Genetic Studies of Serum Cholesterol Level in the Chicken1

PRE-INCUBATION TREATMENT OF EGGS

ted by differences in the two breeds involved. ACKNOWLEDGMENT The authors are indebted to Dr. G. C. Ashton of the Physics Department of the Ontario Agricultural College for the experimental design used in this experiment.

development of fresh laid hens eggs. Poultry Sci. 13: 74-90. Hays, F. A., 1937. Hatchability as related to season and hour of laying. Poultry Sci. 16: 85-89. Hutt, F. B., and A. M. Pilkey, 1930. Studies in embryonic mortality in the fowl. IV: Comparative mortality rates in eggs laid at different periods of the day. Poultry Sci. 9: 194-203. Kosin, I. L., 1956. Studies on pre-incubation warming of chicken and turkey eggs. Poultry Sci. 35: 1384-1392. McNally, E. H., and T. C. Byerly, 1936. Variation in the development of embryos of hens' eggs. Poultry Sci. 15: 280-283. Taylor, L. W., and C. A. Gunns, 1935. Size of unincubated embryo in relation to hatchability. Poultry Sci. 14: 294.

Genetic Studies of Serum Cholesterol Level in the Chicken 1 FRANK L. CHERMS, JR. 2 , F. H. WILCOX AND C. S. SHAFFNER Department of Poultry Husbandry, University of Maryland, College Park, Maryland (Received for publication October 9, 1959)

T

HERE is little doubt as to the importance of cholesterol in metabolism. Many reports are available in the literature concerning the biosynthesis of cholesterol and the effect of diet on cholesterol level. However, the influence of heredity in the regulation of serum cholesterol level has been studied only to a limited extent. Most of the information available on the influence of heredity on this trait has been obtained from studies with humans, in which the presence of a dominant gene has been suggested (Wilkinson et al., 1948; Schaefer

1 Scientific Article No. A807, Contribution No. 3079 of the Maryland Agricultural Experiment Station (Department of Poultry Husbandry). This study was conducted as part of a Northeastern Regional Project (NE-6): a cooperative study involving agricultural experiment stations in the Northeast Region and supported in part by regional funds of the United States Department of Agriculture. 2 Present address: Poultry Department, University of Wisconsin, Madison.

et al., 1953; Piper and Orrild, 1956; Harris-Jones et al., 1957). There are very few reports on studies of this type with experimental animals. In a study of 4 strains of rats, Kohn (1950) reported that when a strain with high serum cholesterol was crossed with a strain with low serum cholesterol the Fj. progeny had intermediate levels. Bumgardner (1955) reported a heritability estimate of .42 for the serum cholesterol level in the New Hampshire breed of chickens. In this study the influence of heredity on the serum cholesterol level of chickens has been studied by analyzing for differences between dam and sire families, by estimating the heritability of this trait, and by breeding for high and low serum cholesterol lines. EXPERIMENTAL PROCEDURE

In February, 1956, 709 eggs representing 50 sire and 160 dam families were obtained from the random-bred White Leg-

Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015

REFERENCES Becker, W. A., and G. E. Bearse, 1958. Pre-incubation warming and hatchability of chicken eggs. Poultry Sci. 37: 944-948. Funk, E. M., 1934. Relation of the time of laying to hatchability. Poultry Sci. 13: 184-187. Hays, F. A., and C. Nicolaides, 1934. Variability in

889

890

F. L. CHERMS, JR., F. H. WILCOX AND C. S. SHAFFNER TABLE 1.—Composition of University of Maryland

all-mash starting ration used Ingredient Ground yellow corn Wheat standard middlings Fish meal (60% protein) Meat and bone scrap (50% protein) Soybean oil meal (50% protein) Dehydrated alfalfa meal (17% protein) Dried whey Dried grain fermentation solubles (BY-100) DL-methionine Limestone Defluorinated rock phosphate (14% P, 34% Ca) Salt, iodized Manganese sulfate (24%Mn) Vitamin D supplement (1,500 I.C.U. Ds/gm.) Vitamin A supplement (4,500 I.U. A/gm.) Antibiotic and vitamin B12 supplement (2 gms. penicillin, 3 mgs. Bu/lb.) Choline chloride (25% premix) Nitrofurazone (11% mix) Riboflavin mix (1 gm./oz.) Furoxone (5 mgs./3 oz.) Niacin Calcium pantothenate

Lbs./ton 1,168.0 100.0 50.0 50.0 480.0 50.0 25.0 20.0 0.6 15.0 25.0 8.0 0.4 1.0 2.0 2.0 2.0 1.0 Gms./ton 56.0 126.0 20.0 3.0

family differences were found for the level of immature birds but not for them at maaturity. The ration used throughout the study for the young chicks is shown in Table 1. RESULTS To determine the effect of age on serum cholesterol level during the growing period, measurements were made weekly (except for the fifth and ninth weeks) starting at 1 day of age and continuing up to 10 weeks of age. Chicks used were from a cross between females of a flightless strain and White Leghorn males. Results of this test (Table 2) agree with the work of Rodbard et al. (1951) who also found that at hatching the serum cholesterol level is about three times as high as at any other time during the experimental period. There was no significant variation in levels between 1 to 10 weeks of age, indicating that determinations made any time during this period are comparable. Analysis of both the 1956 random-bred White Leghorn population and another one obtained in 1957 showed significant damfamily within sire-family differences for this trait in immature stock (Table 3). A heritability estimate was made for this trait

Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015

horn flock maintained at Cornell University. This stock was used since it is maintained in sufficient size to serve as a control and has a large number of sires and dams (King, et al, 19S9). At 9 weeks of age 3 ml. of blood was drawn by heart puncture and incubated at 37°C. for lj4 hours after which the serum was separated and then frozen until cholesterol determinations were made. In suceeding years 2 ml. of blood was drawn at 6 weeks of age. Cholesterol was analyzed in duplicate by the method of Zlatkis et al. (1953) in 1956 and 1957 and by the method Of Zak (1957) thereafter. For all determinations a Coleman Universal Spectrophotometer, model 14, was used. Using the serum cholesterol level as the only criterion of selection, extreme individuals within extreme families were selected and mated together in an attempt to produce two lines differing markedly in cholesterol level. The number of males used for mating for each line was 4, 8, and 1216 in 1957, 1958, and 1959, respectively. An average of 29 females was used each year for each line for mating. Except for the introduction of one male in the high line and two males in the low line in 1958 from a second random-bred population, no other stock was introduced into the high and low lines after the initial matings in 1957. Two hatches of chicks were obtained each year. Serum cholesterol was analyzed from all female progeny, but in the case of males only from selected families of the second hatch. Similar measurements were made at this time (first hatch only) on random-bred White Leghorns obtained as hatching eggs from Cornell University, and reared among high and low line chicks. The serum cholesterol level at 6 to 9 weeks of age was used as a basis of selection because it was felt that there would be less environmental variation at this age than at maturity. Also, significantly dam-

SERUM CHOLESTEROL TABLE 2.—Effect

SOI

of age on serum cholesterol levels in chicks of a Flightless-White Leghorn cross

1 day Cholesterol levels (mg.%)

759

Age in weeks 1

2

3

4

6

7

8

10

266

258

260

219

227

260

226

239

were not statistically significant in 1957, they were highly significant (P.01) in 1958 and 1959. In addition, highly significant differences were present between hatches in 1958 and 1959, between sirefamilies within lines in 1958, and between dam-families within sire-families and lines in 1957. The difference between dam-families within sire-families and lines in 1959 was statistically significant (P.05). DISCUSSION The data presented provide evidence from several sources that the level of cholesterol in serum is under genetic control. This is shown by significant differences between families and by lines differing markTABLE 4.—Heritability estimates of serum cholesterol levels of the 1956 random-bred White Leghorns at 9 weeks of age Estimate of variance components 4S

E-HD+S

TABLE 3.—Analysis of variance of serum cholesterol levels of random-bred White Leghorns d.f.

Mean sqs.

Hatched in 1956; blood drawn at 9 weeks of age Sex 1 7,987 22.185** Sire-family 49 642 1.354 Dam-family within sire-family 114 474 1.317* Error 262 360 Hatched in 1957; blood drawn at 6 weeks of age Sex 1 18,509 143.481** Sire-family 40 771 .656 Dam-family within sire-family 64 1,175 9.109** Error 139 129 * Significant at 5 % level. ** Significant at 1% level.

.19

4D Dams

Source of variation

Heritability

.41

E+D+S 2(D+S) .30

Combined

E+D+S S = the component of variance resulting from differences between groups of half sisters from different sires, containing one-quarter of the genetic variance. D = the component of variance resulting from differences between groups of half sisters from dams mated to the same sire, containing onequarter of the genetic variance and all of the variance due to maternal effects. E = the component of variance resulting from differences between full sisters, containing the remainder of the genetic variance and all of the environmental variance.

Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015

in the 1956 random-bred population (Table 4); however, a heritability estimate could not be calculated for the 1957 random-breds because the dam-family within sire-family mean square was larger than the sire-family mean square. This is apt to occur in analyses where small numbers of individuals are involved (King and Henderson, 1954). The combined estimate of .30 does not greatly differ from the estimate of .42 found for New Hampshires by Bumgardner (1955). According to King and Henderson (1954) a higher dam estimate may be due to the presence of a maternal effect. The combined estimate obtained here indicates that there is some genetic influence on this trait but that there is also rather high environmental effect. After three generations of selection (Table 5), lines differing markedly in serum cholesterol were developed. Although differences between the high and low lines

892

P. L. CHERMS, JR., F. H. WILCOX AND C. S. SHAFFNER TABLE 5.—Average serum cholesterol levels for 6-week old females of high and low lines and random-bred controls Line

Generation"

Year

Hatch High

Random-bred

Low

Si

1957

1 2

184 175

169

178 169

S2

1958

1 2

155 135

134

124 116

S°

1959

1 2

168 151

151

131 122

edly in their cholesterol level. This is of particular interest since all other variables have been held constant, especially diet; in genetic studies with humans, this has not been possible. Other data (Wilcox and Shaffner, unpublished) indicate that the differences between the high and low lines also exist in adult serum cholesterol, but not in yolk cholesterol. Investigations are in progress to elucidate biochemical processes involved in the line differences in serum cholesterol. SUMMARY

Serum cholesterol levels were measured at 6-9 weeks of age in random-bred White Leghorns as well as lines selected for differences in cholesterol. Significant differences between dam-families were noted in two successive years and heritability was estimated to be .30. After three generations of selection two lines have been developed which differ markedly and significantly in their serum cholesterol level. ACKNOWLED GMENTS

The authors are indebted to Mrs. Dorothy Smith, Miss Carmen Rodriguez-Mettee, and Mr. Richard Seagrave for technical assistance. REFERENCES Bumgardner, H. L., 1955. Studies on the serum

protein bound iodine levels and serum cholesterol levels of the chicken. Ph.D. thesis. Univ. of Maryland. Harris-Jones, J. N., E. G. Jones and P. G. Wells, 1957. Xanthomatosis and essential hypercholesterolaemia. The Lancet, 272: 855-857. King, S. C , and C. R. Henderson, 1954. Variance components analysis in heritability studies. Poultry Sci. 33: 147-154. King, S. C , J. R. Carson and D. P. Doolittle, 1959. The Connecticut and Cornell randombred populations of chickens. World's Poultry Sci. J. 15: 139-159. Kohn, H. I., 1950. Changes in plasma of the rat during fasting and influence of genetic factors upon sugar and cholesterol levels. Am. J. Physiol. 163: 410-417. Piper, J., and L. Orrild, 1956. Essential familial hypercholesterolemia and xanthomatosis. Am. J. Med. 2 1 : 34-46. Rodbard, S., L. N. Katz, C. Bolene, R. Pick, M. Lowenthal and G. Gros, 1951. The age factor in hypercholesterolemia and atheromatosis in the chick. Circulation, 3 : 867-874. Schaefer, L. E., S. R. Drachman, A. G. Steinberg and D. Aldersberg, 1953. Genetic studies on hypercholesterolemia : frequency in a hospital population and in families of hypercholesteremic index patients. Am. Heart J. 46: 99-116. Wilkinson, C. F., E. A. Hand and M. T. Fliegelman, 1948. Essential familial hypercholesterolemia. Ann. Int. Med. 29: 671-686. Zak, B., 1957. Simple rapid microtechnic for serum total cholesterol. Am. J. Clin. Path. 27: 583-588. Zlatkis, A., B. Zak and A. J. Boyle, 1953. A new method for the direct determination of serum cholesterol. J. Lab. Clin. Med. 4 1 : 486-492.

Downloaded from http://ps.oxfordjournals.org/ at Oakland University on June 2, 2015

a Si = first generation of chicks whose parents were selected for differences in cholesterol level, etc. Number of chicks involved in the Si, S2, and S3 generations was 221, 131, and 188 for the high line; 152, 50, and 50 for the random-bred line; and 195, 128, and 159 for the low line, respectively.