ENZYME ACTIVITY AND GROWTH RATE
of embryonic and adult rat, sheep and domestic fowl. Biochem. J. 104: 378-384. Weber, G., G. Banerjee, D. Bixler and J. Ashmore, 1961. Role of enzymes in metabolic homeostasis. II. Depletion and restoration of avian liver carbohy-
1597
drate-metabolizing enzymes. J. Nutr. 74: 157-160. Williamson, J. R., 1966. Response of hepatic glucokinase and glucose-6-phosphatase activities in juvenile and adult hyperthyroid mice. Endocrinology, 87: 124-128.
Antibiotic Dipping Studies in Relation to Uptake, Embryonic Development and Arizona Hinshawii Recoveries from Turkey Hatching Eggs 12 S. C . N I V A S , M . D . YORK AND B . S. POMEROY
Department of Veterinary Biology, University of Minnesota, St. Paul, Minnesota 55108 (Received for publication January 13, 1975)
ABSTRACT Antibiotic dipping with gentamicin sulfate by means of the temperature-differential method was effective in reducing but not completely eliminating Arizona hinshawii (7: 1, 7, 8) from artificially infected turkey hatching eggs. Embryonic development was well maintained. The antibiotic dip solution intake was variable from egg to egg. Removal of cuticle by means of either 0.2 N HCl or a 10% disodium salt of ethylene diamine tetraacetic acid enhanced egg weight gains and maintained good embryonic development. Complete elimination of A. hinshawii was achieved in one experiment out of four. Correlation studies between egg weight gains and gentamicin concentrations of the contents of the turkey hatching eggs indicated a statistically significant relationship between these two parameters. POULTRY SCIENCE 54: 1597-1604, 1975
al, 1962; Hall et al., 1963; Stuart and Bruins, 1963; Yoder and Hofstad, 1965; Kumar et N recent years, a number of papers on al, 1966; Newman, 1967; Bigland, 1970). antibiotic egg dipping of chicken and turkey Recent publications have used egg-dipping hatching eggs with or without certain quaterprocedures for the control of Salmonellanary ammonium compounds in the dip soluArizona organisms in chicken hatching eggs tion have been published. Earlier papers have (Stuart and Keenum, 1970; Snoeyenbos and reported on egg dipping with different antibiCarlson, 1973) and turkey hatching eggs otics in relation to Mycoplasma organisms (Lucas et al., 1970; Saif et al., 1971; Saif using the temperature-differential method. and Shelby, 1973; Ernst et al, 1974). The The results showed a definite reduction of temperature-differential method of egg-dipMycoplasma from dipped eggs but not comping was again used. Except in one report plete elimination; hatchability usually was (Saif et al., 1971) where gentamicin was the well maintained (Chalquest and Fabricant, antibiotic used for egg-dipping, complete 1959; Levine and Fabricant, 1962; Olson et elimination of Salmonella-Arizona organisms was not obtained, though a reduction in the number of recoveries were made. Fertility 1. Presented in part at the North Central Poultry and hatchability usually were again well Disease Conference, Champaign, 111. June 12-13, 1974. maintained. 2. Published as Paper No. 8959 Scientific Journal The cuticle, shell and shell membranes Series of the Minnesota Agricultural Experiment Staprobably are instrumental in bringing about tion. INTRODUCTION
I
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S. C. NIVAS, M. D. YORK AND B. S. POMEROY
variations between eggs contributing to the differences in antibiotic liquid uptake and resulting in incomplete elimination of Salmonella-Arizona organisms from the egg contents of turkey hatching eggs. When Pseudomonas fluorescens was used to experimentally infect chicken eggs, Lifshitz et al. (1964), noted that the inner shell membrane was the most important barrier against the penetration of this organism followed by the shell and then the outer shell membrane. Board and Fuller (1974), reviewed the nonspecific antimicrobial defenses of the avian egg, embryo and neonate. Removal of the cuticle with chemicals like ethylene diamine tetra-acetic acid (EDTA) and HCl increased the penetration of microorganisms like Pseudomonas aeruginosa and thus increased spoilage (Brown et al, 1966). These results were also supported by Williams and Dillard (1973) using chicken eggs when S. typhimurium was the infecting organism and 0.1 NHC1 and 10% Na 2 EDTA were the chemicals used for cuticle removal. In a recent review article, Board and Halls (1973), noted that removal of the cuticle of chicken eggs by means of EDTA, NaOH or N a 2 S - 9 H 2 0 enhanced liquid uptake when compared to eggs with the intact cuticle. Alls et al. (1964), observed that on removing the cuticle with HCl, the median Erythromycin drug level was increased from 3.81 fj-g./ml. to 6.4 ^.g./ml. The purpose of this study was to evaluate the effect of cuticle removal with either 0.2 N HCl or 10% disodium salt of EDTA and
TABLE 1.—Effect of cuticle removal with 0.2 N HCl on weight gains and gentamicin uptake of turkey hatching eggs after dipping with gentamicin sulfate at a concentration of 1,000 p.p.m. Dipped with Dipped with intact cuticle cuticle removed Av. wt. gain (gms.) Range Diff.
0.0864 (32 eggs) 0.395 (16 eggs) -0.005 to 0.270 0.270 to 0.480 0.275 0.210
subsequent temperature-differential dipping of turkey hatching eggs with gentamicin sulfate at a concentration of 1,000 p.p.m. on (a) uptake as measured by weight gains, (b) Arizona hinshawii recoveries from inner contents of artificially-infected eggs with A. hinshawii!: 1, 7, 8 and (c) embryonic development of these eggs. MATERIALS AND METHODS Eggs. Turkey hatching eggs of the Wrolstad strain were obtained from a commercial hatchery. Four trials were conducted and the eggs were all obtained from the same flock. The number of eggs used for each trial was variable and this is shown from Tables 1 through 4. Four groups per trial were usually used, as shown in Tables 2 through 4, except in the first trial (Table 1), where there were only two groups. The formaldehyde-fumigated eggs were stored in the cooler (12° C.) and experiments were conducted within 48 hours after the eggs were laid. Artificial Infection. One out of four batches of eggs served as controls, and the other three were artificially infected with A. hinshawii 7: 1, 7, 8 organisms. Initially, the eggs were brought out of the cooler and warmed at room temperature (25° C.) for about an hour. These were immersed in a pressure cooker containing normal saline plus an 18 hour veal-infusion broth culture of A. hinshawii organisms at a concentration approximately between 1.0 to 4.0 x 10 7 /ml. of the dip solution. Vacuum was then used at 17 inches of mercury for 5 minutes and the eggs next left in the pressure cooker at atmospheric pressure for 12 minutes to allow the dip solution to penetrate the cuticle and shell of the eggs for infection of the membranes and egg contents to take place. The eggs were removed, air-dried and returned to the cooler for 24-48 hours. The control group was artificially treated with normal saline using the above procedure.
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ANTIBIOTIC DIPPING OF TURKEY EGGS
TABLE 2.—Effect of cuticle removal with 0.2 N HCl on weight gains, embryonic development and Arizona hinshawii recoveries from turkey hatching eggs after artificial infection with Arizona hinshawii 7: 1, 7, 8 and dipping with 1,000 p.p.m. gentamicin sulfate
Av. wt. gain Range Diff. % living embryos at 12-14 days
Non-infected non-dipped ~* — — 83.3 (54 eggs per group) 22.22 (54 eggs per group)
Infected non-dipped — — — 59.3
Infected dipped 0.047 (20 eggs) -0.055 to 0.410 0.465 92.6
Infected, cuticle removed, dipped 0.518 (20 eggs) 0.360 to 0.620 0.260 87.3
94.42
27.82
3^6 (S. schwarzengrund)
% Arizona and Salmonella recoveries from all eggs examined 1 —Not weighed. 2 —A. hinshawii and S. schwarzengrund.
TABLE 3.—Effect of cuticle removal with 10% disodium salt of EDTA on embryonic development and Arizona hinshawii recoveries from turkey hatching eggs after artificial infection with Arizona hinshawii 7: 1, 7, 8 and dipping with 1,000 p.p.m. gentamicin sulfate
% living embryos at 12-14 days % Arizona recoveries from all eggs examined
Non-infected non-dipped 73T (26 eggs per group) 0
Infected non-dipped 19^2
Infected dipped 652
Infected, cuticle removed, dipped 57/7
92.3 (26 eggs per group)
17.4
19.2
TABLE 4.—Effect of cuticle removal with 10% disodium salt of EDTA on weight gains, embryonic development and Arizona hinshawii recoveries from turkey hatching eggs after artificial infection with Arizona hinshawii 7: 1, 7, 8 and dipping with 1,000 p.p.m. gentamicin sulfate
Av. wt. gain Range Diff. % living embryos at 12-14 days % Arizona recoveries from all eggs examined 1 —Not weighed.
Non-infected non-dipped ~' — — 95.5 (23 eggs per group) 0
Infected non-dipped — — — 81.8
Infected dipped 0.073 (23 eggs) -0.140 to 0.460 0.600 86.4
Infected, cuticle removed, dipped 0.722 (23 eggs) 0.605 to 0.810 0.205 91.3
86.4 (23 eggs per group)
8.7
0
Shell Treatments. Twenty-four hours after infecting the turkey hatching eggs, the eggs whose curticle were to be removed were taken out of the cooler and warmed at room temperature for about an hour. Two chemicals in separate experiments were used to remove
the cuticle. 0.2 N HCl was used in the first two experiments whereas a 10% disodium salt of EDTA was used in the next two experiments. For the HCl treatments, the eggs were submerged in a hot (43° C.) distilled water solution of 0.2 N HCl with constant
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S. C. NIVAS, M. D. YORK AND B. S. POMEROY
stirring for a period of 2 mins. The cuticle was manually removed by means of a soft brush with gentle rubbing of the surface under running tap water. For the first experiment with EDTA, the same procedure as above for HC1 was used except that the eggs were submerged in the^hot Na 2 EDTA solution for 15 mins. For the second experiment, the eggs were again submerged in a hot (43° C.) distilled water solution of Na 2 EDTA as above, the eggs were taken out and dipped next in hot (43° C.) distilled water with constant stirring for 5 minutes. The cuticle was removed by gentle rubbing with a soft brush while still submerged in the hot distilled water. All such treated eggs were air-dried at room temperature before placing them back into the cooler for 24 hours. The control and experimental (untreated) eggs were left in the cooler during this period. Egg Dipping. Two days after artificial infection with A. hinshawii organisms, all eggs (with or without cuticle) were taken out of the cooler and placed in the incubator (37° C.) for about six hours. The eggs were next removed and individually weighed. This usually took an hour. Eggs were again placed back in the 37° C. incubator for another hour, so as to attain the incubator temperature as close to as possible. The eggs to be dipped were now removed from the incubator and dipped for 15 minutes in a tank containing cold (4-5° C.) gentamicin sulfate solution (powder obtained from Schering Corp., Allentown, New Jersey) at a concentration of 1,000 p.p.m. The eggs were next taken out from the antibiotic tank, air and towel dried, individually reweighed and the weight gains noted. All eggs were then placed in either a small Humidair rotary electric incubator, model 100 (New Madison Incubators, Madison, Ohio), or a Jamesway model 252B (James Manufacturing Co., Fort Atkinson, Wisconsin). Temperatures in the incubators were held at about 37° C. with a relative humidity
of approximately 55%. The eggs were incubated for 12-14 days. Bacteriological and Embryonic Development Analyses. At the end of the incubation period, the egg contents were cultured for A. hinshawii organisms to note recoveries in eggs with or without cuticle after dipping in contrast to the eggs which were not dipped in gentamicin sulfate dip solution. The cultural technique employed for isolation was as follows: swabs were taken from egg contents (the albumen and yolk were mixed together) and placed into selenite brilliant green sulfapyridine (SBGS) enrichment media and incubated at 37° C. for 24 hours; this was next transferred onto brilliant green agar plates and incubated for the same time and temperature; next, colonies appearing pinkish-white and surrounded by a red media were picked and transferred into triple sugar iron agar slants (TSI). After another 24 hours incubation, the TSI showing an acid butt with H 2 S and gas and an alkaline slant were used and the growth transferred into dulcitol, salicin and lysine iron agar. For the results to be accurate, the dulcitol and salicin should remain unfermented and lysine decarboxylase utilized with production of H 2 S . In the case of the second trial (Table 2) where salmonella recoveries were also made, the cultural technique was the same as above except that only dulcitol was fermented and lysine iron decarboxylase was again utilized with production of H 2 S . S. schwarzengrund was identified by serotyping using the SpicerEdwards method (Spicer, 1956). Previous Experiments. Earlier experiments included pressure and temperature-differential methods of egg dipping with 1,000 p.p.m. gentamicin sulfate to note the relationship between egg weight gains and gentamicin concentration of inner contents of dipped eggs with intact cuticles. Twenty eggs for each storage time was used for the vacuum
ANTIBIOTIC DIPPING OF TURKEY EGGS
method and ten eggs again for each storage time for the temperature-differential method. The values for the egg weight gains and gentamicin uptake were statistically analyzed by running correlation coefficients studies. The gentamicin assay of the egg contents was conducted by Schering Corporation, Allentown, New Jersey. RESULTS Table 1 shows the results on weight gains of eggs with and without cuticle after dipping with gentamicin sulfate at a concentration of 1,000 p.p.m. The cuticle was removed by treating eggs with 0.2 N HC1. The values for average weight gains after dipping show a much higher value in cuticle-less eggs (0.395 gram) when compared to eggs with intact cuticle (0.0864 gram). One egg in the latter group even lost weight after dipping. The differences between the largest and the smallest eggs favor the batch of eggs without any cuticle in relation to variability of intake of dip solution. Table 2 shows the effect of cuticle removal with 0.2 N HC1 on average weight gains, percent living embryos and percent Arizona and Salmonella recoveries of non-infected non-dipped, infected non-dipped, infected dipped and infected cuticle-less dipped groups of turkey hatching eggs. The average weight gains are again much superior for cuticle-less eggs in comparison to eggs with intact cuticle and the values for the range differences are also better in the former group. When compared to the non-infected non-dipped batch of eggs, the value for percent living embryos is not lowered after dipping eggs either with the intact cuticle or with the cuticle removed. The data on percent Arizona and Salmonella recoveries shows that control eggs, that is eggs which were not artificially infected or dipped, gave recoveries of Arizona hinshawii and S. schwarzengrund (22.2%). Similarly, the values of
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94.4% and 27.8% for the infected non-dipped and infected dipped group of eggs also includes S. schwarzengrund recoveries. The last group of eggs, namely 3.6% was exclusively due to S. schwarzengrund. Some of the flocks belonging to the hatchery were naturally infected with A. hinshawii and S. schwarzengrund and so the eggs laid by the hens for both the control and experimental groups could have come from such a flock. If one considers percent living embryos in relation to Arizona and/or Salmonella recoveries, the results show that only in the infected, cuticle-less dipped group the two eggs from which S. schwarzengrund were recovered had dead embryos. The effect of cuticle removal by a 10% disodium salt of EDTA in relation to embryonic development and percent A. hinshawii recoveries is shown in Table 3. In this batch of eggs, for unexplained reasons, a lower percentage of living embryos were noted in the control group with a similar pattern in the last two experimental groups, namely, infected dipped and infected, cuticle removed, dipped. No recoveries of A. hinshawii were made from control eggs, and the number of eggs showing A. hinshawii from infected non-dipped, infected dipped, and infected, cuticle-less, dipped groups respectively were 92.3%, 17.4% and 19.2%. Recoveries of Arizona from the infected dipped and infected cuticle-less dipped groups were also made from eggs that had living embryos. The respective numbers were two with living embryos and two showing early embryonic deaths from the former group and one and four respectively from the latter. Table 4 depicts the effect of a 10% disodium salt of EDTA on weight gains, percent living embryos and percent A. hinshawii recoveries. The average weight gains of eggs after cuticle removal is considerably increased—0.722 gram vs. 0.073 gram—and the differences in the range of weight gains are minimized. The data on percent living embryos shows that
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S. C. NIVAS, M. D. YORK AND B. S. POMEROY
this is not affected in each of the three groups, that is control, infected dipped and infected cuticle-less dipped eggs. No recoveries of A. hinshawii were made from control eggs as opposed to infected non-dipped eggs. Dipping infected eggs considerably reduced A. hinshawii recoveries, and the eggs from which recoveries were made had dead embryos present. Removal of the cuticle after artificial infection and subsequent dipping completely eliminated A. hinshawii from turkey hatching eggs, as shown in the last group of eggs. Previous experiments conducted to evaluate dipping of turkey hatching eggs with gentamicin sulfate at 1,000 p.p.m. by the vacuum method to note the relationship between egg weight gains and gentamicin uptake of the egg contents gave the following results: a) for eggs stored in the walk-in cooler (12° C.) for 24 hours before dipping, the correlation coefficient between egg weight gains and gentamicin uptake was r = +0.831; b) similarly, for eggs stored in the cooler for 48, 72 and 96 hours before dipping, the correlation coefficients were r = +0.902, r = +0.889 and r = +0.925 respectively. When the temperature-differential method of egg dipping for different storage times were studied, the results obtained were the following: a) for eggs stored in the cooler for 24 hours before dipping, the correlation coefficient between egg weight gains and gentamicin uptake was r = +0.990; b) similarly, the correlation coefficient values for eggs stored in the cooler for 48, 72 and 96 hours; 7, 10, and 14 days before dipping were r = +0.924, r = +0.534, r = +0.952, r = +0.809, r = +0.993 and r = +0.882 respectively.
DISCUSSION Artificial infection of turkey hatching eggs with A. hinshawii organisms using the pressure differential (vacuum) method consistently gave recoveries of better than 85% on a qualitative basis. Most of the time more than 90% of the eggs that were artificially-infected, gave recoveries from their inner contents when these were cultured. These results support the findings of Saif et al. (1971). They used the vacuum method of egg infection at 15 inches of mercury for 5 minutes, and then the eggs remained in the dipping solution for 10 minutes under atmospheric pressure. The results on egg weight gains after dipping by the temperature-differential method (Tables 1, 2 and 4) would indicate an increase in gentamicin uptake after cuticle removal especially when 10% Na 2 EDTA was used after artificial infection. The work of Alls et al. (1964), which utilized 0.2 N HC1 for cuticle removal before dipping the chicken eggs with erythromycin and showed almost a two-fold increase in the concentration of the antibiotic, would support our observations that the cuticle is one of the barriers to egg shell penetration of the antibiotic. The high correlation coefficients between egg weight gains and gentamicin uptake for each batch of eggs dipped either by the pressure differential (vacuum) or temperature-differential methods would indicate a direct relationship between these two parameters. The range differences between the smallest and the largest weight gains for eggs with and without the cuticle would indicate that removal of the cuticle reduced variability between eggs. Alls et al. (1964), also noted this reduction in variability. Larsen (1974), noted variations between eggs for antibiotic intake after dipping, and on the basis of his experimental data proposed that the primary barrier to the penetration of antibiotic dipping solutions into the turkey egg is the shell membrane and not the shell.
ANTIBIOTIC DIPPING OF TURKEY EGGS
The author did not conduct any studies to remove the cuticle before coming to this conclusion. On the basis of experiments conducted here to remove the cuticle, and no studies on the shell membranes, we propose that the cuticle is a major barrier to the penetration of antibiotic dipping solutions. This barrier can be overcome purely by chemical means without recourse to changes in genetics, diet, or the environment of the laying hen turkeys. At the same time it should be noted that though the cuticle is a barrier to egg shell penetration of antibiotic dip solutions, it is also a barrier to pathogenic microorganisms like Pseudomonas sp. (Alls et al., 1964) and S. typhimurium (Williams and Dillard, 1973). These authors removed the cuticle of chicken eggs with 0.2 N HC1, and 0.1 N HC1 and 10% Na 2 EDTA and observed a significant increase of these bacteria from the contents of such eggs in comparison to the control group of eggs with an intact cuticle. The data on percent living embryos, at 12-14 days post incubation, would indicate that dipping with or without the cuticle had no effect on this parameter when compared to the control group of undipped eggs. The exception probably were eggs dipped with or without cuticle with 10% Na 2 EDTA as shown in Table 3. This result probably could be attributed due to chance since eggs Na 2 EDTA-treated and dipped in a subsequent experiment (Table 4), maintained good embryonic development when compared to the controls. These results on percent living embryos have been supported by the work done by Saif et al. (1971). Although complete elimination of Arizona hinshawii and in one case S. schwarzengrund could not be achieved in earlier experiments (Tables 2 and 3) after chemical treatment of turkey eggs either with 0.2 N HC1 or 10% Na 2 EDTA and subsequent dipping, the last experiment (Table 4) shows such an effect, when no recoveries of Arizona hinshawii
1603
organisms could be made. This positive result may be due to a change in the procedure for cuticle removal, whereby these eggs after treatment with 10% Na 2 EDTA had their cuticle removed in a container containing circulating hot water with gentle rubbing with a soft brush. This procedure could have removed the cuticle even more thoroughly. In the previous experiments after treatment with either HC1 or Na 2 EDTA, the cuticle was removed under running tap water with gentle rubbing with a soft brush. ACKNOWLEDGMENTS This investigation was supported by grants in part from the Minnesota Turkey Growers Association; Schering Corporation, Allentown, New Jersey; and U.S.D.A., A.R.S. grant no. 12-14-100-9087(45). REFERENCES Alls, A. A., M. S. Cover, W. J. Benton and W. C. Krauss, 1964. Treatment of hatching eggs for disease prevention—factors affecting permeability and a visual detection of drug absorption. Avian Dis. 8: 245-256. Bigland, C. H., 1970. Experimental control of Mycoplasma meleagridis in turkey by the dipping of eggs in Tylosin and Spiramycin. Canad. J. Comp. Med. 34: 26-30. Board, R. G., and N. A. Halls, 1973. The cuticle: a barrier to liquid and particle penetration of the shell of the hen's egg. Brit. Poultry Sci. 14: 69-97. Board, R. G., and R. Fuller, 1974. Non-specific antimicrobial defenses of the avian egg, embryo and neonate. Biol. Rev. 49: 15-49. Brown, W. E., R. C. Baker and H. B. Naylor, 1966. Shell treatments as affecting microbial egg spoilage. Poultry Sci. 45: 276-279. Chalquest, R. R., and J. Fabricant, 1959. Survival of PPLO injected into eggs previously dipped in antibiotic solutions. Avian Dis. 3: 257-271. Ernst, R. A., J. P. Schroeder, R. E. Pfost and R. F. A. Holte, 1974. Field studies to evaluate commercial disinfectants for turkey hatching egg sanitation. Poultry Sci. 53: 149-156. Hall, C. F., A. I. Flowers and L. C. Grumbles, 1963. Dipping of hatching eggs for control of Mycoplasma gallisepticum. Avian Dis. 7: 178-183. Kumar, M. C , S. Kumar, R. E. Dierks, J. A. Newman
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S. C . N I V A S , M . D . YORK AND B . S. POMEROY
and B. S. Pomeroy, 1966. Airsacculitis in turkeys. II. Use of tylosin in the control of the egg transmission of Mycoplasma spp. other than Mycoplasma gallisepticum in turkeys. Avian Dis. 10: 194-198. Larsen, C. T., 1974. Variation in dip intake by turkey hatching eggs. Abs. 15th World Poultry Congress, New Orleans. Pp. 335-356. Levine, P. P., and J. Fabricant, 1962. Effect of dipping eggs in antibiotic solutions on PPLO transmission in chickens. Avian Dis. 6: 72-85. Lifshitz, A., R. C. Baker and H. B. Naylor, 1964. The relative importance of chicken egg exterior structures in resisting bacterial penetration. J. Food Sci. 29: 94-99. Lucas, T. E., M. C. Kumar, S. H. Kleven and B. S. Pomeroy, 1970. Antibiotic treatment of turkey hatching eggs preinfected with Salmonella. Avian Dis. 455-462. Newman, J. A., 1967. The detection and control of Mycoplasma meleagridis. Ph.D. Thesis, University of Minnesota. Olson, N. O., T. R. Hash, J. O. Heishman and A. Campbell, 1962. Dipping of hatching eggs in Erythromycin for the control of Mycoplasma. Avian Dis. 6: 191-194.
Saif, Y. M., L. C. Ferguson and K. E. Nestor, 1971. Treatment of turkey hatching eggs for control of Arizona infection. Avian Dis. 15: 448-461. Saif, Y. M., and S. M. Shelly, 1973. Effect of Gentamicin dip on Salmonella organisms in experimentally infected turkey eggs. Avian Dis. 17: 574581. Snoeyenbos, G. H., and V. L. Carlson, 1973. Gentamicin efficacy against Salmonellae and Arizonae in eggs as influenced by administration route and test organism. Avian Dis. 17: 673-682. Spicer, C. C , 1956. A quick method of identifying " H " antigens. J. Clin. Path. 9: 378-379. Stuart, E. E., and H. W. Bruins, 1963. Preincubation immersion of eggs in erythromycin to control chronic respiratory disease. Avian Dis. 7: 287-293. Stuart, E. E., and R. D. Keenum, 1970. Preincubation treatment of chicken hatching eggs infected with Salmonella pullorum. Avian Dis. 14: 87-95. Williams, J. E., and L. H. Dillard, 1973. The effect of external shell treatments on Salmonella penetration of chicken eggs. Poultry Sci. 52: 1084-1089. Yoder, H. W., Jr., and M. S. Hofstad, 1965. Evaluation of Tylosin in preventing egg transmission of M. G. in chickens. Avian Dis. 9: 291-301.
The Contribution of Intramuscular Collagen to the Tenderness of Meat from Chickens with Different Ages R. NAKAMURA, S. SEKOGUCHI AND Y . SATO
Laboratory of Food Science and Technology (Animal Products), Faculty of Agriculture, University, Nagoya, Japan
Nagoya
(Received for publication January 13, 1975)
ABSTRACT The contribution of intramuscular collagen to the tenderness of meat from chickens of different ages was investigated. Although the total collagen content of meat increased a little with advancing age, it did not correlate significantly with the tenderness of meat. The amount of the collagen rich fraction of meat decreased with advancing age, but the collagen content of this fraction increased significantly with age. The percentage of both the thermal residual collagen and the unextractable collagen increased largely with advancing age. All these results clearly show that changes in the collagen molecule affect the age-related tenderness of chicken meat greatly. POULTRY SCIENCE 54: 1604-1612, 1975
INTRODUCTION
T
HERE is a considerable accumulation of work concerning the relation between age-related changes in intramuscular collagen and the tenderness of meat (Wilson et al., 1954; Hill, 1966; Herring et al, 1967; Goll
et al., 1963, 1964a, b, c; Carmichael et al., 1967a, b; Field et al., 1969, 1970; Kruggel et al., 1970; Shimokomaki et al., 1972; Cross et al., 1973; Wangen et al, 1968). However, these works are mainly about bovine muscles and very little work has been done with poultry muscles. Although many studies have
of embryonic and adult rat, sheep and domestic fowl. Biochem. J. 104: 378-384. Weber, G., G. Banerjee, D. Bixler and J. Ashmore, 1961. Role of enzymes in metabolic homeostasis. II. Depletion and restoration of avian liver carbohy-
1597
drate-metabolizing enzymes. J. Nutr. 74: 157-160. Williamson, J. R., 1966. Response of hepatic glucokinase and glucose-6-phosphatase activities in juvenile and adult hyperthyroid mice. Endocrinology, 87: 124-128.
Antibiotic Dipping Studies in Relation to Uptake, Embryonic Development and Arizona Hinshawii Recoveries from Turkey Hatching Eggs 12 S. C . N I V A S , M . D . YORK AND B . S. POMEROY
Department of Veterinary Biology, University of Minnesota, St. Paul, Minnesota 55108 (Received for publication January 13, 1975)
ABSTRACT Antibiotic dipping with gentamicin sulfate by means of the temperature-differential method was effective in reducing but not completely eliminating Arizona hinshawii (7: 1, 7, 8) from artificially infected turkey hatching eggs. Embryonic development was well maintained. The antibiotic dip solution intake was variable from egg to egg. Removal of cuticle by means of either 0.2 N HCl or a 10% disodium salt of ethylene diamine tetraacetic acid enhanced egg weight gains and maintained good embryonic development. Complete elimination of A. hinshawii was achieved in one experiment out of four. Correlation studies between egg weight gains and gentamicin concentrations of the contents of the turkey hatching eggs indicated a statistically significant relationship between these two parameters. POULTRY SCIENCE 54: 1597-1604, 1975
al, 1962; Hall et al., 1963; Stuart and Bruins, 1963; Yoder and Hofstad, 1965; Kumar et N recent years, a number of papers on al, 1966; Newman, 1967; Bigland, 1970). antibiotic egg dipping of chicken and turkey Recent publications have used egg-dipping hatching eggs with or without certain quaterprocedures for the control of Salmonellanary ammonium compounds in the dip soluArizona organisms in chicken hatching eggs tion have been published. Earlier papers have (Stuart and Keenum, 1970; Snoeyenbos and reported on egg dipping with different antibiCarlson, 1973) and turkey hatching eggs otics in relation to Mycoplasma organisms (Lucas et al., 1970; Saif et al., 1971; Saif using the temperature-differential method. and Shelby, 1973; Ernst et al, 1974). The The results showed a definite reduction of temperature-differential method of egg-dipMycoplasma from dipped eggs but not comping was again used. Except in one report plete elimination; hatchability usually was (Saif et al., 1971) where gentamicin was the well maintained (Chalquest and Fabricant, antibiotic used for egg-dipping, complete 1959; Levine and Fabricant, 1962; Olson et elimination of Salmonella-Arizona organisms was not obtained, though a reduction in the number of recoveries were made. Fertility 1. Presented in part at the North Central Poultry and hatchability usually were again well Disease Conference, Champaign, 111. June 12-13, 1974. maintained. 2. Published as Paper No. 8959 Scientific Journal The cuticle, shell and shell membranes Series of the Minnesota Agricultural Experiment Staprobably are instrumental in bringing about tion. INTRODUCTION
I
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S. C. NIVAS, M. D. YORK AND B. S. POMEROY
variations between eggs contributing to the differences in antibiotic liquid uptake and resulting in incomplete elimination of Salmonella-Arizona organisms from the egg contents of turkey hatching eggs. When Pseudomonas fluorescens was used to experimentally infect chicken eggs, Lifshitz et al. (1964), noted that the inner shell membrane was the most important barrier against the penetration of this organism followed by the shell and then the outer shell membrane. Board and Fuller (1974), reviewed the nonspecific antimicrobial defenses of the avian egg, embryo and neonate. Removal of the cuticle with chemicals like ethylene diamine tetra-acetic acid (EDTA) and HCl increased the penetration of microorganisms like Pseudomonas aeruginosa and thus increased spoilage (Brown et al, 1966). These results were also supported by Williams and Dillard (1973) using chicken eggs when S. typhimurium was the infecting organism and 0.1 NHC1 and 10% Na 2 EDTA were the chemicals used for cuticle removal. In a recent review article, Board and Halls (1973), noted that removal of the cuticle of chicken eggs by means of EDTA, NaOH or N a 2 S - 9 H 2 0 enhanced liquid uptake when compared to eggs with the intact cuticle. Alls et al. (1964), observed that on removing the cuticle with HCl, the median Erythromycin drug level was increased from 3.81 fj-g./ml. to 6.4 ^.g./ml. The purpose of this study was to evaluate the effect of cuticle removal with either 0.2 N HCl or 10% disodium salt of EDTA and
TABLE 1.—Effect of cuticle removal with 0.2 N HCl on weight gains and gentamicin uptake of turkey hatching eggs after dipping with gentamicin sulfate at a concentration of 1,000 p.p.m. Dipped with Dipped with intact cuticle cuticle removed Av. wt. gain (gms.) Range Diff.
0.0864 (32 eggs) 0.395 (16 eggs) -0.005 to 0.270 0.270 to 0.480 0.275 0.210
subsequent temperature-differential dipping of turkey hatching eggs with gentamicin sulfate at a concentration of 1,000 p.p.m. on (a) uptake as measured by weight gains, (b) Arizona hinshawii recoveries from inner contents of artificially-infected eggs with A. hinshawii!: 1, 7, 8 and (c) embryonic development of these eggs. MATERIALS AND METHODS Eggs. Turkey hatching eggs of the Wrolstad strain were obtained from a commercial hatchery. Four trials were conducted and the eggs were all obtained from the same flock. The number of eggs used for each trial was variable and this is shown from Tables 1 through 4. Four groups per trial were usually used, as shown in Tables 2 through 4, except in the first trial (Table 1), where there were only two groups. The formaldehyde-fumigated eggs were stored in the cooler (12° C.) and experiments were conducted within 48 hours after the eggs were laid. Artificial Infection. One out of four batches of eggs served as controls, and the other three were artificially infected with A. hinshawii 7: 1, 7, 8 organisms. Initially, the eggs were brought out of the cooler and warmed at room temperature (25° C.) for about an hour. These were immersed in a pressure cooker containing normal saline plus an 18 hour veal-infusion broth culture of A. hinshawii organisms at a concentration approximately between 1.0 to 4.0 x 10 7 /ml. of the dip solution. Vacuum was then used at 17 inches of mercury for 5 minutes and the eggs next left in the pressure cooker at atmospheric pressure for 12 minutes to allow the dip solution to penetrate the cuticle and shell of the eggs for infection of the membranes and egg contents to take place. The eggs were removed, air-dried and returned to the cooler for 24-48 hours. The control group was artificially treated with normal saline using the above procedure.
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ANTIBIOTIC DIPPING OF TURKEY EGGS
TABLE 2.—Effect of cuticle removal with 0.2 N HCl on weight gains, embryonic development and Arizona hinshawii recoveries from turkey hatching eggs after artificial infection with Arizona hinshawii 7: 1, 7, 8 and dipping with 1,000 p.p.m. gentamicin sulfate
Av. wt. gain Range Diff. % living embryos at 12-14 days
Non-infected non-dipped ~* — — 83.3 (54 eggs per group) 22.22 (54 eggs per group)
Infected non-dipped — — — 59.3
Infected dipped 0.047 (20 eggs) -0.055 to 0.410 0.465 92.6
Infected, cuticle removed, dipped 0.518 (20 eggs) 0.360 to 0.620 0.260 87.3
94.42
27.82
3^6 (S. schwarzengrund)
% Arizona and Salmonella recoveries from all eggs examined 1 —Not weighed. 2 —A. hinshawii and S. schwarzengrund.
TABLE 3.—Effect of cuticle removal with 10% disodium salt of EDTA on embryonic development and Arizona hinshawii recoveries from turkey hatching eggs after artificial infection with Arizona hinshawii 7: 1, 7, 8 and dipping with 1,000 p.p.m. gentamicin sulfate
% living embryos at 12-14 days % Arizona recoveries from all eggs examined
Non-infected non-dipped 73T (26 eggs per group) 0
Infected non-dipped 19^2
Infected dipped 652
Infected, cuticle removed, dipped 57/7
92.3 (26 eggs per group)
17.4
19.2
TABLE 4.—Effect of cuticle removal with 10% disodium salt of EDTA on weight gains, embryonic development and Arizona hinshawii recoveries from turkey hatching eggs after artificial infection with Arizona hinshawii 7: 1, 7, 8 and dipping with 1,000 p.p.m. gentamicin sulfate
Av. wt. gain Range Diff. % living embryos at 12-14 days % Arizona recoveries from all eggs examined 1 —Not weighed.
Non-infected non-dipped ~' — — 95.5 (23 eggs per group) 0
Infected non-dipped — — — 81.8
Infected dipped 0.073 (23 eggs) -0.140 to 0.460 0.600 86.4
Infected, cuticle removed, dipped 0.722 (23 eggs) 0.605 to 0.810 0.205 91.3
86.4 (23 eggs per group)
8.7
0
Shell Treatments. Twenty-four hours after infecting the turkey hatching eggs, the eggs whose curticle were to be removed were taken out of the cooler and warmed at room temperature for about an hour. Two chemicals in separate experiments were used to remove
the cuticle. 0.2 N HCl was used in the first two experiments whereas a 10% disodium salt of EDTA was used in the next two experiments. For the HCl treatments, the eggs were submerged in a hot (43° C.) distilled water solution of 0.2 N HCl with constant
1600
S. C. NIVAS, M. D. YORK AND B. S. POMEROY
stirring for a period of 2 mins. The cuticle was manually removed by means of a soft brush with gentle rubbing of the surface under running tap water. For the first experiment with EDTA, the same procedure as above for HC1 was used except that the eggs were submerged in the^hot Na 2 EDTA solution for 15 mins. For the second experiment, the eggs were again submerged in a hot (43° C.) distilled water solution of Na 2 EDTA as above, the eggs were taken out and dipped next in hot (43° C.) distilled water with constant stirring for 5 minutes. The cuticle was removed by gentle rubbing with a soft brush while still submerged in the hot distilled water. All such treated eggs were air-dried at room temperature before placing them back into the cooler for 24 hours. The control and experimental (untreated) eggs were left in the cooler during this period. Egg Dipping. Two days after artificial infection with A. hinshawii organisms, all eggs (with or without cuticle) were taken out of the cooler and placed in the incubator (37° C.) for about six hours. The eggs were next removed and individually weighed. This usually took an hour. Eggs were again placed back in the 37° C. incubator for another hour, so as to attain the incubator temperature as close to as possible. The eggs to be dipped were now removed from the incubator and dipped for 15 minutes in a tank containing cold (4-5° C.) gentamicin sulfate solution (powder obtained from Schering Corp., Allentown, New Jersey) at a concentration of 1,000 p.p.m. The eggs were next taken out from the antibiotic tank, air and towel dried, individually reweighed and the weight gains noted. All eggs were then placed in either a small Humidair rotary electric incubator, model 100 (New Madison Incubators, Madison, Ohio), or a Jamesway model 252B (James Manufacturing Co., Fort Atkinson, Wisconsin). Temperatures in the incubators were held at about 37° C. with a relative humidity
of approximately 55%. The eggs were incubated for 12-14 days. Bacteriological and Embryonic Development Analyses. At the end of the incubation period, the egg contents were cultured for A. hinshawii organisms to note recoveries in eggs with or without cuticle after dipping in contrast to the eggs which were not dipped in gentamicin sulfate dip solution. The cultural technique employed for isolation was as follows: swabs were taken from egg contents (the albumen and yolk were mixed together) and placed into selenite brilliant green sulfapyridine (SBGS) enrichment media and incubated at 37° C. for 24 hours; this was next transferred onto brilliant green agar plates and incubated for the same time and temperature; next, colonies appearing pinkish-white and surrounded by a red media were picked and transferred into triple sugar iron agar slants (TSI). After another 24 hours incubation, the TSI showing an acid butt with H 2 S and gas and an alkaline slant were used and the growth transferred into dulcitol, salicin and lysine iron agar. For the results to be accurate, the dulcitol and salicin should remain unfermented and lysine decarboxylase utilized with production of H 2 S . In the case of the second trial (Table 2) where salmonella recoveries were also made, the cultural technique was the same as above except that only dulcitol was fermented and lysine iron decarboxylase was again utilized with production of H 2 S . S. schwarzengrund was identified by serotyping using the SpicerEdwards method (Spicer, 1956). Previous Experiments. Earlier experiments included pressure and temperature-differential methods of egg dipping with 1,000 p.p.m. gentamicin sulfate to note the relationship between egg weight gains and gentamicin concentration of inner contents of dipped eggs with intact cuticles. Twenty eggs for each storage time was used for the vacuum
ANTIBIOTIC DIPPING OF TURKEY EGGS
method and ten eggs again for each storage time for the temperature-differential method. The values for the egg weight gains and gentamicin uptake were statistically analyzed by running correlation coefficients studies. The gentamicin assay of the egg contents was conducted by Schering Corporation, Allentown, New Jersey. RESULTS Table 1 shows the results on weight gains of eggs with and without cuticle after dipping with gentamicin sulfate at a concentration of 1,000 p.p.m. The cuticle was removed by treating eggs with 0.2 N HC1. The values for average weight gains after dipping show a much higher value in cuticle-less eggs (0.395 gram) when compared to eggs with intact cuticle (0.0864 gram). One egg in the latter group even lost weight after dipping. The differences between the largest and the smallest eggs favor the batch of eggs without any cuticle in relation to variability of intake of dip solution. Table 2 shows the effect of cuticle removal with 0.2 N HC1 on average weight gains, percent living embryos and percent Arizona and Salmonella recoveries of non-infected non-dipped, infected non-dipped, infected dipped and infected cuticle-less dipped groups of turkey hatching eggs. The average weight gains are again much superior for cuticle-less eggs in comparison to eggs with intact cuticle and the values for the range differences are also better in the former group. When compared to the non-infected non-dipped batch of eggs, the value for percent living embryos is not lowered after dipping eggs either with the intact cuticle or with the cuticle removed. The data on percent Arizona and Salmonella recoveries shows that control eggs, that is eggs which were not artificially infected or dipped, gave recoveries of Arizona hinshawii and S. schwarzengrund (22.2%). Similarly, the values of
1601
94.4% and 27.8% for the infected non-dipped and infected dipped group of eggs also includes S. schwarzengrund recoveries. The last group of eggs, namely 3.6% was exclusively due to S. schwarzengrund. Some of the flocks belonging to the hatchery were naturally infected with A. hinshawii and S. schwarzengrund and so the eggs laid by the hens for both the control and experimental groups could have come from such a flock. If one considers percent living embryos in relation to Arizona and/or Salmonella recoveries, the results show that only in the infected, cuticle-less dipped group the two eggs from which S. schwarzengrund were recovered had dead embryos. The effect of cuticle removal by a 10% disodium salt of EDTA in relation to embryonic development and percent A. hinshawii recoveries is shown in Table 3. In this batch of eggs, for unexplained reasons, a lower percentage of living embryos were noted in the control group with a similar pattern in the last two experimental groups, namely, infected dipped and infected, cuticle removed, dipped. No recoveries of A. hinshawii were made from control eggs, and the number of eggs showing A. hinshawii from infected non-dipped, infected dipped, and infected, cuticle-less, dipped groups respectively were 92.3%, 17.4% and 19.2%. Recoveries of Arizona from the infected dipped and infected cuticle-less dipped groups were also made from eggs that had living embryos. The respective numbers were two with living embryos and two showing early embryonic deaths from the former group and one and four respectively from the latter. Table 4 depicts the effect of a 10% disodium salt of EDTA on weight gains, percent living embryos and percent A. hinshawii recoveries. The average weight gains of eggs after cuticle removal is considerably increased—0.722 gram vs. 0.073 gram—and the differences in the range of weight gains are minimized. The data on percent living embryos shows that
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S. C. NIVAS, M. D. YORK AND B. S. POMEROY
this is not affected in each of the three groups, that is control, infected dipped and infected cuticle-less dipped eggs. No recoveries of A. hinshawii were made from control eggs as opposed to infected non-dipped eggs. Dipping infected eggs considerably reduced A. hinshawii recoveries, and the eggs from which recoveries were made had dead embryos present. Removal of the cuticle after artificial infection and subsequent dipping completely eliminated A. hinshawii from turkey hatching eggs, as shown in the last group of eggs. Previous experiments conducted to evaluate dipping of turkey hatching eggs with gentamicin sulfate at 1,000 p.p.m. by the vacuum method to note the relationship between egg weight gains and gentamicin uptake of the egg contents gave the following results: a) for eggs stored in the walk-in cooler (12° C.) for 24 hours before dipping, the correlation coefficient between egg weight gains and gentamicin uptake was r = +0.831; b) similarly, for eggs stored in the cooler for 48, 72 and 96 hours before dipping, the correlation coefficients were r = +0.902, r = +0.889 and r = +0.925 respectively. When the temperature-differential method of egg dipping for different storage times were studied, the results obtained were the following: a) for eggs stored in the cooler for 24 hours before dipping, the correlation coefficient between egg weight gains and gentamicin uptake was r = +0.990; b) similarly, the correlation coefficient values for eggs stored in the cooler for 48, 72 and 96 hours; 7, 10, and 14 days before dipping were r = +0.924, r = +0.534, r = +0.952, r = +0.809, r = +0.993 and r = +0.882 respectively.
DISCUSSION Artificial infection of turkey hatching eggs with A. hinshawii organisms using the pressure differential (vacuum) method consistently gave recoveries of better than 85% on a qualitative basis. Most of the time more than 90% of the eggs that were artificially-infected, gave recoveries from their inner contents when these were cultured. These results support the findings of Saif et al. (1971). They used the vacuum method of egg infection at 15 inches of mercury for 5 minutes, and then the eggs remained in the dipping solution for 10 minutes under atmospheric pressure. The results on egg weight gains after dipping by the temperature-differential method (Tables 1, 2 and 4) would indicate an increase in gentamicin uptake after cuticle removal especially when 10% Na 2 EDTA was used after artificial infection. The work of Alls et al. (1964), which utilized 0.2 N HC1 for cuticle removal before dipping the chicken eggs with erythromycin and showed almost a two-fold increase in the concentration of the antibiotic, would support our observations that the cuticle is one of the barriers to egg shell penetration of the antibiotic. The high correlation coefficients between egg weight gains and gentamicin uptake for each batch of eggs dipped either by the pressure differential (vacuum) or temperature-differential methods would indicate a direct relationship between these two parameters. The range differences between the smallest and the largest weight gains for eggs with and without the cuticle would indicate that removal of the cuticle reduced variability between eggs. Alls et al. (1964), also noted this reduction in variability. Larsen (1974), noted variations between eggs for antibiotic intake after dipping, and on the basis of his experimental data proposed that the primary barrier to the penetration of antibiotic dipping solutions into the turkey egg is the shell membrane and not the shell.
ANTIBIOTIC DIPPING OF TURKEY EGGS
The author did not conduct any studies to remove the cuticle before coming to this conclusion. On the basis of experiments conducted here to remove the cuticle, and no studies on the shell membranes, we propose that the cuticle is a major barrier to the penetration of antibiotic dipping solutions. This barrier can be overcome purely by chemical means without recourse to changes in genetics, diet, or the environment of the laying hen turkeys. At the same time it should be noted that though the cuticle is a barrier to egg shell penetration of antibiotic dip solutions, it is also a barrier to pathogenic microorganisms like Pseudomonas sp. (Alls et al., 1964) and S. typhimurium (Williams and Dillard, 1973). These authors removed the cuticle of chicken eggs with 0.2 N HC1, and 0.1 N HC1 and 10% Na 2 EDTA and observed a significant increase of these bacteria from the contents of such eggs in comparison to the control group of eggs with an intact cuticle. The data on percent living embryos, at 12-14 days post incubation, would indicate that dipping with or without the cuticle had no effect on this parameter when compared to the control group of undipped eggs. The exception probably were eggs dipped with or without cuticle with 10% Na 2 EDTA as shown in Table 3. This result probably could be attributed due to chance since eggs Na 2 EDTA-treated and dipped in a subsequent experiment (Table 4), maintained good embryonic development when compared to the controls. These results on percent living embryos have been supported by the work done by Saif et al. (1971). Although complete elimination of Arizona hinshawii and in one case S. schwarzengrund could not be achieved in earlier experiments (Tables 2 and 3) after chemical treatment of turkey eggs either with 0.2 N HC1 or 10% Na 2 EDTA and subsequent dipping, the last experiment (Table 4) shows such an effect, when no recoveries of Arizona hinshawii
1603
organisms could be made. This positive result may be due to a change in the procedure for cuticle removal, whereby these eggs after treatment with 10% Na 2 EDTA had their cuticle removed in a container containing circulating hot water with gentle rubbing with a soft brush. This procedure could have removed the cuticle even more thoroughly. In the previous experiments after treatment with either HC1 or Na 2 EDTA, the cuticle was removed under running tap water with gentle rubbing with a soft brush. ACKNOWLEDGMENTS This investigation was supported by grants in part from the Minnesota Turkey Growers Association; Schering Corporation, Allentown, New Jersey; and U.S.D.A., A.R.S. grant no. 12-14-100-9087(45). REFERENCES Alls, A. A., M. S. Cover, W. J. Benton and W. C. Krauss, 1964. Treatment of hatching eggs for disease prevention—factors affecting permeability and a visual detection of drug absorption. Avian Dis. 8: 245-256. Bigland, C. H., 1970. Experimental control of Mycoplasma meleagridis in turkey by the dipping of eggs in Tylosin and Spiramycin. Canad. J. Comp. Med. 34: 26-30. Board, R. G., and N. A. Halls, 1973. The cuticle: a barrier to liquid and particle penetration of the shell of the hen's egg. Brit. Poultry Sci. 14: 69-97. Board, R. G., and R. Fuller, 1974. Non-specific antimicrobial defenses of the avian egg, embryo and neonate. Biol. Rev. 49: 15-49. Brown, W. E., R. C. Baker and H. B. Naylor, 1966. Shell treatments as affecting microbial egg spoilage. Poultry Sci. 45: 276-279. Chalquest, R. R., and J. Fabricant, 1959. Survival of PPLO injected into eggs previously dipped in antibiotic solutions. Avian Dis. 3: 257-271. Ernst, R. A., J. P. Schroeder, R. E. Pfost and R. F. A. Holte, 1974. Field studies to evaluate commercial disinfectants for turkey hatching egg sanitation. Poultry Sci. 53: 149-156. Hall, C. F., A. I. Flowers and L. C. Grumbles, 1963. Dipping of hatching eggs for control of Mycoplasma gallisepticum. Avian Dis. 7: 178-183. Kumar, M. C , S. Kumar, R. E. Dierks, J. A. Newman
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S. C . N I V A S , M . D . YORK AND B . S. POMEROY
and B. S. Pomeroy, 1966. Airsacculitis in turkeys. II. Use of tylosin in the control of the egg transmission of Mycoplasma spp. other than Mycoplasma gallisepticum in turkeys. Avian Dis. 10: 194-198. Larsen, C. T., 1974. Variation in dip intake by turkey hatching eggs. Abs. 15th World Poultry Congress, New Orleans. Pp. 335-356. Levine, P. P., and J. Fabricant, 1962. Effect of dipping eggs in antibiotic solutions on PPLO transmission in chickens. Avian Dis. 6: 72-85. Lifshitz, A., R. C. Baker and H. B. Naylor, 1964. The relative importance of chicken egg exterior structures in resisting bacterial penetration. J. Food Sci. 29: 94-99. Lucas, T. E., M. C. Kumar, S. H. Kleven and B. S. Pomeroy, 1970. Antibiotic treatment of turkey hatching eggs preinfected with Salmonella. Avian Dis. 455-462. Newman, J. A., 1967. The detection and control of Mycoplasma meleagridis. Ph.D. Thesis, University of Minnesota. Olson, N. O., T. R. Hash, J. O. Heishman and A. Campbell, 1962. Dipping of hatching eggs in Erythromycin for the control of Mycoplasma. Avian Dis. 6: 191-194.
Saif, Y. M., L. C. Ferguson and K. E. Nestor, 1971. Treatment of turkey hatching eggs for control of Arizona infection. Avian Dis. 15: 448-461. Saif, Y. M., and S. M. Shelly, 1973. Effect of Gentamicin dip on Salmonella organisms in experimentally infected turkey eggs. Avian Dis. 17: 574581. Snoeyenbos, G. H., and V. L. Carlson, 1973. Gentamicin efficacy against Salmonellae and Arizonae in eggs as influenced by administration route and test organism. Avian Dis. 17: 673-682. Spicer, C. C , 1956. A quick method of identifying " H " antigens. J. Clin. Path. 9: 378-379. Stuart, E. E., and H. W. Bruins, 1963. Preincubation immersion of eggs in erythromycin to control chronic respiratory disease. Avian Dis. 7: 287-293. Stuart, E. E., and R. D. Keenum, 1970. Preincubation treatment of chicken hatching eggs infected with Salmonella pullorum. Avian Dis. 14: 87-95. Williams, J. E., and L. H. Dillard, 1973. The effect of external shell treatments on Salmonella penetration of chicken eggs. Poultry Sci. 52: 1084-1089. Yoder, H. W., Jr., and M. S. Hofstad, 1965. Evaluation of Tylosin in preventing egg transmission of M. G. in chickens. Avian Dis. 9: 291-301.
The Contribution of Intramuscular Collagen to the Tenderness of Meat from Chickens with Different Ages R. NAKAMURA, S. SEKOGUCHI AND Y . SATO
Laboratory of Food Science and Technology (Animal Products), Faculty of Agriculture, University, Nagoya, Japan
Nagoya
(Received for publication January 13, 1975)
ABSTRACT The contribution of intramuscular collagen to the tenderness of meat from chickens of different ages was investigated. Although the total collagen content of meat increased a little with advancing age, it did not correlate significantly with the tenderness of meat. The amount of the collagen rich fraction of meat decreased with advancing age, but the collagen content of this fraction increased significantly with age. The percentage of both the thermal residual collagen and the unextractable collagen increased largely with advancing age. All these results clearly show that changes in the collagen molecule affect the age-related tenderness of chicken meat greatly. POULTRY SCIENCE 54: 1604-1612, 1975
INTRODUCTION
T
HERE is a considerable accumulation of work concerning the relation between age-related changes in intramuscular collagen and the tenderness of meat (Wilson et al., 1954; Hill, 1966; Herring et al, 1967; Goll
et al., 1963, 1964a, b, c; Carmichael et al., 1967a, b; Field et al., 1969, 1970; Kruggel et al., 1970; Shimokomaki et al., 1972; Cross et al., 1973; Wangen et al, 1968). However, these works are mainly about bovine muscles and very little work has been done with poultry muscles. Although many studies have
