+ 0.00 ¢.0~+ 0.0- 0.0+ 0.00 mm 010+ 0.0+ 0.¢¢ 0.0 + 0.0+ ~.¢+ 0.00 02 0 - - 100 - - - 0.00 0m 0 00.7 0.0+ 0.00 0.0 - 0.0+ 0.0+ ¢.0¢ 04 00+ 0.0+ 0.00 0.0~+ 0.0+ 0.0- 0.0¢ 03 “0.0+ ~.¢+ 0.00 0.0 + 0.0+ 0.0+ 0.¢¢ mZ _ - - 0.00 - - - 0.0¢ 0m 0 3:33:03 0% 59¢ c002 ~03G0H0H§Q 0389030 Um 50.3 502 500 0&4 £00 00500000 coEatSQ mgummflo Gowuo0~0m comudw>0Q G033>0Q ~3G0udnm 000009 00030 503 300.30 A02: 33.533000300 0003 00.2.3 2: 0G0 $0.3 no.0 unmanohomfiv nomuo0~0m\0mnomm0u >£ 0G: ADM: gown—sou 500:0." 000 no G005 0:» 88.3 2530100 030: 5:53:03 00s200u 00020800 .00 030p. 63 differential (intensity) for the particular line and generation. These estimates (Table 17) were quite variable from generation to genera- tion and many calculated estimates of realized heritability go beyond one, and, these values cannot be accepted as valid since unity is the theoretical maximum of heritability. For the duration of the experi- ment (P to G4), the accumulative deviation response divided by the 0 accumulative selection differentials for the D/C percent parameter, as shown in Table 17, provided realized heritability estimates of +0.26, +0.10 and -0.35 for lines NS, HS and LS, respectively. Accumulative selection differentials, as shown in Table 17, were +30.4 for the NS line, +83.9 for the high growth rate selected (HS) line and -l6.2 for the low selected (LS) growth rate line. In view of the fact that the empirical data for this experiment were obtained as grams of weight gained (three weeks minus one day weight), it was considered essential to estimate realized heritability for the gain in weight measurement. Since the trait (weight gain) was not the criteria for selection of individuals for breeding, esti- mations of realized heritability computed in the usual way [response (gain) divided by selection differential (intens ity)] could not be made with validity because gain in weight does not meet the restrictions as described by Lush ( 1945) and redefined by Magee (1965). These authors point out that frequently the concept and use of selection differential is in error and that the term, properly used, 64 applies only when there is mass selection for one trait (t1) and when the response or change from generation to generation is for that same trait (t1) being selected. Magee (1965) identifies "secondary selec- tion differential" as an appropriate term for the situation where one looks at a second trait for differences between means of the breeders and the population as a whole of (t2) which is different than the one used as selection criteria (t1) where "t1" represents the trait being selected and ”t2" identifies a second trait observed. In the cases for proper use of "t2" the breeder must not have considered the second trait in the selection process . Computed estimates of realized heritability were made for a second trait (t2), three week gain in weight, using the secondary selection differential. The secondary selection differential was computed directly using the mean of the breeders (15) minus the mean of the population (5) in which they were born. These estimates were made from the deviation of the HS (high) and LS (low) line from the control (RC) line as observed on the 0.5 percent lysine diet. Realized heritability estimates were made using the secondary selection differential for the NS line which was reproduced by random matings among survivors in the low (0.5% lysine) dietary stress nutritive environment . Shown in Table 18 on a generation by generation basis are the realized heritability estimates for the second trait (t2), three week 65 00.0- 00- 0 + m4 00.0+ 00+ 0 + mm 00.0+ 0 + 0 + m2 0mnon000m 03000580004 00.0- 0 + 00 0 - 0 + 0 + 0¢ m4 000- 0 + 00 ¢ + ¢ - 0 + 0¢ 00.0 00.0- 0 + 00 0 + 0 - 00+ 0¢ 02 ¢ - - 0¢ - - - 00 0m 0 00.0- 0 + 0¢ 0 - 0 + 0 - 0¢ m4 0¢.0+ 0 + 0¢ 00+ ¢0+ 0 - 0¢ mm 00.0+ 00+ 0¢ 0 + 0 + 0 + 0¢ 070 0 - - 00 - - - 0¢ 0m 0 00.0+ 0 - 0¢ 00- 0 - ¢ + 0¢ m4 00.0- 0 - 0¢ 0 + 00- 0 + 00 00.0 00.0+ 0+ 0¢ 0+ 0 - 0 + 0¢ 02 0 - - 0¢ - - - 0¢ 000 O 00.0- ¢ + 0¢ 0 - 0 + 0 + 00 010 00.0+ 0 + 00 0 + 0 + 0 + 00 mm 00.0- 0 + 0¢ 0 - 0 + 0 00 m2 0 - - 0¢ - - - 00 000 O 300000000000 0m 50.0.0 c0002 .mon0 00000 .000 035000000 0m 5900 00030 .000 05.0 .000 0000000000 00000000000 000800.000 0.0095000 05000007000 “500005.09 000:0u0n0 00000 000000 0.00.0 0000 0000 0000 0000oon0 00030 0003 0080.000 0000 00 00: 03» 000.3 no.0 000000000000 050000000 0.0000500300080000.“ 0n. 0000 00000 00.3050 500200.“ 05 00 0008 05 89G 0050003000 00005 3000000090: 00000000 000020500 .00 0030.0. 66 gain (grams) in weight. These estimates were as variable and un- realistic as those computed for the first trait (t1) the D/C percent parameter. The accumulative response in comparison to the accumulative secondary selection differential used to estimate realized heritability for the second trait (t2) was in partial agreement with the estimates from the D/C parameter. For a total time span of to G , both the high and low growth rate five generations from P0 4 selected lines declined with regard to their mean three week gain performance while the NS line changed very slightly. An accumulative secondary selection differential of six grams was realized for the NS line but the mean gain for P0 and G4 offspring were identical (55 grams) on the 0.5 percent lysine diet. For the high selected (HS) line, the accumulative selection intensity accounted for +21 grams while mean performance declined six grams (Table 18). With reference to the low selected (LS) line, the accumulative secondary selection differential was -27 grams with a very slight change in mean growth of minus three grams per chick on the average. If a difference in genetic ability exists, a specific expression in the low 0.5 percent lysine nutritive environment was not detectable with reference to a change in mean performance (Table 14). Further, there was no indication of genetic involvement from the numerous estimates of realized heritability (Tables 17 and 18). Since 67 contemporary full-sib groups were fed a control diet with 1.0 percent lysine, growth rate data for these individuals, as well as the full-sibs on the 0.5 percent lysine diet, were available for further analysis. The component of variance procedure, Henderson (1953), King and Henderson (1954) and Harvey (1960), was employed for estimating heritability for gain in weight (grams), from one day to three weeks of age, from inter-related sets of sibs. A numeric example of the analysis of variance and expected mean squares (EMS) is presented in Table 19 . Additional calculations from these values allow one to obtain variance component estimates for the effect due to sire (0:), dam (0;), and for the variance of all offspring (63V). ‘ A numeric example has been worked through to show the pro- cedure for computing variance component for sires, (0:) heritability estimate (hi) for sires and the standard error of the estimate of heritability [S.E. (1:2)] for sires (Table 20). This example repre- sents the actual data for the sire variance component of the low selected line (LS) in the fourth generation on the 0. 5 percent lysine diet, and it reveals a value of 0.24 as the estimate for heritability of three week gain in weight due to sire effect with 0.20 for the standard error of the heritability estimate. Heritability estimates computed by the component of variance method for progeny fed the control (1.0% lysine) diet appear in Table 21. Some deviation exists from one estimate to another; 68 .000 039900 $009302 00cm 33.00330 of c0 poucomonm one? mon u mx 00cm 00 .o n N00 .Nm.m u 0V0 30800000000 mGSSQEOU no.0 mponuoz * .3 . . N b Nm m0N 2. Noon va mou0m\ mEmQ\mn0nmmuO O . 3 . . N bNm m + N b mo moN om 0©wN0 ow mou0m\mEmQ n0 3 Mbmo.N0~+Nb00.o+ Nb 0N.oww mN.0mNm o mafim 0.012600. mom 0.308 *mzm m0>0 mm 000 :o0um0nm> mo meadow 3% 2:9: «$5 2: BEER $13 9000 0000000“; 300 of 00 0030. 9.900 no0uduonmw no.0 mvnmsvm 536 00000033 3.3.0.0909: on: £03 :w0mo0u 39009.8 0309330000 of H3 0003 60030.3; .00 3900.98 00 3&033 00.38570 . d0 3an 69 wmvbMN u 6+ 3 Q m N N N b + b 60 £033.09, 0093000209 0.30» 90H. on .o n nNHb 60 8500 .80 unocomgoo 0000.60.16». .208 00 03.6.0. 000 603.9, m0>0m of who 600000320800 02:00 300.0500 0» £030 * 3 Q m. b + b + b m ON.o u wooMN N N N AvaAmd 3003300000 . 0 0000000 no so» Max:201 ~036wa N a +Wl II W02 $0 N Nm N woomN 36+Qb+mb m . . 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P0 RC -.166 .204 1.030 .562 .432 .405 NS .537 .480 .393 .420 .465 .356 HS -.113 .227 1.173 .574 .530 .412 LS -.009 .111 -.234 .313 -.122 .157 G1 RC .612 .406 .219 .201 .416 .145 NS .493 .359 .397 .233 .445 .240 HS .365 .311 .475 .267 .420 .239 LS .642 .437 .295 .217 .468 .263 oz RC .664 .448 -.188 .291 .238 .265 NS .667 .496 .238 .422 .453 .354 HS .043 .274 .835 .557 .439 .396 LS .963 .642 .238 .305 .600 .373 (33 RC -.162 .198 .265 .450 .018 .296 NS .685 .578 .734 .433 .710 .424 HS .004 .262 .559 .496 .281 .345 LS .126 .223 .153 .376 .139 .246 G4 RC .357 .264 .454 .216 .405 .197 NS .614 .369 .067 .133 .340 .200 HS .142 .201 .501 .274 .322 .210 LS .590 .379 .101 .148 .345 .209 Mean .353 .344 .385 .344 .369 .287 71 however, 85 percent of the 60 estimates representing 3612 progeny are within the mathematical limit of unity. From the component of variation analysis, estimates of heritability for three week gain in weight on the average were as follows: sires, (’11:) = .35 :l: 34; dams, (’11:) = .39 :h .34; and combined sires plus dams, (fi:+d) = .37 :I: .29. These estimates are in relatively good agreement with other pub- lished heritability estimates (Godfrey, 1968, and Kinney, 1969). The estimates of heritability from 3753 offspring fed the 0.5 percent lysine diet were somewhat lower than those computed for growth performance on the 1.0 percent lysine diet. From among the 60 estimates of heritability for three week gain in the low (0.5% lysine) dietary nutrient environment, 88 percent appeared to be realistic heritability estimates and none of the estimates approached l .0, the upper limit of heritability. Estimates made within the 0.5 percent lysine dietary group (Table 22) were less variable than those from the 1.0 percent lysine dietary treatment environment (Table 21). The paternal half—sib component of four times the sire compo- nent of variance (40 2) divided by the total phenotypic variance 2 2 2 2 . . . . . "2 [(O'P) — US + GB + 0W] estimated heritability for Sires (he) — .231: .26, Z Z the maternal half-sib component (40 D/cr P) estimated heritability (hi) = .26 :1: .31, and from the full-sib analysis of two times the joint 2 Z 2 . . . . S + 0 D)/0 P] the heritabihty estimate effects of sires plus dams [2(0 A2 (h8+d) = .25 :l: .24 (Table 22). 72 Table 22. Heritability estimates from component of variance for three week weight gain (grams), with sexes pooled on the 0.5% lysine diet Gen. Line 112 S.E. fig S.E. A2 S.E. s d s+d P0 RC .116 .128 .318 .398 .101 .262 NS .218 .100 .164 .386 .027 .235 HS .535 .390 -.159 .290 .188 .242 LS .343 .295 .111 .282 .227 .221 G1 RC .310 .215 -.136 .153 .087 .128 NS .020 .119 .432 .242 .226 .177 HS .183 .177 .102 .193 .143 .147 LS .050 .101 .476 .264 .213 .191 G2 RC .443 .363 .035 .342 .239 .268 NS .214 .231 -.064 .298 .075 .195 HS .144 .317 .723 .524 .433 .382 LS .680 .484 . 198 .287 .439 .300 03 RC .524 .517 .720 .461 .622 .410 NS .298 .374 .647 .418 .473 .359 HS .204 .331 .443 .438 .323 .331 LS .354 .352 .351 .359 .352 .288 G4 RC .131 .156 .345 .228 .238 .168 NS .258 .190 .063 .150 .160 .127 HS .189 .204 .181 .232 .185 .176 LS .241 .201 .158 .179 .200 .147 Mean .234 .262 .256 .306 .245 .238 73 The data in Tables 21 and 22 were reorganized and presented in Table 23 for greater ease in comparing heritability among the lines. It may be observed that the heritability estimates were quite similar from each level of the variance component analysis and agreed very well between lines. For the 1.0 percent lysine dietary control fed situation, the best estimates of heritability with gener— ations pooled, were .31, .48, .40 and .29 for lines RC, NS, HS and LS respectively. In the 0.5 percent lysine environment, the com- bined sire + dam estimates of heritability were .26, . 18, .26 and .29 for lines RC, NS, HS and LS respectively. One essential consideration of all selection work, whether experimental or commercial in nature and of particular importance in small populations, is the consequence of inbreeding. Inbreeding in general terms is the degree of relationship among individuals. More specifically, the coefficient of inbreeding refers to the homozygous condition or the probability that two genes at any particular locus in an individual are identical by descent. Wright ( 19 34) described the path coefficients method of calculating the degree of inbreeding by saying that the level of inbreeding is equal to one half the coefficient of relationship of its common ancestors. Wright's path coefficient procedure was used to estimate the coefficient of inbreeding for the males in each of the selected lines (HS and LS) in this experiment, Fx=%2[(21')n(1+ FCAH 74 Table 23. Summary of calculated heritability estimates by line for diets separately and generations pooled for each level of the component of variance Line Source RC NS HS LS Control 1.0% lysine diet Sire (’12:) .261 .599 .088 .462 A2 Dam(hd) .356 .366 .709 .111 A2 Combined (h ) .309 .483 .398 .286 s+d Deficient 0.5% lysine diet , AZ Slre (ha) .258 .114 .251 .314 A2 Dam (hd) .256 .248 .258 .259 A2 Combined (hs+d) .257 .181 .255 .287 75 where: Fx is percent inbreeding of individual "x"; n is the number of paths; FCA is the inbreeding of the common ancestor. A brief review of the design of this experiment as it influences the breeding relationships within each line is important. First, each sire was to be represented in the next generation by a son, either by random choice for lines RC and NS or by selection as determined by the criteria for the HS and LS line respectively. Second, for lines RC and NS, each of eight sires was mated at random to females from within the closed line. For the high selection (HS) and low selection (LS) lines, a pedigree mating plan was used as a means of avoiding close matings. Neither full nor half—sib matings were allowed. All matings were completed by artificial insemination for individually caged females. The number of males and females used during this experiment are present by line in Table 24 and by generation in Table 25. Because of the potential consequence of low numbers in the effective breeding population which could contribute to an inbreeding depression, the number of dams mated to each sire was increased in the fourth generation. The increase in the number of dams had the effect of slowing down the rate of increase of "F", the coefficient of inbreeding. The actual number of males and females that contributed ~offspring to the breeding population of the next generation are 76 0500802020 magvonn 000000000 co0umnosow 008: 0:» 00 0.30505 0000500920 ways: on 00050000 melon o>0uoo00mxn Swap: 0.30.0. «Nam! 6.3 or} 6.6: m4 ww0nom 00.010 m.mn0 «.00 mm oONnov N.mn0 w.mu0 0.90 mZ oONuow N.m”0 w.mu0 0.90 000 .aom 0300000000 *donm umpoonm wan—0.200000 .pounm .Qon0 .3000on 0:010 00 SSW 638600.10 06 638m .5 0o 6:30 0.3000 mm 00 000.30 0G 080.0 2008 000» $00.30. 0:00 .3 000......" 00.680060900 ommnocfiw .wN 03.6.0“. 77 00000802020 90000093. 060000006 cofimuocom 008: 9.00 00 mama—Eon» 000360.20 matron mm 06050000 9309. 0300000000 * Swap: 0.30M. llll . . 0. oNNuom m T0 020.0 00 m0 0 oM0u0m 04.010 m.mn0 ob: m0 0m0uNm 01.010 m.mn0 00.90 NO mfumm 0.90 Nd; o8: 00 m0~0nNm m.010 N.mu0 0.90 onw .Qom 030000000 *.n00n0 00000ch 90020000 .poun0 .monm Hopoonm .G00 00 55m 0300000000 00 000.30 .nm 00 000.30 00000000w 00 000mm 000080.398 000 900.300 co0uohvcow >0 000d.“ 01800535 mmm~o>< .mN 000.68 78 tabulated in Table 26, along with the coefficient of "F" or the estimate of the percent of inbreeding at each generation interval and by line. With no more than eight percent for the highest degree of inbreeding for any line, it is concluded that an inbreeding depression is unlikely as a major factor influencing the course of this experiment. Further, there must be reasons other than "inbred" for lack of progress in the direction of selection, whether it be for the high or for the low gain (grams) in weight. In theory, the bi-directional selection scheme will separate two lines from each other with regard to the parameter under selection, and the amount of dispersion is influenced by two factors. The first of these factors is the intensity (i) or selection differential (S) applied for the trait. The second factor is the heritability (hz) of the trait being selected. Application of this concept allows the researcher to predict genetic change or progress from one generation to another due to selection. The formula used for this prediction was: where: GA = the amount of genetic change; i = intensity or selection differential; h2 = heritability of the trait selected. 79 Table 26. Coefficient of inbreeding "F" by line and generation interval Actual Actual Mating No. No. Coefficient Gen. Line system Males Females of ”F" P RC Restricted 8 36 . 000 0 Random Cl 8 40 . 015 G2 8 40 . 030 G3 8 30 . 046 G4 8 63 . 059 P NS Restricted 8 32 . 000 0 Random G1 8 43 . 015 G2 8 38 . 030 G3 8 34 . 045 G4 8 59 . 059 P0 HS Pedigree 8 37 . 000 G1 Selection 8 40 . 000 GZ 8 37 . 000 G3 8 32 . 068 G4 7 42 . 078 PO LS Pedigree 8 40 . 000 G1 Selection 8 42 . 000 G2 8 36 . 000 G3 7 40 . 049 G4 7 56 . 066 Total N 157 817 80 Predictions for direction and progress using this model for this experiment did not provide any estimates of genetic change (GA) similar to the behavior observed for either measurement, D/C per- cent or three week gain (grams) as parameters. From the lack of divergence in the mean performance (after four generations of selection) of the high (HS) and low ( LS) selected lines, and in view of the relatively large selection differential which was calculated, one may say that generally the trait being selected did not exhibit additive gene action. To further test for genetic change (GA) among the selected lines (also called pure lines), a series of reciprocal line crosses were made and their cross line Fl progeny were measured for growth rate on both diets; these diets being the control with 1.0 percent lysine and the deficient with 0. 5 percent lysine by formulation. The diets fed to the pure lines and the F cross lines progeny l were the same diets from the same batch of feed mixed on an annual basis. Least squares analysis of variance computations were made on data representing the second, third and fourth generations to test for diet, line, and diet by line interaction effects. The statistical model for this analysis was: Yijk = p. + Di + Lj + DLij + eijk 81 where: th . . . . .th . Y.. = the ean observed for the k 1nd1v1dual 1n the J line on 13k .t . the 1 diet; p. = the overall common mean; .th . . Di = the effect of the 1 d1et, 1 = l, 2; .th . . Lj = the effect of the J line, J = l . .4; DLi' = the two-way interaction associated with the designated J subclasses; eijk = the random error among observation. The analysis of variance for these data pooled for generations (Table 27) show highly significant differences for diet line and diet by line (DxL) interation effects. Included as lines for this analysis were the high (HS) and low (LS) selected lines and F cross line progeny from 1 HS d'x LS? and LS 6'): H82. The diet by line interaction was also highly significant (P < .005), a result not previously observed among the pure line alone. An examination of the means by diet and line, whether pure line HS and LS or cross line progeny from HS 1: LS and LS x HS (Table 28), show that the cross line progeny on the control 1.0 percent lysine diet were always superior in three week gain in weight as com- pared to the pure line progeny. For growth response observed under dietary deficient 0. 5 percent lysine fed conditions, generally, the pure line progeny grew more rapidly than the cross line progeny. The lack of a uniform response characteristic for full-sibs tested on two 82 Table 27. Least squares analysis of variance for three week gain (grams) for pure line HS and LS progeny and for F cross line progeny with generations 02' G3 and G4 pooled Source of Variation df V MS F Sig. Total 3883 Diet 1 5302628.96 11249 .40 <. 005 Line 3 9412.69 19.97 <.005 DxL 3 36270.01 76.95 <. 005 Error 3876 471 . 37 83 000000000 08400 000 0003 00.005 000 .00000 .300 00 msoum 000000000w 0000 400000000 0000 000003 0mmo0 40000000 3000000000 00 00>00 0000000 030 000 00 0000000000 4000000000030 00: 0.3 000000 0 6N4 n 3. N: 2.4 a 6N4 NE 00604 4 6 N44 a 3. aNN $4 6 6.2 EN 04600 A 6.: 0 $5 6 mm +8 6N4 a N4 mNm 04804 4. 34 6 N6 EN 664 a 4N4 emN 006.00 0 6N4 6 46 6N4 $4 6 N2 42 mmqu 4 m 84 6 me a: £4 6 24 m2 mqumm A .5 0 6N4 6 3 3.0 CTN 6 NS oi 04004 N 06.4 6 S 6: MEN 0 84 84 $000 0 £4 a 6 we 3 NvN 6 :4 cm 00x04 4 N 34 6 N6 mo 34 6 m: 2: qumm A E o A6N4 6 m4. :4 66:6. 0 £6 6N4 04604 N 664 46 64. 2: SN 40 :6 84 006.0: 0 .m.m mo. v0 686% z .m.m mo. v60 .mfiwm z 6&4 .060 6:463 .3. .o 686: so 4 0000000 00800 0003 09,0005 000000000066 0000 00000 >0 >G0m0§0 0000 000.00 0000 0000 0.23 .000 008003 000m 0000? 00.20» 00030 . wN 0000B 84 dietary levels of lysine shown in Table 28 indicate the existence of the diet by line interaction which was verified by the analysis of variance (Table 27). To more easily visualize the interaction effect, a series of two-way contingency tables (appearing in Table 29) were assembled to show the diet by generation by line of sire and line of dam effects . Heterosis as a behavioral phenomenon has been reported for many different traits in animals and plants. The expression of hybrid vigor results from recombinations of many hereditary factors. Gowen ( 1964), with the help of many colleagues, published an entire book summarizing the information available at that time on heterosis. One type of heterosis (Gowen, 1964) which may be identified by crossing inbred lines is an expression of interaction between allelic genes. Another kind is hybrid vigor resulting from interaction effects at the chromosomal and/or cytoplasmic sites. Of specific concern in this experiment was a test for hybrid vigor, ”heterosis", through Fl cross line progeny from the high (HS) and low (LS) selected lines and, further, to measure the relative advantage of selecting in a poor nutritional environment for optimum performance of F cross line progeny in different nutritional regimes. l A study and analysis of the performance data for the pure line vs. cross line progeny was made by estimating the percent heterosis 85 Table 29 . Two-way contingency tables for cell means showing diet by line interaction effect for line of sire and line of dam by diet and generationl. 7- 1. 0% lysine 0. 5% lysine Gen. Dam Dam G2( F1) Sire HS LS HSHS HSLS HS 43 LSHS LSLS LS 45 HSHS HSLS HS 45 LSHS LSLS LS 41 ; G4( Fl) HSHS HSLS HSHS HSLS HS 40 LSHS LSLS LS 40 15:3 lPure line genotype sources are shown as HSHS and LSLS Cross line genotype sources are shown as HSLS and LSHS 86 for the cross line progeny. Estimates of percent heterosis were calculated by generation according to the following formula: Cross line Pure line Percent mean - mean 100 heterosis ' Pure line x mean where: Percent heterosis is the degree of response; Pure line mean is the average of the parental types HS x HS plus LS x LS; Cross line mean is the average of F progeny types HS x LS plus LS x HS. 1 The result of these estimations have been tabulated in Table 30 according to the response in each nutritive environment. Measure- ments for growth were recorded in either the control 1.0 percent lysine or the deficient 0. 5 percent lysine nutritive environment. The average percent heterosis favored the cross line progeny in the control nutritive environment by 17 percent (Table 30); however, in the case of the deficient nutritional plane, there was no consistent response as generation G showed a positive 14 percent heterosis 2 while G3 and G4 were each negative, -4 and -25 percent respectively. One aspect of the nutritional environment not yet considered is the overall effect of the low level 0.5 percent lysine (an imbalanced protein diet) on livability of experimental chicks. A specific line (NS) was incorporated in this study to observe the effect of the 0. 5 percent 87 Table 30. The influence of nutritional environment on heterosis for three week gain (grams) of chicks from the high (HS) and low ( LS) lines selected for growth on a 0.5 percent lysine diet 1.0% lysine diet Cross line Pure line Percent Gen mean mean heterosis G2 115 97 +19 G3 135 106 +27 G4 129 123 + 5 Mean +17 0.5% lysine diet C2 50 44 +14 G3 43 45 - 4 -2 C4 40 53 5 Mean - 5 88 lysine dietary environment as a force of natural selection. This treatment effect was called "natural selection" since reproduction from one generation to the next was by random choice of the breeders; however, random selection of breeders was permitted only among survivors on the poor nutritional environment. Presented in Table 31 are percent mortality data by diet, sex, generation and line. Con- siderable variation was evident with no consistent trend. The diet by sex interaction is believed to exist because the 0. 5 percent lysine diet contributed to a higher mortality rate among the males averaging 17. 12 percent on the deficient diet, but only 8. 12 percent on the control diet. For the females on the deficient diet the mortality rate was 15.15 percent and 10.14 percent on the control diet (Table 31). The higher mortaltiy rate of males in the deficient lysine environment is considered to be due to an inherent growth rate differential favoring the males, thus, they were stressed much more, resulting in the higher death rate, than the females when fed the lysine deficient diet. Also there was a marked difference in mortality rates as influenced by the dietary environment. Throughout the five generations involved, the average mortality rate was excessively high with 9 .28 percent for progeny on the control diet ( l. 0% lysine) and 16.14 percent for those fed the deficient diet (0. 5% lysine) during the critical three week growth period for this study. The question as to how well a population of laying hens produce eggs is always of interest to investigators; consequently, the average 89 Table 31. Percent mortality from one day to three weeks of age by diet, sex, generation and line * I. 070 lysine * 0. 570 lysine Number Gen . Line offspring Male Female Male Female P0 RC 281 2.90 5.71 18.57 7.89 NS 258 1.85 5.26 10.45 10.45 HS 330 8.77 15.79 14.71 18.89 LS 335 8.33 8.22 15.07 8.79 61 RC 536 4.80 8.96 12.67 15.25 NS 603 7.53 6.20 9.26 11.39 HS 561 9.49 13.11 14.74 14.89 LS 607 12.03 13.11 14.20 14.58 G2 RC 335 13.11 9.59 18.56 19.78 NS 315 1.92 8.20 15.22 8.42 HS 306 5.36 11.67 26.97 18.68 LS 343 14.47 8.70 9.80 15.22 G3 RC 271 8.57 5.88 30.00 15.00 NS 305 0.00 9.09 21.05 20.00 HS 265 6.90 14.29 35.29 17.14 LS 325 15.28 17.65 25.00 27.50 C4 RC 753 16.85 12.44 18.04 16.17 NS 823 10.70 13.27 12.76 12.84 HS 508 7.97 10.74 6.47 18.02 LS 710 8.56 10.86 13.53 12.15 Means sex/diet 8.12 10.14 17.12 15.15 Diet .14 :5: Percent lysine in starter diet; all birds were fed the same rations throughout the remainder of the study. 90 number of eggs produced by survivors of 280 days (ten, 28 day periods) was summarized (Table 32). Statistical evaluation for differences was completed and a ranking of means is denoted at the five percent level of probability (Duncan, 19 55). The data, average number of eggs per hen (Table 32), have been converted to percent production for diet, generation and line variables of the experiment and these percents are recorded in Table 33. Some variation exists from one generation interval to another but the direction of change and deviations within and between diets are nearly identical. From one generation to the next, the percent change was -4, -4, and +1 for the birds grown on the 1.0 percent lysine diet and -7, -2 and +2 for those fed the 0.5 percent lysine diet. An examination of the. overall mean (Table 33) for each diet reveals no significant differences (P > .05). From a commercial and economic point of view, mortality rate during the laying period is tremendously important. The mortality records for birds housed in this experiment have been summarized in Table 34 according to diet as a factor during the early growth period and for generation and line sources of variation. From these data, one may conclude that, in the adult p0pulation as a whole, livability of survivors of the dietary stress situation during growth was no different than that of the non-stressed (RC) line. The mortality rates were quite variable and no particular pattern was 91 0000000500 000 0000w00000 0000000 0500 000 0000 0003 000000 000 000000 0000000 00 0000>0 000000n0 000 000 000500000 >0000000 000003 000000000m 0000000000 0 000 000000 0500 000 000.3 000030 .320 00000600 3000000000 00 00>00 0000000 030 000 .00 0000000000 >000000000w00 0 . >000: 000 00 * 8.0 6 64.004 N0 00.0 0 3.2.4 .3. 04. 00.0 6 004.64 0.6 0040 6. A6.0.04.4 : 00 00.0 6 NN.02 $1 00.0 0 6 2.404 .64. 02 m 84. 6 04.02 E. 0N0 6 0N4: 0N 00 0 06:0 0 6 3.000 00 0.0.0 0 6 004.2 mm 04 00.0 0 No.02 3 4.0.0 0 6 $4.3 N 00 00.0 0 6 49.6.2 00. 0.0.0 0 24.2 N 02 N 06.0 6 0N.o04 06 2.0 6 0.0.0004 00. 00 0 8.0. 0 6 00.02 am :0 0 8.02 am 04 00.0 0 6 $4.04 0.0 00.0 6 26.0: mm 00 0:6. 0 8.2.4 00 00.0 0 8.004 4.0 0z 4 004. 6 0.4.004 00 00.0 6 0+4: 2. 00 0 :64. 0 0.0: 2 N40 6 8.004 3 04 004. 6 NN.004 00 00.0 6 00.0: N0 00 N010 0 09m: 00 00.0 6 $004 an 02 o NN.0 40 2.4.: 00 2.0 46 404.: 0. 00 0 .00 0000 6006 60.00 .00 0000 6006 60.00 6&4 .060 .000 .oz .62 .000 .62 .oz 050% 60m .0 * 00000: 6&9 . 0 * 0000 00 00>000 owNv 000003 ow. m00000u 00o>0>000 000 0000000000 0ww0 00 000500 0w000>< . Nm 00009 Table 33 . (280 days) on test 92 Percent egg production for survivors during 40 weeks * l. 0% lysine 0 . 5% lysine* Gen. Line No. birds % prod. No. birds % prod. P0 RC 41 62.33 59 63.36 NS 39 60.21 55 62.14 HS 42 62.79 50 67.58 LS 112 59.64 Z_1_ 62.65 171 61.19 235 63.76 Gl RC 73 61.23 50 59.34 NS 64 53.58 83 53.71 HS 55 61.12 54 58.73 LS _5_9_ 53.71 §_9_ 56.00 261 57.55 246 56.50 G2 RC 46 57.45 46 57.24 NS 27 47.90 45 54.75 HS 25 51.73 41 48.22 LS 2 55.30 fl 56.41 131 53.85 182 54.36 G3 RC 28 61.16 44 56.49 NS 43 54.25 49 55.44 HS 31 52.38 34 52.64 LS _4_3_ 52.76 52. 59.33 145 54.74 179 56.12 Overall totals and mean 708 842 57.98 57.17 3): Percent lysine in starter diet; all birds were fed the same rations throughout the remainder of the study. 93 Table 34. Laying house mortality during 40 weeks (280 days) on tests * =1: 1.0% lysine 0.5% lLsine No. No. % No. No. % Gen. Line housed dead mort. housed dead mort. P0 RC 47 6 12.8 65 6 9.2 NS 46 7 15.2 60 5 8.3 HS 49 7 14.3 57 7 12.3 LS _56 _7_ 12.5 _71 _6 7.8 198 27 13.6 259 24 9.3 Gl RC 80 7 8.6 55 5 9.1 NS 82 8 9.8 99 16 16.2 HS 67 12 17.9 64 10 15.6 LS _6_1 _8 11.9 _61 _8 11.9 296 35 11.8 285 39 13.7 (32 RC 52 6 11.5 51 5 9.8 NS 33 6 18.2 57 12 21.1 _ HS 30 5 16.7 50 9 18.0 LS __3_8_ _§_ 13.2 _5_6 _6 10.7 153 22 14.4 214 32 15.0 G3 RC 35 7 20.0 50 6 12.0 NS 51 8 15.7 58 9 15.5 HS 36 5 13.9 46 12 26.1 LS i6 _3 6.5 _59_ _1 11.9 168 23 13.7 213 34 16.0 Overall totals and means 815 107 13.1 971 129 13.3 * Percent lysine in the starter diet: all birds were fed the same rations throughout the remainder of the study. 94 evident. The rates were apparently not influenced by line and the mean adult mortality was 13.1 percent for birds started on the con- trol l. 0 percent lysine diet while the chicks started on the deficient O. 5 percent lysine diet had an overall mortality rate of 13.3 percent (Table 34). Fertility rate by generation and line as well as the percent hatchability was computed for the pure lines RC, NS, HS and LS and the results appear in Table 35. Artificial insemination was prac- ticed for all matings during the tenure of this experiment. Fertility rate, while having some variation from line to line within generation interval, did not establish a consistent trend; therefore, this trait was not considered to have an influence on the overall experiment. The percent of fertile eggs hatched was very erratic from generation to generation but quite uniform among lines per generation. The overall weighted mean percent hatch (Table 35) shows generation three to be depressed as compared with all other generations among which there was little variation. An examination of the data for fertility and hatchability among reciprocal line crosses (Table 36), compared with the pure lines (Table 35) shows the HS line of sire as having contributed to a higher percent fertility than line LS. The effect of line of dam was incon- sistent and assumed to be random. The overall mean for percent hatched at the third generation interval for F cross line progeny was 1 95 Table 35 . Percent fertility and hatchability by generation for pure lines Mean % Gen. Line No. eggs % fertile % hatched hatched P0 RC 501 80.64 69.55 NS 474 74.26 73.30 HS 532 87.59 70.82 1 LS 533 79.92 78.64 73.13 (31 RC 988 83.91 64.66 NS 1045 79.81 72.30 HS 929 84.39 71.56 LS 1039 77.96 74.94 70.93 G2 RC 534 88.76 70.68 NS 526 78.90 75.90 HS 546 89.38 62.70 LS 471 90.45 80.52 72.14 G3 RC 799 78.60 43.15 NS 658 86.47 53.60 HS 801 81.40 40.64 LS 952 69.64 40.02 46.41 G4 . RC 1235 84.53 72.13 NS 1208 89.57 76.52 HS 814 82.92 75.26 LS 1005 86.17 81.99 76.30 lWeighted mean 96 Table 36 . Percent fertility and hatchability by generation for reciprocal line crosses Gen. Line No. eggs % fertile % hatched GZ(F1) HSX LS 275 92.73 81.96 LSXHS 319 86.21 72.00 G3(Fl) HSX LS 710 67.18 76.10 LS X HS 662 76.59 72.78 G4(Fl) HSX LS 383 85.64 80.79 LS X HS 278 70.14 63.59 97 approximately 74 percent, a value in good agreement with those of other generations for both pure line and cross line breeding. This indicates that there was nothing particular for that year which con- tributed to the low rate for hatchability among the pure lines of generation three. The low hatchability of 46.41 percent for the pure lines at generation three (Table 35) is accountable only to a variable introduced by man causing the high degree of error or due to equip- ment in the hatchery. CHAPTER V CONCLUSIONS Mass selection pressure was applied to a population of egg-type chickens to evaluate the genetic parameters of the lysine require- ment, estimate heritability and to evaluate the latent consequences of the early growth diet (one day to three weeks of age) upon subsequent reproductive performance. The rearing environment was deficient in lysine with O. 5 percent available. This bi-directional selection experiment for pedigreed high and low growth rate lines indicated that the two-way selection was rela- tively inefficient in separating the lines when they were fed the lysine deficient diet; however, each selected line exhibited growth rate improvement as compared to the unselected RC (random control) line. Line NS (meaning natural selection) was maintained as sur- vivors only of the deficient dietary environment, and no selection pressure other than dietary stress was applied. The high growth rate line designated as HS was intensely selected for gain in weight from one day to three weeks of age. The LS line represented the influence of maximum selection pressure for low (gain in weight) growth in the dietary lysine deficient environment. 98 99 Realized heritability estimates computed on deviations from the random bred control line were quite variable, 0.26, 0.10 and -0.35 for the lines NS, HS and LS, respectively. Estimates of heritability from the component of variance method were more consistent for each line. These estimates were to 0.23, 0.26 and 0.25 for the sire, dam and combined sire plus dam sources of variance with lines NS, HS and LS pooled. Considering the combined sire plus dam estimate of heritability as the best estimate, a comparison of the high (HS) and the low (LS) selected lines in the bi-directional selection experiment showed heritability for growth in the lysine deficient environment to be 0.26 and 0.29 for lines HS and LS, respectively. Coefficients of inbreeding were estimated for each line in the study and the level of inbreeding was not found to be high enough to contribute significantly to an inbreeding depression. Cross line breeding was practiced among the HS and LS selected line producing Fl progeny during the second, third and fourth genera- tions. These progeny were also tested in the two nutritional environ- ments and they exhibited a high degree of positive heterosis when grown in the control dietary environment with l. 0 percent available lysine and a negative heterosis for the contemporary full-sibs grown in the 0.5 percent lysine dietary environment. The expression of differential heterosis by cross line progeny in the two different nutritional environments needs further study and may have commercial applicability . 100 The associated traits of egg production, adult livability and reproductive capacity as measured by fertility and hatchability were not influenced by nutritional background common to the individual or selected line. This experiment has demonstrated a need for further research as to the genetic parameters for appetite control, patterns of feed consumption and feed efficiency among lines for various levels of lysine in the diet. It is probable that larger populations of a more rapidly growing stock would be more productive in studies of growth rate and adaptability of genetic stock to malnutrition type environ- ments. Since heritability was found to be low, 0.26, a more intense selection design, as opposed to mass selection, should be employed for more rapid progress. B IB LIOGRAPHY Almquist, H. J., 1957. Protein and Amino Acids in Animal Nutrition. Fourth Ed., U. S. Ind. Chem. Co., New York. pp. 1-32. Bartlett, M. S., 1937. Properties of sufficiency and statistical tests. J. Royal Soc. London. 160: 273-275. Beadle, G. W. and E. L. Tatum, 1941. Experimental control of deveIOpment and differentiation; genetic control of developmental reactions. Amer. Nat. 75: 107-116. Becker, W. A., 1967. Manual of Procedures in Quantitative Genetics. Second Ed., Wash. State Univ. Press. 130 pp. Boorman, K. N. and H. Fisher, 1966. The arginine-lysine inter- action in the chick. Brit. Poultry Sci., 7: 39-44. Colombo, J. P., R. Richterich. A. Spahr, A. Donath. and E. Rossi, 1964. Congenital lysine intolerance with periodic ammonia intoxication. Lancet. 1: 1014. Crow, J. F., 1952. Heterosis. Iowa State College Press. pp. 282- 297. ’7 Dean, W. F. and H. M. Scott, 1965. The development of an amino acid reference diet for the early growth of chicks. Poultry Sci. 44: 803- 808. Dean, W. F. and H. M. Scott, 1968. Ability of arginine to reverse the growth depression induced by supplementing a crystalline amino acid diet with excess lysine. Poultry Sci. 47: 341-342. Duncan, D. B., 1955. Multiple Range and Multiple F Test. Biometrics. 11: 1-42. Dickerson, G. E., 1960. Techniques and Procedures in Animal Production Research. Amer. Soc. of An. Prod. pp. 57-96. Enos, H. L. and R. E. Moreng, 1965. Evidence of genetic varia- bility for lysine utilization. Poultry Sci. 44: 964-971. /0/ 102 Falconer, D. S., 1953. Selection for large and small size in mice. J. Gen. 51: 470-501. Falconer, D. S., 1960. Quantitative Genetics. The Ronald Press Co., New York. pp. 254-263. Falconer, D. S. and M. Latyszewski, 1952. The environment in relation to selection for size in mice. J. Gen. 51: 67-80. Friedman, M. and S. O. Byers, 1948. J. Biol. Chem. 175: 727. Garrad, A. E., 1902. The Incidence of Alkaptonuria: a study in chemical individuality. Lancet. Ghadimi, H., V. Binnington and P. Pecora, 1965. Hyperlysinemia associated with retardation. New Eng. J. Med. 273: 723-729. Godfrey, E. F., 1968. Ten generations of selection for lysine utilization in Japanese Quail. Poultry Sci. 47: 1559-1566. Gordon, R. S., 1963. Growth arrest through tryptophan deficiency in the very young chicken. Proc. Sixth Int. Cong. of Nutr., Edinburgh. Gowen, J. W., 1964. Heterosis. Hefner Publishing Company, New York. 552 pp. Griminger, P., 1955. The amino acid requirement of chicks as influenced by their genetic ability grow. Poultry Sci. 34: 1198- 1199. Griminger, P. and H. Fisher, 1962. Genetic differences in growth potential on amino acid deficient diets. Proc. Soc. Exp. Biol. and Med. 111: 754-756. Harvey, W. R., 1960. Least squares analysis of data with unequal subclass frequencies. USDA, ARS, Beltsville. 20-8. Henderson, C. R., 1953. Estimation of variance and covariance components. Biometrics. 9: 226-252. Hess, C. W., H. M. Edwards, Jr., and E. F. Dembnicki, 1962. Growth- rate selection on a methionine deficient diet. Poultry Sci. 41: 1042-1047. 103 Hill, D. C., E. M. McIndoo and E. M. Olson, 1961. Influence of dietary zein on the concentration of amino acids in the plasma of chicks. J. Nutr. 74: 16-22. Hill, D. C., J. Singh and G. C. Ashton, 1966. A chick bioassay for lysine. Poultry Sci. 45: 554-560. Hill, D. C. and T. Shao, 1968. Effect of arginine on weight gain of chicks consuming diets first-limiting in lysine or tryptophan. J. Nutr. 95: 63-66. Hutt, F. B., and Nesheim, M. C., 1966. Changing the Chick's requirement of arginine by selection. Can. J. Gen. Cytol. 8: 251-259. Hutt, F. B., and Nesheim, M. C., 1967. Genetic variation in the utilization of arginine by chicks. Poultry Sci. 46: 1274. Jones, J. D., 1961. Lysine toxicity in the chick. J. Nutr. 73: 107- 112. Jones, J. D., 1964. Lysine-arginine antagonism in the chick. J. Nutr. 84: 313-321. Jones, J. D., S. J. Petersburg and P. C. Burnett, 1967. The mechanism of the lysine-arginine antagonism in the chick: Effects of lysine on digestion, kidney arginase, and liver transamidinase. J. Nutr. 93: 103-116. King, S. C. and C. R. Henderson, 1954. Variance component analysis in heritability studies. Poultry Sci. 33: 147-154. Kinney, T. B., Jr., 1969. A summary of reported estimates of heritabilities and of genetic and phenotypic correlations for traits of chickens. USDA, ARS, Beltsville. 363: 1-49. Klain, G. J., H. M. Scott and B. C. Johnson, 1960. The amino acid requirement of the growing chick fed a crystalline amino acid diet. Poultry Sci. 39: 39-44. Lamoreux, W. F. and F. B. Hutt, 1948. Genetic resistance to deficiency of riboflavin in the chick. Poultry Sci. 27: 334-341. Lerner, I. M., 1958. The Genetic Basis of Selection. John Wiley and Sons, New York. 273 pp. ' ' 104 Lush, J. L., 1945. Animal BreedingPlans. Third Ed., Iowa State College Press, Ames. 439 pp. Lush, J. L., 1948. The Genetics of Populations. Mimeo Notes, Iowa State College, Ames. 381 pp. Magee, W. T., 1965. Estimating response to selection. J. An. Sci. 24: 242-247. Maloney, M. A., Jr., J. C. Gilbreath and R. D. Morrison, 1963. Two-way selection for body weight in chickens. I. The effective- ness of selection for twelve-week body weight. Poultry Sci. 42: 326-334. Martin, G. A., and A. E. Bell, 1960. An experimental check on the accuracy of prediction of response during selection. Biometrical Gen. Pergamon Press, London. pp. 178-187. Mendel, L. B., 1915. Nutrition and growth. J. Am. Med. Assn. 64: 1539. Morton, R. A. and E. C. Amoroso, 1967. Protein Utilization by Poultry. Oliver and Boyd, Ltd., Edinburgh. 210 pp. Merck. 1961. Lyamine: A Technical Bulletin on Lysine in Animal Nutrition. Merck and Co., Inc., Rahway. 77 pp. Mitchell, H. B. and M. B. Houlahan, 1946. Am. J. Botany. 33: 31-35. National Research Council, 1960. Nutrient requirements for poultry. Publ. 827, Wash. D. C. 1-23. Nutritional Biochemicals Corp., 1964-68. Standard Amino Acid Supplies. N°B' C, Cleveland. 1-10. Nesheim, M. C., 1966. Genetic Variation in Nutrient Requirements. World's Poultry Sci. 22: 290-298. Nesheim, M. C., 1969. Personal communication. Nesheim, M. C. and F. B. Hutt, 1962. Genetic differences among White Leghorn chicks in requirements of arginine. Sci. 137: 691-692. 105 NOPCO, 1962. Feed Egredient analysis table. NOPCO Chem. Co.,, Park Place, Newark. 1 p. Nyhan, W. L., 1967. Amino Acid Metabolism and Genetic Variation. McGraw-Hill Book Co., New York. 490 pp. O'Dell, B. L. and J. E. Savage, 1966. Arginine-lysine antagonism in the chick and its relationship to dietary cations. J. Nutr. 90: 364-370. Pearson, E. S. and H. O. Hartley, 1954. Biometrika. Tables for Statistics. Cambridge Univ. Press. 1: 179-180. Ralston Purina Co., 1964-68. Analytical Reports, "Spinco Amino Acid Analysis," Checkerboard Square, St. Louis. 4 pp. Ryan, W. I. and I. C. Wells, 1964. Homocitrulline and homoarginine synthesis from lysine. Sci. 144: 1122. Schwartz, H. G., M. W. Taylor and H. Fisher, 1958. The effects of dietary energy concentration and age on the lysine require- ment of growing chicks. J. Nutr. 65: 25-37. Siegel, P. B., 1962. Selection for body weight at eight weeks of age. 1. Short term response and heritabilities. Poultry Sci. Singsen, E. P., J. Nagel, S. G. Patrick and L. D. Matterson, 1965. The effect of a lysine deficiency on growth characteristics, age at sexual maturity, and reproductive performance of meat- type pullets. Poultry Sci. 44: 1467-1473. Smith, R. E., 1968. Effect of arginine upon the toxicity of excesses of single amino acids in chicks. J. Nutr. 95: 547. Smith, G. H., and D. Lewis, 1966. Arginine in poultry nutrition. 3. Agent and target in amino acid interactions. Brit. J. Nutr. 20: 621-631. Snedecor, G. W. and W. G. Cochran, 1967. Statistical Methods, Sixth Ed., Iowa State Univ. Press, Ames. 575 pp. Squibb, R. L., 1968. Effect of a dietary imbalance of lysine on protein metabolism in the chick. Poultry Sci. 47: 199-204. Wagner, R. P. and H. K. Mitchell, 1955. Genetics and Metabolism. John Wiley and Sons. Inc., New York. 444 pp. 106 Wagner, R. P. and H. K. Mitchell, 1964. Genetics and Metabolism, Second Ed., John Wiley and Sons, Inc., New York. 657 pp. Wallace. H. W., K. Moldave and A. Meister, 1957. Studies on con- version of phenylalanine to tyrosine in phenylpyruvic oligOphrenia. Proc. Soc. Exp. Biol. Med. 94: 632. Williams, R. J., 1951. Nutrition and Alcoholism. Univ. of Okla- homa Press, Norman. Williams, R. J., 1956. Biochemical Individuality. John Wiley and Sons, Inc., New York. 141-163. Williams, M. A. and G. R. Grau, 1956. Food intake and utilization of lysine deficient protein by the chick in relation to the digestible energy concentration of the diet. J. Nutr. 59: 243- 254. Wilson, S. P., 1967. Selection procedures for altering specific nutrient requirements in poultry. Poultry Sci. 46: 773-774. Wilson, S. P. and C. W. Hess, 1968. Selection for three week body weight on normal and methionine deficient diets. Poultry Sci. 47: 919-924. Wright, S., 1934. The method of path coefficients. Ann. of Math. Stat. 5: 161-215. Typed and Reproduced by TYPE-INK Fort Collins (11111111 5” R“ ”I l 1'3””133056 1876 (Tallinn)! 3 129