- Email: [email protected]
Transmission of Aspergillosis During Incubation
USE OF RECLAIMED EGG CASES
studies are indicated, to evaluate hidden costs in extra labor required in processing plants because of inferior materials used, the cost of empty positions on automatic
727
conveyors in both table egg and egg products operations, and in the extra handling required where the plant is highly automated.
Transmission of Aspergillosis During Incubation M. L. WRIGHT, G. W. ANDERSON AND J. D.
MCCONACHIE
Departments of Microbiology and Poultry Science, Ontario Agricultural College, Guelph, Canada (Received for publication July 29, 1960) .
INTRODUCTION
Aspergillus jumigatus on the 10th day of incubation. EXPERIMENTAL
Two experiments were conducted. In the first experiment, embryonating eggs were placed alternately adjacent to cracked infertile eggs. The eggs were cracked to enhance mold penetration and infertile eggs were used to preclude embryo development in any of the eggs in the cracked group to ensure uniform treatment of embryonating eggs. The second experiment comprised only sound fertile eggs. In both experiments, spores of Aspergillus jumigatus were dusted over all of the eggs using a cotton ball without removing them from the setter trays. This was accomplished on the 10th day of incubation. Normal incubation was continued and on the 18th day in each case only the sound fertile eggs were transferred to hatching trays. This was determined by candling each egg separately. All eggs that appeared moldy at this time were indexed and included in the hatching stage. After the fertile eggs were placed in the hatching trays, they were placed in the hatching compartment and cotton filters were placed in the ventilation holes of the incubator. In each experiment, the hatching compartment containing the embryonating eggs was fumigated in a manner to destroy all the superficial mold elements, according
Downloaded from http://ps.oxfordjournals.org/ by guest on April 7, 2015
T
HE susceptibility of young chicks and poults to respiratory infections by inhaled microorganisms is well established. Clemmer et al. (1960) reported that hematogenous infections may occur following inhalation of salmonella bacteria. Aspergillosis has been experimentally produced in hatching chicks (O'meara et al., 1959). This coincided with a report (Wright et al., 1959) that aspergillosis was being transmitted by some commercial hatcheries. The implication of hatcheries in the transmission of aspergillosis was based on the recovery of Aspergillus spp. from cracked eggs, living embryos, fluff from hatching compartments and unthrifty chicks and polts under ten days of age, all from the same hatchery. The isolation of Aspergillus spp. was surprisingly frequent and was not limited to fluff samples from Canadian hatcheries. Recently, Wright et al. (1960) successfully transmitted aspergillosis to incubating embryos which resulted in infected chicks. Mold penetration of embryonating eggs under ordinary incubation occurred within six days of contamination of an adjacent cracked egg with a spore-albumen mixture. This report concerns the transmission of aspergillosis during incubation when embryonating eggs were dusted with spores of
728
M. L. WRIGHT, G. W. ANDERSON AND J. D.
MCCONACHIE
cised together with the adjacent dorsal section of the rib cage. The excised material was planted onto acidified potato dextrose agar (Difco) and incubated at 40°C. for 18 hours before macroscopic examination for the mold. After 10 days in isolated confinement, all the survivors were killed and examined as outlined above. During the confinement, tap water and feed free of Aspergillus spp., based on cultural examination of a tengram sample, were given ad libitum.
to methods previously published by Wright et al. (1958), except that 2 ml. of formalin were used instead of 1.5 ml. per cubic foot. This was performed to limit the mold stress on the chicks to embryo infection and to inspiration of mold elements released by infected embryos through the process of hatching. On the 21st day of incubation, the chicks were removed from the hatching compartment and placed into sections of a battery brooder and enclosed in an isolation unit (Figure 1). They were observed daily for clinical symptoms of respiratory ailments and for mortality. All unhatched eggs were candled to detect moldiness or were broken out individually and aseptically into sterile jars. These were examined for mold growth after 3 days at 40°C. At two days of age, 6 chicks were selected at random from experiment one and subjected to post mortem and cultural examination for the presence of Aspergillus jumigatus. This was accomplished by killing the chicks with chloroform followed by immersion for three minutes in a 1:40 solution of Chloro-o-fen.* They were removed and drained; the skin was stripped from the dorsal region and the lungs ex* Trade name of disinfectant supplied by the Canadian Germicide Co. Toronto, Canada.
RESULTS AND DISCUSSION
In the first experiment, 17 chicks were placed in confinement and brooded at 34°C. Gasping, flicking of head and restlessness increased to the 2nd day. At this time, 10 chicks exhibited respiratory distress. By the 3rd day gasping was less severe and by the 4th day no respiratory distress was evident. Post mortem cultures of the lungs and rib cage of the 6 two-day-old chicks selected at random revealed Aspergillus jumigatus in 5 of the 6. After 10 days' brooding, the remaining 11 chicks were similarly examined. No evidence of mold infection was found in any of them. This substantiated the clinical evidence that spontaneous remission apparently occurred. This also apparently occurs under commercial conditions as evidenced by the relatively small number of chronic cases of aspergillosis as compared to the large number of chicks hatched in mold infested hatcheries. This suggests that other environmental factors may play an important role in the chronicity of aspergillosis. Aspergillus jumigatus was recovered from 22 and 10 percent respectively of cracked and unhatched fertile eggs remaining after the chicks were removed. The mold was recovered from 2 embryos which were alive when broken out as well as from 5 dead, 15- to 18-day-old embryos. The dead-inshell embryos were comprised of some dead
Downloaded from http://ps.oxfordjournals.org/ by guest on April 7, 2015
FIG. 1. Isolation units of wooden and pliofilm construction with cotton niters.
TRANSMISSION OF ASPERGILLOSIS
FIG. 2. Embryos infected with Aspergillus jumigatus died during hatching. Note: The beaks have penetrated the inner membranes of 2 and the other 2 have pipped completely. The air cells of all eggs have been manually opened to show the presence of Aspergillus jumigatus.
hatched from a total of 145 fertile sound eggs which had been dusted with spores of Aspergillus jumigatus on the 10th day of incubation. The chicks were divided into 3 groups
FIG. 3. Mold colonies resulting from dorsal contact of 2 chicks picked at random.
Downloaded from http://ps.oxfordjournals.org/ by guest on April 7, 2015
before the 19th day, some which had pierced the inner membrane and some which had completely pipped before death (Figure 2). On the other hand, some chicks emerged from eggs which had been indexed as containing mold and a living embryo in the same egg when they were candled on the 18th day of incubation. Since the free mold elements in the hatching compartment had been killed by fumigation before hatching commenced, the infected embryos which hatched and the moldy, dead-in-shell embryos that pipped must have been the source of contamination during the hatching process. The presence of Aspergillus jumigatus in the hatching compartment after the hatch was demonstrated by contacting the chicks superficially with acidified potato dextrose agar medium in petri plates. After 24 hours' incubation at 40° C. the mold colonies were visible on the medium (Figure 3). The presence of molds on the down of chicks hatched in these conditions may implicate the hatchery in the transmission of this disease. In the 2nd experiment, 116 chicks were
729
730
M. L. WRIGHT, G. W. ANDERSON AND J. D.
MCCONACHIE
Downloaded from http://ps.oxfordjournals.org/ by guest on April 7, 2015
incubation. Only one egg of the grossly moldy group was infertile. The 14 unhatched eggs, apparently not moldy at candling time, were also cultured. Aspergillus jumigatus was recovered from 3 of 9 infertile eggs, 3 dead-in-shell embryos and 2 late-hatched chicks comprising this group. These results further substantiate those of experiment 1 and the previous findings of Wright et al. (1959, 1960) which indicate that hatcheries can be implicated in the spread of aspergillosis. Furthermore, the embryo mortality caused by the obscure presence of this mold in hatchFIG. 4. Aspergillus jumigatus growing on lungs eries indicates that it can become an economic factor by decreasing the hatchability after 18 hrs. at 40°C. as well as causing deaths and unthriftiness and placed in confinement similar to experi- in chicks. Therefore, more emphasis should ment 1. Respiratory stress was applied to be placed on the microbiological aspects of 2 of the groups by exposure to a formalde- hatchery sanitation. hyde fumigation of 0.2S ml. per cubic foot. SUMMARY Typical clinical symptoms of respiratory distress were observed in all groups but with Two experiments conducted to demongreater severity in the stressed groups. There strate infection and transmission of Asperappeared to be no difference between the gillus jumigatus during incubation indiformaldehyde stressed groups, therefore cated the following:— they have been considered as a single 1. Mold penetration of embryonating group. At 10 clays of age, the mortality was eggs within 8 days after dusting them with 30 and 74 percent in the non-stressed and spores of Aspergillus jumigatus was evistressed groups respectively. dent by ordinary candling methods. At 10 days of age, all the survivors were 2. Chicks hatched from eggs indexed as killed and the lungs excised, as before, for internally moldy on the 18th day of incubacultural study. Aspergillus jumigatus was;, tion. • recovered from 29 and 54 percent of the 3. Chicks selected at random from the individuals in the non-stressed and stressed hatching compartment exhibited on their groups respectively. The mold was macro- down contamination with the infective scopically evident on many lung samples agent. within 18 hours at 40°C. (Figure 4). 4. Lov level formaldehyde stress on The 29 unhatched eggs remaining after chicks exposed to A. jumigatus increased the hatch was taken off were examined for respiratory symptoms as well as the prevathe presence of Aspergillus jumigatus. Fif- lence of the mold in the lungs at 10 days of teen of these eggs had been marked as age. moldy when candled on the 18th day of REFERENCES incubation and three of them had pipped Clemmer, D. I., J. Hickey, J. F. Bridges, D. J. before the embryos died. Eleven others conSchliessmann and M. F. Shaffer, 1960. Bacteriotained embryos which died at 15—18 days' logic studies of experimental air-borne sal-
TRANSMISSION OF ASPERGILLOSIS monellosis in chicks. J. Infect. Dis. 106: 197210. O'meara, D. C , and H. L. Chute, 1960. Aspergillosis experimentally produced in hatching chicks. Avian Dis. 3 : 404-406. Wright, M. L., G. W. Anderson and N. A. Epps, 1959. Hatchery sanitation. Can. J. Comp. Med. 33: 288-290.
731
Wright, M. L., G. W. Anderson and N. A. Epps, 1960. Hatchery sanitation as a control measure for aspergillosis in fowl. 32nd Northeastern Conference on Avian Diseases, U. of Maine, Orono. Wright, M. L., and N. A. Epps, 1958. Hatchery sanitation. Can. J. Comp. Med. 22: 396-399.
Nutritional Investigations with Turkey Hens 1 2. REQUIREMENT FOR UNIDENTIFIED FACTORS
(Received for publication, August 2, 1960)
D
URING the past decade, workers at the Texas Agricultural Experiment Station have published reports showing that turkey breeder hens require certain dietary unidentified factor supplements for optimum reproductive performance (Atkinson et al., 1951, 1953, 1955; Ferguson et al, 1956; Feldman et al., 1957; and Whiteside et al., 1960). Addition of such supplements as dried brewer's yeast, condensed fish solubles, fermentation residue, and dehydrated alfalfa meal to a milo-corn-soybean oil meal basal diet, markedly improved hatchability of fertile eggs, (Whiteside et al., 1960). An experiment to study the effect of adding several unidentified factor supplements to a corn-soybean oil meal diet on the performance of Broad Breasted Bronze hens is reported in this paper. EXPERIMENTAL
Twelve pens of 10 Broad Breasted Bronze turkey hens in an enclosed house with mechanical ventilation were used. The birds had been on a protein requirements study (Jensen and McGinnis, 1961). For eleven weeks, they were fed a diet similar 1
Scientific Paper No. 1982 Washington Agricultural Experiment Stations, Pullman. Project No. 71S.
to the basal diet used in the present experiment, except that the corn and soybean oil meal levels were varied to obtain different levels of protein. No sources of unidentified factors used in the present experiment had been included in the diet. The percentage composition of the diet was: yellow corn, 69.8; soybean oil meal (44% protein), 22.0; steamed bone meal, 5.0; limestone, 2.0; iodized salt, 0.5. The following were added per kilogram of diet: vitamin A, 6600 I.U.; vitamin D 3 , 1100 I.C.U.; vitamin E, 22 I.U.; menadione sodium bisulfate, 2.2 mg.; riboflavin, 3.3 mg.; vitamin B12, 6.6 meg.; Ca pantothenate, 19.6 mg.; folic acid, 6.6 meg.; niacin, 33 mg.; choline chloride, 660 mg.; BHT, 125 mg.; MnS0 4 (70%), 125 mg. Each of the experimental diets was fed to two groups as shown in Table 1. With addition of unidentified factor supplements, adjustments were made in the levels of corn, soybean oil meal and bone meal so that similar levels of crude protein, calcium and phosphorus were present in all experimental diets. The following supplements were added individually at the 5% level: dehydrated alfalfa meal (17% protein), dried whey product, delactosed (18% protein), herring fish meal (73% protein) and dried
Downloaded from http://ps.oxfordjournals.org/ by guest on April 7, 2015
LEO S. JENSEN AND JAMES MCGINNIS Department of Poultry Science, Washington State University, Pullman
studies are indicated, to evaluate hidden costs in extra labor required in processing plants because of inferior materials used, the cost of empty positions on automatic
727
conveyors in both table egg and egg products operations, and in the extra handling required where the plant is highly automated.
Transmission of Aspergillosis During Incubation M. L. WRIGHT, G. W. ANDERSON AND J. D.
MCCONACHIE
Departments of Microbiology and Poultry Science, Ontario Agricultural College, Guelph, Canada (Received for publication July 29, 1960) .
INTRODUCTION
Aspergillus jumigatus on the 10th day of incubation. EXPERIMENTAL
Two experiments were conducted. In the first experiment, embryonating eggs were placed alternately adjacent to cracked infertile eggs. The eggs were cracked to enhance mold penetration and infertile eggs were used to preclude embryo development in any of the eggs in the cracked group to ensure uniform treatment of embryonating eggs. The second experiment comprised only sound fertile eggs. In both experiments, spores of Aspergillus jumigatus were dusted over all of the eggs using a cotton ball without removing them from the setter trays. This was accomplished on the 10th day of incubation. Normal incubation was continued and on the 18th day in each case only the sound fertile eggs were transferred to hatching trays. This was determined by candling each egg separately. All eggs that appeared moldy at this time were indexed and included in the hatching stage. After the fertile eggs were placed in the hatching trays, they were placed in the hatching compartment and cotton filters were placed in the ventilation holes of the incubator. In each experiment, the hatching compartment containing the embryonating eggs was fumigated in a manner to destroy all the superficial mold elements, according
Downloaded from http://ps.oxfordjournals.org/ by guest on April 7, 2015
T
HE susceptibility of young chicks and poults to respiratory infections by inhaled microorganisms is well established. Clemmer et al. (1960) reported that hematogenous infections may occur following inhalation of salmonella bacteria. Aspergillosis has been experimentally produced in hatching chicks (O'meara et al., 1959). This coincided with a report (Wright et al., 1959) that aspergillosis was being transmitted by some commercial hatcheries. The implication of hatcheries in the transmission of aspergillosis was based on the recovery of Aspergillus spp. from cracked eggs, living embryos, fluff from hatching compartments and unthrifty chicks and polts under ten days of age, all from the same hatchery. The isolation of Aspergillus spp. was surprisingly frequent and was not limited to fluff samples from Canadian hatcheries. Recently, Wright et al. (1960) successfully transmitted aspergillosis to incubating embryos which resulted in infected chicks. Mold penetration of embryonating eggs under ordinary incubation occurred within six days of contamination of an adjacent cracked egg with a spore-albumen mixture. This report concerns the transmission of aspergillosis during incubation when embryonating eggs were dusted with spores of
728
M. L. WRIGHT, G. W. ANDERSON AND J. D.
MCCONACHIE
cised together with the adjacent dorsal section of the rib cage. The excised material was planted onto acidified potato dextrose agar (Difco) and incubated at 40°C. for 18 hours before macroscopic examination for the mold. After 10 days in isolated confinement, all the survivors were killed and examined as outlined above. During the confinement, tap water and feed free of Aspergillus spp., based on cultural examination of a tengram sample, were given ad libitum.
to methods previously published by Wright et al. (1958), except that 2 ml. of formalin were used instead of 1.5 ml. per cubic foot. This was performed to limit the mold stress on the chicks to embryo infection and to inspiration of mold elements released by infected embryos through the process of hatching. On the 21st day of incubation, the chicks were removed from the hatching compartment and placed into sections of a battery brooder and enclosed in an isolation unit (Figure 1). They were observed daily for clinical symptoms of respiratory ailments and for mortality. All unhatched eggs were candled to detect moldiness or were broken out individually and aseptically into sterile jars. These were examined for mold growth after 3 days at 40°C. At two days of age, 6 chicks were selected at random from experiment one and subjected to post mortem and cultural examination for the presence of Aspergillus jumigatus. This was accomplished by killing the chicks with chloroform followed by immersion for three minutes in a 1:40 solution of Chloro-o-fen.* They were removed and drained; the skin was stripped from the dorsal region and the lungs ex* Trade name of disinfectant supplied by the Canadian Germicide Co. Toronto, Canada.
RESULTS AND DISCUSSION
In the first experiment, 17 chicks were placed in confinement and brooded at 34°C. Gasping, flicking of head and restlessness increased to the 2nd day. At this time, 10 chicks exhibited respiratory distress. By the 3rd day gasping was less severe and by the 4th day no respiratory distress was evident. Post mortem cultures of the lungs and rib cage of the 6 two-day-old chicks selected at random revealed Aspergillus jumigatus in 5 of the 6. After 10 days' brooding, the remaining 11 chicks were similarly examined. No evidence of mold infection was found in any of them. This substantiated the clinical evidence that spontaneous remission apparently occurred. This also apparently occurs under commercial conditions as evidenced by the relatively small number of chronic cases of aspergillosis as compared to the large number of chicks hatched in mold infested hatcheries. This suggests that other environmental factors may play an important role in the chronicity of aspergillosis. Aspergillus jumigatus was recovered from 22 and 10 percent respectively of cracked and unhatched fertile eggs remaining after the chicks were removed. The mold was recovered from 2 embryos which were alive when broken out as well as from 5 dead, 15- to 18-day-old embryos. The dead-inshell embryos were comprised of some dead
Downloaded from http://ps.oxfordjournals.org/ by guest on April 7, 2015
FIG. 1. Isolation units of wooden and pliofilm construction with cotton niters.
TRANSMISSION OF ASPERGILLOSIS
FIG. 2. Embryos infected with Aspergillus jumigatus died during hatching. Note: The beaks have penetrated the inner membranes of 2 and the other 2 have pipped completely. The air cells of all eggs have been manually opened to show the presence of Aspergillus jumigatus.
hatched from a total of 145 fertile sound eggs which had been dusted with spores of Aspergillus jumigatus on the 10th day of incubation. The chicks were divided into 3 groups
FIG. 3. Mold colonies resulting from dorsal contact of 2 chicks picked at random.
Downloaded from http://ps.oxfordjournals.org/ by guest on April 7, 2015
before the 19th day, some which had pierced the inner membrane and some which had completely pipped before death (Figure 2). On the other hand, some chicks emerged from eggs which had been indexed as containing mold and a living embryo in the same egg when they were candled on the 18th day of incubation. Since the free mold elements in the hatching compartment had been killed by fumigation before hatching commenced, the infected embryos which hatched and the moldy, dead-in-shell embryos that pipped must have been the source of contamination during the hatching process. The presence of Aspergillus jumigatus in the hatching compartment after the hatch was demonstrated by contacting the chicks superficially with acidified potato dextrose agar medium in petri plates. After 24 hours' incubation at 40° C. the mold colonies were visible on the medium (Figure 3). The presence of molds on the down of chicks hatched in these conditions may implicate the hatchery in the transmission of this disease. In the 2nd experiment, 116 chicks were
729
730
M. L. WRIGHT, G. W. ANDERSON AND J. D.
MCCONACHIE
Downloaded from http://ps.oxfordjournals.org/ by guest on April 7, 2015
incubation. Only one egg of the grossly moldy group was infertile. The 14 unhatched eggs, apparently not moldy at candling time, were also cultured. Aspergillus jumigatus was recovered from 3 of 9 infertile eggs, 3 dead-in-shell embryos and 2 late-hatched chicks comprising this group. These results further substantiate those of experiment 1 and the previous findings of Wright et al. (1959, 1960) which indicate that hatcheries can be implicated in the spread of aspergillosis. Furthermore, the embryo mortality caused by the obscure presence of this mold in hatchFIG. 4. Aspergillus jumigatus growing on lungs eries indicates that it can become an economic factor by decreasing the hatchability after 18 hrs. at 40°C. as well as causing deaths and unthriftiness and placed in confinement similar to experi- in chicks. Therefore, more emphasis should ment 1. Respiratory stress was applied to be placed on the microbiological aspects of 2 of the groups by exposure to a formalde- hatchery sanitation. hyde fumigation of 0.2S ml. per cubic foot. SUMMARY Typical clinical symptoms of respiratory distress were observed in all groups but with Two experiments conducted to demongreater severity in the stressed groups. There strate infection and transmission of Asperappeared to be no difference between the gillus jumigatus during incubation indiformaldehyde stressed groups, therefore cated the following:— they have been considered as a single 1. Mold penetration of embryonating group. At 10 clays of age, the mortality was eggs within 8 days after dusting them with 30 and 74 percent in the non-stressed and spores of Aspergillus jumigatus was evistressed groups respectively. dent by ordinary candling methods. At 10 days of age, all the survivors were 2. Chicks hatched from eggs indexed as killed and the lungs excised, as before, for internally moldy on the 18th day of incubacultural study. Aspergillus jumigatus was;, tion. • recovered from 29 and 54 percent of the 3. Chicks selected at random from the individuals in the non-stressed and stressed hatching compartment exhibited on their groups respectively. The mold was macro- down contamination with the infective scopically evident on many lung samples agent. within 18 hours at 40°C. (Figure 4). 4. Lov level formaldehyde stress on The 29 unhatched eggs remaining after chicks exposed to A. jumigatus increased the hatch was taken off were examined for respiratory symptoms as well as the prevathe presence of Aspergillus jumigatus. Fif- lence of the mold in the lungs at 10 days of teen of these eggs had been marked as age. moldy when candled on the 18th day of REFERENCES incubation and three of them had pipped Clemmer, D. I., J. Hickey, J. F. Bridges, D. J. before the embryos died. Eleven others conSchliessmann and M. F. Shaffer, 1960. Bacteriotained embryos which died at 15—18 days' logic studies of experimental air-borne sal-
TRANSMISSION OF ASPERGILLOSIS monellosis in chicks. J. Infect. Dis. 106: 197210. O'meara, D. C , and H. L. Chute, 1960. Aspergillosis experimentally produced in hatching chicks. Avian Dis. 3 : 404-406. Wright, M. L., G. W. Anderson and N. A. Epps, 1959. Hatchery sanitation. Can. J. Comp. Med. 33: 288-290.
731
Wright, M. L., G. W. Anderson and N. A. Epps, 1960. Hatchery sanitation as a control measure for aspergillosis in fowl. 32nd Northeastern Conference on Avian Diseases, U. of Maine, Orono. Wright, M. L., and N. A. Epps, 1958. Hatchery sanitation. Can. J. Comp. Med. 22: 396-399.
Nutritional Investigations with Turkey Hens 1 2. REQUIREMENT FOR UNIDENTIFIED FACTORS
(Received for publication, August 2, 1960)
D
URING the past decade, workers at the Texas Agricultural Experiment Station have published reports showing that turkey breeder hens require certain dietary unidentified factor supplements for optimum reproductive performance (Atkinson et al., 1951, 1953, 1955; Ferguson et al, 1956; Feldman et al., 1957; and Whiteside et al., 1960). Addition of such supplements as dried brewer's yeast, condensed fish solubles, fermentation residue, and dehydrated alfalfa meal to a milo-corn-soybean oil meal basal diet, markedly improved hatchability of fertile eggs, (Whiteside et al., 1960). An experiment to study the effect of adding several unidentified factor supplements to a corn-soybean oil meal diet on the performance of Broad Breasted Bronze hens is reported in this paper. EXPERIMENTAL
Twelve pens of 10 Broad Breasted Bronze turkey hens in an enclosed house with mechanical ventilation were used. The birds had been on a protein requirements study (Jensen and McGinnis, 1961). For eleven weeks, they were fed a diet similar 1
Scientific Paper No. 1982 Washington Agricultural Experiment Stations, Pullman. Project No. 71S.
to the basal diet used in the present experiment, except that the corn and soybean oil meal levels were varied to obtain different levels of protein. No sources of unidentified factors used in the present experiment had been included in the diet. The percentage composition of the diet was: yellow corn, 69.8; soybean oil meal (44% protein), 22.0; steamed bone meal, 5.0; limestone, 2.0; iodized salt, 0.5. The following were added per kilogram of diet: vitamin A, 6600 I.U.; vitamin D 3 , 1100 I.C.U.; vitamin E, 22 I.U.; menadione sodium bisulfate, 2.2 mg.; riboflavin, 3.3 mg.; vitamin B12, 6.6 meg.; Ca pantothenate, 19.6 mg.; folic acid, 6.6 meg.; niacin, 33 mg.; choline chloride, 660 mg.; BHT, 125 mg.; MnS0 4 (70%), 125 mg. Each of the experimental diets was fed to two groups as shown in Table 1. With addition of unidentified factor supplements, adjustments were made in the levels of corn, soybean oil meal and bone meal so that similar levels of crude protein, calcium and phosphorus were present in all experimental diets. The following supplements were added individually at the 5% level: dehydrated alfalfa meal (17% protein), dried whey product, delactosed (18% protein), herring fish meal (73% protein) and dried
Downloaded from http://ps.oxfordjournals.org/ by guest on April 7, 2015
LEO S. JENSEN AND JAMES MCGINNIS Department of Poultry Science, Washington State University, Pullman