THE EFFECT OF RAW SOYBEAN DIETS AND CERTAIN SANITANY MEASURES UPON DISEASE INCIDENCE AND NUTRITIONAL PERFGRMANCE 0F CHICKENS Thesis for Ike Degtea of F21. D. MICHIGAN STATE UNIVERSITY Harbhaian Singh 1964 THESIS LIBRAR y Michigan State nivcrsity This is to certifg that the thesis entitled THE EFFECT OF RAW SOYBEAN DIETS AND CERTAIN SANITARY MEASURES UPON DISEASE INCIDENCE AND NUTRITIONAL PERFORMANCE OF CHICKENS presented by HARBHAJ AN S INGH has been accepted towards fulfillment of the requirements for Ph. D. degree in Poultry Science professor 0-169 R n.” um no; 9.; E 0:421 ABSTRACT THE EFFECT OF RAW SOYBEAN DIETS AND CERTAIN SANITARY MEASURES UPON DISEASE INCIDENCE AND NUTRITIONAL PERFORMANCE OF CHICKENS by Harbhajan Singh Eight preliminary experiments were conducted using chickens of different breeds and varieties to determine the effects of chlorination of water on its consumption, weight of birds and egg production. Experiments were than undertaken with commercially-hatched White Leghorn-type hens to study the effects of certain sanitary management practices as well as dietary treatments on egg production and rate of mortality. The sanitary measures applied were: chlorinated drinking water and/or frequent litter change. Disinfectant pads were placed at the entrance to each experimental pen. Dietary treatments consisted of the use of soybean meal, whole raw soybeans, whole cooked soybeans, or the combination of raw and cooked soybeans in isocaloric and isonitrogenous rations. The management measures were followed in an attempt to see if experimental birds kept in a cleaner environment would be healthier and produce better. Birds dying during the experiment were taken promptly to the laboratory for necropsy in order to investigate the cause of death. At the end of the experiment, blood was collected for the following laboratory tests: differential leukocytic counts, coagulation time, urea nitrogen and sugar. The criteria used for evaluation were rate of mortality, relative infectiveness of the litter to baby chicks, rate of egg production, gain or loss in body weights of birds, egg size, fertility and hatchability of eggs from the hens receiving various dietary and management treatments. In the preliminary experiments, the following levels of chlorine in water did not affect the feed or water consumption. 50 ppm for 3-week-old chicks 75 ppm for 10-week-old chicks 25 ppm for laying hens Chlorination of the water at 100 to 1,000 ppm for 5-week~old chickens or 50 to 500 ppm for old hens lowered water intake but did not affect feed consumption or body weights. Water and feed consumption, as well as gain in body weight were depressed by chlorination of water at levels above: 100 ppm for baby chicks 1,000 ppm for S-week-old chickens 500 ppm for laying hens Chlorination at levels of 50 ppm depressed egg production in laying hens. Analysis of the data showed no significant differences among the various management practices as far as the mean egg production or the mortality rates were concerned. A significant difference (P.( .01), however, occurred in egg production from different dietary treatments. The soybean oil meal containing ration produced the best results but the rate of production of birds on this ration was not significantly better than that of birds on the experimental diet containing raw soybeans with supplementation of methionine and antibiotic, Pro-strep. There was less mortality among birds fed soybean meal containing diets than among those fed raw soybeans with supplemental methionine, Pro-strep with or without detergent; however, this difference was not significant. Cannibalism, leukosis, ruptured yolks and peritonitis were the primary causes of death. Other pathological conditions diagnosed were sinusitis, nephritis, hepatitis, lymphoid mesenteric tumor, synovitis and impacted oviduct. Infections associated with reproductive systems appeared to be more common than others. Differential leukocytic counts, coagulation time, plasma proteins and blood urea nitrogen showed little variation among birds of different groups. Blood sugar levels were significantly (P < .01) higher in birds given both chlorinated water and litter change treatments than in birds given tap water with litter change treatments. Various dietary and management treatments had similar effect on average egg weight, Haugh scores and egg shell thickness. Improvement in hatchability of eggs produced by birds kept on built-up litter was not evident. Baby chicks brooded on litter previously used by hens kept under different management treatments did not differentiate between relative infectiveness of different litter systems. There were no significant differences in bacterial counts in samples examined from different litter treatments. During winter the moisture content of the litter was higher in the treatments receiving frequent litter change than that of built-up litter. Different litter treatments had similar effects on coccidial oocysts. The results of these studies suggest that chlorination of drinking water and better housekeeping such as more frequent litter changes or both may not be advantageous over the usual tap water and "built-up" litter. practices, when judged on basis of mortality. This conclusion may not be applicable during an outbreak of disease when better housekeeping may be a primary step towards the control of such an outbreak. THE EFFECT OF RAW SOYBEAN DIETS AND CERTAIN SANITARY MEASURES UPON DISEASE INCIDENCE AND NUTRITIONAL PERFORMANCE OF CHICKENS by Harbhajan Singh A THESIS SUBMITTED TO Michigan State University in partial fulfillment of the requirements for the degree of Doctor of PhilOSOphy Department of Poultry Science 1964 To ACKNOWLEDGEMENTS Dr. Philip J. Schaible for the guidance and advice that made this work possible. Dr. T. H. Coleman, whose suggestions were most helpful and whose association will be a pleasant experience of my stay at Michigan State University. Dr. Howard C. Zindel, Chairman of Department of Poultry Science and Professor J. A. Davidson for making this work possible in the form of a research assistantship. Michigan State University, Department of Poultry Science and the people of Michigan for the use of farm facilities and poultry science laboratories. Drs. H. c. Zindel, '1‘. H. Coleman, 0'. c. Ellis and w. w. Snyder for constructive review of this manuscript. Dre. C. C. Ellis, D. A. Schmidt, Dept. of Veterinary Pathology, Dr. L. G. Harmon, Department of Food Science and Dr. W. L. Mallmann, Department of Microbiology and Public Health for their assistance in this study. My wife, Sharanjet, for her constant interest and encouragement throughout the course of study for this degree. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS................. TABLE OF CONTENTS . . . . . . . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . . . . . . . LIST OF APPENDICES . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . . . . . . . Mortality in contest flocks . . . . . . . Mortality of young chickens . . . . . . . . Mortality in farm flocks . . . . . . . . . Bactericidal effect of chlorine . . . . . . Deep litter 0 I O C O C C O O O O C O O O 0 Effect of deep litter on incidence of mortality Addition of lime in built-up floor litter . . . Deep floor litter as source of dietary supplements Effect of deep litter on hatchability . . . . . . . Intestinal flora of chickens . . . . . . . Bacterial flora of chicken drOppings . . . The microflora of drOppings and its relation to litter management . . . . . . . . . . . . EXPERIMENTAL PLAN AND STATISTICAL PROCEDURES . . Experiment 8.]- plan 0 O O O O O O O O C O O O O 0 Statistical procedures . . . . . . . . . . . . . . . . PART I. INTRODUCTION TO EXPERIMENTS I THROUGH VIII. Procedure and Results . . . . . . . . . . . Experiments I - II. The effect of chlorination of drinking water on the performance of baby chicks . . . . . . . . . . . . . . Experiment I . . . . . . . . . . . . . . Experiment II o o o o o o o o o o o o 0 Experiments III, IV and V. The effect of chlorination of water on its consumption by growing chickens Experiment III . . . . . . . . . . . . . . . Experiment IV . . . . . . . . . . . . . Experiment V . . . . . . . . . . . . . . Experiments VI, VII and VIII . . . . . . . . . . . . . Effect of chlorinated water on performance of laying hens . . . . . . . . . . . iii Page ii iii ix ' 18 l9 19 19 20 20 22 23 23 31 32 32 32 37 37 Experiment VI . Experiment VII Experiment VIII Results . . . . . . Experiment VI . Experiment VII Experiment VIII PART II. INTRODUCTION TO EXPERIMENTS IX and X Experiment IX. laying hens The effect of chlorination of drinking water and frequent change of litter on egg production and incidence of disease in the 38 . 38 38 38 42 44 Experiment X. The effect of various diets and litter treatments upon the incidence of mortality in laying hens DISCUSSION . . . . . . . Part I O O O O O O 0 Part II . . . . . . SUMMARY AND CONCLUSIONS Part1 .0000. Part II . . . . o 0 LITERATURE CITED . . . . APPENDH O O O O O O O 0 iv 67 96 96 98 105 106 107 116 LIST OF TABLES Table Page 1-1 Feed intake, water consumption and feed efficiency of White Leghorn cockerels with different levels of chlorination of drinking water . . . . . . . . . . 25 1-2 Analysis of variance of three-week body weights of White Leghorn cockerels used in Experiment I . . . . 26 1-3 Weights of liver, heart and gizzard of three-week- old cockerels sacrificed during Experiment I . . . . 27 1-4 Bacterial population of intestines of three-week- old cockerels used in Experiment I . . . . . . . . . 28 2-1 Effect of chlorination of water on feed and water consumption as well as gain in weight of five-week- old White Leghorn cockerels used in Experiment I . . 29 2-2 Analysis of variance of body weights of five-week- old White Leghorn cockerels used in Experiment II . . 30 3-1 Chlorinated water consumption by ten-week-old White Rock chickens during a period of 15 days . . . . . . 33 3-2 Free chlorine levels in drinking water after standing 24 hours in drinking fountain . . . . . . . . . . . . 34 4-1 Feed intake, water consumption and gain in weight of five-week-old Cornish Cross chickens during a period of 35 days . . . . . . . . . . . . . . . . . . 35 5-1 Feed intake, water consumption and gain in weight of five-week-old White Rock chickens during a period of 35daYSOOOOOOOOOOOOOOOOOOOOOOO 36 6-1 Effect of different levels of chlorination of drinking water on feed and water consumption, as well as egg production in laying hens during a 21-day period . . 39A 6-2 Statistical analysis for eggs produced by birds used in Experiment VI . . . . . . . . . . . . . . . . . . 39B 7-1 Chlorinated water consumption, feed intake, water consumption and egg production for laying hens during a 30-day period . . . . . . . . . . . . . . . 40A 7-2 Analysis of variance for eggs produced by birds used in Experiment VIII . . . . . . . . . . . . . . . . . 40B 8-1 Chlorinated water consumption, feed intake, water consumption and egg production in DeKalb hens during aBO-dayperiOdooooococo...coo-coo 4']- Table 9-1 9-2 9-3 9-4 9-5 9-6 9-7 9-8 9-10 9-11 9-12 9-13 9-14 9-15 9-16 Percentage composition of the diets used in Experiment IX . . . . . . . . . . . . . . . . . Egg production percentage with respect to diet and management treatment . . . . . . . . . . . Analysis of variance of egg production data with respect to diet and management treatments (168-day trap period) . . . . . . . . . . . . . Incidence and cause of mortality under various diets and treatments . . . . . . . . . . . . . Analysis of variance of mortality data for birds in Experiment m 0 O O O O O O O O O O O O O O O 0 Percent egg production under various diets and management treatments computed on basis of total number of live birds present at the beginning of each month . . . . . . . . . . . . . . . . . . . Average body weights (lbs) of birds on different diet and litter treatments . . . . . . . . . . . Analysis of variance of final body weights of birds on different treatments . . . . . . . . . Percent fertile and percent hatch of fertile eggs set from birds given litter change and built-up littertreatments..o............. Summary of production and quality of eggs from hens under different treatments' . . . . . . . . . . . Effect of different diets and treatments on egg weights 0 O O O O O O O O O O O O C O C O O 0 Summary of results obtained from agglutination test using salmonella antigen 0 group D . . . . Summary of results of agglutination test using E4 C0121. aIltj-gens O O I O O C O O O O O O O O O 0 Differential leukocytic counts (percentages) from birds kept under different management treatments . Blood coagulation time for birds kept on different diet and management treatments . . . . . . . . . . Summary of blood glucose levels in birds receiving different management treatments . . . . . . . . . vi Page 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Table 9-17 9-18 9-19 10-1 10-2 10-3 10-4 lO-SA lO-SB 10-6 10-7A lO-7B 10-8 10-9 Analysis of variance of blood sugar levels among birds kept on different management tre atment S O O O C O O C 0 O O O O O O O O O 0 Summary of blood urea nitrogen levels in birds receiving different management treatments . . Summary of plasma protein levels in birds receiv- ing different management treatments . . . . . Experimental plan and composition of original six experimental rations for Experiment X. . . Composition of layer-breeder rations for Experiment X . . . . . . . . . . . . . . . . . Incidence of mortality under various diets and management treatments . . . . . . . . . . . . Analysis of variance of mortality data for birds in Experiment X . . . . . . . . . . . . Summary of postmortem findings showing incidence of leukosis and reproductive disorders under various diets and management treatments . . . Monthly incidence of mortality in various experimental pens under different diets and treatments . . . . . . . . . . . . . . . . . . Summary of egg production and mortality (percentage) with respect to diet and management treatment . . . . . . . . . . . . Percent egg production under various diets and management treatments computed on the basis of total number of birds live at the beginning of each month . . . . . . . . . . . . . . . . . Percent egg production under various diets and management treatments computed on basis of total number of birds in production . . . . . . . . Analysis of variance of egg production data with respect to diets and management treatments . . Summary showing quality of eggs from hens receiving different experimental diets and management treatments . . . . . . . . . . . . . vii 64 65 66 75 76 77 78 79 80 81 82 83 84 85 Table 10-10 10-11 10-12 10-13 10-14 10-15 10-16 10-18 10-19 Average body weights (lbs.) of birds kept on different diets and litter treatments during Experiment X C O C C O C O I O O O C O O O O D Percent fertile and percent hatch of fertile eggs set from birds on different litter management treatments in Experiment X . . . . . . . . . . Bacterial counts from litter changed frequently . Bacterial counts from litter unchanged during ExperimentX.................. Moisture content of litter used for housing birds on Experiment X . . . . . . . . . . . . . Summary of examination of litter and droppings for coccidial oocysts and ova from.Ascaridi§ lineata and HEterakiS gallinae o o o o o o o o o o o o 0 Feed intake, feed efficiency, gain in weight and mortality rate of three-week-old White Leghorn chicks used in Experiment X for determination of relative infectiveness of litter under different management treatments . . . . . . . . . . . . . . Analysis of variance of three-week body weights of chicks used in Experiment X for determining relative infectiveness of litter under different litter treatments . . . . . . . . . . . . . . . . Summary of results obtained from chicks sacrificed at different intervals for determining relative infectiveness of litter resulting from various management practices . . . . . . . . . . . . . . viii 86 87 88 89 90 91 93 94 95 LIST OF APPENDICES Table Page 1 Composition of standard chick starter (M.S.U. 63-8) used in Experiments I and II . . . . . . . . . 116 2 Residual chlorine determinations in filtrate from mixture of feed and chlorinated water . . . . . . . 117 3 Composition of ration (amounts in lbs./200 lbs.) used in Experiments III, IV and V . . . . . . . . . . . . 118 f? 4 Composition of basal ration used in Experiments VI, VII and VIII 0 C O O O C C O O O O O O O O O O O O O 119 5 Composition of ration for baby chicks used in Experiment x O O I C O O O O O O O I C O I O 0 O O O le/Iv’y'l 6 Composition of vitamin-trace mineral mixes . . . . . 121»“73 7 Postmortem diagnosis sheet . . . . . . . . . . . . . 123 ix INTRODUCTION AND REVIEW OF LITERATURE Domestic fowl are susceptible to various ailments that impair their health and interfere with normal growth or egg production. The loss of chicken meat and eggs due to disease, parasitism and nutritional defici- encies amounts to approximately one-quarter billion dollars a year in the United States. This loss includes not only the market fowl value but lowered egg production, wasted feed and labor as well as reduced returns on the overall investment. In 1962, the loss from laying flocks alone was estimated to be 80 million dollars (Snyder.g£.§l., l962).7' _:. From 15 to 25 percent of all the pullets housed succumb to illness or parasitism before they complete their first year of egg production. WOrm infestation or other systemic disorders is responsible for poor feed conversion, loss of body weight, depressed egg production and an excessive number of culls. Profits on a poultry farm can be increased, therefore, through control of disease and other causes of mortality. Excellent drugs and vaccines are available to the poultry farmer to combat these losses. However, prevention of disease is more economical than medical treatment after diseases have developed. Disease organisms or parasites and their eggs may be found in the drOppings or in the discharges from the eyes, nostrils and mouths of infected or carrier birds. The infective agent can be picked up by a flock from the litter, feed or water that is contaminated With the discharges of the sick birds. Outbreaks of parasitism or contagious diseases are believed to result from imprOperly maintained waterers, spilling of feed into the litter, or the contaminated condition of the litter itself. Water is the least expensive nutrient birds get and one that becomes contaminated easily. It seems reasonable to expect that certain parasitic and infectious diseases could be prevented or their incidence lessened by maintenance of a healthy environment and sound management practices. From the poultryman's standpoint, of course, it is necessary to find practical means of improving conditions in the hen house. With these thoughts in mind, the hypothesis was developed that perhaps disease incidence could be lowered through use of disinfectants in the drinking water and/or frequent introductions of clean litter. Consequently, the purpose of the studies reported herein was to determine the effect of better sanitation practices on the mortality rate and egg production of laying hens fed diets that differed substantially in the character of certain ingredients. The sanitation and antibiotic practices used in these experiments are over and above those practiced in many modern operations. REVIEW OF LITERATURE According to Winton (1932), a carefully planned sanitation program on a poultry farm would aid in the control and eradication of certain infectious diseases and parasites. He came to these conclusions as the result of a practical experiment and flock survey in which litter was removed daily to avoid dampness and contamination. Winton cites an experiment conducted under farm conditions at Kansas Agricultural Experi- ment Station in which a flock of chickens given very little attention other than to provide food and water, had a total mortality of 42 percent throughout the year; whereas, a second flock, where the quarters were kept reasonably clean and sanitary during the year had an annual loss of only seven percent. Barger gt al.(1958) state that the health and performance of any flock depend mainly upon how closely one follows the principles of sani- tation and good management rather than the use of medicines. The diseases of poultry are quite different in nature from those of other livestock. Of greatest importance are those considered mass disorders rather than ailments of individual birds. Therefore, the authors stress preservation of the health and vigor of the flock as a whole rather than devoting energies to a few individual sick birds. Mortality in "contest" flocks Dunnicliff (1913) in New South Wales analyzed mortality data from a total of 5,448 hens during a lO-year egg laying contest and reported a death loss of 6.3 percent a year. During the seventh Storrs Competition, Card and Kirkpatrick (1919) obtained mortality of 15 percent among 1,000 pullets. An average annual death rate of 25.4 percent was reported by Anderson (1928) during the first five years of the New York state egg laying contest at Farmingdale. In the above contest, the mortality was 3 28 percent among White Leghorns and 45.6 percent among Rhode Island Reds. Among 10,000 pullets entered in eight successive laying contests in Michi- gan, Stafseth and Weisner (1931) reported an average mortality of 19 per- cent a year. The fourth, fifth and sixth years of the contest showed an increase over the first three years. During the seventh and eighth years even a higher mortality occurred than in all previous years. More than 20 percent of the mortality during these years was due to laryngotracheitis, commonly called infectious bronchitis. Stafseth and Weisner further report- ed an increase in the incidence of peritonitis, sarcamatosis, bronchitis, pericarditis, tumors, leukosis, ruptured oviduct and salpingitis. On the other hand, they found decreased incidence of fowl cholera, tuberculosis, fowl typhoid and leg paralysis. An analysis of mortality data throughout the year showed a rather marked seasonal shift in the mortality curve. Highest mortality was observed in the months of March to July. The death loss in December and January was higher than that in November. The authors are of the Opinion that higher death rate follows heavy egg production. During seven successive egg laying contests in Utah, Alder (1934) obtained mortality percentages of 13, 35, 33, 27, 18, 20, and 58, respectively. He attributed the higher death rate in the last year to an outbreak of infectious laryngotracheitis. Dudley (1928) during a period of 15 years found a death loss of 7.2, 6.8 and 5.7 percent for White Leghorn, White wyandottes and Rhode Island Reds, respectively. These breeds showed a different pattern of mortality during the year. The death rate of White Leghorn and Rhode Island Reds was similar during December, although it was considerably different in magnitude. From January to July the death rates of White wyandottes seemed behind that of the other two breeds. The maximum death rate occurred in August with the White Leghorns, September with the White Wyandottes and March with the Rhode Island Reds. Mortality rates of the White Leghorns and of the Rhode Island Reds reached a high level in March, fell to a low level in April, rose again in May and fell in June and then both increased. Harris and Boughton (1927) have studied for a period of ten years the death rate among the birds entered for the International Egg Laying Contest conducted at Storrs, Connecticut. The death rate of White wyandotte fowl during the first year of lay was significantly higher than that of the Rhode Island Red and White Leghorn fowl, with the former somewhat higher than the latter. They noted that death loss in different breeds was not the same during each month of the year, but showed a gradual increase from November to July. In all these breeds the death rate for May was lower than that for April. Harris and Boughton (1927), like Stafseth and Weisner (1931) expressed the opinion that higher death loss follows heavy egg production. They concluded that the strain of heavy egg production tends to eliminate the weaker birds. Harris and Boughton attempted to show a correlation between the monthly death loss and the average monthly egg production of each breed. Brunson and Godfrey (1952) summarized autopsy records of the Oklahoma egg laying tests for a period of 14 years. They found an annual mortality rate of 13.84 percent to 26.3 percent with a 14-year average of 20.27 percent. Death rate among White Plymouth Rocks and White Leghorns was higher than that of New Hampshire or Rhode Island Reds. Major causes of mortality reported were reproductive disorders, lymphomatosis and respiratory infections. New York random sample test (1953) report showed an average annual mortality of 35.5 percent in hens hatched as chicks. Lymphomatosis was responsible for 14.5 percent of this loss, two-thirds of which was caused by the visceral form of the disease. Random Sample test report XX (1963) showed an average mortality rate of 10.86 percent among laying hens. The minimum and maximum death loss ranged between 0.00 and 30.60 percent. Mortality of Young_Chickens Voorhies and Read (1931) made an extensive study of daily mortality of 6,000,000 chicks for the first 14 days of the brooding period in the years 1927, 1928 and 1929. Their data showed highest mortality on the fifth day, then a rapid fall off from the 6th to the 14th day, after which the rate of loss was again at the same level as on the firstday. Barrett (1929) studied the causes and distribution of mortality among a total of 4,806 chicks in seven hatches for a period of the first eight weeks and found a total loss of 11 percent. Voorhies and Read (1931) have reported a relatively high mortality, unassociated with any disease. Broadfoot _£._l. (1957) reported an outbreak of infectious bronchitis in 14,000 susceptible pullet chicks in 35 flocks during the first 5 to 7 days of life. The chicks of high-producing strains suffered death losses varying from 5 to 35 percent. Newcastle disease virus during the period of active involvement restricted feed and water intake and depressed weight gains of White Leghorn cockerels (Squibb, 1961). Vitamin therapy did not affect weight gains, water intake, feed efficiency or mortality of cockerels. Hays and Spear (1952) determined that chick mortality from young parents was significantly higher during the first eight weeks of life than was observed in chicks from older parents. The data collected by Hays (1955), over a 5-year period on pedigreed Rhode Island Reds bred for high fecundity did not indicate that age of parents significantly affected either viability of pullets in the laying house or the viability of males and females during the growing period. Dardiri and Zaki (1955) reported oomphalitis in 135,000 chicks shipped from New York to Cairo, Egypt. Highest mortality occurred on the second to sixth day of infection. Pseudomonas aeruginosa, Proteus morgani, Aerobacter agrogenes and clostridia species were isolated from dead chicks. Reid gt al.(l961) isolated Escherichia coli, enterobacteriaceae, clostridium, Pseudomonas, streptococcus and Bacillus from dead embryos of chicks involving 25 hatcheries. §,_£Qli isolated varied in pathogene- city showing from 20 to 100 percent mortality in baby chicks. Chute and Mear (1957) considered litter as the source of infection in most cases of aspergillosis. Wright _t _l. (1960, 1961) successfully transmitted aspergillosis to incubating embryos which resulted in infected chicks. Penetration of sound and cracked eggs by Aspergillus fumigatus occurred within six days of incubation. He further added that the presence of certain molds, particularly Aspergillus species markedly increased chick and pullet mortality during the first ten days of life. Todd (1948) examined 1014 chicks for parasitic worms and found 972 (95.8 percent) parasitized by an average of 125.6 worms representing an average of 3.5 species. Tyzzer _£._l. (1932) reported that young birds were less susceptible than older birds to Eimeria necatrix. Contrary to this, Brackett and Bliznick (1952) found that following inoculation of equal number of oocysts, young birds were found more severely affected than old birds. Gardiner (1955) was of the Opinion that chicks were most resistant to cecal cocci- diosis at two weeks of age than at any other period during the first six weeks of their life. Benzanson and Stephenson (1948) reported that an increase in depth of litter greatly reduced the incidence of breast blister conditions in broilers. Wet litter tends to result in more breast blisters on broilers than dry litter (Smith, 1956). According to recent reports from the Inspection Service, the greatest single cause for condemnation during postmortem inspection is airsacculitis. Following close behind and on the increase is leukosis. Next in importance is septicemia or toxemia (Morris, 1963). Helmboldt et a1. (1963) reported cutaneous lymphomatosis in nine-week-old broilers condemned for human use. Mortality in Farm Flocks Poultrymen who house their flocks in winter quarters at the right time, normally experience a low death rate in their flocks. Buster (1928) reported 24 percent mortality in 35 flocks having a total of 65,264 hens in the Petaluma district of California in 1926. During a period of three years, 1926 to 1928 in Oregon, there was an average death loss of 13 percent among flocks totaling 271,337 hens (Scudder g£_§i., 1931). Thomas and Clawson (1933) in Utah noted a period of three years -- 1929 to 1931, a mortality of twenty percent in 315,577 Leghorn hens kept in commercial flocks of about 1,000 each. Misner (1932) recorded 27 percent mortality on 108 New York State poultry farms during the years 1930-1931. The average number of hens per farm in this group was 959. The average mortality by years was 19, 24, 27, 37, 36 and 39 percent. Darcel._t._l. (1960) determined 13 and 16 percent mortality over a period of two years in a White Leghorn flock of 9,442 birds. They observed a very low incidence of neOplastic diseases in general, and leukosis in particular. Most prevalent disease reported in this flock was histomoniasis. Takashi and Reid (1961) also made a survey and found an increasing trend of histomoniasis in chicken flocks. Jaquette and Fogg (1962) posted 5,816 chickens from 1145 flocks and concluded that CRD complex was most widely spread disease on the Del Marva Peninsula. The other most prevalent diseases reported were coccidiosis and worm infestations. Other causes of mortality diagnosed were lympho- matosis, synovitis, enteritis, hemorrhagic syndrome, laryngotracheitis, avian encephalomyelitis and mycosis. Olesiuk and Van Rockel (1960) reported that CRD caused a rapid decline in egg production and long-lasting effects on both external and internal egg quality. The egg abnormalities reported were rough and thin shelled, bleached shell color and watery albumen. Raggi‘_£._l. (1961) found that infectious laryngotracheitis had only temporary effects on egg production and shell thickness. Taylor g£.al. (1955) reported an outbreak of avian encephalomyelitis in a breeding flock of chickens. Eggs produced just before and during the period of depressed egg production showed decreased hatchability and an increased embryonic mortality during the last three days of incubation. Prolapse in the chicken is defined as the eversion of the oviduct with the cloaca through the vent (Barger and Card, 1935). Stafseth _§._l. (1932) suggested that prolapse may result from atony associated with toxic action of products incident to worms, coccidia or secondary microbial invaders. Wheeler and Hoffmann (1948) reasoned that prolapse and pickouts in chickens may be either a sudden increase in estrogens in the blood or a protracted high estrogen blood level. Contrary to this hypotheses, Burmester (1948) found that females given estrogen (diethylstilbestrol) had the lowest incidence of prolapse although not statistically different from the controls. Neal (1956) reported that methionine when supplemented at a higher level suppressed cannibalism and pickouts. Gross (1961) isolated from litter and feed samples, Escherichia coli pathogenic serotypes. Calnek and Levine (1957), Fabricant _£__l, (1959) and Gross (1961) found these serotypes associated with myc0p1asma (pleuro- pneumonia-like organisms or PPLO), infectious bronchitis virus (IBV) and Newcastle disease virus (NDV) in cases of air sac disease complex. Olesiuk 10 and Van Rockel (1960) reported that chickens may become infected with pleuropneumonia organisms (PPLO) by egg transmission or by close contact with infected birds. Van Rockel (1955) was of the Opinion that environ- mental factors such as over-crowding, low or irregular brooding temperature may increase the transmission and severity of disease. Calnek and Levine (1957), Olesiuk and Van Rockel (1960) reported that viruses such as IBV and NDV can greatly increase the transmission and severity of PPLO infection. Chalquest and Fabricant (1959) suggested the possible control of the transmission of pleuro-pneumonia-like organisms by dipping hatching eggs into cold antibiotic (erythromycin) solution. Waters and Prickett (1944), Waters and Bywaters (1949) suggested egg transmission of leukosis virus which was supported further by Gross (1951, 1952), Cottral (1952) and Cottral t 1. (1954). Contrary to this hypothesis, Hutt._§-al. (1944), Cole (1949), Cole and Hutt (1951) and Hutt (1951) found that some forms of lymphomatosis were not egg transmissible. Patterson _£‘_l. (1932), Kennard and Chamberlin (1934) successfully trans- mitted lymphomatosis in groups of chicks inoculated With nasal washings from both infected and normal mature hens. Burmester and Waters (1955) are of the Opinion that lymphomatosis virus which is deposited in eggs laid by hens having a latent infection, is transmitted to the embryo and chicks which hatch from such eggs. These maternally infected chicks may be a source of infection for chicks from other parents not similarly infected and hence are lacking in anti- bodies. They further state that the transfer of such infection appears to take place during the brooding period. Burmester._§-_l. (1955) found 17 of the 22 hens of the infected population shedding virus into their eggs. Cole and Hutt (1956) reported variations in the amount of lymphomatosis virus in the eggs Of the same hen. Burmester and Waters (1956) found a ,. J decree in bi: E‘VV‘. ll ‘4.( . . CF‘K" Qt. I h 11 decrease in the shedding of the lymphomatosis virus into eggs as hens became Old. Burmester and Gentry (1954) produced lymphomatosis in birds given the virus via the trachea (83%) or as an aerosol spray (39%). Sevoian _£;_J, (1963) determined that lymphomatosis agents can be transmitted in birds via the air route. The clinical and pathological manifestation of the disease were undistinguishable from those of parenterally induced or natural infection. , 1956) Sevoian and Chamberlin (1963) and Cole and Butt (1954] found that neural form was most prevalent in younger age groups (4-20 weeks) Of chickens and visceral form in mature birds. Burmester _£._l, (1959) reproduced the visceral form of lymphomatosis after long incubation periods. Neural form was rarely Observed by these workers. However, it does not necessarily follow that each form is caused by a different agent. They reported further that the age of the chicken and variation in the dose of virus at infection time is a factor influencing the response of the chickens to tumor viruses. High doses primarily caused erythroblastosis, which occurred in less than four months and low doses resulted in a response that was primarily visceral lymphomatosis, with death occurring after four months. They further state that filtrates of tumors and other materials from 5 Of the 7 propagated lymphoid tumor strains caused osteopetrosis in addition to erythroblastosis and visceral lymphomatosis. Kennard and Chamberlin (1936) and Hutt and Cole (1954) indicated that assistance to leukosis increases with age. Sevoian and Chamberlin (1963) reported that day-old chicks were approximately 1,000 to more than 10,000 times as susceptible as their dams and grand dams. Waters (1951) collected data over a nine-year period on resistance and susceptibility to lymphomatosis among six different inbred lines of White Leghorns maintained for a period of 600 days. He reported a marked increase or decrease in the percentage of 12 mortality from lymphomatosis resulted in certain lines where inbreeding accompanied by rigid selection was practiced. Hutt and Cole (1954) reported that the severity of exposure increases with the proximity of the susceptible hicks to adult hens infected with leukosis. They further state that chickens raised in batteries contracted leukosis just as readily as did other chicks on the floor. Kennard (1933) found the mortality from neurolymphomatosis in hen-bred pullets about half that of pullet-bred pullets. Contrary to these results, Carr (1952) and Cole (1955) found that pullet-bred families were free Of death from lymphomatosis. During the next year, Cole and Hutt (1956) reported that in pedigreed flocks bred for resistance to leukosis, age of the hen had little effect on the resistance Of her daughter. Forgacs and Car11(1955) and Forgacs-gt.al.(l954) isolated toxic fungi (Aspergillus cloyatus, A. flavus, and A. Fumigatus) from feed and litter samples collected from areas where poultry hemorrhagic syndrome was enzootic. Using fungi, which they isolated from litter samples, Forgacs and Carll (1955) produced clinical and pathological conditions Of mycotoxi- cosis in chickens similar to those found in field cases of hemorrhagic syndrome. Later, these findings were confirmed by Forgacs _£._l, (1955, 1958, 1962) who produced toxicosis in chickens housed both in batteries and floor pens. The addition of vitamin K to the toxic fungal diets did not prevent the mycotoxicosis in chickens. Enigh (1935) reported sixty-one cases of blackhead in German flocks caused by "Blastocystis" fungus, later reported to be yeast-like fungus condidalalbicagg. Couch (1955) and Snoeyenbos _£,_l, (1961) found an increase in the incidence of cage paralysis with the rapid increase in the practice Of keeping layers in individual cages. 13 Reports from various diagnostic laboratories (Snyder._§.al., 1962) indicate that leukosis, coccidiosis, chronic respiratory disease and ascariaris are the most prevalent diseases among farm flocks. Other causes of mortality in chickens reported are avian monocytosis, enteritis, synovitis and capillaria worms. Bactericidal Effect of Chlorine One to two ppm of chlorine (Anon., 1949) destroys not only bacteria but also viruses. According to Ehleres and Steel (1927) chlorine possesses selective bactericidal effect, destroying bacteria mostly Of the pathogenic and coli groups. A few harmless bacteria and spores are rather resistant to its action. Ehleres and Steel (1927) stated that chlorine can be applied to water, either as gas or in the form of chlorinated lime. Chlorine gas is preferable to chlorinated lime because it does not lose its activity as readily as the latter and is more convenient to handle. Chlorinated lime contains 30 to 35 percent by weight of available chlorine. Disinfection by chlorine (Anon., 1949) depends on the concentration of residual chlorine, the temperature of the water, and the amounts of organic impurities present in it. A reasonably higher temperature and acidic media favor bactericidal action. Ehleres and Steel (1927) found that in water containing little or no organic matter, 0.2 ppm of available chlorine is sufficient to kill bacteria. According to them, chlorine in water forms unstable hypochlorous acid which readily breaks down into hydrochloric acid and atomic Oxygen which possess bactericidal property. The orthotolidine test, when properly applied, measures the amount of residual chlorine. Deep Litter Deep litter, if properly managed, can keep the house warm and dry. As described by Lippincott and Card (1946), the usual practice with deep or l4 built-up litter is to start in the fall with 2 to 4 inches Of dry litter, and then add more gradually until the floor is covered 8 to 12 inches deep by about the middle of December. After this no more litter is added, but the upper surface is stirred occasionally to prevent matting. The litter should be spread evenly on the floor, and wet cakes formed around the water fountains removed and replaced with new, dry material. It may be removed and replaced with clean, shallow litter in the late spring or it may be kept until the regular fall cleaning. Effect of Deep Litter on Incidence of Mortality A number of research workers have discussed highly absorbent and insulat- ing properties of deep litter. According to Kennard and Chamberlin (1949), as well as MOore and Chamberlin (1953), the heat generated by the microbial decomposition of the litter has a self-sterilization effect. Biological and chemical reactions in this type of litter can destroy harmful bacteria, protozoa and other infective agents. Kennard and Chamberlin (1948b, 1949, 1950a, 1950b and 1951) reported decreased incidences of cannibalism, cecal coccidiosis and pullorum disease in birds maintained on built-up litter. They reared 26 consecutive broods of chickens on the same litter and found that whether limed or unlimed, it contained fewer yeasts, molds and bacteria than new litter changed frequently. Koutz (1952), however, demonstrated that coccidial oocysts were not killed in deep litter. Boughton (1939) advocated the use of deep litter as one method for control of coccidiosis in young chicks. He found that rapid drying or extreme wetness reduced the number of sporulating oocysts; a limited amount of moisture was highly desirable for microbial action and composting of the litter and drOppings. Contrary to the results of the previous investigators, Koutz (1952, 1953) found that all birds on built-up litter were heavily infested with de \l. 15 parasites such as Eimeria tenella, Eimeria acervulina, Eimeria maxima, .éfiEQEiQLQ lineata, Heterakis gallinae and Capillaria retusa. He made periodic checks on this litter and found numerous ova and oocysts of these parasites. The control birds were free from intestinal nematodes although some coccidia were present in a few birds. Klein of the University of Massachusetts (Hinshaw, 1952) felt that deep litter increased the flea problem in poultry houses. Rats, cats and dogs were blamed as constant reservoirs of these fleas. Cotterill and Winter (1953) reported that litter which becomes too wet may result in excessive ammonia production and impair the health Of the poultry flock. Faddoul and Ringrose (1950) Observed that avian keratoconjunctivitis in young chicks developed as results of ammonia liberated from built-up litter. Addition of Lime in Built-up Floor Litter The use of built-up litter systems became a well established practice in the late forties. Subsequently, lime was added to the litter to avoid dampness. The New Jersey Agricultural Experiment Station reported that hydrated lime could be used safely as a deodorizer and preservative of nitrogen in bird droppings. Lime was recommended for floor litter (Kennard and Chamberlin, 1947) in brooder houses as a control measure against the spread of coccidiosis. They cite an experiment at Western Washington Agricultural Experiment Station where the use of lime in floor litter was found favorable. When liming litter, hydrated lime is scattered over the litter at the rate of 12 to 15 pounds per 100 square feet Of floor space or one pound per layer. The newly added lime is covered with l" to 2" Of fresh litter. Both are stirred into the Old floor litter at intervals of 2 to 4 weeks. Moore and Chamberlin (1953) emphasized that, depending upon the weather and local conditions, care must be taken to keep litter dry by frequent stirring. 16 Deep Floor Litter_as Source Of Dietary Supplements Hammond (1942) and Whitson gt a1. (1945) reported that cow manure, when added to a diet deficient in riboflavin, had marked beneficial effects, as measured by livability of young embryos, hatchability of eggs and rate of chick growth. Rubin.g£;_l, (1946) and Bird (1947) demonstrated that chick growth factors in cow manure were also present in the droppings of hens. Kennard and Chamberlin (1948a, 1948b) showed that deep litter was a potent source of dietary supplements including the unidentified animal protein or vitamins necessary to supplement an all-plant protein diet for higher egg production and hatchability. Halbrook (1950a) isolated 140 different organisms from built-up litter capable of synthesizing vitamin B12. During the same year in another publication, Halbrook (1950b) and Halbrook._§'_l. (1950) reported 250 mu Of vitamin B12 per gram of built-up litter as compared to 1 mu in unused control litter. Kennard and Chamberlin (1951) found that there was a direct correlation Of the rate of growth, feed efficiency, and rate Of mortality with the age of the floor litter, whether the chickens received the all-plant diet with or without supplements. Experimental evidence has been accumulating during the past thirty years to establish the fact that the synthesis Of vitamin or vitamin-like factors is accomplished after the feces are voided. Lamoreux and Schumacher (1940) reported that the feces of laying hens contained no more riboflavin at the time of defecation than was present in the feed which the hens were receiving. McGinnis 25 _l. (1947) and Lamoreux and Schumacher (1940) found a 100 percent increase in the riboflavin content of feces when held at room temperature for 24 hours and a 300 percent increase when held for one week. The authors were of the Opinion that riboflavin synthesis did not occur in the digestive tract Of the fowl but resulted after the feces were voided. (1") W: 17 t a1.(1961), Marr t al. (1961) and Singsen t l. (1961) Crowley reported that the calcium-phosphorus requirement is higher for laying hens maintained in cages than for those maintained on litter in floor pens. They suggested perhaps hens housed on litter may acquire part of their calcium-phosphorus requirements through COprOphagy. Effect of Deep Litter on Hagghability Kennard and Chamberlin (1948a) reported 78 percent hatchability of eggs from hens that were on old, built-up litter and that received an all-plant diet versus 32 percent hatchability of eggs from the same ration given to similar hens on litter that was removed and renewed every two weeks. Sunde _£__l. (1951) also found that the hatchability of eggs from birds on a basal, all-vegetable diet increased with the age of the built-up litter. Intestinal Flora of Chickens Kern (1897), King (1905) and Gage (1911) have reported that Escherichia .391; andeerobact r aerogenes are the predominant organisms Of the intestinal flora. §;.£21$ is a constant inhabitant Of the intestinal tract and tends to replace other organisms whose presence depends upon the food supply. King (1905) and Gage (1911) further reported the presence of Lactobacilli, Micrococci, pseudomonaga, bacilli, garciaaa clostridia and yeast in the chick intestines. BasteriallFlora of Chicken DrOppings Emmel (1930) reported that the bacterial flora of the feces was approximately the same in both chickens and hens -- Escherichia coli and Escherichia Communior were the predominant organisms in the feces of both two-week-Old chicks and of hens. Schumacher and Heuser (1941) have demonstrated the presence of E;.£Qll along with sporeformers, non-gelatin liquifiers, gram-positive cocci, molds and Bacillus Megatherium in chicken feces. 18 The Microfloragpf Dr0ppings and Its Relation to Litter Management Halbrook._£._l, (1951) have suggested that the microflora of fresh droppings has a trend similar to that of the litter on which the chicks run. They obtained lower counts from droppings of chickens on built-up litter than from droppings of those kept on litter that was changed every eighth week. They further stated that the drOppings of birds on wire floor have counts as high as those of chicks on litter. The authors were of the Opinion that the flora in birds was mainly established through feed and water and suggested that perhaps alkalinity of built-up litter had a depressing effect on the intestinal microflora of the bird. Halbrook a; 1.(l951) have reported an increase in molds and yeast with the age of the litter for the first eight weeks. Cob litter used for chick brooding but changed weekly contained more bacteria, mold and yeast than new cobs; litter unchanged for eight weeks contained more than the weekly changed litter. Built-up litter which had been in use for over one year contained fewer yeast, molds and coliform bacteria than either weekly-changed or unchanged litter. These researchers have also made counts on the bacterial pOpulation of litter used for chick brooding for eight weeks and found a hundred times more molds and yeast, fifty times more coliform, fifteen times more Lactobacilli than was found in built-up litter. However, total counts between unchanged and built-up litter did not vary to a great extent. The aerobic bacterial count was higher in built-up litter than in new, unused litter by 1,000 to 2,000 times. They reported coliform and yeast counts higher in built-up cob litter than in new cobs. . . a..." u L.‘ -. bu ‘Afi' ¥ EXPERIMENTAL PLAN AND STATISTICAL PROCEDURES Experimental Flag The study reported herein includes ten experiments conducted over a period of two years. The report is divided into two main parts with the first consisting of eight experiments on chlorination; the second part consisting of two experiments relating to better sanitation practices. A preliminary study of chlorination of drinking water was carried out to determine how much chlorination chickens can tolerate without curtailing water intake and performance. Growing chicks and laying hens of different breeds and varieties were supplied chlorinated water with chlorine levels ranging from 25 to 2,000 ppm. Experiments with commercially-hatched Leghorn-type hens were undertaken in order to determine the effects of better sanitation practices on egg production and rate of mortality. ‘§£atistica1 Procedures Data from the experiments were subjected to statistical analysis. Data for individual birds were analyzed by the analysis of variance as outlined by Snedecor (1956). The standard error Of the mean was then calcu- lated and Duncan's (1955) multiple range test employed to determine which means were significantly different at both the .01 and .05 levels of probability. Egg production and mortality data for treatments are expressed in percentages, but the actual number of eggs each hen laid over the period was subjected to analysis of variance and Duncan's multiple range tests. The are sine trans- formation Of mortality data was carried out before it was subjected to analysis of variance and Duncan's multiple range tests. 19 PART I INTRODUCTION TO EXPERIMENTS I THROUGH VIII Water is an important nutrient which may easily become contaminated with bacteria, viruses, protozoa and algae (Fair gtmal., 1954). These may sometimes be responsible for an outbreak of disease in poultry. The presence of these organisms in the bird's water supply seems objectionable and consequently their destruction or prevention of growth may be of some signi- ficance for flock owners, but convincing proof of their causation of disease under practical conditions is lacking. Chlorine at levels from 1 to 2 ppm in water is not only bactericidal but also destroys viruses (Anon., 1949). Thus chlorination of water might prevent the spread of any contagious water-borne infections. There is very little information at present as to how much chlorinating chickens can tolerate without affecting water intake and performance. Such information would be necessary if the poultry industry is to take advantage of chlorinated drink- ing water for the improvement of poultry sanitation. Having this idea in mind, chlorinated water of different concentrations was Offered to experimental birds. Data collected were gain or loss in body weight, feed intake, water consumption, rate Of mortality, and egg production. In Experiments III through VIII, group weights were taken and this explains why statistical analyses were not performed. These studies were conducted May through August using 25 to 2000 ppm of chlorine in water. A five percent solution of sodium hypochlorite was used as the source of chlorine. Available Ehlezies-ie-yatss-ve§-ts§Es§-hr-ths-9£tb°t°1idine reaction with the W and 1* * Wallace and Tiernan Comparator, Belleville 9, N. J. 21 comparator. Before actual experimentation with birds, a laboratory study was undertaken to determine whether ch10rine was available in filtrates from chlorinated water-feed mixtures. This was felt desirable because drinking water is usually contaminated to a greater or less degree by feed from the beaks Of the birds. Varying amounts Of the all-mash feed mixture (Appendix Table 1) was weighed in clean, dry Erlenmeyer 500 ml flasks and water having 500 to 1000 ppm of chlorine, was added in amounts as shown in Appendix Table 2. The flasks were shaken vigorously for three minutes and, at intervals, these mixtures were filtered. The filtrates were tested for residual free chlorine by the orthotolidine reaction. In order to get a filtrate from a.mixture of feed and chlorinated water, the amount of water had to be double that of the feed. On shaking 50 grams of a11- mash feed with 70 mls of water having 500 ppm of available chlorine, no residual chlorine was Obtained at ten minutes. When the same amount of feed was shaken with 80 mls of water containing 1000 ppm Of chlorine, the filtrate showed 50 ppm of chlorine after five minutes but there was none at 15 minutes. On shaking vigorously 32 grams of feed with 120 mls «of water containing 1000 ppm Of chlorine, the orthotolidine test revealed 250 ppm of chlorine at five minutes. This availability of chlorine decreased to one ppm within half an hour and there was no available cflnlorine at 45 minutes (Appendix Table 2). PROCEDURE AND RESULTS Experiments I - II. The Effect of Chlorination of Drinking Water on the Performance of Babnghicks Two experiments were conducted each with a different level of chlorination. One-day-Old Single Comb White Leghorn cockerels were divided into equal weight groups and from these groups randomly distributed into the various experimental pens of chick starting batteries. Control replicates were Offered tap water while experimental groups were given chlorinated water Of different concentra- tions. Feed consisted Of a standard chick starter (Appendix Table 1) fed aa libitum. Feed for each group was weighed at the beginning and end Of the experimental periods and the feed consumption and mortality data recorded. Stainless steel water troughs were attached to batteries. Each morning the troughs were thoroughly cleaned and refilled with chlorinated water for the rest of the day. For a period of one week, the unconsumed water in the troughs was weighed and the average water consumption per bird calculated. Water samples were collected daily in sterilized glass bottles containing a. few crystals of sodium thiosulphate and taken immediately to the laboratory for plating on nutrient agar to determine total bacterial counts. An attempt was made to maintain chlorination at its initial levels by adding more chlorinated water twice a day. Dead birds were taken immediately to the diagnostic laboratory for autOpsy. As far as possible the cause of death was determined. On the day the first experiment was terminated, control birds as well as birds from groups where 1500 ppm chlorination levels in water were administered were killed. The intestines including ceca were transferred aseptically to sterilized glass bottles and taken immediately to a laboratory. The intestines were homogenized for two minutes in a sterilized blender with an amount of water double the amount of their weight. The homogenized material was made into a number of 22 23 of serial dilutions so as to have count ranges from 30 to 300 colonies per plate. Agar Pour Plate Method Test -- The purpose of this test was to ascertain the total bacterial population of the intestinal tract. The basic media for the agar plate consisted of: 1. Bacto* - Agar, 15 grams 2. Bacto - nutrient broth, 5 grams 3. Distilled water, 1000 cc. Autoclaved at 15 pounds pressure for 15 minutes. Just prior to plating, the agar media was liquified by heating and cooled to 42° C and then poured over a measured quantity Of intestinal material on the petri dishes. When the agar became solidified, the petri dishes were kept inverted in the incubator for 24 to 48 hours. At 24 to 48 hours the colonies were counted. Experiment I: One hundred and fifty-four day-Old cockerels were alloted to different groups and except controls were supplied different levels of chlorination (100 to 1500 ppm) for a period of three weeks. Experiment II: One hundred and twenty birds were selected at random from Experiment I and redistributed so that each lot had an equal number of birds with approximately the same live weight. Experimental groups were Offered lower levels of chlorinated water (25 to 150 ppm) for a period of another two weeks. The experimental design, body weights, feed and water consumption, as well as feed efficiency data are summarized in Tables 1-1 and 2-1, while the analysis of variance for these are shown in Tables 1-2 and 2-2. Significant differences (P < .01) were found in body weights of different groups when analyzed statistically. Rate of growth Of control birds was significantly * Available from Difco Laboratories, DetrOit, Michigan 24 higher (P ( .01) than experimental birds provided chlorine (250 ppm or more) in drinking water. Chlorination up to 150 ppm had slight but non- significant effect on growth rate and feed efficiency. Depression in growth increased as the levels of chlorine in water increased above 100 ppm. During the third week three birds were found dead in each lot where 1,000 and 1,500 ppm of chlorine was made available in water. Autopsy on these birds did not show any lesion except emaciation and empty crops. The weights of heart, liver and gizzard were proportional to body weights (Table 1-3). Water samples were then plated on nutrient media. No growth of bacterial colonies was Observed. Higher concentrations of chlorine in drinking water had a depressing effect on the bacterial flora of the intestinal tract (Table 1-4). 25 OHN om.N om.m HmH HNH HNH .mm o.oo coma m NmN om.N mN.N mom 00H .mmH .moH m.mo oooH m qom Hm.N mH.N omq omm .ONN HOH o.mw comm Q Hum wN.N oa.m Ono Hmm .NON .mqa o.ow 0mm 0 N¢m mN.N NH.N moq mmm .qom .mqa o.mw OOH m omq NN.N NH.N moo CON .oom .ama m.ww m.o < .me .me .me Ema mwmm B How .mx3 muo .mxB «no .mx3 Mno .mx3 N10 .33 m .x3 N .xB H mHO>mH muCOEumOHH muwn\.mcoo Gwmw\wmmm phan\.mcoo poem munmwmz ammum>< maHHOHso uwum3 .>< Houmz wcwxawun mo cowumcHHOHno mo mam>mq ucmumwwfin :uflz mamnmxooo anonwmq muflnz mo moaowowmmm pooh pow aoHuneamnoo nouns «OxmucH pooh .HnH ofimme 26 Table 1-2. Analysis of Variance of Three-week Body Weights of White Leghorn Cockerels Used in Experiment I Source Degrees Sum of of of Mean F variation freedom squares sgpare ragio F .01 Total 147 498,080 Treatments 5 396,576 79,314 111 3. 02 Error 142 101,504 715 Standard error of mean = 5.4 Treatments: A B C D E F 260 246.5 242 ' 226.5 157 121 Means not underscored by the same line are significantly different (P < .01). 27 m.O 0.NH mm. H.N 0.0 0.0 00N : 0.0 0.HH 0m. 0.N N.N 0.N NmN : H.O 0.0a m0. H.N O.N 0.0 0ON : N.O 0.NH H0. 0.N 0.N 0.0 00N : m.O 0.HH an. m.H 0.N 0.N NON : 0.0 0.0a mm. 0.N 0.N 0.N mmN : N .O 0 .3 mm . H .N 0 .m 0 .0 moN : m.O 0.HH m0. H.N 0.N 0.N RON : H.O 0.NH N5. H.N HaN 0.0 NmN : H.O 0.NH m0. 0.N N.N 0.0 OmN 0.0 H.m m.O 00. 0.0 0.N 0.N 00 : 0.0 0.0 00. 0.0 0.N 0.N mm : 0.0 0.0 Om. H.H 0.N 0.0 0HH : m.O 0.N 0m . H .H 0.N N .m OOH : 0.0 n.0H 00. N.H N.N 0.0 NOH : 0.0 0.0 05. 0.H 0.N 0.0 MON : N.m 0.0 00. 0.N 0.N 0.0 ONH .. .- 0 .m 0 .0 00 . 0 .H 0 .N 0 .O OOH : H6 NK Om. HJ m.N 0.0 m: : n.m 0.0 BO. 0.0 0.N 0.0 00H 00mH mam mam mam m80 800 .umwwvom N mwnm< .us mwom N mfiumd (Jam >000 N mwum< .u3 mHO>OH .u3 wawnmwo .u3 ummwm .uB um>HA %0Om maHnoHso H u:mEHHmaxm moansm woufimanomm mamumxooo OHOuxmmzummunH mo 0HONNN0 0cm uummm anm>wg mo munwwmz .mna wanna 28 Table 1-4. Bacterial Population of Intestines of Three-week-Old Cockerels Used in Experiment I Chlorination Body Bacterial colonies/m1 of levels weight intestinal materia1i ppm gms Thousands 0.3 (control) 292 6,000 " 247 100 " 294 2,000 " 273 640 " 265 6,000 " 242 4,000 " 246 2,000 " 283 a 500 " 252 4,000 " 268 6,000 ” 243 ‘ 8,000 " 287 45,000 " 251 760 1500 105 60 n 117 440 n 144 120 " 174 4:500 n 143 60 n 182 80 n 140 60 " 116 3,500 I! 95 80 " 88 65 ” 192 74 " 117 570 n 132 160 29 00H NH.N H00 000 0.NNN 00H 0 NmH ma.m 0nm NHO 0.NNN 00H 0 00H No.0 O00 0NO 0.NNN om 0 Non oo.m ooo was o.NNN AnonucouV m.o < mam wx3 mum mam mxB mum mxB 0:0 Ema mww\0ufin\msoo cwmwldwmm vufin mmamv 0u00\u3 mao>oa muflmfiMfiWuH umuws Owwum>< \.maou 0mmm meum>< mcHuOqu HH uamEHudem OH 0mm: maonmxoou anonwmq muanz 0HOuxom3um>Nm mo unwwmz cfl CNOQ mm Ham: mm GOHOQEOchU umuwz 0cm 0mmm so Houmzmo cowuwawuoHno mo uommmm .HnN magma 30 Table 2-2. Analysis Of Variance of Body Weights of Five-week-Old White Leghorn Cockerels Used in Experiment II Source Degrees Sum of of of Mean F variation freedom sguares sguare ratio F .01 Total 119 620,483 Treatments 3 11,012 3,671 .7 3.95 Error 116 609,471 5,254 Treatments: D C B A 396 417 420 418 Means underscored by the same line are not significantly different (P<’.05) EXPERIMENTS III, IV and V The Effect of Chlorination of water on Its Consumption by Growing Chickens General procedures for the following experiments were the same, except that the chlorine levels in drinking water were varied. Experiment III was of 15 days duration while Experiments IV and V were continued for 35 days. The chickens in different lots were weighed at the beginning and end of each experiment. The all-mash growing ration formula used is found in Appendix Table 3. Feed was added to the hoppers as necessary to keep an ample supply before the birds at all times. The feed for each lot was weighed at the beginning and end of the experimental periods and the feed consumption and mortality data were recorded. To supply the chlorinated water, an inverted waterer of approximately four-quart capacity was placed in each pen of a battery. This type of waterer exposed a minimum of surface to atmospheric evaporation and contamination. Each day a weighed quantity of chlorinated water was prepared and placed in each vessel; at the end of 24 hours, the unconsumed water was weighed and the daily water consumption per bird calculated. The evaporation was not taken into account because of the fact that only a very small area of water surface was exposed to atmospheric evaporation and all waterers had Openings of the same dimensions. On alternate days, a water sample was collected in a sterilized glass bottle containing a few crystals of sodium thiosulphate. This water sample was immediately taken to the laboratory where it was tested for the presence of coliform and Salmonella organisms. Three times a day a water sample was taken from each waterer and tested for the residual free chlorine by the orthotolidine reaction. 31 32 Experiment III. -- One hundred fifty ten-week-old White Plymouth Rock chicks were distributed equally in five lots each being confined in a separate pen of a finishing battery. Levels of chlorination ranged from 25 to 100 PPm- Experiment IV. -- One hundred twenty five-week-old Cornish Cross chickens were divided into six groups and except control were Offered 100 to 500 ppm of chlorine in drinking water. Experiment VI -- Seventy five five-week-old White Rock chicks were divided into five lots each alloted a separate pen of a battery. Chlorine levels in drinking water varied from 500 to 2,000 ppm. Chlorine up to 75 ppm did not affect the daily water consumption or feed intake. There was a slight reduction in the daily water consumption of chickens at a level of 100 ppm Of chlorine (Tables 3-1 and 4-1). There was a gradual decrease in water intake with increasing levels of chlorine above 100 ppm (Table 4-1). FrOm 100 to 1,000 ppm of chlorine decreased the water consumption but had no appreciable effect on the average daily gain in weight or the feed consumption per bird. Chlorine levels at 1500 and 2000 ppm in water decreased water and feed consumption as well as gain in weight. These results are shown in Table 5-1. Water samples taken during each experiment 24 hours after watering were negative to coliform and Salmonella organisms. The chlorine levels in water after 24 hours depended upon the original chlorine levels as well as upon the amount of the feed added in the waterers by the beaks Of the birds (Table 3-2). 33 Table 3-1. Chlorinated Water ConsumptiOn by Ten-week-Old White Rock Chickens* During a Period of 15 Days Chlorine Water consumed/ Feed consumed/ levels bird/day bird/day ppm gms gms 0.3 147 110 25 145 111 50 148 109 75 148 111 100 134 109 * 30 birds in each group 34 Table 3-2. Free Chlorine Levels in Drinking water After Standing 24 Hours in Drinking Fountain Initial chlorine Chlorine levels after levels 24-hour period* 500 25 H 20 II 0 I! O H 5 II 0 II 35 " 50 1000 150 " 100 ll 10 ll 20 " 600 " 100 " 100 " 500 1500 100 " 800 " 1000 H 20 " 800 " 500 " 1000 " 100 2000 200 " 1000 " 1200 ll 10 " 1200 " 1500 " 500 " 100 ‘* Variable amounts Of feed were found in water troughs. Possibly this might have been responsible for lower levels of chlorine in wate r. 35 Table 4-1. Feed Intake, Water Consumption and Gain in Weight of Five-week-old Cornish Cross Chickens* During a Period of 35 Days Chlorine Gain in wt/ Av. feed intake/ Av. water cons/ level§_1i chick[day chick/day chick/day Ppm gms 1 ng gms 0.3 22.0 66 143 100 23.8 67 134 200’ 21.8 68 115 300 20.3 68 110 400 22.3 66 106 500 20.4 65 93 * 20 birds in each group Table 5-1. 36 Feed Intake, water Consumption and Gain in weight Of Five-week-Old White Rock Chickens* During a.Period Of 35 Days Chlorine Gain in wt/ Feed intake/ Water cons/ leve1a7 chick/day chick/day chick/day PPm Ems gms gms 0.3 27.27 81 282 500 29.35 82 170 1000 27.42 79 144 1500 10.6 65 116 2000 4.9 38 113 * 15 birds in each group EXPERIMENTS VI, VII and VIII Effect of Chlorinated Water on Perfpgmance of Laying Hens Three experiments were performed using different levels ranging from 25 to 2,000 ppm of chlorine in water. For each experiment, White Leghorn- type hens of approximately the same weight that had been in production for a period of five months were selected. Birds were confined individually in wire cages and fed an all-mash egg mash (Appendix Table 4). A11 birds were weighed at the start and end of each experiment. Feed and water consumption and mortality data were collected. The interiors of the galvanized water troughs were covered with polyethylene sheets. More than enough water to satisfy daily needs was provided and unconsumed water from each group at the end of each day was decanted and weighed. Fresh chlorinated water was then provided for the night. Water evaporation was not taken into consideration because all watering troughs were of similar dimensions. The feed hoppers were filled only about half full in order to keep wastage due to "billing out" at a.minimum. Each group was given a weighed quantity of feed which was reweighed at the end of the experiment. Eggs were gathered four times a day and marked for identification. After seven days the eggs were weighed individually. “gaperiment V; -- Forty-eight hens Of approximately the same weight were distributed at random in six groups. To each group a different concentration of chlorine in water varying from 500 to 2,000 ppm was made available. Expepiment V11 -- Seventy-two hens were selected at random and distributed into four groups. Two groups were supplied drinking water with chlorine levels of 25 and 50 ppm. An equal number of birds served as control and were supplied with tap water. 37 38 Experiment VIII -- The groups Of birds consuming water with 25 and 50 ppm of chlorine in Experiment VII were reversed. RESULTS Experiment VI -- Daily feed and water consumption per bird was reduced in all groups supplied chlorinated water. In fact, there was a gradual decrease in feed and water consumption as the levels of chlorine in water increased. This was reflected in body weight changes -- the birds consuming water containing chlorine, 1000 ppm or more, lost 15 to 17 percent of their body weight. Birds in all groups excluding the control, started to molt on the tenth day of the experiment. The severity of molt depended upon the concentrations of chlorine in water and the decrease in feed and water consumption with the increased levels of chlorine. Egg production of birds receiving the chlorinated water was much lower than in the control birds. The average weights of the eggs in all groups for a period Of one week before the experiment and during the experimental period was about the same. These results are shown in Table 6-1 and statistical analysis for this data are given in Table 6-2. Experimeng VII -- Daily feed and water consumption as well as egg production was not affected by 25 ppm of chlorine in water. When the chlorine levels were increased to 50 ppm, a decrease in feed and water intake was obtained. These results are shown in Table 7-1 and statistical analysis for egg production data are given in Table 7f2. Experiment VIII. Chlorine at a level Of 25 ppm increased .daily feed intake or water consumption as well as egg production. On the other hand, chlorination at 50 ppm depressed feed and water intake as well as egg production. It appeared as if there was an improvement trend in the performance of birds consuming chlorinated water at 25 ppm (Table 8-1). 39 H0 u HH msouo an 0NOH wwwm 00 u H msouw m0 0HOH wwwm «k 0HOH wumB wwwm on no 3mm mnm> % x 0.0m 0N H.N N0 Om 000N 0 H> 0N.Om O.mm 0N 0.0 m0 m0 000H 0 > 0.00 0.0m 0N 0.0 00 an 000H 0 >H % 0.00 0N 0.0 00 On 000 0 HHH N.em m.em steam a. own sea m.o on HH .H 080 mam mnH Ema xom3 0H0 as .uqu muommn 000a .u3 avon 0mom uwum3 umumB m0Hw0 aaouu 00m MOO .um|;>< mmwm .02 a“ mmOH vmadmooo mmm. mewxafiuw .Oz Hmuoa mm0\0ufln\mau cw O:HHOH00 vofiwmm mmvnaN w weapon mama 0amhmq a“ ooauoawonm 000 mm Ham: mm «OOHuaEOmcoo umumz 0cm wean no nouns 0awxafiu0 mo :Ofiuwaauoazu mo mHm>OA udoummman mo uommmm .Hn0 macaw 39A Table 6-2. Statistical Analysis for Eggs Produced by Birds Used in Experiment VI Source Sum Degrees of of of Mean F variation square freedom square value F .01 Total 675 39 Treatments 385 5 77 9.1* 3.70 Error 290 34 8.5 * Significant at P (.01 Treatments: Chlorine levels in water (ppm) .3 .3 1500 2000 500 1000 Av. eggs/hen 11.1 10.1 3.6 3.5 3.1 2.9 Means not underscored by the same line are significantly different (P<(.Ol) muonm some aw mvuwn 0H % 40 m.Om O0H 0. + 00 mON 0.0 >H N.nm m0 O.H + 00 m0H 0N HHH 0.0m om 0. + mm 00H 0.0 HH 0.00 0O H.H a on mOH 00 H mam mnH mam new 800 wwu vfima .us 0000 :H hmv\0nwn hmv\0uan Momma wafixcwuw knaouu \u3 .>< ammo .02 OH anew \mooo ween \maoo wmumz OH mHO>OH no mmoq HmuOH cowumm mmvnm m 0cfln50 mama wawmmg you GOHOOOOOHH 000 was cowumadmaoo nouns «mxmuaH 00mm «sowumasmaoo umumz woumaHuOHno .an OHQOH 40A Table 7-2. Analysis Of Variance for Eggs Produced by Birds Used in Experiment VII Source Sum Degrees of of of Mean F variation squares freedom square ratio F .01 Treatments 209 3 69.6 Error 319 41 7.8 8.9* 4.31 Total 528 44 * Significant at P ( .01 Treatments: Chlorine levels in water (ppm) .3 .3 25 50 Av. eggs/hen 5.8 5.0 4.8 2.5 Means not underscored by the same line are significantly different (P ( .01) 41 05090 sumo OH mvuwn 0H % mm O.H + O0 mHH 00 HH 00 N.m + Hm NOH 0N H OQH 8am 0mmw .uB an 0mmw ponds momma kasouu wwwo .Oz damw no mmoa hmw\0uwn pawl. 0cwxcwu0 cmafimnou .mau cw mnHuoHno COHuoswoum wwm 0cm wofiumm mmwuom m wafiuan mamm QHOMOQ a“ cofiuassmzoo momma «OxmunH wwmm «coaumabmcoo noun: woumcfiuoaso .Hu0 manma PART II INTRODUCTION TO EXPERIMENTX IX AND X Floor litter, for the comfort of chickens, has gone through an interesting evolution over the last thirty years. Before World War II, the usual practice was to remove old litter and add new litter to laying and brooding houses every week or so. The purpose of frequent change of litter was mainly to avoid dampness so as to prevent spread of coccidiosis and other infections. As a result of a study made on 26 consecutive broods of chickens, Kennard and Chamberlin (1947, 1948a, 1948b, 1949, 1950b, 1950c, 1951) advocated the use of built-up litter over frequently changed litter. Boughton (1939) and Kennard and Chamberlin (1947, 1949) reported that deep litter was successful for the control of coccidiosis in young chicks. On the other hand, Koutz (1952, 1953) found that all birds on "built-up" litter were heavily infested with parasites such as Eimeria tene11a, Eimeria acervalina, scaridia lineata, Heterakis gallinae and gapilloria retusa. Faddoul and Ringrose (1950), Cotteril and Winter (1953) blamed "built-up" litter as a source of excessive ammonia production and cause of kerato- conjunctivitis in young chicks. For some people, especially flock owners who are interested in the health of their flocks, the conception of "built-up" litter seems contrary to the principles of hygiene and sanitation. SO, it seems that the problem of litter systems for a poultry house is still open for further research. The idea of frequent removal of litter is closely associated with prin- ciples of sanitation. Therefore, a study with laying hens was undertaken in which both treatments, chlorination of water and frequent litter change, were employed singly and in combination. 42 43 The results of this experiment indicate that the use of chlorine in the drinking water and better management practices or both may not be advantageous over the usual tap water and "built-up" litter when judged on the basis of mortality. Since there was considerable variability of diets and/or chlorination, these might have affected the results of better sanitation practices, a study was undertaken the following year when treat- ments included: (1) "built-up" litter and (2) its frequent removal and replacement. Blood chemistry from birds kept on different litter treatments in Experiment IX was similar and, therefore, it was not repeated in Experiment X. The birds used in these experiments had been properly vaccinated against infectious bronchitis, fowl pox and lNewcastle; disease, Management measures were followed in an attempt to keep the experimental birds in a cleaner, healthier environment. The criteria used for evaluation were incidence and rate of mortality, gain or loss in body weights, egg production, egg weight and egg shell thickness measured at regular intervals. EXPERIMENT IX The Effect of Chlorination Of the Drinking watergand Frequent Change of Litter on Egg Production and Incidence of Disaase in Laying Hens At the beginning of the experiment, the floor, walls and other equipment such as water fountains and hanging feeders in the experimental pens were thoroughly cleaned, scrubbed well with two percent lysol solution and dried completely. Control pens had some fresh litter added at intervals of eight weeks. About a half-inch layer or approximately ten pounds of dry, soft wood shavings were spread on the floor in each experimental pen and after every third day this litter was removed and replaced by new litter. At the start of the experiment, all equipment used for handling the litter was new. The birds were held on top of the covered roosts by two wooden partitions during the change of the litter. Fresh litter was stored in an adjacent pen previously disinfected. Droppings and used litter were trans- ported away from the poultry house. The feed hOppers were of the hanging type. Wire guards were provided to keep the birds from getting into them or perching on top, or billing-out the feed. Water fountains, rather than open troughs were used to reduce exposure of the water to contamination. For two weeks before the treatment, the birds to be given treated water were accustomed to drinking chlorinated water from this type of water fountain with levels of chlorine from 5 to 15 ppm. On the day this experiment was started, chlorine levels in the treated pens were raised to 25 ppm and maintained at that point thereafter. At regular intervals water samples were collected for determination of residual chlorine by the W and T Comparator. When the chlorine levels in water were reduced to five ppm, the water fountains were cleaned and refilled with fresh chlorinated water. The control birds were supplied tap water. A foot pad soaked in a disinfectant solution was placed in front of the entrance to each experimental pen. This was to prevent the possible transmission of any 44 45 infective agent via the caretaker's shoes. Plastic sheets separated each treatment group to reduce possible contamination via dust. Every second week, birds were examined by clinical as well as microscopic means to determine any subclinical infectious -- principally coccidiosis, tapeworms and round worms. Any diseased birds were kept in isolation to prevent infection of others. Birds dying during the experiment were immediately taken to the laboratory for necropsy in order to investigate the origin Of any infection. A11 birds were trapnested and their eggs marked for identification. Four-hundred 25-week-old Leghorn-type pullets were randomized into four groups and fed diets containing soybean meal, raw soybeans, cooked soybeans, or a combination of raw and cooked soybeans. These experimental rations are shown in Table 9-1. Each feed was supplied to four replicate pens which were given the following treatments: drinking water chlorinated, litter changed frequently, both water chlorination and frequent litter change, tap water and "built-up" litter (control). On the day this experiment was started the droppings of birds were examined for presence Of coccidiosis and worm infestation. Male birds housed in pens with these pullets were sacrificed and examined. No specific Inacroscopic or microscopic lesions were noted in the sacrificed birds. The coccidial oocysts and nematode ova from feces of birds were found not significant. Hence, no medical treatment was given to these birds before the experiment. At the end of the experiment, blood was collected for the following 1£flx3ratory tests: differential leukocytic count, plasma protein, coagulation tinma, urea nitrogen, and sugar. For differential leukocyte counts, the blcmxi smears were stained by Wright's method and 200 white blood cells counted per? field. The results are expressed as percentages. Five ml Of fresh blood 46 was withdrawn in a centrifuge tube containing heparin and centrifuged. Plasma samples were run on an autoanalyzer* for the determination of sugar** and urea nitrogen**. Plasma proteins were determined with a Hitatchi Refracto- meter**. The plasma was stored at 4° C for three days before it was tested for coagulation time by recalcification of plasma as outlined by Caldwell (1957). The agglutination titre of serum against Salmonella 0 group D*** and §;_g211 antigens O and H was determined both by the tube and rapid slide tests as are reported by Mackie and McCartney (1950). The data on egg production and mortality for the various groups are shown in Tables 9-2 and 9-4. Statistical analysis of this data are given in Tables 9-3 and 9-5. Different diets had a significant effect on egg production, both at the .05 and .01 levels of probability.. The overall egg production was higher (P < .01) on the control diet but nonsignificant differences were obtained between the birds fed the experimental diets. There was a drastic drop in egg production with the hens which received the pelleted concentrate and whole grain. This occurred whether the soybeans were cooked or raw. These results are shown in Tables 9-2 and 9-6. Different management treatments had no significant effect on the incidence Of mortality. However, there appeared to be less (nonsignificant) mortality among the birds fed the basal diet. As shown in Table 9-4, a very high incidence of pickouts occurred among all lots of birds but the number was especially high among those lots fed the experimental diets. Ruptured yolk and/or peritonitis and leukosis ranked second and third, respectively of the causes of mortality. * Technocon Auto Analyzer, Technical Instruments Corp., Chauncery, N.Y. ** These determinations were made by Dr. D. A. Schmidt and his staff of the Veterinary College. *** Difco Laboratories, Detroit, Michigan 47 Final body weights of birds did not show any significant difference among different diets or management treatments. However, there appeared to be greater gain in body weights among birds fed the basal diet or kept on builtlup litter. These results are shown in Table 9-7 and the analysis Of variance for this data are given in Table 9-8. In general, poor fertility and hatchability were maintained by birds fed various experimental diets and given litter change or built-up litter treatments (Table 9-9). There was no difference due to diets and management treatments on average egg weight or egg shell thickness (Tables 9-10 and 9-11). Sera from birds in all groups showed no agglutination with antigens of £1.921; and Salmonella type 0 Group D (Tables 9-12, 9-13). Differential leukocytic counts among birds of each group were similar (Table 9-14). Blood coagulation times did not differ significantly (Table 9-15). Blood sugar levels were significantly higher (P ( .01) among birds having both chlorinated water and frequent litter change. These results are shown in Table 9-16 and statistical analysis of this data are given in Table 9-17. Plasma protein and blood urea nitrogen showed little variation among birds of different groups (Table 9-18 and 9-19). Table 9-1. Percentage Composition Of the Diets Used in Experiment IX Ingredient 1 2 3 4 Soybean Oil meal, 45% protein 21.4 -- -- -- Soybeans, whole raw -- 25.9 12.95 -- Soybeans, whole cooked -- -- 12.95 25.9 Fat, animal 4.50 -- -- -- Corn, ground yellow 59.9 -- -- -- Corn, whole grain gallow -- 59.9 59.9 59.9 Fish meal with solubles, 50% prot. 2.0 2.0 2.0 2.0 Meat and bone scraps, 50% prot. 2.0 2.0 2.0 2.0 Alfalfa leaf meal, 20% prot. 2.50 2.50 2.50 2.50 Limestone, ground 1.64 1.64 1.64 1.64 Dicalcium phosphate 1.20 1.20 1.20 1.20 Salt, iodized 0.25 0.25 0.25 0.25 Vitamin trace mineral mix* 0.25 0.25 0.25 0.25 Antibiotic“ + + + + * NOpcosol M-4, NOpco Chemical CO., Harrison, New Jersey. ** Lincomycin at 50 mt/ton Calculated analyses: Ingredients % Vitamins Amt/1b Protein 17.67 A . IU 4021 Fat 7.27 D3 added ICU 750 Fiber 3.42 B12 mcg 6.26 Calcium 3.0 Riboflavin mg 2.37 Phosphorus .68 Niacin mg 19.89 Arginine 1.02 Pantothenic acid mg 5.79 Glycine .90 Choline mg 537.00 Cystine .25 Prod. energy Cal/1b 983 Methionine .35 Folic acid mg .23 Lysine .90 Tryptophan .19 Calorie-protein 55.5 .— na A; I. . 1 11 Ill . . .w . ..‘ , , . . a l o N . W ‘ . . . i . a o q . o o a , . a . o g . o o o O . Q . Q a ' 49 Table 9-2. Egg Production Percentage With Respect to Diet and Management Treatment Litter Chlorine Litter change Control change in water and chlorine Diet (C) CL) (W) in water (CL) 1 75.5 72.7 66.0 70.0 2 58.4 46.1 49.8 50.0 3 48.2 53.4 57.7 53.3 4 42.2 43.7 62.5 65.5 Diet 1 2 3 4 Average value (%) 71.0 51.0 53.1 53.4 Treatments C 1 W CL Average value 56.1 53.9 59.0 59.7 iMeans underscored by the same line are not significantly different (P ( .01) 50 Table 9-3. Analysis of Variance of Egg Production Data With Respect to Diet and Management Treatments (168-day trap period) Source Sum Degrees of of of Mean F .xapgation squages freedom sqpare _£atio F L01 Diets 32,196 .3 10,732 25.1 3.88 Treatments 207 3 69 .16 3.88 D X T 7,319 9 813.20 1.9 1.92 Error 99,296 232 428 Total 139,018 247 Diets 1 2 3 4 Av. values (%) 71.0 51.0 53.1 53.4 ‘Management treatments C L W LW Av. values (%) 56.1 53.9 59.0 59.7 Means not underscored by the same line are significantly different (P ( .01) 51 Nm>HH mo cofiumhmamwmv huumm H mm mHuHunamz u z mwusvmoonm huoumuoan How OOOHMHNOmm u m mHuHcoowuom 05m 3H0» wousumam u muwm momma OHMHOOmchO Hmsuo u N mHuHmzomHH 0am mHuHmach u Hum uosuH>O wmuommEH n 01H manoso H300 u om mHmOxde u H musoxowm n m ”monwch Ou hmm «k 00. m um unmomecmecoz s OH N N O N O NH O H m0 mHmuoa H H H oH N.O ONm .Ho - mmuuHH m H H H H m m.NH o 0 .Ho H H O N.O umuuHH H 0 N.O Houuaou O H H H m m.NH on.Ho - umuuHH H H N O.w ON: .Ho m m H H aH O.N umuuHH H H H H O 0.mN Honucoo m N H H H o m.m ON: .Ho - mmuuHH N 0.0 0 0 .H0 H H O O.N umuuHH H N 0H n.0H Houunoo N H N N.O 0N: .HO . umuuHH H H H N.@ ONO .Ho H H m n.m NmuuHH H H H 0.0 Houucoo H m x o-H H on 2 N-wm a-m on N Hausuocsa unmusmmrx unmawmmus umHn Awmmmov sawdeaHm Emuuofiumom mupoxOHa mmmHv «NOHHmuuoz_N muGOEumOHH 05m mumfin moowum> Hows: huHHmuHoz_mo mmsmo 0am moamvwoGH .Onm mHnma 52 Table 9-5. Analysis Of Variance of Mortality Data for Birds in Experiment IX Source Sum Degrees of of of Mean F variation square freedom square ratio F p.01 Treatments 110.52 3 36.84 2.3 6.9 Diets 142.89 3 47.63 3.01 6.9 Error 142.20 9 15.8 Total 395.61 15 Diets 1 2 3 4 Av. value (%) 13.7 18.2 20.8 14.02 Management treatments C L W LW Average value (%) 13.6 17.2 20.7 15.2 Means underscored by the same line are not significantly different (P < .01) 53 Table 9-6. Percent Egg Production Under Various Diets and Management Treatments Computed on basis of Total Number of Live Birds Present at the Beginning Of Each Month Diet Treagment* Dec. Jan. Feb. Mar. April May 1 L 75.2 82.1 70.0 71.1 70.1 68.0 W 57.6 68.0 64.3 72.4 66.9 66.2 WL 70.6 72.0 70.2 69.4 67.6 66.2 C 71.4 64.0 67.5 81.1 84.0 84.2 2 L 45.6 47.9 35.7 44.1 58.6 64.7 W 44.6 41.2 47.0 40.1 64.6 61.3 WL 43.9 32.8 28.2 60.1 66.7 68.4 C 94.8 36.6 11.3 20.8 57.7 90.3 3 L 42.8 33.8 30.5 39.9 83.3 90.3 W 64.3 54.5 48.6 54.2 59.5 65.0 WL 55.0 47.5 47.5 52.2 57.4 60.1 C 48.6 41.2 28.2 55.1 58.2 58.0 4 L 53.7 41.2 37.0 44.5 42.5 43.0 W 66.3 61.4 47.7 56.5 67.1 75.4 WL 63.9 54.9 59.4 84.1 60.0 70.8 C 43.5 22.2 31.6 55.0 53.3 47.0 * W = Chlorinated water L = Litter change WL = Chlorinated water and litter change C = Control 54 »Table 9-7. Average Body Weights (lbs) of Birds on Different Diet and Litter Treatments Diet Treatment* Initial Fina1 Change 1 L 4. 2]. 3. 90 - o 3]- C 4.00 4.60 + .60 2 L 4.10 4.05 - .05 C 3 92 4 03 - .ll 3 L 4.02 3.85 - .17 C 4.05 4.26 + .21 4 L 4.02 3.79 - .23 C 3.85 4.00 + .15 litter change ,1. 1" II II Built-up litter 55 Table 9-8. Analysis of Variance of Final Body Weights of Birds on Different Treatments Source Degrees Sum of of Of Mean F variation freedom square sqgare ratio F .01 Total 85 25.79 Treatment 7 7.28 1.4 .59* 2.95 Error 78 18.51 .24 * Since F ratio was not significant both at .01 and .05 level of probability, the further analysis of cell totals was not performed. Diet 1 2 3 4 Average value 4.28 3.9 4.2 3.77 Treatments C L Average value 4.03 3.9 Means underscored by the same line are level of probability. not significant at 0.01 or 0.05 56 0.00 O 5.00 Owamzo HmuuHH 0.00 m N.m5 N O.m5 Ownmno HouuHH 0.00 fi 0.00 m 5.05 N m.mw Houucoo mudeumOuH O.NN HNV msHm> .>< H mumHn "NHHHHHweoumm 0.05 ARV OsHm> .>< HOHuGOU "mudmfiummHH 0.0s HevmaHm> .>< H mumHn “NNHHHOHON NMOEEOm .mmum0 uoquMMHc um umm wwwm mo nonapd Osu mmumowvcH mHmOnucOuma OH amass: one o.HN O.oN HONV o.Nm o.mN HONV o.mN N.m HHOV «Nessa umuuHH a.mm N.NN Hmsv N.NN o.ow Hosv o.ooH N.Om HNHV Houucoo O 0.NN N.NN ANNV o.os o.HN HNV H.NN 0.Ns Hssv «Names umuuHH N.ma 0.NN HOmV o.NN a.mN Hmmv o.ooH O.N HNOV Hopscoo N -- -- 0.40 o.mN HHmv 0.Nm O.Nw Hva magmau umuuHH H.om N.Nm Ammv -- -- HNV N.Nm o.ms HQOV Houucoo N N.Oa 0.0N HONV o.Om o.om HONV a.mm o.om HNoHV mwcmao HouHHH N.NN N.Om Homv o.Hm o.om Hmsv N.oa N.NN Home Houucou H Soumm N .unm N Soumm N .flWWm N .Soumm N111WMWWW N quEMMWHH HOMO NomH .N .HQO NONH qHH eons: NomHlaHN .qme muamaummua umuuHH 0:1uHH50 0am Owcmno NOOOHH cm>H0 mwuHm Eoum uom mmwm OHHuHmm mo noumm unmoumm 0am OHHqum unmowmm .mum mHan 57 HHO.v mv uoOHO00H0 hHucmOHmHamHm no: mum uOquH HmuHmmO mama 0GH>mn mums: % umum3 mo cOHuOGHHOHSO O.NH m.ON N.Om no HOV o.oN was swamno umuuHH a.mH N.ON N.om mm HOV o.so amass emumcHHoHeu N.NH m.ON N.Nm mN HOV N.NN mwcmeu smuuHH 5.mH 0.05 N.mm 00 A40 0.05 Houuaoo H HagaH Hoo.v Hmamv mmmchHau muoom .u3 .>¢‘ wwwo «Oz t>m0\ams uaOEAmOHH uOHQ HHonm swam: .OOHQ unwoumm HHEm umm;NuHHmuu;.mNHm mucOaumOHH ucmummmHa HO0OD mam: aoum wwwm mo muHHmdo 05m aoHuOO0oum mo Nwmaasm .0Hnm OHan Table 9 ll Diet (‘2 Diet ‘* E 58 Table 9-11. Effect of Different Diets and Treatments on Egg Weights* Litter change Chlorine in change and No. Litter drinking chlorine in Diet eggs Control chapge water water (3%) (ng) (ng) (gm) 1 160 55.2 53.3 56.7 54.2 2 150 54.8 55.1 53.3 53.0 3 180 48.2 52.4 55.2 55.9 4 140 51.5 56.1 56.0 56.9 Treatments 52.4 54.2 55.3 55.0 Diets 54.8 54.0 52.9 55.1 * Expressed in grams 59 Table 9-12. Summary of Results Obtained From Agglutination Test Using Salmonella antigen 0 Group D* Total Treagments* birds Chlorine Reaction Diet tested Litter water Both Control (+ or -) 1 8 x . - 2 6 x - 3 5 x - 4 1 x l l 3 x - 2 3 x - 3 5 x - 4 4 x - 1 6 x - 2 6 x - 3 8 x - 4 5 x - 1 5 x - 2 1 x - 3 9 x - 4 5 x - .Available from Difco Laboratories, Detroit, Michigan .X 7:; **' represents treatment 60 Table 9-13. Summary of Results Of Agglutination Test Using1§1 coli Antigens* No. serum Reaction samples Management with aptigen Diet tested treatment H 0** 1 8 Chlorine water + litter change 1 - 8 Control 1 - * Supplied by Dr. D. E. Schoenhard and his staff, Dept. Of Microbiology and Public Health. Test tube consisted of 0.5 ml of serum and 0.5 m1 of antigen. Test tubes were incubated for 12 hours and refrigerated afterwards for 4 hours. ** Prepared from H antigen by boiling for 1/2 hour in a water bath, centrifuged, washed bacterial sediment with normal saline. Reconstituted with same amount of normal saline. 61 Table 9-14. Differential Leukocytic Counts (percentages) From Birds * Kept Under Different Management Treatments Treagment Lymphocyte Monocyte Heterophils Eosinophils Basophils Control 70.25 2.0 23.1 1.87 2.75 Litter change 68.10 2.5 25.2 1.90 2.25 Litter change and 67.10 2.0 27.7 1.37 1.75 chlorine in water * Birds fed diet 1 62 Table 9-15. Blood Coagulation Time for Birds Kept on Different Diet and Management Treatments No. of Average time Diet sample Treatment (seconds) 1 6 Chlorinated water and litter change 56 6 Control 70 3 5 Litter change , 66 5 Chlorinated water 80 63 Table 9-16. Summary of Blood Glucose Levels in Birds Receiving Different Management Treatments NO. samples Av. blood Diet Treatment examined glucoae (mg_%2 1 Control 22 226 (A) Litter change 22 222 (A) Litter change and chlorinated water 22 242 (B) ‘Means not indicated by the same capital latter are significantly different (P ( .01) 64 Table 9-17. Analysis of Variance of Blood Sugar Levels Among Birds Kept on Different Management Treatments Source Degrees Sum of Of of Mean F variation freedom squares square rapio F .01 Total 66 19,134 Treatments 2 4,991 2,496 11.3 4.98 Error 64 14,143 221 Treatments C L LW Average Values 226 222 242 Means not underscored by the same line are significantly different (P <.01). 65 Table 9-18. Summary of Blood Urea Nitrogen Levels in BirdskReceiving Different Management Treatments No. of Av. blood urea Trgatment samples nitrogen (mg %) Control 22 1.89 Litter change 22 1.86 Litter change and 22 1.61 chlorination of water * Birds fed diet 1 66 Table 9-19. Summary of Plasma Protein Levels in Birds Receiving Different Management Treatments NO. of Av. plasma protein gms/ Diet Traatment samples 100 ml of blood 1 Control 22 6.75 Litter change 22 7.13 Litter change and chlorinated water 22 7.52 EXPERIMENT X The Effect of Various Diets and Litter Treatments Upon The Incidence of Mortality in Laying Hens. All the experimental procedures were the same as described for Experiment IX with the following additions or modifications. Six-hundred 24-week-Old, commercially-hatched White Leghorn-type hens were randomized into six groups, based on weight and egg production, and fed the control or experimental egg-laying diets. The dietary treatments consisted of soybean Oil meal and raw ground soybeans with and without supplementation of methionine, sodium carbonate, detergent and antibiotic (Pro-strep, a combination Of one part procaine penicillin and three parts streptomycin). There were four replicate pens on each diet and pens having odd and even numbers were given different litter treatments. The compositions of the basal rations and experimental plans are shown in Tables 10-1 and 10-2. Different litter treat- ments employed were as follows: A. Frequent change Of litter. From pens having Odd numbers, old shavings were removed and replaced by a %-inch thick layer Of new shavings once a week. B. Built-up litter: At the start Of the experiment, pens having even numbers were provided 8-10 inches of new shavings. At intervals of eight weeks, some fresh litter was added and stirred lightly into old litter. Chlorination of drinking water as used in previous Experiment (IX) was not attempted. For control of pickouts, specks were used. Two replicate pens from diet 1, with different litter treatments were SGIected for bacteriological, coccidiosis and moisture determination studies. 67 68 Samples of litter and droppings were examined to determine whether any oocysts present were sporulating. An attempt was made to differentiate pathogenic species of coccidia, as they existed in these samples. For this purpose, the sugar flotation concentration method (Morgan and Hawkins, 1953) was used as follows: The collected samples were mixed with water, allowed to stand for one-half hour, mixed, strained through a layer of cheesecloth, and poured into two 15 ml, round-bottom centrifuge tubes (approximately one-quarter full). Saturated sugar solution was added until the tubes were filled. These tubes were then centrifuged for one minute at approximately 1,000 r.p.m. Using a beaded glass rod, the material from the surface solution was transferred to a glass slide and covered with a coverslip. An examination Of this material was made using a compound microsc0pe (lOQX), for the presence of oocysts. The oocyst counts from litter and drOppings were made as reported by Young (1929) with the following additions or modifications: five grams Of fresh droppings were collected and carefully weighed into a lOO-ml Erlenmeyer flask. Forty-five milliliters of distilled water were added and the contents of the flask shaken vigorously until the total mass became thoroughly emulsi- fied. Ten milliliters of the emulsion which contained one gram of the sample were transferred to a round-bottomed 15 ml centrifuge tube. An electric centrifuge at a low speed of 1,000 r.p.m. for a period Of one minute was used to throw down the oocysts to the bottom of the tube, and the supernatant fluid ‘was decanted. To the residue, a sufficient amount of saturated sugar solution ‘was added to bring the level of the liquid to the upper edge of the centrifuge tube. A thick coverslip (No. 3) was placed over it in such a way that no air bubbles appeared below the coverslip. A low centrifuge speed Of about 1,000 r.p.m. continued for about one minute to force the oocysts to the top of the tube where they were collected on the undersurface of the coverslip. {rhe coverslips were removed and mounted on a slide. The oocyst counts were 69 made using the low power lens of the microscope. This process was repeated until no oocysts were found in the specimen preparation. The total of the amounts Obtained from individual coverslips gave the number Of oocysts in one gram of the sample. Dead birds from these pens were especiallychecked for coccidiosis. For this purpose, smears were made of intestinal scrapings to detect presence of oocysts. To pursue further any infectiveness of different litter treatments, a study was undertaken with baby chicks as follows: 200 one-day-Old, incubator hatched White Leghorn, straight-run chicks were distributed into two brooder pens each with a different source of floor litter. Used shavings from two replicate pens receiving different litter treatments were spread on the floor of two brooder pens. The remaining two replicates continued as originally planned. This experiment was continued for a period of 21 days. During this period the droppings of these young chicks were closely examined for blood or mucous discharge. At regular intervals, feces from these chicks were collected and examined for presence of oocysts. At the end of the first and second week of the experiment, 10 birds per treatment were sacrificed and intestines including ceca were examined for coccidiosis. A chick starter containing no antibiotic or coccidiostat (Appendix Table 5) was fed.ag libitum. Each group was given a weighed quantity of feed which was reweighed at the end of the experimental period. All birds were regularly fed and watered every day. When birds died, a necrOpsy was performed to ascertain whether or not coccidia could have caused the death. Group weights were taken as soon as the birds were put on experiment and at the end of the second week. Individual weights were recorded at the end of the experiment. At this time, 25 birds per group were removed to an avian necropsy room and sacrificed and their intestines including ceca checked for coccidiosis lesions. Any lesions were further checked by either of the following methods depending on their nature. 1.61. 70 l. Intestinal epithelium was scraped and smear preparations were examined using a low power of microscope. 2. Intestines including ceca were removed and cut lengthwise. The epithelium was scraped and contents were drained into a 50-ml Erlenmeyer flask which was one-half filled with tap water. This material was placed in a Wering blender and subjected to its action for 1.5 minutes. The material from the blender jar was poured into two 15-ml centrifuge tubes and centrifuged at approximately 1,000 r.p.m. for two minutes. The supernatant was poured off and the tubes refilled with tap water. This procedure was repeated until the supernatant was clear. The supernatant was then removed and saturated sugar solution was added. The rest of the procedure was the same as described on page 68. To determine the moisture content, samples Of both types of litter were collected and twenty-gram samples were weighed into well-cleaned and dry beakers. These were dried completely in an oven at 100° C. for 48 hours and reweighed. For bacteriological studies, ten grams of the well-mixed representative litters were placed in a sterile Erlenmeyer flask and 90 grams of sterile distilled water added. This material was then shaken vigorously (about 150 times) and filtered through sterile cheesecloth into another sterile container. .Filtrate was made into a number Of serial dilutions before plating so as to have count ranges from 30 to 300 colonies per plate. The method of preparation of nutrient media used in this study has already been described under Experiment I on page 23. The criteria used for evaluation of different litter treatments were :rate of mortality, relative infectiveness of the litter to chicks, rate of snag production, egg size, fertility and hatchability of eggs from the hens 71 receiving different management treatments. Initial and final body weights Of the experimental birds were also determined. During each month one week's eggs were broken out in order to determine differences in the number of bloodspots, weight, Haugh scores and shell thickness of eggs-produced by the experimental birds kept on different management treatments and fed diets having different soybean variables. Various litter management practices under different diet treatments had statistically nonsignificant (P <..05) effect on egg production and 'mortality rate. Egg production for birds kept on litter change treatment was higher than for those kept on built-up litter. There was significantly (P ( .05) less mortality among the birds receiving diets 19-1, 10-2 and 10-3. These results are summarized in Tables 10-3, 10-5 and 10-6. Egg production in these birds for January was higher than that for February and March (Table lO-Zaand 10-7b). Death rate for a period of four months (December to'March) ‘was relatively higher than during October and November when these birds were not in production (Table lO-SA and lO-SB). Leukosis, ruptured yolk and peritonitis were the primary causes of death. Other isolated cases which occurred included sinusitis, nephritis, synovitis, CRD, fatty degeneration of liver, lymphoid mesenteric tumor and impacted oviduct. Once again, the incidence of the visceral form Of leukosis 'was predominantly higher than its other forms. Infections associated with the reproductive system were more common than others. Mortality data are also shown in Tables 10-3 and 10-5 and the analysis of variance in Table 10-4. The egg production records were maintained for five months, with ‘trapping for 110 days during the period (November through March). Variability in diets had a significant effect (P ( .01) on egg production. During this five-month period of time, egg production was significantly depressed (P (.01) tnr'ration 10-2. The soybean Oil meal containing ration (Diet 10-1) produced timz best results but this rate of production was not significantly better 72 from that produced by the experimental diet (10-4) containing raw soybeans with supplemental methionine and antibiotic, Pro-strep. The egg production on diet 10-3 which contained 0.16 percent methionine plus 0.1 percent of sodium carbonate was not statistically different from that produced by diets 10-1, 10-4 and 10-6. These results are shown in Tables 10-6, 10-7 and the analysis of variance are given in Table 10-8. The percentage of bloodspots in eggs examined was very low and the inclusion of various soybean variables in the ration under different management practices did not produce an increase in their incidence. There were no differences due to diets and/or management treatments in average egg weight, Haugh scores or egg shell thickness (Table 10-9). The weight of the birds kept on litter change treatment was not statistically different from the weight of the birds housed on the built-up litter floor. The birds on each diet gained weight. The birds receiving diet 10-4 gained more than any other group. The differences in final body weights reflected to an extent the adequacy Of the diets being fed. The results are summarized in Table 10-10. Eggs laid during a five-day period in December, January and February were incubated and the percentage of fertile eggs and the percentage hatched of fertile eggs determined. Satisfactory fertility and hatchability were maintained on most of the experimental rations. However, the fertility and hatchability produced by diet 10-1 was very low. This depression was due mainly to one replicate pen Of this diet. No explanation seems apparent for these results. Perhaps the males present in these pens lacked fertility CTable 10-11). Total bacterial counts per gram of litter did not differ significantly among the different treatments (Tables 10-12, 10‘13). However, the moisture content of the litter changed frequently was higher than that of built-up litter. The third day after changing litter, the moisture content was as high 73 as that of built-up litter. In pens where litter was changed, the floor litter appeared damp and in poor condition throughout the winter months. In the adjacent pens with built-up floor litter, the litter was in good condition. Of course, temperature at the top of the litter or down at the floor did not vary in pens having different litter treatments. These results are shown in Table 10-14. Various litter treatments had similar effects on oocyst contents of litter and drOppings. However, slightly lower counts were Observed in droppings of birds housed on built-up litter.'§1 Acervulina was most widespread specie of coccidia found in a greater number of the samples of litter and drOppings examined in both litter treatments. Ascarid ova were seen only in two samples examined from built-up litter floor pens. These results are shown in Tables 10-15 and 10-16. There were non-statistical (P‘< .05) differences in feed conversion and gain in body weights of chicks reared on different types of litter treatment. Greater gains in body weights and better feed efficiency resulted with chicks reared on litter transferred from pens where it was changed frequently than in chicks raised on litter from built-up litter pens. These results are shown in Table 10-17 and the analysis of variance for these data are given in Table 10-18. During the first weeks of the experiment, when birds were sacrificed, the number Of infected birds and the presence of hemorrhagic ceca varied with the group. At the end of the experiment, the sacrificed birds failed to show any evidence of coccidiosis infection, except for a few oocysts present in the cecal contents (Table 10-19). During the first three days of the experiment, nine chicks were found dead in both experimental groups. Autopsy on these birds showed absence of hemorrhages in ceca, as well as absence of merozoites in cecal and 74 intestinal scrappings. Pathological conditions Observed in these birds were air sacculitis, peritonits and other nonspecific conditions. 75 Table 10-1. Experimental Plan and Composition of Original Six Experimental Rations for Experiment X Ration Pen “ppmber number* Composition** 10-1 9, 18, 37, 46 Positive control ration using 44% protein soybean meal 10-2 4, 11, 36, 43 Corn, raw ground soybeans plus .16% methionine 10-3 10, 17, 38, 45 As 10-2 plus 0.1% sodium carbonate 10-4 8, 15, 40, 47 As 10-2 plus Pro-strep (50 gms/ton) 10-5 5, 14, 33, 42 As 10-2 plus .05% detergent (Solar F-221, Swift & Co.) 10-6 7, 16, 39,48 As 10-5 plus Pro-strep (50 gms/ton) * Pens having Odd numbers were given litter change treatment. ** See Table 10-2 for the exact amounts of ingredients used. 76 Table 10-2. Composition of Layer-breeder Rations for Experiment X Percent of ration No. lygredients 10-1 lO-gfi 10;;1 10-4 10-5 10-6 Corn, ground yellow 59.90 59.90 59.90 59.90 59.90 59.90 Raw soybeans, ground -- 21.40 21.40 21.40 21.40 21.40 Soybean oil meal, 44% prot. 21.40 -- -- -- -- -- Fish meal and solubles, 60% 2.0 2.0 2.0 2.0 2.0 2.0 prot. Alfalfa leaf meal, 20% prot. 2.50 2.50 2.50 2.50 2.50 2.50 Limestone, ground 6.0 6.0 6.0 6.0 6.0 6.0 Dicalcium phosphate, ground 1.20 1.20 1.20 1.20 1.20 1.20 Salt, iodized .25 .25 .25 .25 .25 .25 Fat, animal 4.50 4.50 4.50 4.50 4.50 4.50 Vitamin-trace mineral mix* .25 .25 .25 .25 .25 .25 DL-methionine -- .16 .16 .16 .16 .16 Sodium carbonate -- -- 0.1 -- -- -- Detergent (Solar F-221) -- -- -- -- .05 .05 Antibiotic** -- -- -- + -- + ** Nopco Chemical CO., Harrison, New Jersey ** Pro-strep 50 gms/ton Calculated analysis for ration: Protein % 17.67 Glycine % 0.90 Fat % 7.27 Methionine % 0.35 Fiber % 3.42 Cystine % 0.25 Calcium % 3.00 Lysine % 0.90 Phosphorus % 0.68 Tryptophan % 0.19 iArginine % 1.02 Vitaminjs: Amt/lb Vitamins Amt/lb B IU 4021 Niacin mg 19.89 D3(added) I.C.U. 750 Pantothenic acid mg 5.79 E mg 5.04 Choline mg 537.00 Bl mcg 6.26 Folic acid mg 0.23 Rigoflavin mg 2.37 Prod. energy Cal/1b 983. 77 mommo OHHHOOOOOOO HOSOO mHuH>Ocmm OHOHueaOz mHuHumamm moan» OHuouanoE OHonaezH musoxOHm mHmoumaosgamHuousoz Egnagifi:2:g;cp OHOOOOEOHQBNH HOHOOOH> u H> uO=0H>O OOOOOOEH n 02H OHOHOOOHHOQ HO\0:O xHO% wmnaumdm u annm OOOOOHO NHOOOHHOOOH OHcouso u 000 OHuHonomuu 0cm mHuHmaon I Hm "OmchcHw ou >OM ttx HOOOHH as-uHH=N m Owcmno HOOOHH u o «% mo.o um HOOOHOHOOHOO H N H m H mH N H H Hmuoe H O m H m H OH O O H N N OH O H H 0 NH 0 m H H H H H NH O m H oH O O H N m H O 0 m H N O 0 N H H O m H H o O H am 0 H2O m (Hz H> muwmml HMO Hm ON! sauces umHn *ktmon0aHm Emuuoaumom 3H Hmunoz nummua mucmeumwuH Hassommamz 0cm mumHn msoHum> nova: NOHkuuoz mo mocmeucH .mnoH OHHOH 78 Table 10-4. Analysis of Variance of Mortality Data for Birds in Experiment X Sum Degrees of of Mean F Source squares freedom square ratio F .05 Treatments 14 1 14.0 3.9 6.61 Diets 194 5 38.8 10.8* 5.05 Error 18 5 3.6 Total 226 11 Standard error of the mean = 1.33 * Significant at P ( .05 Treatments: C B Means 7.66 6.00 Diets: 4 5 6 l 2 3 Means 22 22 14 10 8 6 IMeans not underscored by the same line are significantly different at 0.05 level of probability. 79 Table 10-5A. Summary of Postmortem Findings Showing Incidence of Leukosis and Reproductive Disorders Under Various Diets and Management Treatments Leukosis Diseases including associated other with reproductive Diet Treatment* tumors system Others 1 C 1 1 1 B 1 1 - 2 C - - 2 B l - 1 3 C - B - 1 - 4 C 3 1 1 B 2 2 2 5 C 3 - 3 B 2 3 - 6 C 3 2 - B l - 1 Total 17 ll 13 Bitter change 3(- 0 II II Built-up litter 80 Table 10-5B. Monthly Incidence of Mortality in Various Experimental Pens Under Different Diets and Treatments Pen Monthly av. Diet no.* Oct. Nov. Dec. Jan. Feb. Mar. mortality (7.) 1 9 - - - - - 1 .67 37 - - - l l - 1.33 18 - - - - - - 0 46 - - - - 1 1 1.33 2 11 1 - - - - - .67 43 - l - - - - ..67 4 - - - - - - 0 36 - - - - - 2 1.33 3 17 - - - - - 1 .67 45 - - - - l - .67 10 - - 1 - -~ - .67 38 - - - - - - 0 4 15 1 - - l - 1 2.0 47 - - - - 1 1.35 8 - - - l - - .67 40 - - 2 2 1 - 3.33 5 5 - - - 2 2 1 3.33 33 - - - ~ - .67 14 - - - - 1 - .67 42 1 l l - 1 - 2.67 6 7 - - 2 - 1 - 2.0 39 - - - - l 1 1.33 16 - - - - l - .67 48 - - - - - 1 .67 ‘* Odd number pens were given litter change treatment. Each experimental pen had 25 birds at the start. 81 Table 10-6. Summary of Egg Production and Mortality (percentage) With Respect to Diet and Management Practices Av. egg production Diet Treagment* (Nov. - Mar.) Mortality (7.) (7°) 1 C 57.4 6 B 58.2 4 2 C 45.6 4 3 C 56.6 4 B 50.9 2 4 C 56.5 10 B 57.2 12 5 C 51.4 12 B 56.4 10 6 C 58.7 10 B 53. 8 4 * C = change of litter B = Built-up litter Diets l 4 6 3 5 2 Av. prod. (%) 57.8 56.8 56.7 53.8 53.7 46.6 Treatments: C B Av; prod. (%) 54.4 54.0 IMeans not underscored by the same line are significantly (P ( .01) 82 Table 10-7A. Percent Egg Production Under Various Diets and Management Treatments Computed on the Basis of Total Number of Live Birds Present at the Beginning Of each month Average Treat- production Diet ment* Nov. Dec. Jan1 Feb. March Dec.-March l C 12.5 66.5 71.0 67.6 62.6 66.9 B 9.0 67.8 68.0 70.0 71.3 69.3 2 C 7.0 56.4 57.8 47.1 55.2 54.1 B 9.2 55 8 57 2 52.9 59 2 56 3 3 C 11.9 64.7 68.3 64.0 69.4 66.6 B 15. 63 0 59.3 56 4 57 6 59 0 4 C 12.5 64.9 69.8 61.6 70.6 66.7 B 12 9 71 0 67.7 60 7 66 4 66 5 5 C 11.9 57.2 61.1 62.2 61.7 60.6 B 8.5 61 9 69.9 64 2 68 3 66 0 6 C 11.5 71.1 68.2 63.1 71.8 68.6 B 10.0 65.3 61.4 60 8 65 1 63 2 * C litter change U5 II II built-up litter 83 Table lO-7B. Percent Egg Production Under Various Diets and Management Treatments Computed on Basis of Total Number of Birds in Production Each Month Diet Treapment* Nov. Dec. Jan:= Feb March 1 C 22.9 82.1 79.0 78.0 75.5 B 16.2 88.8 80.9 83.2 87.7 2 C 19.0 85.1 78.2 68.8 79.5 B 12.3 76.1 64.7 65.5 71.1 3 C 20.8 96.5 72.9 70.4 75.5 B 16.2 82.9 72.8 71.0 68.8 4 C 24.0 89.9 83.0 72.4 79.4 B 22.4 93.8 78.2 75.5 81.1 5 C 15.9 76.1 68(2 68.3 75.4 B 16.9 79.9 76.0 66.8 75.1 6 C 19.9 84.1 76.6 73.0 80.8 B 17.7 85.5 72.4 77.0 76.3 * C = Change Of litter B = Built-up litter 84 Table 10-8. Analysis of Variance of Egg Production Data With Respect to Diets and Management Treatments Source Degrees Sum of Of of Mean .gapgation freedom_ squagas square F F .01 Diets (D) 5 4673 934.6 7.3* 3.06 Treatment (T) 1 492 492 3.84 6.76 Interaction (D X T) 5 823 164.6 1.28 Subtotal 11 5988 Error 408 52239 128.0 Total 419 58227. * Significant both at .01 level of probability Diets 2 5 3 6 4 1 Means 46.6 53.7 53.8 56.7 56.8 57.8 Treatments C B Means 54.4 54.0 Means not underscored by the same line are significantly different (P < .01) 85 umuuHH mauuHHom u 0 HOOOHH mo mwcmno u 0 x H N.OH N.NN N.N N.NN NO N amuuw-0HN N N.NH N.HN N.O N.ON NO O aou\mew ON + N-oH OOHO O-OH H O.NH N.NN N.N N.ON NO N HOHHsN .HNN-H HmHomO N o.NH N.NN N.O a.mm NO O HOONHOHOO NNO. + N-OH OOHO N-oH N N.NH o.HN 0.0 H.ON NO N amuum-oum H N.NH N.HN N.O N.ON NO O aou\meN ON + N-oH OOHH O-oH N N.NH N.NN o.O H.ON NO N N N H N.OH N.NN H.O o.ON HO O OO «2 NH.O + N-oH OOHO N-oH o o.NH N.NN 0.N N.NN NN N OOHOOHHHOE NOH. maHa H N.NH N.ON N.O N.NN NO O .cnoo .Heasouwv .:OOHNOO awe N-oH o a.mH N.ON N.N 0.0N NN N HmOE GOOQNOO H 0.0H N.NN H.O N.NN NO O .auou .Houucou O>HHHmom H-oH HHOOH Hoo.v _ OOOQOOOOHQ mmochH£u OHOOO uanmn mmev ammo tucofi GOHuHmOQEOU OOHQ umnaaz HHmnm swamm aoaanH< .u3.>< .Oz numONH HHmnm 0cm NOHHmaulaomHm mucmaummua Osmammmcmz.0cm mumHn HmucmeHuwmxm ucmummmHn 0OH>HOOOM Odom Eonm mmwm mo huHHmso 0CH30£m mumeaom .mnOH OHHOH 86 Table 10-10. Average Body Weights (lbs.) of Birds Kept on Different Diets and Litter Treatments During Experiment X Diet Treatment* Initial Final Change 1 C 2.81 3.99 +-1.l8 B 2.85 4.08 + 1.23 2 C 2.94 3.88 + .94 B 2 86 3.73 + .87 3 C 2.98 3.93 +- .95 B 2 82 3.90 + 1.08 4 C 2.98 4.22 + 1.24 B 2 91 4.08 + 1.17 5 C 2.78 3.76 + .98 B 2 86 3.90 + 1.04 6 C 2.84 4.08 + 1.24 B 2 91 4.11 + 1.20 Built-up litter x. 0 II II Litter change H.HN N.NN N.NN N.NO N.NN N.NO HHO mosHm> .>O N N O N N H NOOHN "NOHHHNNHOOON O.HN N.NN HNO OOHO> .>O U 0 "uamaummua HNO N.NN 0.0N N.NO N.ON o.NN H.NN mgHg .>< N N O N N H .OOHN "NOHHHOHON HOOOHH NFHHHON u N NMOEEOm umuuHH mo Owcmnu u 0 « mmumv ucmuwwwHO um uom m00o mo Nomads Onu OOOOOHOOH mHmOnucmuma OH HOOEOO O08 87 o.ON N.NO ANOO N.NN N.NO HONHO N.NN N.OO HNHHO O O.HN o.NN HOOO H.NN N.NN HONHO N.om N.NO HHNHV N N N.NO N.NO HNNO N.NN N.NN HNNHO N.NN N.NO HNHHO O N.NO o.NN HNNO N.NO N.NN HONHO N.NN N.ON HONHO N N o.Hm N.NN HNNO H.0N o.ON HONHO N.NN O.ON HOOHO O N.NN o.ON HNNV N.NO H.NN ANNHO N.NN N.NN HNNHO N O N.NO o.ON ANOO O.ON a.ma HHNO N.NN N.NN HNoHO O o.NN N.ON ANNO N.NN N.NN HNNHO H.ON O.NN HONHO N N o.ON N.NO HONO O.HN N.NN HHHHO N.NN H.NN HHoHO O N.NN N.NN HNNO N.NN N.NN HOHHO H.0N N.NN HNOHO N N N.NO N.NO HONO O.ON N.NO HOOHO N.NN N.NN HNNHO O o.OO N.NO ANNV N.NO N.NO ANNHO N.NN N.NO HNNHV N H Noam: N, .OHON N NOON: N .OHON N NOON: N .OHON N «Osmaumaua HOHO ONNH .oH .NON OONH .NH .OON NONH1.NH .OON x unmeHumaxm OH muOmEumOuH udwEmwmamz HOOOHH ucouomea so NOHHm Eoum umm N000 OHHqum mo nuumm udooumm 05¢ OHHqum ucmuumm .HHuoH OHHOH 88 Table 10-12. Bacterial Counts From.Litter Changed Frequently - ‘1 Fresh litter Litter samples Bacterial colonies/ Added Obtained from gm of litter (days) (thousandths) 1 Center of floor 3,120 1 Center of floor 66,000 1 Near water fountain 14,000 1 Near water fountain 38,000 1 Floor under laying nests 158,000 2 Center of floor 384,000 2 Near water fountains 304,000 2 Center of floor 84,000 2 Floor under laying nests 1,024,000 2 Floor under laying nests 402,000 2 Near water fountains 734,000 3 Floor under laying nests 176,000 3 Center of floor 434,000 3 Near water fountains 202,000 3 Floor under laying nests 596,000 3 Near water fountains 135,000 3 Near entrance 382,000 4 Center of floor 440,000 4 Near water fountains 172,000 4 Under laying nests 434,000 4 Floor 1,426,000 5 Near water fountains 696,000 5 Near entrance door 1,978,000 5 Center of floor 220,000 5 Floor under laying nests 230,000 5 Near water fountains 1,724,000 5 Floor adjacent to drOpping pfi: 140,000 7 Near entrance door 888,000 7 Near water fountains 4,150,000 7 Floor under laying nests 652,000 7 Floor adjacent to dropping pit 172,000 7 Center of floor 1,278,000 7 Floor under laying nest 725,000 7 Near entrance door 1,525,000 7 Near water fountains 725,000 89 Table 10-13. Bacterial Counts From Litter Unchanged During Experiment X Litter samples Obtained from Floor Floor adjacent Center Near under Near to the Bacterial Of water laying entrance drOpping colonies/gm of floor fountains nests door pits litter (thousandths) x 324,000 x 1,384,000 x 1,840,000 x 170,000 x 576,000 x 4,600 x 13,600 x 126,000 x 36,000 x 748,000 x 69,200 x 125,000 x 1,354,000 x 132,000 x 78,000 x 1,400,000 x 1,248,000 x 131,000 x 376,000 x 145,000 x 125,000 x 1,850,000 x 720,000 x 680,000 x 172,000,000 x 85,000 x 180,000 x 725,000 90 Table 10-14. Moisture Content of Litter Used for Housing Birds in Experiment X Interval Number Average after samples moisture cleaning, examined content (Z) (daYS) 2 5 23 ,3 5 29 4 5 41 5 9 44 Built-up litter 10 30.5 91 uammmua mummooo vmumHouoam ON 0H10H mHan 00m mucaoo OONOOO mom t H 0 N H N 0 HOOOHH manuHHam mwcHamoun mwaHamou0\3 H o N 0 «ON O HOOHHH OO-OHHON NOxHa OOOOHH uOuuHH mo ummmH o o o o o N OOOOHH NO-HHHON OOON NOOH-NHON 0 0 H H H m uOuuHH AHHHIOHHOHH HOOOHH mo HONOH HouuHH 0uscmno o o N N N N OOONO NON NON ONOHNOOHN HOOOHH 0OH0GOLO mwchaou0\3 o o N H NON N OOONO NON NON NOxHa OOOOHH HOOOHH wcHwamno o o o H H N OOOOO NON NON OOOOHH No OONOH mmmOaHH OOOHHHON OOHOOOO ,mHHOOOu .0 NOONOOO OOOHNMWO OOOEOOONH OHQEmw OHOHHOON< meOuOuOm OOHOOQO 0 xHHuooa .m HOHOHOOOU OOHOEOO nonuo 0 Honsdz OEHsz .0 OOHHs>uoo<.m O>0 New OumNooo HOHNHOOOO uom mwchOoun 0am HOOOHH mo OOHOOOHamxm mo Newsasm NOOOHHHOM NHxOuOuOm 0cm OOOOOHH OHOHHOON< Boum .mHaoH OHan 92 Table 10-16. Coccidial Oocyst Counts from Litter and Droppings No. of oocysts gram of §amp1e Treagment sample Litter mixed with 5th day after changing 67 drOppings litter Litter mixed with 5th day after changing 47* droppings litter Fresh droppings 5th day after changing 212 litter Fresh droppings 5th day after changing 144 litter Litter mixed with drOppings Built-up litter 42* Litter mixed with droppings Built-up litter 37* Fresh droppings Built-up litter 92 Fresh drOppings Built-up litter 86 * Sporulated oocysts present 93 Table 10-17. Feed Intake, Feed Efficiency, Gain in Weight and Mortality Rate of Three-week-old White Leghorn Chicks Used in Experiment X for Determination of Relative Infectiveness of Litter Under Different Management Treatments M =========== Feed Treat- Av. wt./ consumed/ Feed/ Morta- ment Source of litter bird bird gain lity (8M) (8M) (7.) B Upper layer of "built-up" 183.1 445 3.11 6 litter used for housing laying hens in Experiment X C From pens where litter was 187.2 466 2.98 3 changed frequently during Experiment X ' Treatments B C Av. wt. /bird 183. 1 187. 2 Means underscored by the same line are nonsignificantly different at 0.05 level of probability 94 Table 10-18. Analysis of Variance of Three-week Body weights of Chicks Used in Experiment X for Determining Relative Infectiveness of Litter Under Different Litter Treatments Source Degrees Sum of of of Mean Variation freedom squares sqgare F F .05 Total 149 138,371 Treatments 1 638 638 .68* 3.91 Error 148 137,733 .931 * Nonsignificant at .05 level of probability Treatments B C Av. wt. /bird 183. 1 187. 2 Means underscored by the same line are not significantly different (P < .05) 95 x unmeHumaxm 0OHH50 meOO3 OOGOOHO mama Eoum HOOOHH 000cmso NHucOOOOHw Onu mm? Osmaummuu mHsu OH HmuuHH mo condom u 0 Owen Oust mummo HOHOHOOOO tum 4 u t .N OGOEHHOmxu OH OUHHA 0ch505 you 00m: :msuuHHanz mmz umuuHH mo mousom u 0 «.N .. N m N 0 ON 1 m m mN 0 N m m N 0H 0 N N N N 0H 0 N N m H OH to O 0 0 H OH *0 p 23 mwchmuom HmcHumOucH 85000 :H memo mo Hm>HOOcH O0HHH *ucmEummuH no Hmooo Ocu OH muoamuooHp HO unoamwanam .Oz NOOHONOHOE no mucmucoo Newmsuuoemm OOOHmeOmm HOOOO SH NOONOOO HOHOHOOOU OOOHuomum Osmaomwamz OOOHHO>.EOHm 0cHuH5mOm HOOOHH mo Ommcm>HuOOmcH O>HOOHOM 0oHaHahOuOn How OHO>HOOOH OCOHOMMHQ um OOOHMHNOOm OxOHno scum voonuoo mqumOm mo Numeaam .mHuoH OHan DISCUSSION PART I From the review of literature and results reported herein, it is Obvious that chlorine possesses bactericidal effect (Ehleres and Steel, 1927), and destroys many bacteria of the pathogenic and coli groups. Disinfection by chlorine (Anon, 1949) depends on the concentration of residual chlorine, and thus the amount of organic impurities that get in the water is important. Feed added to chlorinated water is oxidized and the nascent Oxygen no longer is present. However, if the chlorinated water remains free from contamination, it would kill bacteria, especially gram-negative ones, and certain viruses if present in feed when the birds consumed it. Thus, chlori- nation Of drinking water may prevent the transmission in chickens of bacterial and viral infections originating from food or water. In an attempt to determine effective levels of chlorine in drinking water, filtrates from chlorinated water-feed mixtures were tested in a preliminary study. This study was conducted in the laboratory using variable levels of chlorine in water combined with a measured quantity of feed. Results of this study indicate that chlorine levels in filtrates depend upon the level of the chlorine used and presence Of feed in the mixture. Results from Experiment I showed that higher concentrations of chlorine in drinking water had a depressing effect on the intestinal bacterial flora. Possibly chlorination practiced from.the time of hatching would inhibit the establishment Of normal flora in baby chicks. Halbrook._§._1. (1951) were of the Opinion that flora is established mainly through feed and water. Perhaps chlorination of the drinking water for mature birds where flora has already been established may not have similar effects. 96 97 In Experiments I and II, growth rate in control birds was significantly higher (P ( .01) than in experimental birds supplied chlorine 250 ppm or more in drinking water. Slightly better growth resulted in control birds when compared to birds drinking chlorinated water having 100 to 250 ppm of available chlorine. Depression in growth rate increased as the levels of chlorine in drinking water increased. Whether this depression was a result Of reduced feed and water intake or an effect of chlorination on intestinal microbial flora, especially §;.£211 and a; communior. is unknown. However, autopsy on birds did not show any pathological lesion except emaciation and empty crops. This suggests that probably it was not chlorination but starvation which resulted in their death. Of course, depression in feed and water consumption occurred as a result of higher levels of chlorination. Results from Experiments III, IV and V conducted with young, growing chicks and increasing levels of chlorine in water show that chlorination up to 75 ppm had no depressing effect on daily water consumption although some birds consumed more water than others. Any restriction in feed and water consumption usually lowers egg production. However, the average size of the eggs does not vary. This might be the reason that some Of the experimental groups of birds stopped laying altogether during the first week while others did not. These birds definitely performed better when the chlorine levels were reduced. Severe molting was observed in all experimental birds consuming 500 ppm or higher levels of chlorination. Birds in production are very sensitive to any external stimuli and stress from thirst or insufficient feed could cause the molting Observed in laying birds. Free chlorine levels in water after 24 hours depended upon the original levels as well as upon the amount of feed added to the waterers by the beaks of the birds. PART II It is generally believed that the maintenance of a clean environment will reduce mortality in laying flocks (Snyder a£.a1., 1962). Data collected in these experiments, however, did not support this view. Results from Experiment IX showed no significant differences among the various manage- ment practices as far as the mean egg production or the mortality data was concerned. A significant difference (P < .01), however, occurred in egg production due to different dietary treatments. Soybean meal in the diet resulted in significantly greater egg production then did the other soy bean variables. This depression in egg production may be attributed to the experimental nature of the soybean products used. There was less mortality among birds fed the control diet and this may help explain why the performance of these chickens was better. A higher incidence Of pickouts occurred among birds fed the experimental diets. Among these lots, there were ration inadequacies presently being studied. Ruptured yolks, and/or peritonitis, and leukosis ranked second and third, respectively after pickouts as causes of mortality. The visceral form of leukosis was predominantly greater than its other forms. Higher but nonsignificant final body weights were obtained with birds fed the basal diet or kept on "built-up" litter. These results lend support to the hypothesis put forward by Halbrook a§.a1. (l950)that chickens may Obtain from the litter nutritional products such as vitamin B12 and ribo- flavin as a result of bacterial fermentation. In general, poor fertility and hatchability were Obtained by various experimental diets and management practices. The increase in percent hatchability of eggs from.hens on "built-up" litter as reported by Kennard and Chamberlin (1948a, 1948b) and Sunde a§.a1. (1951) was not found in 98 99 this experiment. No explanation is offered for these results. Differential leukocytic count, plasma protein and blood urea nitrogen showed little variation among birds of different groups. Blood sugar levels were higher (P < .01) in birds given both chlorinated water and litter change treatments. Stress, stemming from diet and litter treatments may have been responsible for these increased values but, of course, these results were obtained on a relatively small number of birds. Since blood sera was not agglutinated with Salmonella and Eg‘ggli, specific strain antigens, it is likely that these birds had little exposure to these specific infections. Certain inhibitors might be responsible for an anticoagulant activity in blood of chickens provided raw soybeans. In Experiment X, a ;significant difference (P < .01) resulted in egg production from various diet treatments. The .16 percent level of methionine appeared to be of little or no benefit when added singly or in combination appeared to be of little or no benefit when added singly or in combination with .05 percent detergent to a ration containing raw soybeans. These results are, however, in contrast to that already reported by Yates (1963) who found that .16 percent level of methionine when added in combination with .05 percent of surface-active agent to the ration containing raw soybeans significantly improved egg production. There were differences in basal rations used and moreover long-term effects of this diet is still under investigation. Antibiotic, Pro-strep, when added with supplemental .16 percent methionine to the ration containing raw soybeans (Diet 10-4) significantly improved egg production. However, when this antibiotic was added in combination with .05 percent detergent and .16 percent methionine to a ration, egg production was apparently but not significantly depressed from that produced by the diet 10-4. These results suggest that supplementation of antibiotic in rations containing raw soybeans improved egg production. The weights of the birds housed on built-up litter were neither significantly nor apparently different from the weights of those birds kept on lOO pens where litter was changed frequently. This is not consistent with the results obtained previously (Experiment IX) where higher body weights were found for birds housed in built-up litter floor pens. This difference in final body weights apparently reflected to an extent the adequacy of the diet being studied. Once again, the results of hatchability suggest that built-up litter floor pens have no effect in increasing hatchability of eggs laid by birds housed in these pens. Apparently, there were no differences in bacterial counts of litter and droppings samples examined from different litter treatments. However, isolation and characterization of various bacteria was not attempted. Perhaps bacterial flora of built-up litter may be different in nature than that of litter which is changed frequently. There appeared to be less (P < £05) mortality among the birds fed the experimental diets 10-1, 10-2 and 10-3. High death rate was observed among birds fed high producing experimental diets containing raw soybeans with supplemented methionine, Pro-strep with or without detergent. Whether this occured as a result of stress of high egg production on these diets is unknown. Various management practices had similar effects on the incidence of mortality. Death rate among birds was higher during periods of heavy egg production. During six months period of time only 41 birds died out of a total of 600; one-third of these deaths were caused by leukosis or other tumors. Individual egg records indicated that the birds that died during Gotober, November and early December were poor layers. Perhaps this is another of nature's ways of culling weaker birds. All birds that died after December 15 were in high production. Possibly the resistance of a producing bird is lowered by stress created as a result of high egg production. This may also help to explain the increased incidence of ruptured yolks and peritonitis which may result as a consequence of some disease, hormonic lOl disturbance or mechanical obstructions followed by bacterial infection. Some basic work is desirable to investigate further the cause of this infection which appears predominantly in high-producing birds. Kennard and Chamberlin (1947, 1949) have advocated the use of built- up floor litter as one of the measures for control of coccidiosis. 0n the other hand, Koutz (1948, 1953) found built-up floor litter a reservoir of coccidial oocysts and parasitic ova. However, the results of this study did not show differences between different litter treatments when judged on basis of coccidial oocysts and mortality data collected from baby chicks. There were statistically nonsignificant (P ( .05) differences in feed conversion and gain in body weights of chicks reared on different types of litter treatments. Greater gains in body weights and better feed efficiency resulted with chicks receiving litter from pen where it was changed freque- ntly than in chicks reared on litter transferred from builtlup litter pen. During the first two weeks of the experiment, when birds were sacrificed, the number of infected birds and the presence of hemorrhagic ceca varied with the group. This is possibly due to the nature of the coccidial organisms and the relative number of sporulated oocysts present in the litter. At the end of the experiment, the sacrificed birds failed to show any evidence of coccidiosis infection, except for a few oocysts present in cecal contents. Probably the birds were immunized resulting in discontinuation of the coccidial life cycle. During the first three days of this experiment nine chicks were found dead in both experimental groups. Autopsy on these birds revealed absence of hemorrhages in ceca, as well as the absence of merozoites in cecal and intestinal scrappings suggesting that coccidiosis was not responsible for their death. Such results are possible because one 102 would anticipate mortality at the end of four to five days had infection occurred. The remaining young chicks did not develop hemorrhages and consequently showed no symptoms of coccidiosis. Probably l - 2-week-old birds show higher resistance‘gg‘§& tenella infection (Gardiner, 1955). In addition to this, resistance of the host, breed of chickens, virulence of oocysts and whether sporulated or not, are some of the factors which may offer some explanation for these results (Herrick, 1934). These results were obtained on young chicks whether this may apply to mature birds is unknown. Oocyst counts were relatively higher in drOppings of birds housed in pens where litter was changed frequently, than in droppings of birds kept on built-up litter. Perhaps these birds had less exposure to infection, thus lacked immunity. Birds on built-up litter might have developed immunity and thus eliminated less oocysts in their feces. Of course, these results were obtained on a relatively small number of fecal samples. This variability may further be explained by the individuality of each bird as well as the stage of coccidia life cycle. Eimeria acervulina oocysts were found comparatively in large numbers of samples of litter and drOppings examined from either kind of litter treatment. Perhaps this specie can withstand considerable adversity which it encounters outside the body. [2; acervulina being responsible for chronic infections is possibly discharged with the feces for a longer period. Edgar (1954, 1955) reported that sporuhtion of‘§& tenella oocysts (time involved from oocysts' passage until it becomes infective) was achieved in approximately 18 to 24 hours at 29° C. and relative humidity 60 percent. During this study, sporulated oocysts were found in samples of litter and droppings examined from pens cleaned frequently. In these pens, possibly sufficient moisture and aeration was present to sporulate coccidial oocysts. The moisture content of the litter, where it was changed frequently, was higher than that of built-up litter. The presence 103 of moisture in the litter not only tends to sporulate oocysts but also increases livability of nematode eggs (Koutz, 1952). These results suggest that weekly removal of litter from poultry houses may not be advantageous judged on the basis of control of cocci- diosis infection. Perhaps this might be of some significance if practiced at a much higher frequency to inhibit sporulation. Naturally, this will result in more labor and a proportional increase in expenditure. Young (1929) even has claimed that all viable oocysts whether segmented or not were potentially capable of causing infection. If this is true, then it seems unnecessary to clean the pens as often as has been recommended in the past speciafly for control of coccidiosis and possibly other diseases transmitted through feces. Higher mortality observed during Experiment IX may be attributed to the following: 1. Pickouts -- This was eliminated in this experiment by successful use of specks. 2. Inadequacy of experimental diets such as inclusion of whole raw and cooked soybeans in feed formula. In this experiment, however, whole raw or cooked soybeans were not used. 3. Severe cold during winter of 1962-63 might have further lowered the resistance of the experimental birds. The results of Experiments IX and X suggest that sanitation practices for poultry may not be advantageous over ordinary management practices with built-up floor litter when judged on the basis of mortality and mean egg production. This may be true, because most of the diseases on a poultry farm such as this one may be nonspecific and nontransmissible in origin. The occurrence of such infections may not be prevented by better housekeeping or applying 104 other measures of sanitation. Infectious disease outbreaks may occur but seems to be very rare. In a modern hen house, the average mortality per month is more than one percent (Sheppard, 1964). The results of this study showed monthly mortality of one percent. This range of mortality is probably a normal occurrence due to certain nonspecific causes and may not be prevented by sanitation practices. Sanitary management is most likely to be useful during an outbreak of infectious diseases. Of course, during the entire period of this study, no infectious outbreak was seen on this poultry research farm. SUMMARY AND CONCLUSIONS PART I Eight experiments were conducted to determine homeuch chlorination chickens can tolerate in the drinking water without curtailing water intake and rate of growth. Two experiments were undertaken using Leghorn-type hens to investigate the effects of chlorinated water and litter management practices on production and rate of mortality. The following levels of chlorine in water did not affect the feed or water consumption. 50 ppm in 3-week-old chicks 75 ppm in lO-week-old chicks 25 ppm in laying hens Lowered water intake did not reduce feed consumption or gain in body weights by chlorination at 100 to 1,000 ppm in .5-week-old chickens 50 ppm to 500 ppm in old hens Water and feed intake as well as gain in body weights were depressed by chlorination above 100 ppm in baby chicks 1000 ppm in .5-week-old chickens 500 ppm in laying hens Thus, the consumption of chlorinated water may depend upon the individual nature of each bird. Some birds may consume more than others. When feed and water consumption was depressed, it was reflected in body weight changes in growing chicks as well as lower egg production and molt in laying birds. The egg production in laying hens was depressed by chlorination at 50 ppm. 105 106 PART II Frequent litter change treatments had no effect on egg production and incidence of mortality. Cannibalism, leukosis, ruptured yolks and peri- tonitis were the primary causes of mortality in these experiments. Other pathological conditions diagnosed were hepatitis, nephritis, sinusitis, synovitis, chronic respiratory disease and impacted oviduct which may be associated with heavy egg production. The incidence of visceral lympho- matosis was greater than that of other forms of leukosis. Infections of the reproductive system were more common than others. Rations containing soybean oil meal produced more eggs but the rate of production of birds on these rations was neither apparently nor signi- ficantly better than that produced by the experimental diet containing raw soybeans with supplementation of methionine and antibiotic, Pro-strep. Hatchability of eggs was not improved when birds were kept on "built-up" litter. Baby chicks brooded on litter previously used by hens kept under different management treatments did not differentiate between relative infectiveness of different litter systems. There were no significant differences in bacterial counts of litter and droppings samples examined from different litter treatments. The moisture content of the litter was higher in the treatments.reCeiving frequent litter change than that of "built-up" litter. Different litter treatments had similar effect on coccidial oocysts. These results suggest that chlorinated drinking water and frequent change to clean litter or both may not offer any advantages over the usual tap water and "built-up" litter, judged on the basis of mortality. This conclusion may not be applicable during an outbreak of disease. The removal of litter at such a time may be a primary step towards the control of such an outbreak. LITERATURE CITED Alder, B., 1934. Results of seven years of egg laying contests. Utah Agr. Exp. Sta. Bul. 248: 1. Anderson, C. D., 1928. Report of the New York State Egg Laying Contest Nov. 1 to Oct. 23, 1927. p. 34. Issued by State Inst. of Applied Agriculture, Farmingdale, N. Y. Anon, 1949. Editorial. Lancet 256: 1056. Barger, E. H., Card, L. E. and Pomeroy, B. S., 1958. Diseases and Parasites g§_Poult§y. Lea and Febiger, Philadelphia. Barrett, F. N., 1929. Mortality as a factor in poultry production. Thesis, Univ. of Illinois, pp. 1-30. Bezanson, J. M. and E. L. Stephenson, 1948. Factors affecting a breast blister condition in broilers. Poultry Science Ass'n. 47th Annual Meeting. p. 6. Bird, H. R., 1947. A chick growth factor in cow manure. IV. Poultry Sci. 26: 439. Boughton, D. C., 1939. Studies on the control of poultry coccidiosis. I. The sporulation of oocysts in various types of litter. Bul. Univ. of Georgia 39: 9. Brackett, S. and A. Bliznick, 1952. The relative susceptibility of chickens of different ages to coccidiosis caused by Eimeria nec§trix. Poultry Sci. 31: 148. Broadfoot, D. I., B. S. Pomeroy and W. M. Smith, 1957. Effects of infective bronchitis on egg production. Jour. Amer. Vet. Med. Ass'n. 124: 128. Brunson, C. C. and G. F. Godfrey, 1952. A fourteen-year summary of mortality in the Oklahoma Egg Laying Test. Poultry Sci. 31: 149. Burmester, B. R., 1948. The influence of sex hormones upon the occurrence of prolapse in chickens. Poultry Sci. 27: 745. Burmester, B. R. and R. F. Gentry, 1954. A study of possible avenues of infection with the virus of avian lymphomatosis. Proc. A.V.M.A. 311. Burmester, B. R. and R. F. Gentry, 1955. The propagation of the virus of visceral lymphomatosis in embryonated eggs. Poultry Sci. 34: 669. Burmester, B. R., R. F. Gentry and N. F. Waters, 1955. The presence of viruses of visceral lymphomatosis in embryonated eggs of normal appearing hens. Poultry Sci. 34: 609. Burmester, B. R., A. B. Gross, W. G. Walter and A. K. Fontes, 1959. Patho- genecity of a viral strain (RPL-12) causing avian visceral lymphomatosis and related neoplasms. J. Nat'l. Cancer Inst. 22: 103. 107 108 Burmester, B. R. and N. F. Waters, 1955. The role of the infected egg in the transmission of visceral lymphomatosis. Poultry Sci. 34: 1415. Burmester, B. R. and N. F. Waters, 1956. Variation in the presence of the virus of visceral lymphomatosis in the eggs of the same hen. Poultry Sci. 35: 939. Burmester, B. R., N. F. Waters, W. R. Bryan and V. Groupe, 1960. The response of several inbred lines of White Leghorns to inoculation with the viruses of strain RPL-12 visceral lymphomatosis-erythro- blastosis and of Rous sarcoma. Poultry Sci. 39: 199. Buster, M. W., 1928. What California's cost of production studies reveal concerning poultry management. Poultry Sci. 7: 49. Caldwell, M. J., 1957. Detecting blood coagulation defects in the clinical laboratory. Am. J. M. Tech. 23: 277. Calnek, B. W. and P. O. Levine, 1957. Studies on experimental egg transmission of pleuro-pneumonia-like organisms in chickens. Avian Diseases 1: 208. Card, L. E. and W. F. Kirkpatrick, 1919. Egg laying contests. Storrs Agr. Exp. Sta. Bul. 100. Carr, J. G., 1952. The avian leukosis complex. World's Poultry Science Journal 8: 276. Chalquest, R. P. and J. Fabricant, 1959. Survival of PPLO injected into eggs previously dipped in antibiotic solution. Avian Diseases 3: 257. Chute, H. L. and D. C. Meara, 1957. A bibliography of avian mycosis. Maine Agr. Exp. Sta. Misc. Publ. 631. Cole, R. K., 1949. The egg and avian leukosis. Poultry Sci. 28: 31. Cole, R. K., 1955. Some observations on breeding fowl resistant to lymphomatosis. Poultry Sci. 34: 312. Cole, R. K. and F. B. Hutt, 1951. Evidence that eggs do not transmit leukosis. Poultry Sci. 30: 205. Cole, R. K. and F. B. Hutt, 1956. Parents age unrelated to leukosis in progeny. Poultry Sci. 35: 731. Cotterill, O. J. and A. R. Winter, 1953. Some nitrogen studies of built-up litter. Poultry Sci. 32: 365. Cottral, G. E., 1952. Endogenous viruses in the egg. The chick embryo in biological research. Ann. N. Y. Acad. Sciences 55: 221. 109 Cottral, G. E., B. R. Burmester and N. F. Waters, 1954a. The transmission of visceral lymphomatosis with tissue from embryonated eggs and chicks of normal parents. Poultry Sci. 33: 1174. Cottral, G. E., B. R. Burmester and N. F. Waters, 1954b. Egg transmission of avian lymphomatosis. Poultry Sci. 33: 1174. Couch, J. R., 1955. Cage layer fatigue. Feed Age May, 88: 55. Crowley, T. A., A. A. Kurnick and A. R. Kemmerer, 1961. Phosphorus and laying hen performance. Poultry Sci. 40: 1391. Darcel, C. LeO, L. Niilo, E. V. Langford and R. Connell, 1960. Mortality in White Leghorn females on a farm in Southern Alberta. Avian Dis. 4:258. Dardiri, A. B. and O. Zaki, 1955. Mortality due to oomphalitis following air shipment of baby chicks. Poultry Sci. 34: 327. Dawson, L. E. and J. P. Watts, 1952. Effect of nest litter on cleanliness of eggs. Poultry Sci. 31: 913. Dudley, F. J., 1928. The death rate of three standard breeds of fowl during the pullet year. Poultry Sci. 7: 245. DuncanvD. B., 1955. ‘Multiple range and multiple F tests. Biometrics 11: l. Dunnicliff, A. A., Jr., 1913. Ten years egg laying tests at Hawkesbury Agricultural College and Experimental Farm. Richmond, New South Wales. New South wales Dept. Agr. Farmers Bul. 66: 1-96. Edgar, S. A., 1954. Effect of temperature on the sporulation of oocysts of the protozoan, Eimeria tenella. Trans. Amer. Micros. Soc. 73: 237. Edgar, S. A., 1955. Sporulation of oocysts at specific temperatures and notes on the prepatent period of several species of avian coccidia. Jour. Parasit. 41: 214-216. Ehleres, V. M. and W’ F. Steel, 1927. Municipal and rural sanitation. McGraw-Hill Book Co. p. 87. Emmel, M. W., 1930. Bacterial flora in feces of the normal fowl. J. Infectious Diseases 46: 293. Enigh, C. F., 1935. Arch. Wiss. u. Prakt Tierheilk 69: 410. Fabricant, J., P. P. Levine, B. W. Calnek, H. E. Alder and J. R. Berg, 1959. Studies of egg transmission of PPLO in chickens. Avian Diseases 3: 197. Faddoul, G. P. and R. C. Ringrose, 1950. Avian keratoconjunctivitis. Vet. Med. 45: 492. Fair, M. G., J. C. Geyer and J. C. Morris, 1954. Water supply and waste disposal. John Wiley and Sons, Inc., New York. 110 Forgacs, J. and W. T. Carll, 1955. Preliminary mycotoxic studies on hemorrhagic diseases in poultry. Vet. Med. 50: 172. Forgacs, J., H. Koch and W T. Carll, 1955. Further mycotoxic studies on poultry hemorrhagic disease. Poultry Sci. 34: 1194. Forgacs, J., W. T. Carll, A. S. Herring and B. G. Mahlandt, 1954. A toxic Aspergillus clavatus isolated from feed pellets. Am. J. Hyg. 50: 15-26. Forgacs, J., H. Koch, W. T. Carll and R. H. White-Stevens, 1958. Additional studies on relationship of mycotoxicoses to the poultry hemorrhagic syndrome. Am. J. Vet. Res. 19: 744-753. Forgacs, J., H. Koch, W. T. Carll and R. H. White-Stevens, 1962. Myco- toxicoses. 1. Relationship of toxi fungi to moldy feed toxicosis in poultry. Avian Dis. (accepted for publ. Feb. 7, 1962). Gage, G. E., 1911. A study showing bacterial and animal organisms deter- mined in the feces and intestinal mucosa of healthy chickens. Md. Agr. Exp. Sta. Bul. 153, p. 201. Gardiner, J. L., 1955. The severity of cecal coccidiosis infection in chickens as related to the age of the host and the number of oocysts ingested. Poultry Sci. 34: 415. Gross, L., 1951. The "Vertical" transmission of mouse mammary carcinoma and chicken leukemia. Its possible implications for human pathology. Cancer 4: 626. Gross, L., 1952. Mouse leukemia. Viruses as causative agents in cancer. Ann. N. Y. Acad. Sci. 54: 1184. Gross, W. B., 1961. The development of "Air sac disease". Avian Dis. 5: 431. ‘ Halbrook, E. R., 1950a. Vitamin B12 synthesis by microorganisms in poultry house litter. Ph.D. Thesis, Ohio State Univ. Halbrook, E. R., 1950b. Proc. 39th Ann. Meeting of Poultry Sci. Ass'n. p. 16. Halbrook, E. R., T. S. Sutton and A. R. Winter, 1950. The effect of manage- ment on the vitamin B1 content of poultry house litter. 11. As determined by microbiological assay. Poultry Sci. 29: 679. Halbrook, E. R., T. S. Sutton and A. R. Winter, 1951. The microflora of poultry house litter and drOppings. Poultry Sci. 30: 381. Hammond, J. C., 1942. Cow manure as a source of certain vitamins for growing chickens. Poultry Sci. 21: 544. Harris, J. A. and C. D. Boughton, 1927. The death rates of three standard breeds of fowl. Poultry Sci. 7: 120. v74". ' - ii} »-‘ ‘.- t O. 9T. a I'u' -¢ (all: 111 Hays, F. A., 1955. Age of parents and mortality in off-spring. Poultry Sci. 34: 473. Hays, F. A. and E. W. Spear, 1952. Relation of age of parents to maturity and sex ratio of chicks to eight weeks. Poultry Sci. 31: 792. Helmboldt, C. F., F. K. Wills and M. N. Frazier, 1963. Field observations of the pathology of skin leukosis in Gallus Callus. Avian Dis. 7: 402. Herrick, C. A., 1934. The development of resistance to the protozoan parasite, Eimeria tenella by the chicken. 1. Parasit. 20: 329. Hinshaw, W. R., 1952. How diseases become established in poultry flocks. Vet. Med. 47: 164. PIA-— Hutt, F. B., 1951. The control of leukosis in the fowl. WOrld's Poultry Sci. J. 7: 16. Hutt, F. B., and R. K. Cole, 1954. Problems concerning leukosis and its , control. Proc. 10th World's Poultry Congr., Edinburgh, Scotland 197. ' Hutt, F. B., R. K. Cole, M. Ball, J. H. Bruckner and R. F. Ball, 1944. E“ A relation between environment to two weeks of age and mortality from lymphomatosis in adult fowls. Poultry Sci. 23: 396. Jaquette, D. S. and D. E. Fogg, 1962. Survey of poultry diseases on Del Marva Peninsula. Broiler Producer, Mar., 1962, p. 18. Jull, M. A., 1934. The mortality problem in poultry. A reprint from the July and August, 1934 issue of the U. S. Egg and Poultry Mag. pp. 11- Kennard, D. C., 1933. Pullet mortality. Poultry Sci. 12: 335. Kennard, D. C. and V. D. Chamberlin, 1934. Poultry mortality. Ohio Agr. Expt. Sta. Bi-monthly Bul. 19: 137. Kennard, D. C. and V. D. Chamberlin, 1936. Eight years experiences with losses of pullet layers. Ohio Agr. Expt. Sta. Bi-monthly Bul. 21: 63. Kennard, D. C., R. M. Bethke and V. D. Chamberlin, 1948. Built-up floor litter a source of dietary factors essential for hatchability of chicken eggs. Poultry Sci. 27: 477. Kennard, D. C. and V. D. Chamberlin, 1947. Lime treatment of floor litter for chickens. Ohio Agr. Expt. Sta. Farm and Home Res. 32: 11. Kennard, D. C. and V. D. Chamberlin, 1948a. Built-up litter as a source of dietary factors essential for the growth of chickens. Poultry Sci. 27: 240. Kennard, D. C. and V. D. Chamberlin, 1948b. Built-up floor litter to date. Ohio Agr. Expt. Sta. Farm and Home Res. 33: 130. 112 Kennard, D. C., V. D. Chamberlin, 1949. Built-up floor litter sanitation and nutrition. Ohio Agr. Expt. Sta. Farm and Home Res. 34: 162. Kennard, D. C., V. D. Chamberlin, 1950a. Proc. 39th Ann.. Meeting Poultry Sci. Ass'n. Kennard, D. C. and V. D. Chamberlin, 1950b. Mortality of chicks as affected by floor litter. Wbrld's Poultry Sci. Jour. 6: 183. Kennard, D. C., 1950c.‘ Floor litter management as a factor in poultry nutrition. World's Poultry Sci. Jour. 6: 177. Kennard, D. C. and V. D. Chamberlin, 1951. Growth and mortality of chickens as affected by the floor litter. Poultry Sci. 30: 47. Kern, H., 1897. Beitrag zur kenntris der im drama und magender vagel. Vorkommender Bactrien.Afheiten Bact. Ins. Tech. Hochschule Karlsruke 1: 379. King, W. E., 1905. The bacterial flora of the intestinal mucosa and conjunctiva of the normal chicken. J.A.V.M.A. 10: 400. Koutz, F. R., 1948. Immunity studies in avian coccidiosis. Poultry LE Sci. 27: 793. Koutz, F. R., 1952. The effect of built-up litter on the parasitic ova and oocysts of poultry. I. Poultry Sci. 31: 123. Koutz, F. R., 1953. The effect of built-up litter on the parasitic ova and oocysts of poultry. II. Poultry Sci. 32: 320. Lamoreux, W. F. and W’ Schumacher, 1940. Is riboflavin synthesized in the feces of fowls. Poultry Sci. 19: 418. Lippincott, W. A. and L. E. Card, 1946. Poultry Production. Lea and Febiger, Philadelphia, p. 292. Mackie, T. J. and J. E. McCartney, 1950. Handbook‘gf Practical Bacterio- 1 gy. Eighth ed. E. and S. Livingstone, Ltd. Edinburgh. p. 233. Marr, J. E., C. W’ POpe, H. L. Wilcke and R. M. Bethke, 1961. Re-evalua- tion of the phosphorus requirement of the laying hen. Poultry Sci. 40: 1427. McGinnis, J; J., ML Stevens and K. Groves, 1947. The‘ig vitro synthesis of a chick growth promoting factor in hen's feces. Poultry Sci. 26: 432. Misner, E. C., 1932. Economic studies of poultry farming in New York. IV. Cornell Agr. Expt. Sta. Mimeo Reports. Moore, E. N. and V. D. Chamberlin, 1953. Compost litter. Nulaid News 31: 18 (Sept.) 113 Morgan, B. B. and P. A. Hawkins, 1952. Veterinary Protozoology. Burgess Publishing Co., Minneapolis, Minn. 187 pp. 23 plates. Morris, D., 1963. Condemnations in poultry. The Merck Agricultural Memo. 8(4): Nov. Neal, W. M., 1956. Cannibalism, pickouts and methionine. Poultry Sci. 35:10. New York Random Sample Poultry Tests, 1953. (Three-year average report) College of Agriculture, Cornell Univ. Olesiuk, O. M. and H. Van Rockel, 1960. Transmission of chronic respiratory disease in chickens. Avian Disease 4: 348. f“ Patterson, F. D., H. L. Wilcke, C. Murray and E. W. Henderson, 1932. . So-called range paralysis of the chicken. Am. Vet. Med. Ass'n. J. 3 34: 747. V Profitable Management, 1963. A series of Beacon research, May 14. I Beacon Feeds, Beacon Division of Textron, Cuyaga, N. Y. Raggi, L- G-: J. R- Brownell and G. F. Stewart, 1961. Effects of C infectious laryngotracheitis virus on egg production and quality, Poultry Sci. 40:134. Random sample egg production test report. United States and Canada (1963). Agr. Res. Service, U. 8. Dept. of Agr. Reid, W. M. T. A. Maag, M. B. Frank, A. L. Kleckner and S. C. Schmittle, 1961. Embryo and baby chick mortality and morbidity induced by a strain of.§L coli. Poultry Sci. 40: 1501. Rubin, M., H. R. Bird and I. Rothchild, 1946. A growth promoting factor for chicks in feces of hens. Poultry Sci. 26: 439. Schumacher, A. E., G. F. Heuser, 1941. Bacterial flora in chicken feces. Poultry Sci. 20: 272. Scudder, H. D., A. S. Burrier, A. G. Lunn and F. L. Knowlton, 1931. Cost and efficiency in commercial egg production in Oregon. Ore. Agr. Expt. Sta. Bul. 287: l. Sevoian, M. and D. M. Chamberlin, 1963. Avian lymphomatosis. III. Incidence and manifestations in experimentally infected chickens of various ages. Avian Diseases 7: 97. Sevoian, M., D. M. Chamberlin and R. N. Larose, 1963. Avian lymphomatosis. V. Air-borne transmission. Avian Diseases 7: 102. Sheppard, C., 1964. Personal communication. Extension Service Michigan State University. 114 Singsen, E. P., A. H. Spandorf, L. D. Matterson, J. A. Serafin, 1961. Phosphorus in the nutrition of the adult hen. Minimum phosphorus requirements.’ Poultry Sci. 40: 1457. Smith, R. C., 1956. Kind of litter and breast blisters on broilers. Poultry Sci. 35: 595. Snedecor, G. W., 1956. Statistical Methods Appliedigg Experiments in Agriculture and Biology. Fifth ed., Ames Iowa College Press. Snoeyenbos, M., M. Sevoian, D. L. Anderson and H. I. Basch, 1961. Avian Diseases 1: 349. Snyder, J. M., O. A. Rowath, J. C. Schales, C. E. Lee, 1962. Profitable Poultry Management. 24th ed. Beacon Feeds. Squibb, R. L., 1961. Avian diseases virus and nutrition relationships. Poultry Sci. : 425. Stafseth, H. J., W. W. Thompson and C. G. Grey, 1932. Observations on prolapse (Blow-outs). Jour. Am. Vet. Med. Ass'n. 80: 80. Stafseth, H. F. and E. S. Weisner, 1931. Causes of mortality in laying hens. Mich. Agr. Expt. Sta. Quart. Bul. 13: 153. Sunde, M. W., W. W. Cravens and J. G. Halpin, 1951. The effect of vitamin B , antibiotics and deep litter on laying and breeding hens. Poultry Sol. 30: 932. Takashi, O. and W. M. Reid, 1961. Histomoniasis in chickens. Age of greatest susceptibility and pathogenecity studies. 5: 355. Taylor, L. W., D. C. Lowery and L. G. Raggi, 1955. Effect of an outbreak of avian encephalomyelitis (Epidemic tremor) in a breeding flock. Poultry Sci. 34: 1037. Thomas, W. P. and M. Clawson, M., 1933. Economic factors affecting poultry production and marketing in Utah. Utah Agr. Expt. Sta. Bul. 244: 1. Titus, W., D. W. Whitson and H. R. Bird, 1945. The effect of feeding cow manure on egg production and hatchability. Poultry Sci. 25: 52. Todd, A. C., 1948. Worm parasites of Tennessee chickens. Tenn. Agr. Exp. Sta. Bul. 205. Tyzzer, E. E., E. H. Theiler, and E. J. Jones, 1932. Coccidiosis in gallinaceous birds. II. A comparative study of species of Eimeria of the chicken. Am. J. Hyg. 15: 319. Van Rockel, H., 1955. Advances in Veterinary Science 2: 64. Voorhies, E. C. and G. A. Read, 1931. A biometrical study of the mortality of Single Comb White Leghorn chicks. Hilgardia 5: 531. 115 Waters, N. F., 1951. Mortality from lymphomatosis and other causes among inbred lines of White Leghorns. Poultry Sci. 30: 531. Waters, N. F. and C. O. Prickett, 1944. The develOpment of families of chickens free of lymphomatosis. Poultry Sci. 23: 321. Waters, N. F. and J. H. Bywaters, 1949. Influence of age of chickens at contact exposure on incidence of lymphomatosis. Poultry Sci. 23: 321. Waters, N. F. and J. H. Bywaters, 1949. Influence of age of chickens at contact exposure on incidence of lymphomatosis. Poultry Sci. 28: 254. Wheeler, R. S. and E. Hoffmann, 1948. Prolapse and pickouts in diethyl- stilbestrol-treated cockerels. The Cornell Vet. 38: 89. Whitson, D. W., W. W. Titus and H. R. Bird, 1945. The effect of feeding cow manure on egg production and hatchability. Poultry Sci. 25: 52. Winton, B., 1932. Poultry sanitation program for Missouri. Missouri Agr. Expt. Sta. Ext. Cir. Bul. 282. 1959 Wright, M. L., G. W. Anderson and N. A. Epps./ Hatchery sanitation. Canad. Jour. Comp. Med. 23: 288. Wright, M. L., G. W. Anderson and N. A. Epps, 1960. Hatchery sanitation as a control measure for Aspergillosis in fowl. Avian Diseases 4: 368. Wright, M. L., G. W. Anderson and J. D. McConachie, 1961. Transmission of Aspergillosis during incubation. Poultry Sci. 40: 727. Yates, J. D., 1963. The feeding of unheated soybeans to poultry. Ph.D. Thesis. ‘Michigan State University. Young. 3. P. 1929. A quantitative study of poultry coccidiosis. J. Parasit. 15: 241.250. 116 Appendix Table 1. Composition of Standard Chick Starter* (M.S.U. 63-S) Used in Experiments I and II (Amts. in lbs/2,000 lbs.) Ingredient Pounds Soybean oil meal, 45% protein 500 Corn, ground yellow 1060 Bats, ground 100 Wheat flour middlings 100 Alfalfa meal, dehyd., 17% protein 80 Meat and bone meal, 50% protein 50 Whey, dried 40 Fish meal, 55% Vitaproil . 40 Limestone, ground 10 Dicalcium phosphate (24% Ca., 18.5% Phos.) 10 Salt, iodized 6 Vitamin trace mineral 5 Coccidiostat (Zoamix) l Manufactured by King Milling Company, Lowell, Michigan 117 Appendix Table 2. Residual Chlorine Determinations in Filtrate From Mixture of Feed and Chlorinated Water Chlorine Amount Amount levels Residual chlorinejafter time in mins. of feed of water in_water 5 10 15 20* 30‘ 45 (gms) (m1 s) (ppm) 5 10 500 l 0 - - - - 30 60 " - - - - - - 30 70 " 5 - - - - - 32 70 " 1 0 - - - - 3O 70 1000 10 0 - - - - 30 70 " 5 0 - - - - 30 80 " 50 5 0 - - - 32 100 " 100 10 6 1 0 - 32 120 " 250 80 5 l 0 - 0 - No available chlorine 118 ' Appendix Table 3. Composition of Ration (Amounts in lbs/200 lbs.) Used in Experiments III, IV and V Ingredients Pounds Ground corn 1203 Pulverized oats 100 Soybean oil meal, 44% 300 Corn gluten meal 100 Fish meal, 60% 50 Meat and bone scraps 50 Alfalfa meal, dehyd., 17% 60 Dried whey 35 Dried brewers yeast 35 Ground limestone 30 Dicalcium phosphate 25 Salt (iodized) 5 Choline chloride, 25% dry mix 2.5 Vita. A, D & E premix1 2 Coccidiostat2 l Arsanilic acid3 1 Manganese sulfate, feed. grade 0.5 Antioxidant4 0.25 Niacin 18 grams Procaine penicillin 2 grams 1 2 Hamilton Farm Bureau 3 Hess and Clark, potency 50 gms/lb. 4 Abbott Laboratories, Pro-gen 90 gms/lb. BHT 120 Appendix Table 4. Composition of Michigan State University Z-4 Battery All-mash* Used in Experiments VI, VII and VIII (Amounts in lbs./2000 lbs.) Ingredient Pounds Soybean oil meal, 44% protein 50 Corn, ground yellow 690 Oats, ground 400 Bran 300 Alfalfa meal, dehydrated, 17% 60 Wheat flour middlings 200 Milk, dried 40 Fish meal 50 Meat scraps 60 Oyster shell flour 100 Bone meal (steamed) 30 Salt, iodized 12 Fish oil . 8 * Manufactured by King Milling Co., Lowell, Michigan. 121 Appendix Table 5. Composition of Ration for Baby Chicks Used in Experiment X Ingredients Percent Soybean oil meal, 44% protein 31.4 Corn, ground yellow 50.8 Fish meal and solubles, 57% protein 3.0 Alfalfa leaf meal, 20% protein 2.0 Corn oil 6.6 Dried distillers solubles, corn 2.0 Vitamin-trace mineral mix, Nopcosol Mr7 0.25 Whey, dried 2.0 Salt, iodized 0.50 Limestone, ground 0.90 Dicalcium phosphate 0.60 Calculated analysis % Protein 21.92 Fat 9.22 Fiber 3.64 Calcium ~ 0.82 Phosphorus 0.61 Arginine 1.26 Glycine 1.17 Methionine 0,43 Cystine 0.33 Lysine 1.18 Tryptophan 0.25 Productive energy Cal/1b 1024 122 Appendix Table 6. Composition of Vitamin-trace Mineral Mixes* (per 5 1b.) NOpcosol M-4 M-7 Vitamin A, USP units 4,000,000 5,000,000 Vitamin D3, 10 units 1,500,000 1,500,000 Vitamin E, I units 7,500 5,000 Riboflavin 3 3 d-Pantothenic acid, gm 5 5 Niacin, gm 20 30 Choline chloride, gm 200 300 Menadione sodium bisulfite, gm -- ~- Vitamin B12, gm 10 6 Zinc bacitracin, gm -- 4 Butylated hydroxy toluene, gm 113.4 113.4 Manganese , % 2. 4 2. 4 Zinc, % 0.8 2.2 Iron, % 0.8 0.8 Copper, % 0.08 0.08 Iodine, % 0.048 0.048' Cobalt, % 0.008 0.008 * Produced by N0pco Chemical Company, Harrison, New Jersey 123 Appendix Table 7. Post Mortem Diagnosis Sheet* I. Hen Number , Age , Pen Number , No. birds in pen , No. birds affected in each pen , No. bird dead , Symptoms observed, if any Time of death (before post mortem) Physical Examination II. Droopinessn, Lameness, Blindness, Emaciation Discoloration of comb and wattles Condition of nasal sinuses and mouth External parasite, if any Body temperature III. (Visceral Examination) 1. Intestines: Color , Thickness of wall , Tuberculi noduli, if any , Hemorrhages , Wbrms Gizzard and Cr0p: Odor of contents. Congestion of, if any lesions present . Esophagus and Trachea: Color , Noduli, if any Heart: Any abnormality Liver: Color , Any noduli , Fatty degeneration ,Necrosis Kidneys: Color , Enlargement or swelling, if any Presence of calculi . Lungs: Color , Noduli, if any Testes: Ovaries: Oviduct: IV A. Macroscopic Findings B. Microscopic Findings: C. Conclusions: * All birds were autopsied according to this postmortem sheet.