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Estimation of Genetic Diversity Among Strains and Breeds of Chickens by Lymphocyte Increases Following Skin Grafting1,2
ZINC REQUIREMENT Association of Official Agricultural Chemists, 1955. Official Methods of Analysis. P. 843. Washington, D.C. Edwards, H. M., Jr., R. J. Young and M. B. Gillis, 1958. Studies on zinc in poultry nutrition. I. The effect of feed, water and environment on zinc deficiency in chicks. Poultry Sci. 37: 10941099. Mehring, A. L., Jr., J. H. Brumbaugh and H. W. Titus, 1956. A comparison of the growth of chicks fed diets containing different quantities of zinc. Poultry Sci. 35: 956-958.
439
Norris, L. C , R. M. Leach, Jr. and T. R. Zeigler, 1958. Recent research on the mineral requirements of poultry. Proc. Twelfth Distillers Feed Conference 13: 61-71. Young, R. J., H. M. Edwards, Jr. and M. B. Gillis, 1958. Studies on zinc in poultry nutrition. 2. Zinc requirement and deficiency symptoms of chicks. Poultry Sci. 37: 1100-1107. Zeigler, T. R., R. M. Leach, Jr. and L. C. Norris, 1958. Zinc requirement of the chick. Federation Proc. 17, Part 1, No. 1, paper 1956.
J. E. BERRY AND J. V. CRAIG3 Kansas State College, Manhattan, Kansas (Received for publication August 9, 1958)
INTRODUCTION
A
TISSUE homograft generally elicits an immune reaction on the part of the host animal, marked by a rise in the percentage of lymphocytes in the circulating blood, whereas autografts have little or no effect, Blumenthal (1939). Craig and Hirsch (1957), using skin grafts exchanged between chickens, obtained significant negative correlation coefficients, averaging —0.64, between the relative increase in lympho-
Portion of a dissertation presented by the senior author as partial fulfillment of the requirements for the degree Doctor of Philosophy in Genetics at Kansas State College. 1 This investigation was part of the Kansas contribution to the NC-47 Regional Poultry Breeding Project. 2 Contribution No. 240, Department of Poultry Husbandry, Kansas Agricultural Experiment Station, Manhattan, Kansas. 3 Formerly Graduate Research Assistant (present address, Department of Poultry Husbandry, University of Maine, Orono, Maine) and Associate Professor of Poultry Genetics, respectively.
cytes, measured on the fifth postoperative day, and the relationship coefficient between host and donor. Berry et al. (1958) obtained an average correlation coefficient of — 0.49 (P < .05) in a similar set of experiments, using postoperative lymphocyte increases on the third day. Craig and Hirsch (1957), using two breeds, also found that grafts from one breed to the other gave a significantly greater response than grafts exchanged within the breed. These results suggested the possibility of the use of increases in lymphocytes or of postoperative lymphocyte percentages following skin transplantation as a measure of genetic diversity among strains and breeds of chickens. The estimation of genetic diversity of strains and breeds used in crossing would be of value to the extent that heterosis is associated with such diversity. This study was carried out to determine whether a technique utilizing three-day postoperative lymphocyte percentages following skin grafting would adequately measure genetic
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 29, 2015
Estimation of Genetic Diversity Among Strains and Breeds of Chickens by Lymphocyte Increases Following Skin Grafting1'2
440
J. E. BERRY AND J. V. CRAIG
diversity among strains and breeds of chickens.
were analyzed by standard statistical techniques.
MATERIAL AND METHODS
RESULTS AND DISCUSSION
TABLE 1.—Mean preoperative percentage lymphocytes arranged in ascending order by host strains and subjected to "Duncan's New Multiple Range Test'n Host strains Mean Lymphocyte Percentage
R2 2
63.32
'
Ri
R3
63.89
65.24
Li
69.48
L3
L2
70.31
72.58
1 Means underscored by the same line are not significantly different. Means not underscored by the same line are different at the 5 % level of probability. 2 Each mean is based on data from 54 host females.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 29, 2015
Mean preoperative lymphocyte percentThe surgical techniques used in this study were the same as those described by ages for twelve-month-old hens of the six Berry et al. (1958). Skin grafts were made, strains were obtained and subjected to analusing nine males from each of six strains as ysis by Duncan's (1955) new multiple skin donors. Six pieces of skin, of approxi- range test, Table 1. It is apparent from the mately 2 cm. diameter, were taken from the results that the Leghorn strains were signifthigh area of each donor male and one icantly (P < .05) higher in lymphocyte graft each was placed on a female of the percentages than the Rhode Island Red same strain and on hens of each of the other strains. However, differences between five strains. All birds were approximately strains within these two breeds were not twelve months of age at the time of the significant for this trait. The overall mean operation. Differential counts of the leuco- difference between the breeds was 6.64 percytes of the circulating blood were made cent and presumably represents a real breed immediately before transplantation and on difference since all birds were subjected to the same environment. the third postoperative day. Correlation and regression coefficients It was postulated that the preoperative were calculated between preoperative and percentage of lymphocytes might influence postoperative lymphocyte percentages with- an individual's ability to respond to a skin in each of the host strains. The regression co- graft as measured by the postoperative lymefficients were tested for heterogeneity and phocyte percentage. Correlation and regrespostoperative lymphocyte percentages were sion coefficients were calculated on a withinadjusted for differences in preoperative strain basis between preoperative and postlymphocyte levels. operative lymphocyte percentages. Clearly Birds of the six strains used were hatched significant associations were found in four from eggs received from commercial breed- of the six strains (Table 2). Statistical analers who were asked to supply hatching eggs ysis failed to suggest heterogeneity and a from closed flocks. Although there was pooled regression coefficient of 0.33 was probably some relationship among the birds used to adjust for differences in preoperawithin any one strain, the birds within a tive lymphocyte levels of individual host strain were assumed to be unrelated. There hens. Table 3 presents the adjusted threewere three White Leghorn (L) and three day postoperative lymphocyte percentages following exchanges of skin for each of the Rhode Island Red (R) strains. Adjusted three-day postoperative lym- donor-host combinations. Analysis of variance of the adjusted postphocyte percentages following skin grafting
441
3ENETIC DIVERSITY AND LYMPHOCYTE INCREASE T A B L E 2.—Correlation and regression coefficients of percentages by host
Host strain 1
Correlation Coefficient Regression Coefficient1
preoperative and postoperative strains
lymphocyte
Li
L/2
L3
Ri
R2
R3
All strains
0.22 0.24
0.33* 0.38**
0.36** 0.37**
0.11 0.13
0.38** 0.39**
0.44** 0.49**
0.33**
1
E a c h coefficient is based on d a t a from 54 host females. * Significant a t 5 % level. ** Significant a t 1 % level. T A B L E 3.—Three-day postoperative lymphocyte percentages of hosts after for preoperative lymphocyte levels
adjustment
Li U U Ri R2 R3 Means, by Host Strains
Means, by donor strains
L,
U
L3
Ri
R2
R3
72.14 70.71 75.28 71.79 75.00 71.01
75.66 74.89 76.17 75.04 70.89 75.58
78.33 76.93 75.58 77.03 77.35 72.14
69.66 72.58 73.99 72.33 74.75 70.44
72.57 73.45 68.17 67.85 68.33 75.66
76.24 65.58 74.38 74.43 77.65 74.27
74.10 72.36 73.93 73.08 74.00 73.18
72.66
74.71
76.23
72.29
71.01
73.76
73.44
Each mean for a donor-host combination represents readings from nine host females.
operative lymphocyte percentages showed that the only differences approaching significance (P = 0.08) were between the different host strains (Table 4). These results are further illustrated in Table 5 where the three-day postoperative lymphocyte percentages are grouped according to whether the donors and hosts were of the same strain, of different strains of the same breed, or of different breeds. The mean withinstrain value of 72.92 percentage lymphocytes is very close to the mean betweenstrain response of 73.55 percent. 1
T A B L E 4.—Analysis of variance of adjusted erative lymphocyte percentages
Source Donor Strains Host Strains Interaction (DonorsXHosts) Among Hosts Treated Alike
postop-
d.f.
Mean Probsquares ability
5 5 25 287
25.40 187.18 76.37 91.23
0.08
1 Adjusted for p r e o p e r a t i v e percentageof l y m p h o cytes.
In the work of Craig and Hirsch (1957) and of Berry et al. (1958) significant regressions were established for increases in the relative lymphocyte percentage following skin grafting, with known genetic relationship coefficients of donors and hosts. In the present study, using adjusted three-day postoperative lymphocyte percentages as T A B L E 5.—Mean adjusted1 postoperative lymphocyte percentages, grouped according to kind of skin-graft exchange
Kind of exchange
Percentage No. of hosts lymphocytes
A. Unrelated, Same Strain Leghorns Reds
27 27
74.20 71.64
B. Between Strains, Same Breed Leghorns Reds
54 54
75.51 73.46
C. Between Breeds Red Donors, Leghorn Hosts Leghorn Donors, Red Hosts
81 81
73.98 71.85
54 108 162 324 270
72.92 74.49 72.92 73.44 73.55
Weighted Means A B C A, B, C B, C 1
Adjusted for preoperative percentage of lymphocytes.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 29, 2015
Host strains Donor strains
442
J. E. BERRY AND J. V. CRAIG
SUMMARY AND CONCLUSIONS
An attempt was made to use the intensity of the immune reaction evoked by skin grafts, as measured by three-day postoperative lymphocyte percentages adjusted for differences in preoperative lymphocyte levels to estimate the genetic diversity between strains and breeds of the Rhode Island Red and White Leghorn breeds. Three strains represented each breed. White Leghorn
Berry, J. E., J. V. Craig and G. K. L. Underbjerg, 1958. A comparison of the effects of skin implants and skin grafts on the cellular constituents of chicken blood. Poultry Sci. 37: 312-316. Blumenthal, H. T., 1939. Effects of organismal differentials on the distribution of leukocytes in the circulating blood. Arch. Path. 27: 510-545. Craig, J. V., and L. J. Hirsch, 1957. Genetic relationship and the reaction to skin grafts. J. Heredity, 48: 235-237. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42.
JUNE 23-25. WESTERN POULTRY CONGRESS, FAIR GROUNDS, FRESNO, CALIFORNIA JULY 21-23. AMERICAN POULTRY AND HATCHERY FEDERATION CONVENTION, KIEL AUDITORIUM, ST. LOUIS, MO.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 29, 2015
the measure of intensity of the immune re- hens were found to have significantly higher action, the technique failed to indicate the lymphocyte percentages than Rhode Island greater genetic diversity between strains Red hens at approximately twelve months and breeds as compared with that between of age. The preoperative level of lymphopresumably unrelated birds of the same cytes was found to have a significant effect strains. It is difficult to reconcile these re- upon the ability of the bird to respond to sults with the part of the experiment of skin grafts, as measured by the three-day Craig and Hirsch (1957) dealing with be- postoperative lymphocyte percentage. Lymtween-breed responses. There was no pla- phocyte percentages on the third day folteau of response when grafts were ex- lowing skin grafting were no greater when changed between unrelated birds of the skin was exchanged between individuals of same breed in their experiment. In the pres- different breeds than those found after exent experiment such a plateauing of re- changes of skin between unrelated indisponse appears to have occurred. Differ- viduals of the same strains. These results ences in age and genetic constitution of the preclude the use of the present technique, birds used in these different experiments without further modifications, for the purmay be responsible for the divergent results pose of estimating genetic diversity between of these two investigations. In the light of strains and breeds of chickens. the present results, we suggest that the presACKNOWLEDGMENTS ent techniques might be modified to allow The authors wish to express their gratimeasurement of the immune response betude to Drs. A. W. Nordskog and W. H. fore it has reached its maximum level or to Kyle of Iowa State College and the Northelicit less than the maximum possible reCentral Regional Poultry Breeding Laboraaction so that the plateauing of response tory, respectively, for valuable suggestions with increasing genetic diversity between concerning the analysis of the data and to strains and breeds may be avoided. Such Dr. S. Wearden, Statistician at Kansas modification might involve the use of young State College for help in analyzing and inchicks, taking postoperative readings at a terpreting the experiment. shorter interval than three days, or the use of a smaller piece of tissue for transplanting. REFERENCES
439
Norris, L. C , R. M. Leach, Jr. and T. R. Zeigler, 1958. Recent research on the mineral requirements of poultry. Proc. Twelfth Distillers Feed Conference 13: 61-71. Young, R. J., H. M. Edwards, Jr. and M. B. Gillis, 1958. Studies on zinc in poultry nutrition. 2. Zinc requirement and deficiency symptoms of chicks. Poultry Sci. 37: 1100-1107. Zeigler, T. R., R. M. Leach, Jr. and L. C. Norris, 1958. Zinc requirement of the chick. Federation Proc. 17, Part 1, No. 1, paper 1956.
J. E. BERRY AND J. V. CRAIG3 Kansas State College, Manhattan, Kansas (Received for publication August 9, 1958)
INTRODUCTION
A
TISSUE homograft generally elicits an immune reaction on the part of the host animal, marked by a rise in the percentage of lymphocytes in the circulating blood, whereas autografts have little or no effect, Blumenthal (1939). Craig and Hirsch (1957), using skin grafts exchanged between chickens, obtained significant negative correlation coefficients, averaging —0.64, between the relative increase in lympho-
Portion of a dissertation presented by the senior author as partial fulfillment of the requirements for the degree Doctor of Philosophy in Genetics at Kansas State College. 1 This investigation was part of the Kansas contribution to the NC-47 Regional Poultry Breeding Project. 2 Contribution No. 240, Department of Poultry Husbandry, Kansas Agricultural Experiment Station, Manhattan, Kansas. 3 Formerly Graduate Research Assistant (present address, Department of Poultry Husbandry, University of Maine, Orono, Maine) and Associate Professor of Poultry Genetics, respectively.
cytes, measured on the fifth postoperative day, and the relationship coefficient between host and donor. Berry et al. (1958) obtained an average correlation coefficient of — 0.49 (P < .05) in a similar set of experiments, using postoperative lymphocyte increases on the third day. Craig and Hirsch (1957), using two breeds, also found that grafts from one breed to the other gave a significantly greater response than grafts exchanged within the breed. These results suggested the possibility of the use of increases in lymphocytes or of postoperative lymphocyte percentages following skin transplantation as a measure of genetic diversity among strains and breeds of chickens. The estimation of genetic diversity of strains and breeds used in crossing would be of value to the extent that heterosis is associated with such diversity. This study was carried out to determine whether a technique utilizing three-day postoperative lymphocyte percentages following skin grafting would adequately measure genetic
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 29, 2015
Estimation of Genetic Diversity Among Strains and Breeds of Chickens by Lymphocyte Increases Following Skin Grafting1'2
440
J. E. BERRY AND J. V. CRAIG
diversity among strains and breeds of chickens.
were analyzed by standard statistical techniques.
MATERIAL AND METHODS
RESULTS AND DISCUSSION
TABLE 1.—Mean preoperative percentage lymphocytes arranged in ascending order by host strains and subjected to "Duncan's New Multiple Range Test'n Host strains Mean Lymphocyte Percentage
R2 2
63.32
'
Ri
R3
63.89
65.24
Li
69.48
L3
L2
70.31
72.58
1 Means underscored by the same line are not significantly different. Means not underscored by the same line are different at the 5 % level of probability. 2 Each mean is based on data from 54 host females.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 29, 2015
Mean preoperative lymphocyte percentThe surgical techniques used in this study were the same as those described by ages for twelve-month-old hens of the six Berry et al. (1958). Skin grafts were made, strains were obtained and subjected to analusing nine males from each of six strains as ysis by Duncan's (1955) new multiple skin donors. Six pieces of skin, of approxi- range test, Table 1. It is apparent from the mately 2 cm. diameter, were taken from the results that the Leghorn strains were signifthigh area of each donor male and one icantly (P < .05) higher in lymphocyte graft each was placed on a female of the percentages than the Rhode Island Red same strain and on hens of each of the other strains. However, differences between five strains. All birds were approximately strains within these two breeds were not twelve months of age at the time of the significant for this trait. The overall mean operation. Differential counts of the leuco- difference between the breeds was 6.64 percytes of the circulating blood were made cent and presumably represents a real breed immediately before transplantation and on difference since all birds were subjected to the same environment. the third postoperative day. Correlation and regression coefficients It was postulated that the preoperative were calculated between preoperative and percentage of lymphocytes might influence postoperative lymphocyte percentages with- an individual's ability to respond to a skin in each of the host strains. The regression co- graft as measured by the postoperative lymefficients were tested for heterogeneity and phocyte percentage. Correlation and regrespostoperative lymphocyte percentages were sion coefficients were calculated on a withinadjusted for differences in preoperative strain basis between preoperative and postlymphocyte levels. operative lymphocyte percentages. Clearly Birds of the six strains used were hatched significant associations were found in four from eggs received from commercial breed- of the six strains (Table 2). Statistical analers who were asked to supply hatching eggs ysis failed to suggest heterogeneity and a from closed flocks. Although there was pooled regression coefficient of 0.33 was probably some relationship among the birds used to adjust for differences in preoperawithin any one strain, the birds within a tive lymphocyte levels of individual host strain were assumed to be unrelated. There hens. Table 3 presents the adjusted threewere three White Leghorn (L) and three day postoperative lymphocyte percentages following exchanges of skin for each of the Rhode Island Red (R) strains. Adjusted three-day postoperative lym- donor-host combinations. Analysis of variance of the adjusted postphocyte percentages following skin grafting
441
3ENETIC DIVERSITY AND LYMPHOCYTE INCREASE T A B L E 2.—Correlation and regression coefficients of percentages by host
Host strain 1
Correlation Coefficient Regression Coefficient1
preoperative and postoperative strains
lymphocyte
Li
L/2
L3
Ri
R2
R3
All strains
0.22 0.24
0.33* 0.38**
0.36** 0.37**
0.11 0.13
0.38** 0.39**
0.44** 0.49**
0.33**
1
E a c h coefficient is based on d a t a from 54 host females. * Significant a t 5 % level. ** Significant a t 1 % level. T A B L E 3.—Three-day postoperative lymphocyte percentages of hosts after for preoperative lymphocyte levels
adjustment
Li U U Ri R2 R3 Means, by Host Strains
Means, by donor strains
L,
U
L3
Ri
R2
R3
72.14 70.71 75.28 71.79 75.00 71.01
75.66 74.89 76.17 75.04 70.89 75.58
78.33 76.93 75.58 77.03 77.35 72.14
69.66 72.58 73.99 72.33 74.75 70.44
72.57 73.45 68.17 67.85 68.33 75.66
76.24 65.58 74.38 74.43 77.65 74.27
74.10 72.36 73.93 73.08 74.00 73.18
72.66
74.71
76.23
72.29
71.01
73.76
73.44
Each mean for a donor-host combination represents readings from nine host females.
operative lymphocyte percentages showed that the only differences approaching significance (P = 0.08) were between the different host strains (Table 4). These results are further illustrated in Table 5 where the three-day postoperative lymphocyte percentages are grouped according to whether the donors and hosts were of the same strain, of different strains of the same breed, or of different breeds. The mean withinstrain value of 72.92 percentage lymphocytes is very close to the mean betweenstrain response of 73.55 percent. 1
T A B L E 4.—Analysis of variance of adjusted erative lymphocyte percentages
Source Donor Strains Host Strains Interaction (DonorsXHosts) Among Hosts Treated Alike
postop-
d.f.
Mean Probsquares ability
5 5 25 287
25.40 187.18 76.37 91.23
0.08
1 Adjusted for p r e o p e r a t i v e percentageof l y m p h o cytes.
In the work of Craig and Hirsch (1957) and of Berry et al. (1958) significant regressions were established for increases in the relative lymphocyte percentage following skin grafting, with known genetic relationship coefficients of donors and hosts. In the present study, using adjusted three-day postoperative lymphocyte percentages as T A B L E 5.—Mean adjusted1 postoperative lymphocyte percentages, grouped according to kind of skin-graft exchange
Kind of exchange
Percentage No. of hosts lymphocytes
A. Unrelated, Same Strain Leghorns Reds
27 27
74.20 71.64
B. Between Strains, Same Breed Leghorns Reds
54 54
75.51 73.46
C. Between Breeds Red Donors, Leghorn Hosts Leghorn Donors, Red Hosts
81 81
73.98 71.85
54 108 162 324 270
72.92 74.49 72.92 73.44 73.55
Weighted Means A B C A, B, C B, C 1
Adjusted for preoperative percentage of lymphocytes.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 29, 2015
Host strains Donor strains
442
J. E. BERRY AND J. V. CRAIG
SUMMARY AND CONCLUSIONS
An attempt was made to use the intensity of the immune reaction evoked by skin grafts, as measured by three-day postoperative lymphocyte percentages adjusted for differences in preoperative lymphocyte levels to estimate the genetic diversity between strains and breeds of the Rhode Island Red and White Leghorn breeds. Three strains represented each breed. White Leghorn
Berry, J. E., J. V. Craig and G. K. L. Underbjerg, 1958. A comparison of the effects of skin implants and skin grafts on the cellular constituents of chicken blood. Poultry Sci. 37: 312-316. Blumenthal, H. T., 1939. Effects of organismal differentials on the distribution of leukocytes in the circulating blood. Arch. Path. 27: 510-545. Craig, J. V., and L. J. Hirsch, 1957. Genetic relationship and the reaction to skin grafts. J. Heredity, 48: 235-237. Duncan, D. B., 1955. Multiple range and multiple F tests. Biometrics, 11: 1-42.
JUNE 23-25. WESTERN POULTRY CONGRESS, FAIR GROUNDS, FRESNO, CALIFORNIA JULY 21-23. AMERICAN POULTRY AND HATCHERY FEDERATION CONVENTION, KIEL AUDITORIUM, ST. LOUIS, MO.
Downloaded from http://ps.oxfordjournals.org/ at Univ of Iowa-Law Library on June 29, 2015
the measure of intensity of the immune re- hens were found to have significantly higher action, the technique failed to indicate the lymphocyte percentages than Rhode Island greater genetic diversity between strains Red hens at approximately twelve months and breeds as compared with that between of age. The preoperative level of lymphopresumably unrelated birds of the same cytes was found to have a significant effect strains. It is difficult to reconcile these re- upon the ability of the bird to respond to sults with the part of the experiment of skin grafts, as measured by the three-day Craig and Hirsch (1957) dealing with be- postoperative lymphocyte percentage. Lymtween-breed responses. There was no pla- phocyte percentages on the third day folteau of response when grafts were ex- lowing skin grafting were no greater when changed between unrelated birds of the skin was exchanged between individuals of same breed in their experiment. In the pres- different breeds than those found after exent experiment such a plateauing of re- changes of skin between unrelated indisponse appears to have occurred. Differ- viduals of the same strains. These results ences in age and genetic constitution of the preclude the use of the present technique, birds used in these different experiments without further modifications, for the purmay be responsible for the divergent results pose of estimating genetic diversity between of these two investigations. In the light of strains and breeds of chickens. the present results, we suggest that the presACKNOWLEDGMENTS ent techniques might be modified to allow The authors wish to express their gratimeasurement of the immune response betude to Drs. A. W. Nordskog and W. H. fore it has reached its maximum level or to Kyle of Iowa State College and the Northelicit less than the maximum possible reCentral Regional Poultry Breeding Laboraaction so that the plateauing of response tory, respectively, for valuable suggestions with increasing genetic diversity between concerning the analysis of the data and to strains and breeds may be avoided. Such Dr. S. Wearden, Statistician at Kansas modification might involve the use of young State College for help in analyzing and inchicks, taking postoperative readings at a terpreting the experiment. shorter interval than three days, or the use of a smaller piece of tissue for transplanting. REFERENCES