Detection of Cellular Immunity to Rous Tumors of Chickens by the Leukocyte Migration Inhibition Reaction2,3 P . F . COTTER, ' W . M . COLLINS, W . R. DUNLOP AND A. C
CORBETT
Department of Animal Sciences, University of New Hampshire, Durham, New Hampshire, 03824 (Received for publication August 29, 1975)
ABSTRACT Peripheral blood leukocytes obtained from Rous sarcoma-bearing chickens were tested for their ability to be inhibited from migrating in vitro by soluble tumor extract. It was found that the inhibition of leukocytes from chickens with regressing Rous tumors was significantly greater than that of chickens with progressing tumors or non-tumor bearing controls.
INTRODUCTION
MATERIALS AND METHODS
HE specific inhibition of migration of spleen cells in vitro has been shown to be correlated with delayed type hypersensitivity reactions of chickens (Morita and Sokawa, 1971; Zwilling et al., 1972) and other species (David et al., 1964). Moreover delayed type hypersensitivity to tumor specific antigens has been detected by the inhibition of specifically sensitized macrophages in guinea pigs (Bloom et al., 1969; Kroman et al, 1969) and mice (Halliday and Webb, 1969). Fauser et al. (1973) showed that the migration of peripheral blood leukocytes from Marek's disease virus sensitized chickens was specifically inhibited by purified antigen A (Churchill et al., 1969). We undertook this study in order to determine whether a delayed hypersensitivity reaction could be detected in vitro, by the leukocyte migration inhibition reaction, in chickens bearing either progressing or regressing Rous sarcomas.
Chickens. Single Comb White Leghorn chickens of inbred Line 6 were used in this study. They were inoculated in the left wing web at 6 weeks of age with 20 pock-forming units of a Rous sarcoma virus pseudotype, BH-RSV (RAV-1). The virus was obtained from Dr. L. B. Crittenden, Avian Physiology Laboratory, U.S.D.A., Beltsville, Md. All chickens so treated developed wing web tumors which either grew progressively, eventually killing the host, or regressed completely after a short period of growth. Rarely, a tumor would show alternate periods of progressive and regressive growth.
T
1. Department of Biology, Merrimack College, North Andover, Mass. 01845. 2. Published with the Approval of the Director of the New Hampshire Agricultural Experiment Station as Scientific Contribution No. 705. 3. This study was conducted as part of the contributing project of the New Hampshire Agricultural Experiment Station to the Northeast Regional Poultry Breeding Project (NE-60).
Tumor Extract. A crude soluble extract was prepared from Rous tumors induced by BHRSV (RAV-1) using the method of Halliday (1971). It was found that a 1:4 dilution of this extract in Medium Ml99 did not inhibit migration of leukocytes obtained from uninoculated leukosis-free chickens (SPAFAS, Norwich, Connecticut). Preparation of Cells. Buffy coat cells were obtained from 2.0 ml. heparinized cardiac blood by centrifugation. The cells were washed 3 times in M199 and divided into 2 aliquots, one of which was mixed with the tumor extract and the other (control) was mixed with an equal amount of M199. After
1008
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 3, 2015
POULTRY SCIENCE 55: 1008-1011, 1976
1009
CELLULAR IMMUNITY TO ROUS SARCOMA
Measurement of Migration Inhibition.
The
migrating cells formed fan-like images which were magnified and projected onto a sheet of paper by means of an overhead projector (Porta Scribe 1000). The images were traced and their areas measured with a planimeter. The percent migration inhibition was calculated as follows: percent inhibition = (1 — T/C)100, where T = the mean area of 3 replicates in the well containing the tumor extract and C = the mean area of 3 replicates in the well containing M199 only. Statistical Analysis. The data on area of migration were subjected to variance analysis according to type of chicken and presence
TABLE 1.—The results of the migration inhibition test of chickens of Line 6 bearing progressive or regressive tumors and of uninoculated, SPF, controls
Chick wing band no. 2372 2381 (l) c 2381 (2) 4174 4185 4186 4187 4200 4191 2374 2378 none 2385 (l) d 2385 (2) 2385 (3) 1403 2377 1393 4195 SPF-1 SPF-2 SPF-3
Chicken type Regressor Regressor Regressor Regressor Regressor Regressor Regressor Regressor Regressor Regressor Regressor Regressor Progressor Progressor Progressor Progressor Progressor Progressor Progressor Control Control Control
Tumor score at test date" 1 1 0 1 1 1 2 0 2 0 0 0 3 4 3 4 4 4 4
Mean area of migration (cm. 2 ) (antigen absent)
Mean area of migration (cm. 2 ) (antigen present)
2.93(0.26)a 3.57(0.52) 6.23(0.03) 7.90(0.92) 5.63(0.12) 0.87(0.13) 2.97(0.55) 2.47(0.16) 3.03(0.29) 1.97(0.38) 3.77(0.35) 8.16(1.17) 3.23(0.12) 5.10(0.43) 1.70(0.33) 4.26(0.17) 4.23(0.43) 1.47(0.26) 0.50(0.00) 4.10(0.58) 5.30(0.89) 2.90(0.20)
1.60(0.35)" 1.77(0.18) 3.37(0.41) 4.73(0.38) 2.63(0.17) 0.20(0.00) 1.60(0.09) 0.87(0.36) 1.10(0.05) 1.43(0.12) 4.67(0.29) 3.97(0.37) 2.40(0.37) 5.03(0.08) 1.00(0.20) 5.13(0.49) 4.63(0.46) 2.72(0.43) 0.50 5.93(0.67) 6.27(0.35) 2.43(0.29)
Inhibition
(%) 46 51 46 41 53 77 47 65 67 27 -24 51 26 2 41 -20 9 -85 0 -44 -18 16
a tumors were scored weekly based on their size, i.e., 0 = a completely regressed tumor, 4 a large tumor completely filling the wing web area. b ± Standard error. c tested on two successive weeks. d tested on three successive weeks.
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 3, 2015
incubation for 1 hour at room temperature the cells were drawn into hematocrit tubes, which were then flame-sealed at one end, and packed by centrifugation. The hematocrit tubes were then cut at the cell-fluid interface and the extract exposed cells were placed into one well of a Bloom-type cell migration chamber which contained an additional 0.4 ml. of diluted tumor extract. Control cells were placed in the opposite well containing M199 only. Both wells were then filled with M199 containing 15% calf serum, sealed, and incubated at 37° C. for 18 to 24 hours.
1010
P. F. COTTER, W. M. COLLINS, W. R. DUNLOP AND A. C
or absence of antigen. The analysis involved the method of unequal subclass numbers except that the error sum of squares was generated by a preliminary analysis of variance unadjusted unequal subclass numbers (Steel and Torrie, 1960).
RESULTS
Chicken No. 2385 was tested on 3 successive weeks during which time its tumor exhibited alternate regressive and progressive growth patterns. This type of growth has been known to occur in chickens with Rous tumors (Gyles et al., 1967). It is of interest to note that the greatest percentage migration inhibition occurred during the phases of regressive growth while virtually no inhibition was observed during the period of progressive growth in vivo (Table 1). Enhanced migration was observed in 2 of 3 unsensitized controls (Table 1). The reason for this is not clear, but such enhanced migration has been reported for controls tested with old tuberculin antigen and Marek's disease A antigen (Fauser et al., 1973).
DISCUSSION The present study indicated that the in vitro migration of buffy coat cells obtained from chickens with regressing or recently regressed wing web tumors was inhibited by a soluble tumor extract. Inhibition was significantly less when the cells were obtained from chickens with progressively growing wing web tumors or from uninoculated controls. We have previously shown that the incidence of regression of Rous sarcomas is significantly influenced by the particular line of chickens (Cotter et al., 1973a), the presence of thymus tissue (Cotter et al., 1976), and the host age at inoculation (Cotter et al., 1973b). The data suggest that tumor regression in chickens is mediated by a delayed type hypersensitivity reaction in which thymus dependent cells (T cells) become sensitized to and subsequently effect the destruction of tumor cells. Progressive tumor growth, on the other hand, may be facilitated by blocking factors (Halliday, 1971; Hellstrom et al., 1969) which protect tumor cells from a cell mediated immune attack. The use of buffy coat cells in the migration inhibition reaction is a convenient method of investigating immune responses to tumor antigens. Moreover, since buffy coat cells may be obtained from specific chickens over an extended period of time, this method may have potential value in chronologic investigations of the immune response to tumors.
REFERENCES Bloom, B. R., B. Bennett, H. F. Oettgen, E. P. McLean and L. J. Old, 1969. Demonstration of delayed hypersensitivity to soluble antigens of chemically induced tumors by inhibition of macrophage migration. Proc. Nat. Acad. Sci. 64: 11761180. Churchill, A. E., R. C. Chubb and W. Blaxendale, 1969. The attenuation, with loss of oncogenicity, of the herpes-type virus of Marek's disease (Strain HPRS-16) on passage in cell culture. J. Gen. Virol. 4: 557-564.
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 3, 2015
The percent migration inhibition for individual chickens of the 3 types tested, i.e., progressor, regressor, and unsensitized controls is given in Table 1. The percent migration inhibition was generally greater for regressor type chickens than for progressor type or unsensitized controls (P s 0.025). There was one instance where cells obtained from a regressor type chicken were not inhibited (No. 2378, Table 1). In this case, 4 weeks elapsed between the last visible presence of a tumor and the test date. In all other cases the migration inhibition test was performed either while a tumor was visible or within 1 week after its disappearance. Thus, sufficient time may have elapsed for this chicken to become desensitized to the tumor extract.
CORBETT
CELLULAR IMMUNITY TO ROUS SARCOMA
tumor-bearing animals on macrophage migration inhibition with tumor antigens. J. Immunol. 106: 855-857. Halliday, W. J., and M. Webb, 1969. Delayed hypersensitivity to chemically induced tumors in mice and correlation with an in vitro test. J. Nat. Cancer Inst. 43: 141-149. Hellstrom, I., K. E. Hellstrom, C. A. Evans, G. H. Heppner, G. E. Pierce and J. P. S. Yang, 1969. Serum mediated protection of neoplastic cells from inhibition by lymphocytes immune to their tumor specific antigens. Proc. Nat. Acad. Sci. 62: 362-368. Kronman, B. S., H. T. Wepsic, W. H. Churchill, B. Zebar, T. Borsos and H. J. Rapp, 1969. Tumor specific antigens detected by inhibition of macrophage migration. Science, 165: 296-297. Morita, C , and N. Sokawa, 1971. Specific inhibition of splenic cells migration in chickens sensitized to delayed hypersensitivity. Poultry Sci. 50: 1503-1505. Steel, R. G., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw Hill Book Co., Inc., New York. Zwilling, B. S., J. T. Barett and R. P. Breitenback, 1972. Avian delayed type hypersensitivity: Adaption of the migration inhibition assay. Cell. Immunol. 4: 20-28.
Ammoniated Cottonseed Meal as a Protein Supplement for Laying Hens* PARK W. WALDROUP, KENNY R. HAZEN, R. JAMES MITCHELL, JOHN R. PAYNE AND ZELPHA JOHNSON
Department of Animal Sciences, University of Arkansas, Fayetteville, Arkansas 72701 (Received for publication September 2, 1975)
ABSTRACT Ammoniation has been proposed as a means of reducing the level of aflatoxin in contaminated feedstuffs. A lot of cottonseed meal was divided into two portions, one which was subjected to ammoniation and one fed as the untreated meal. These were incorporated into layer hen diets and their effects on performance parameters evaluated. Results indicated that the ammoniation process may result in destruction of part of the cystine present in the cottonseed meal. However, when fed in diets composed of practical-type ingredients where adequate feed intake was maintained, excellent performance was attained on both samples of cottonseed meal. No problems were encountered that would suggest any adverse effects of the ammoniation process on the health of the hens or on the quality of the eggs produced. POULTRY SCIENCE 55: 1011-1019, 1976
C
OTTONSEED meal has been used as a protein supplement in laying hen diets for many years in certain areas of the United *Published with the approval of the Director of the Arkansas Agricultural Experiment Station.
States and in other livestock producing countries where it has an economic advantage over soybean meal. Recent studies from this laboratory (Waldroup and Goodner, 1973) have outlined recommendations for safe and effective usage of this feedstuff to avoid prob-
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 3, 2015
Cotter, P. F., W. M. Collins, A. C. Corbett and W. R. Dunlop, 1973a. Regression of Rous sarcomas in two lines of chickens. Poultry Sci. 52: 799-800. Cotter, P. F., W. M. Collins, W. R. Dunlop and A. C. Corbett, 1973b. Host age dependency of regression of Rous sarcomas of chickens. Cancer Res. 33: 3310-3311. Cotter, P. F., W. M. Collins, W. R. Dunlop and A. C. Corbett, 1976. Progression of Rous sarcomas in thymectomized chickens. Manuscript in preparation. David, J. R., S. Al-Askari, H. S. Lawrence and L. Thomas, 1964. Delayed hypersensitivity in vitro. I. The specificity of inhibition of cell migration by antigens. J. Immunol. 93: 264-273. Fauser, J. S., H. G. Purchase, P. A. Long, L. F. Velicer, W. H. Mailman, H. T. Fauser and G. O. Winegar, 1973. Delayed hypersensitivity and leukocyte migration inhibition in chickens with BCG or Marek's disease infection. Avian Pathol. 2: 55-61. Gyles, N. R., J. L. Miley and C. J. Brown, 1967. The response of resistant and susceptible strains of chickens and their F, and F2 crosses to subcutaneous inoculations with Rous sarcoma virus. Poultry Sci. 46: 465-472. Halliday, W. J., 1971. Blocking effect of serum from
1011
CORBETT
Department of Animal Sciences, University of New Hampshire, Durham, New Hampshire, 03824 (Received for publication August 29, 1975)
ABSTRACT Peripheral blood leukocytes obtained from Rous sarcoma-bearing chickens were tested for their ability to be inhibited from migrating in vitro by soluble tumor extract. It was found that the inhibition of leukocytes from chickens with regressing Rous tumors was significantly greater than that of chickens with progressing tumors or non-tumor bearing controls.
INTRODUCTION
MATERIALS AND METHODS
HE specific inhibition of migration of spleen cells in vitro has been shown to be correlated with delayed type hypersensitivity reactions of chickens (Morita and Sokawa, 1971; Zwilling et al., 1972) and other species (David et al., 1964). Moreover delayed type hypersensitivity to tumor specific antigens has been detected by the inhibition of specifically sensitized macrophages in guinea pigs (Bloom et al., 1969; Kroman et al, 1969) and mice (Halliday and Webb, 1969). Fauser et al. (1973) showed that the migration of peripheral blood leukocytes from Marek's disease virus sensitized chickens was specifically inhibited by purified antigen A (Churchill et al., 1969). We undertook this study in order to determine whether a delayed hypersensitivity reaction could be detected in vitro, by the leukocyte migration inhibition reaction, in chickens bearing either progressing or regressing Rous sarcomas.
Chickens. Single Comb White Leghorn chickens of inbred Line 6 were used in this study. They were inoculated in the left wing web at 6 weeks of age with 20 pock-forming units of a Rous sarcoma virus pseudotype, BH-RSV (RAV-1). The virus was obtained from Dr. L. B. Crittenden, Avian Physiology Laboratory, U.S.D.A., Beltsville, Md. All chickens so treated developed wing web tumors which either grew progressively, eventually killing the host, or regressed completely after a short period of growth. Rarely, a tumor would show alternate periods of progressive and regressive growth.
T
1. Department of Biology, Merrimack College, North Andover, Mass. 01845. 2. Published with the Approval of the Director of the New Hampshire Agricultural Experiment Station as Scientific Contribution No. 705. 3. This study was conducted as part of the contributing project of the New Hampshire Agricultural Experiment Station to the Northeast Regional Poultry Breeding Project (NE-60).
Tumor Extract. A crude soluble extract was prepared from Rous tumors induced by BHRSV (RAV-1) using the method of Halliday (1971). It was found that a 1:4 dilution of this extract in Medium Ml99 did not inhibit migration of leukocytes obtained from uninoculated leukosis-free chickens (SPAFAS, Norwich, Connecticut). Preparation of Cells. Buffy coat cells were obtained from 2.0 ml. heparinized cardiac blood by centrifugation. The cells were washed 3 times in M199 and divided into 2 aliquots, one of which was mixed with the tumor extract and the other (control) was mixed with an equal amount of M199. After
1008
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 3, 2015
POULTRY SCIENCE 55: 1008-1011, 1976
1009
CELLULAR IMMUNITY TO ROUS SARCOMA
Measurement of Migration Inhibition.
The
migrating cells formed fan-like images which were magnified and projected onto a sheet of paper by means of an overhead projector (Porta Scribe 1000). The images were traced and their areas measured with a planimeter. The percent migration inhibition was calculated as follows: percent inhibition = (1 — T/C)100, where T = the mean area of 3 replicates in the well containing the tumor extract and C = the mean area of 3 replicates in the well containing M199 only. Statistical Analysis. The data on area of migration were subjected to variance analysis according to type of chicken and presence
TABLE 1.—The results of the migration inhibition test of chickens of Line 6 bearing progressive or regressive tumors and of uninoculated, SPF, controls
Chick wing band no. 2372 2381 (l) c 2381 (2) 4174 4185 4186 4187 4200 4191 2374 2378 none 2385 (l) d 2385 (2) 2385 (3) 1403 2377 1393 4195 SPF-1 SPF-2 SPF-3
Chicken type Regressor Regressor Regressor Regressor Regressor Regressor Regressor Regressor Regressor Regressor Regressor Regressor Progressor Progressor Progressor Progressor Progressor Progressor Progressor Control Control Control
Tumor score at test date" 1 1 0 1 1 1 2 0 2 0 0 0 3 4 3 4 4 4 4
Mean area of migration (cm. 2 ) (antigen absent)
Mean area of migration (cm. 2 ) (antigen present)
2.93(0.26)a 3.57(0.52) 6.23(0.03) 7.90(0.92) 5.63(0.12) 0.87(0.13) 2.97(0.55) 2.47(0.16) 3.03(0.29) 1.97(0.38) 3.77(0.35) 8.16(1.17) 3.23(0.12) 5.10(0.43) 1.70(0.33) 4.26(0.17) 4.23(0.43) 1.47(0.26) 0.50(0.00) 4.10(0.58) 5.30(0.89) 2.90(0.20)
1.60(0.35)" 1.77(0.18) 3.37(0.41) 4.73(0.38) 2.63(0.17) 0.20(0.00) 1.60(0.09) 0.87(0.36) 1.10(0.05) 1.43(0.12) 4.67(0.29) 3.97(0.37) 2.40(0.37) 5.03(0.08) 1.00(0.20) 5.13(0.49) 4.63(0.46) 2.72(0.43) 0.50 5.93(0.67) 6.27(0.35) 2.43(0.29)
Inhibition
(%) 46 51 46 41 53 77 47 65 67 27 -24 51 26 2 41 -20 9 -85 0 -44 -18 16
a tumors were scored weekly based on their size, i.e., 0 = a completely regressed tumor, 4 a large tumor completely filling the wing web area. b ± Standard error. c tested on two successive weeks. d tested on three successive weeks.
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 3, 2015
incubation for 1 hour at room temperature the cells were drawn into hematocrit tubes, which were then flame-sealed at one end, and packed by centrifugation. The hematocrit tubes were then cut at the cell-fluid interface and the extract exposed cells were placed into one well of a Bloom-type cell migration chamber which contained an additional 0.4 ml. of diluted tumor extract. Control cells were placed in the opposite well containing M199 only. Both wells were then filled with M199 containing 15% calf serum, sealed, and incubated at 37° C. for 18 to 24 hours.
1010
P. F. COTTER, W. M. COLLINS, W. R. DUNLOP AND A. C
or absence of antigen. The analysis involved the method of unequal subclass numbers except that the error sum of squares was generated by a preliminary analysis of variance unadjusted unequal subclass numbers (Steel and Torrie, 1960).
RESULTS
Chicken No. 2385 was tested on 3 successive weeks during which time its tumor exhibited alternate regressive and progressive growth patterns. This type of growth has been known to occur in chickens with Rous tumors (Gyles et al., 1967). It is of interest to note that the greatest percentage migration inhibition occurred during the phases of regressive growth while virtually no inhibition was observed during the period of progressive growth in vivo (Table 1). Enhanced migration was observed in 2 of 3 unsensitized controls (Table 1). The reason for this is not clear, but such enhanced migration has been reported for controls tested with old tuberculin antigen and Marek's disease A antigen (Fauser et al., 1973).
DISCUSSION The present study indicated that the in vitro migration of buffy coat cells obtained from chickens with regressing or recently regressed wing web tumors was inhibited by a soluble tumor extract. Inhibition was significantly less when the cells were obtained from chickens with progressively growing wing web tumors or from uninoculated controls. We have previously shown that the incidence of regression of Rous sarcomas is significantly influenced by the particular line of chickens (Cotter et al., 1973a), the presence of thymus tissue (Cotter et al., 1976), and the host age at inoculation (Cotter et al., 1973b). The data suggest that tumor regression in chickens is mediated by a delayed type hypersensitivity reaction in which thymus dependent cells (T cells) become sensitized to and subsequently effect the destruction of tumor cells. Progressive tumor growth, on the other hand, may be facilitated by blocking factors (Halliday, 1971; Hellstrom et al., 1969) which protect tumor cells from a cell mediated immune attack. The use of buffy coat cells in the migration inhibition reaction is a convenient method of investigating immune responses to tumor antigens. Moreover, since buffy coat cells may be obtained from specific chickens over an extended period of time, this method may have potential value in chronologic investigations of the immune response to tumors.
REFERENCES Bloom, B. R., B. Bennett, H. F. Oettgen, E. P. McLean and L. J. Old, 1969. Demonstration of delayed hypersensitivity to soluble antigens of chemically induced tumors by inhibition of macrophage migration. Proc. Nat. Acad. Sci. 64: 11761180. Churchill, A. E., R. C. Chubb and W. Blaxendale, 1969. The attenuation, with loss of oncogenicity, of the herpes-type virus of Marek's disease (Strain HPRS-16) on passage in cell culture. J. Gen. Virol. 4: 557-564.
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 3, 2015
The percent migration inhibition for individual chickens of the 3 types tested, i.e., progressor, regressor, and unsensitized controls is given in Table 1. The percent migration inhibition was generally greater for regressor type chickens than for progressor type or unsensitized controls (P s 0.025). There was one instance where cells obtained from a regressor type chicken were not inhibited (No. 2378, Table 1). In this case, 4 weeks elapsed between the last visible presence of a tumor and the test date. In all other cases the migration inhibition test was performed either while a tumor was visible or within 1 week after its disappearance. Thus, sufficient time may have elapsed for this chicken to become desensitized to the tumor extract.
CORBETT
CELLULAR IMMUNITY TO ROUS SARCOMA
tumor-bearing animals on macrophage migration inhibition with tumor antigens. J. Immunol. 106: 855-857. Halliday, W. J., and M. Webb, 1969. Delayed hypersensitivity to chemically induced tumors in mice and correlation with an in vitro test. J. Nat. Cancer Inst. 43: 141-149. Hellstrom, I., K. E. Hellstrom, C. A. Evans, G. H. Heppner, G. E. Pierce and J. P. S. Yang, 1969. Serum mediated protection of neoplastic cells from inhibition by lymphocytes immune to their tumor specific antigens. Proc. Nat. Acad. Sci. 62: 362-368. Kronman, B. S., H. T. Wepsic, W. H. Churchill, B. Zebar, T. Borsos and H. J. Rapp, 1969. Tumor specific antigens detected by inhibition of macrophage migration. Science, 165: 296-297. Morita, C , and N. Sokawa, 1971. Specific inhibition of splenic cells migration in chickens sensitized to delayed hypersensitivity. Poultry Sci. 50: 1503-1505. Steel, R. G., and J. H. Torrie, 1960. Principles and Procedures of Statistics. McGraw Hill Book Co., Inc., New York. Zwilling, B. S., J. T. Barett and R. P. Breitenback, 1972. Avian delayed type hypersensitivity: Adaption of the migration inhibition assay. Cell. Immunol. 4: 20-28.
Ammoniated Cottonseed Meal as a Protein Supplement for Laying Hens* PARK W. WALDROUP, KENNY R. HAZEN, R. JAMES MITCHELL, JOHN R. PAYNE AND ZELPHA JOHNSON
Department of Animal Sciences, University of Arkansas, Fayetteville, Arkansas 72701 (Received for publication September 2, 1975)
ABSTRACT Ammoniation has been proposed as a means of reducing the level of aflatoxin in contaminated feedstuffs. A lot of cottonseed meal was divided into two portions, one which was subjected to ammoniation and one fed as the untreated meal. These were incorporated into layer hen diets and their effects on performance parameters evaluated. Results indicated that the ammoniation process may result in destruction of part of the cystine present in the cottonseed meal. However, when fed in diets composed of practical-type ingredients where adequate feed intake was maintained, excellent performance was attained on both samples of cottonseed meal. No problems were encountered that would suggest any adverse effects of the ammoniation process on the health of the hens or on the quality of the eggs produced. POULTRY SCIENCE 55: 1011-1019, 1976
C
OTTONSEED meal has been used as a protein supplement in laying hen diets for many years in certain areas of the United *Published with the approval of the Director of the Arkansas Agricultural Experiment Station.
States and in other livestock producing countries where it has an economic advantage over soybean meal. Recent studies from this laboratory (Waldroup and Goodner, 1973) have outlined recommendations for safe and effective usage of this feedstuff to avoid prob-
Downloaded from http://ps.oxfordjournals.org/ at University of Birmingham on June 3, 2015
Cotter, P. F., W. M. Collins, A. C. Corbett and W. R. Dunlop, 1973a. Regression of Rous sarcomas in two lines of chickens. Poultry Sci. 52: 799-800. Cotter, P. F., W. M. Collins, W. R. Dunlop and A. C. Corbett, 1973b. Host age dependency of regression of Rous sarcomas of chickens. Cancer Res. 33: 3310-3311. Cotter, P. F., W. M. Collins, W. R. Dunlop and A. C. Corbett, 1976. Progression of Rous sarcomas in thymectomized chickens. Manuscript in preparation. David, J. R., S. Al-Askari, H. S. Lawrence and L. Thomas, 1964. Delayed hypersensitivity in vitro. I. The specificity of inhibition of cell migration by antigens. J. Immunol. 93: 264-273. Fauser, J. S., H. G. Purchase, P. A. Long, L. F. Velicer, W. H. Mailman, H. T. Fauser and G. O. Winegar, 1973. Delayed hypersensitivity and leukocyte migration inhibition in chickens with BCG or Marek's disease infection. Avian Pathol. 2: 55-61. Gyles, N. R., J. L. Miley and C. J. Brown, 1967. The response of resistant and susceptible strains of chickens and their F, and F2 crosses to subcutaneous inoculations with Rous sarcoma virus. Poultry Sci. 46: 465-472. Halliday, W. J., 1971. Blocking effect of serum from
1011
