Selection in Chickens for Retrogression of Tumors Caused by Rous Sarcoma Virus

Selection in Chickens for Retrogression of Tumors Caused by Rous Sarcoma Virus

Selection in Chickens for Retrogression of Tumors Caused by Rous Sarcoma Virus N. R. GYLES AND C. J. BROWN (Received for publication November 13, 197...

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Selection in Chickens for Retrogression of Tumors Caused by Rous Sarcoma Virus N. R. GYLES AND C. J. BROWN

(Received for publication November 13, 1970)

investigators have conducted S EVERAL selection studies for resistance and susceptibility of chickens to avian leukosis. Hutt et al. (1941, 1945), Taylor et al. (1943), Waters (1945), McClary et al. (1951) and Bearse et al. (1963) all reported varying degrees of success in selecting divergent lines, from the same original stock, for resistance and susceptibility to avian leukosis complex. The duration of these selection experiments ranged from four to twenty-eight generations. The basis of selection most often used was full-sib family selection, while progeny testing was employed in some studies. The criterion of selection was the percentage mortality from all forms of leukosis during the rearing and laying periods. Natural exposure was usually the sole means of challenge. In a few instances, tumor material was placed in the drinking water. Cole (1968) demonstrated rapid progress in selecting two divergent lines for re-. sistance and susceptibility to Marek's disease. The birds were challenged intraperitoneally with sufficient isolate of Marek's disease to cause approximately 50 percent mortality. The basis for selection was progeny testing of sires and dams. The criterion of selection was the percentage of birds dying from or showing presence of Marek's disease lesions up to eight weeks of age. Greenwood et al. (1948) started with parents that gave retrogressive tumor response to subcutaneous inoculations with Rous sarcoma virus (R.S.V.), and then made selective matings for two generations among birds that gave negative tumor responses. These matings produced offspring

that gave the highest proportion of their tumor response as retrogression. Gyles et al. (1968) reported two different but interrelated genetic mechanisms of resistance in the chicken to R.S.V. One mechanism, characterized by negative tumor response, resists transformation of normal cells to malignancy after the cells have been penetrated by the virus. The other mechanism halts tumor growth and induces complete retrogression of tumors. The purpose of the current selection experiment is to determine the response to selection, within a closed flock, for retrogression of tumors caused by Rous sarcoma virus. MATERIALS AND METHODS

This selection experiment was started in 1965 and to date six generations of selection have been accomplished. The base unselected population was the breeding groups reported in a previous study by Gyles et al. (1967). The numbers of sires and dams of each breeding group that were chosen from the unselected population to produce the first selected generation are as follows: White Leghorns, 1 sire, 1 dam; F a cross (W.L. d X G.J.F. 9 ) 4 sires, 3 dams; Fx cross (G.J.F. c? X W.L. 9 ) 4 sires, 9 dams; F 2 cross (from F x inter se matings W.L. cf X G.J.F. 9 ) 0 sires, 5 dams; F2 cross (from Fj inter se matings G.J.F. d X W.L. 9 ) 2 sires, 3 dams. In producing each generation a sire was mated to not more than five dams in cages by artificial insemination. The eggs were pedigreed, collected for two-week periods, and incubated under

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Department of Animal Sciences, University of Arkansas, Fayetteville, Arkansas 72701

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N. R. GYLES AND C. J. BROWN TABLE 1.—Numbers of hatches, chicks inoculated and breeders chosen in each generation of line selected for retrogression of Rous sarcomas Number of Selected Breeders

Generation

N o . of Hatches

N o . of Chicks Inoculated

Unselected

4 4*

750 102*

1st Selected

3

2nd Selected 3rd Selected 4th Selected

4

688

9

42

5th Selected

7 7*

535 660*

13

60

6th Selected

4 4*

598 589*

Males

Females

11

21

324

17

34

4

647

16

42

4 4*

680 511*

13

32

* Genetic Control.

performance and individual performance within the selected families. The full-sib families selected were those that had the highest percentage of retrogressive tumors based on the number of birds inoculated. Individuals within these selected families were chosen on their ability to cause the larger sizes of tumors to retrogress. Genetic control: In order to demonstrate genetic progress in this selection study, chicks of the Ft cross between Giant Jungle Fowl (G.J.F.) males and White Leghorn (W.L.) females were hatched and placed with chicks of the selected line in each hatch of the unselected, third, fifth and sixth selected generations, respectively. The G.J.F. and W.L. lines are the same as those used in producing the Ft and F 2 crosses that made up the base unselected population at the start of the study. Both of these lines have been maintained as small closed flocks for many years, and the inbreeding of each line is considered high, but the actual values are not known. RESULTS

The numbers of hatches, chicks inoculated with R.S.V. and breeders chosen for each generation are given in Table 1. The number of sires in relation to the number

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standard conditions. The chicks were wingbanded and brooded together under similar conditions for each generation. The chicks were inoculated in the wing-web with R.S.V. between six and seven weeks of age, and the tumors were scored each week when they emerged in the manner previously described by Gyles et al. (1968). The classification of the tumor response was made as described by Gyles et al. (1968). A bird was considered as having a retrogressive tumor response when a definite tumor appeared, and then completely retrogressed with the wing-web returning to a normal appearance and remaining with a score of 0 for a minimum of four weeks. Strain and dosage of virus: Rous sarcoma virus of the Bryan strain (R.S.V.Bryan) was used to challenge the birds in each generation. The identification of the batch of virus and the dilution of virus used per generation are as follows: unselected base generation TV-20 dilution 1: 300; first selected generation TV-40 dilution 10 -3 ; second, third, fourth, fifth and sixth selected generations TV-49 dilution 10 —5. The differences in dilution reflect differences in potency to assure sufficient tumor response based on titration results using the susceptible White Leghorn strain described previously by Gyles et al. (1967). Criterion of selection: Throughout this study, the practice was to challenge all the birds in each generation with R.S.V., and to choose breeders from those birds that had a tumor which retrogressed. Breeders to produce the first, second and third generations of selection were chosen entirely on individual performance with regard to tumor retrogression. Preference was given to those individuals with the larger tumors that retrogressed. Breeders to produce the fourth, fifth and sixth generations were chosen on a combination of full-sib family

Rous

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SARCOMA TUMOR RETROGRESSION

rogression

of dams was kept large in a deliberate attempt to avoid a rapid increase in inbreeding. The numbers and percentages of the various types of tumor responses for each gen- & // eration are given in Table 2. There was an overall increase in the amount of retrogression based on numbers of birds inoculated, S. o /? Percentages based on tumors // from 14.3 percent in the unselected generainitiated // tion to 59.2 percent in the sixth generation § - 10 // Percentages based on birds / inoculated of selection. A comparison between the per/ , i i i i t centages retrogression in the selected line 0 1 2 3 4 5 6 and the genetic control suggests a steady Generations of Selection increase in the percentage of birds with reFIG. 1. Differences between selected line and trogressive tumor response as a result of segenetic control for percentage of birds or tumors lection over six generations. The differences showing retrogression. range from —15.1 percent in the unselected generation to +28.3 percent in the sixth rather than on numbers of birds inoculated generation and are presented graphically in minimize discrepancies due to disproporFigure 1. tional amounts of tumors initiated in relaThe percentage of retrogression of tution to birds inoculated. There was an inmors and other tumor responses based on crease in percentage of retrogressive tumor the numbers of tumors initiated are preresponse from 18.2 percent in the unsesented in Table 3. Comparisons between lected population to 63.7 percent in the percentages retrogressive tumor responses sixth generation of selection. The differbased on numbers of tumors initiated ences between the selected line and the genetic control for the percentages of tumors initiated that later retrogressed are shown TABLE 2.—Percentages of inoculated birds that show various tumor responses by generations in graphically in Figure 1. The differences line selected for retrogression of Rous sarcomas

Gener.,;.-, atIon Unselected

Percentages of Tumor Responses

^tity and Virus Dilution

Negative

TV-20 1:300

21.7 19.6*

Retrogres- Progres- Unclassive sive sified** 14.3 29.4*

58.9 44.1*

1st Selected TV-40 10"' 2nd Selected TV-49 10-5

8.0

3rd Selected TV-49 10-s

5.9 2.5*

35.6 21.7*

49.0 67.7*

4th Selected TV-49 10-5

5.1 6.9*

TABLE 3.—Numbers and percentages of tumors initiated showing various responses by generations in line selected for retrogression of Rous sarcomas Percentages of Tumor Percentages

17.3

Generation

15.6

Unselected

9.5 8.1*

Number of Tumors

Retrogressive

Progressive

587 80*

18.2 36.6*

75.3 54.9*

5.4 2.7*

47.5 24.9*

30.3 59.5*

16.8 12.9*

6th Selected TV-49 10-5

7.0 9.3*

59.2 30.9*

9.7 31.6*

24.1 28.2*

* Genetic Control. ** Includes birds t h a t could not be placed in any of the other three tumor response categories because tumors were present at t h e termination of t h e experiment.

6.5 8.5*

1st Selected

315

27.0

55.2

17.8

2nd Selected

595

37.5

45.5

17.0

3rd Selected

640 498*

37.8 22.3*

52.0 69.5*

10.2 8.2*

4 t h Selected

621

5th Selected

506 642*

50.2 25.5*

32.0 61.2*

17.8 13.2*

6th Selected

556 534*

63.7 34.1*

10.4 34.8*

25.9 31.1*

9.7

5 th Selected TV-49 10-5

Unclassified

* Genetic Control.

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N. R. GYLES AND C. J. BROWN

TABLE 4.—Number of birds with retrogressed

tumors and percentages that retrogressed from various peak sizes by generations in line selected for retrogression of Rous sarcomas Total N o . Birds with Retrogressed Tumors

Unselected

Percentages from Peak Scores** 1 toO

2 toO

3 toO

107 30*

25.2 23.3*

47.7 46.7*

27.1 30.0*

4 toO 0 0* 0

1st Selected

87

33.3

62.1

4.6

2nd Selected

223

34.5

49.3

12.6

3.6

3rd Selected

243 111*

40.7 40.5*

40.7 41.4*

15.6 15.3*

3.0 2.7*

4 t h Selected

291

36.8

46.7

13.1

3.4

5th Selected

254 164*

40.2 42.7*

41.3 40.2*

15.7 14.0*

2.8 3.1*

6th Selected

354 182*

42.9 45.5*

32.9 35.4*

12.0 17.4*

2.2 1.7*

Genetic Control Averages: 37.7, 45.8, 14.4, 2.1. ** Scores represent tumor sizes as follows: zero—no tumor present, 1—very small tumor, 2 and 3—small to intermediate, 4—massive tumor covering entire wing-web of chicken.

range from —18.4 percent in the unselected generation to +29.6 percent in the sixth generation, and are represented graphically in Figure 1. The magnitude of retrogression of tumors, as expressed by a percentage distribution for retrogression of tumors of various sizes, was determined each generation for the selected line and also for the genetic control and are presented in Table 4. The majority of tumors retrogress from scores of 1 and 2, and are about equally divided among these two sizes. The patterns of distribution of retrogression are very similar for the selected line and the genetic control. It may be observed in Table 2, 3 and 4, that there are differences between the unselected population and the genetic control. These differences probably occur because the unselected population was made up primarily of F 2 and F 2 crosses between White Leghorn and Giant Jungle Fowl strains, while the genetic control was the F1 cross between these two strains. Gyles et al. (1967) had previously found that Fi cross chicks gave about twice as many retrogres-

DISCUSSION Substantial progress has been made in selecting for the single trait, retrogression of Rous sarcomas, in a closed population over six generations. The rate of increase in retrogression of Rous sarcomas appears to be faster than that made with quantitative performance traits such as growth rate and egg production, which are influenced by several pairs of genes. However, the progress is not as rapid as that reported by Cole (1968) for resistance to Marek's disease. Gyles et al. (1968) reported some evidence that retrogression of Rous sarcomas may be influenced by a single pair or very few pairs of genes. Bower et al. (1965) estimated that a single pair of genes was responsible for resistance of chicken embryos to pock formations on the chorio-allantoic membranes by Rous sarcomas virus. Gyles et al. (1968) pointed out that the mode of resistance measured by Bower et al. (1965) was probably a second line of defense after penetration of chicken host cells by the virus, and controlled by a-separate pair of genes from the gene pair reported by Crittenden et al. (1964) to stop penetration of the cell membrane by the virus. It is suggested that the rate of progress attained in this selection study for retrogression of Rous sarcomas does not preclude a single pair or a small number of genes with low penetrance. The average frequencies of retrogressions for all seven generations were 37.7, 45.8, 14.4 and 2.1 percent from peak tumor scores of 1, 2, 3 and 4, respectively. It is not known whether retrogression from a particular peak score represents a more forceful form of the phenomenon than from the other scores. The results of this selection experiment

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Generation

sive tumor responses as F 2 cross chicks of these parental strains.

Rous

SARCOMA TUMOR RETROGRESSION

SUMMARY

A closed population of chickens, initiated primarily by Fi and F 2 crosses between Arkansas Experiment Station strains of White Leghorn and Giant Jungle Fowl, was selected for retrogression of Rous sarcomas for six generations. Birds were challenged in the wing-web with Rous sarcoma virus at approximately six weeks of age, scored and observed weekly for size and retrogression of tumors. The percentage of birds inoculated that exhibited retrogression of tumors increased from 14.3 to 59.2, and the percentage of tumors initiated that retrogressed increased from 18.2 to 63.7, over six generations of selection. ACKNOWLEDGEMENTS

The authors are indebted to J. L. Miley, B. R. Stewart, J. Turner, P. Marini, V. Thompson and 0. J. McConnell for their assistance at various times during the selection experiment. REFERENCES Bearse, G. E., W. A. Becker and C. M. Hamilton,

1963. Resistance and susceptibility to the avian leukosis complex in chickens. Poultry Sci. 42: 110-121. Bower, R. K., N. R. Gyles and C. J. Brown, 1965. The number of genes controlling the response of chick embryo chorio-allantoic membranes to tumor induction by Rous sarcoma virus. Genetics, 51: 739-746. Cole, R. K., 1968. Studies on genetic resistance to Marek's disease. Avian Diseases, 12: 9-28. Crittenden, L. B., W. Okazaki and R. Reamer, 1964. Genetic control of responses to Rous sarcoma and strain RPL-12 viruses in the cells, embryos and chickens of two inbred lines. Intern Conf. Avian Tumor Viruses, Mimeo No. 17, Nat'l Cancer Inst. 161-177. Greenwood, A. W., J. S. S. Blythe and J. G. Carr, 1948. Indications of the heritable nature of non-susceptibility to Rous sarcoma in fowls. Brit. J. Cancer, 2: 135-143. Gyles, N. R., J. L. Miley and C. J. Brown, 1967. The response of resistant and susceptible strains of chickens and their Fi and F 2 crosses to subcutaneous inoculations with Rous sarcoma virus. Poultry Sci. 46: 465-472. Gyles, N. R., B. R. Stewart and C. J. Brown, 1968. Mechanisms of genetic reistance in the chicken to Rous sarcoma virus. Poultry Sci. 47 : 430-450. Hutt, F. B., R. K. Cole and J. H. Bruckner, 1941. Four generations of fowls bred for resistance to neoplasms. Poultry Sci. 20: 514-526. Hutt, F. B., R. K. Cole and J. H. Bruckner, 1945. A test of fowls bred for resistance to lymphomatosis. Poultry Sci. 24: 564-571. McClary, C. F., G. E. Bearse, L. R. Berg and C. M. Hamilton, 1951. Prepotency in chickens for susceptibility to natural infection by the avian leukosis complex. Poultry Sci. 30: 923-924. Taylor, L. W., I. M. Lerner, K. B. DeOme and J. R. Beach, 1943. Eight years of progeny-test selection for resistance and susceptibility to lymphomatosis. Poultry Sci. 22: 339-347. Waters, N. F., 1945. Breeding for resistance and susceptibility to avian lymphomatosis. Poultry Sci. 24: 259-269.

AUGUST 25-27. MARKETING CONFERENCE, INSTITUTE OF AMERICAN POULTRY INDUSTRIES, AMBASSADOR WEST HOTEL, CHICAGO, ILLINOIS

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to-date indicate a significant genetic influence on retrogression of Rous sarcomas in the chicken. Selection within a closed flock may substantially increase the percentage of birds exhibiting a retrogressive tumor response. This increase in incidence of the phenomenon may provide sufficient numbers of individuals on which to conduct detailed research into the mechanism of tumor retrogression. Research into this phenomenon has been handicapped in the past by the extremely low frequency of its occurrence.

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