BOSTON PUBLIC LIBRARY 3 9999 063 EXPERIMENTAL STUDIES ON POST-MORTEM CHANGES IN MALLARDS by THOMAS L. MORROW Graduate Research Assistant and FRED A. GLOVER Leader COLORADO COOPERATIVE WILDLIFE RESEARCH UNIT COLORADO STATE UNIVERSITY Boston Public Library Superintendent of Document? OCT 2 2 1970 DEPOSITORY Bureau of Sport Fisheries and Wildlife Special Scientific Report — Wildlife No. 134 Washington, D. C. UNITED STATES DEPARTMENT OF THE INTERIOR, WALTER J. HICKEL, SECRETARY Fish and Wildlife and Parks, Leslie L, Glasgow, Assistant Secretary Fish and Wildlife Service, Charles H. Meacham, Commissioner Bureau of Sport Fisheries and Wildlife, John S, Gottschalk, Director EXPERIMENTAL STUDIES ON POST-MORTEM CHANGES IN MALLARDS by Thomas L. Morrow Graduate Research Assistant and Fred A, Glover Leader Colorado Cooperative Wildlife Research Unit Colorado State University Bureau of Sport Fisheries and Wildlife Special Scientific Report--Wildlife No. 134 Washington, D. C. TABLE OF CONTENTS Page Abstract -^ Introduction ■'- Methods and Results ^ Body Temperature 2 Electrical Resistance 5 Appearance of Eyes ' Muscle Stiffening 8 pH of Muscle Tissue 9 Activity of Cilia 9 Other Criteria 10 Acknowledgments H Literature Cited 12 Appendix 13 List of Tables 13 List of Figures 13 ABSTRACT Post-mortem changes in mallards were studied to develop techniques of estimating time since death in birds killed by hunters. Birds were trapped during the winters of 1965 and 1966, killed and held for obser- vation in controlled-temperature rooms. Body temperature was the most reliable indicator of time since death. Ambient temperature had the most pronounced effect on rate of cooling; weights of the birds had the least effect. Soaking in water after death caused a rapid decrease from the live body temperature, 108 F. The electrical resistance of muscle tissue fell rapidly after death, then more slowly until a base level was reached after 7 to 11 hours. Initially, resistance was greater across the fibers than along them, but the difference decreased steadily after death. Eye changes, particularly configuration of the globe and color of the pupil and iris, generally indicated time since, death. The peak of rigor in muscles of the jaw, neck, and legs was commonly reached by 1 hour after death; rigor development in the flight muscles required about 1.5 hours. Ciliary activity of the tracheal lining declined gradually after death; inactive areas appeared and became larger. The greater part of a tracheal sample was inactive after 32 hours. Body temperature, eye appearance, muscle rigor, and tracheal ciliary activity should prove useful as field methods of estimating time since death. INTRODUCTION The purpose of this study was to develop techniques useful in establishing the time of death of waterfowl killed by hunters. As a necessary limitation, all work was confined to laboratory studies of mallards. These techniques are to be used by game enforcement officers in field-checks of birds taken by hunters. Reliable methods for estimating the time of death would strengthen the investigative position of the officer and offer valuable courtroom evidence. To investigate possible techniques and to gather data, mallards were killed under controlled conditions, and a number of different post-mortem changes observed. Time since death was followed closely in the experiments. Wild mallards were trapped in the vicinity of Fort Collins, Colorado, during the winters of 1965 and 1966; a small number was taken in the spring of 1965 and in the summer of 1966. The live birds were transported in sacks to laboratory facilities at Colorado State University where they immediately became part of an experiment. During the 1965 study period, 18 birds were sacrificed for the experiments; most of these were killed by a sustained thoracic pressure. During the 1966 study period, an additional 118 birds were taken. Of these, 21 were killed by administering intravenously a lethal dose of sodium pentobarbital (1 to 2 cc of a 6.4 per cent sol- ution), 9 were killed with thoracic pressure, and 10 died accidentally through overheating. The rest were asphyxiated with carbon dioxide. Asphyxiation was the most satisfactory method tried; death came con- sistently after about 1.5 minutes of gassing. METHODS AND RESULTS Body Temperature. Body temperature of the human cadaver has long been recognized as a useful indicator of time since death (Marshall and Hoare, 1962). It has been used extensively in investigations of human death, but most often with no clear knowledge of the complexities involved. Re- cent studies have had as their aim a clearer understanding of post- mortem body temperature changes and their effective interpretation in establishing the time of death. O'Meara (1965) has conducted similar investigations on the white-tailed deer. With waterfowl, body temperature was considered an important approach to estimating time of death. It was studied on an exploratory basis in 1965 with several other kinds of post-mortem changes, then intensively during the 1966 study period. The initial series of observations was made on 13 mallards (Table 1, Appendix). All birds were placed in controlled temperature rooms and observations were begun immediately after death. Investigations were made of post-mortem changes in the following: body temperature, electrical resistance of muscle tissues, appearance of the eyes, muscle stiffening in different body parts, pH of muscle tissue, and ciliary activity in the trachea. Eight of the original 13 birds examined were held at ambient temperatures near 40 degrees Fahrenheit (F), while five were kept near 30 degrees. Temperature readings were made at death and each hour thereafter for at least 24 hours. One thermometer with a bimetallic element and a metal probe was positioned in the thoracic cavity and read in place from a dial; it was inserted at a point just in front of the sternum to a penetration of about 3 inches. Another thermometer, a 6- inch mercury- in-glass type, was inserted through the vent to an approximate penetration of 3 inches (referred to here- after as the cloacal position); it had to be partly withdrawn for each reading. Two of the birds held at 40°F. were subjected to a % minute period of wetting, and two held at 30°F. to a 1 minute wetting period. Rates of temperature change and comparisons were made. Each temperature observation was expressed as a difference between the bird temperature and the average ambient temperature at which the bird was kept. These differences for each bird were plotted with hours after death on semi- logarithmic graph paper; the results in each case, two records for each of the 13 birds, seemed close to a linear relation- ship. On this basis, linear regression analysis was performed on each of the 26 sets of data, the natural logarithms of the temperature differences being 7 and hours after death x. Data from the regression coefficients (presented in Table 1) were fitted to the line equation. -2- Covariance analysis was used with the logarithmic temperature data, classifying it by ambient temperature and position of measurement. This analysis demonstrated that cooling was more rapid in birds held at 31 "F. than in those held at 39°F. (Tables 2 and 3, Appendix). Cooling rates at the thoracic position, as expressed by the regression coefficients, seemed in most cases more rapid than those measured cloacally. This difference was related to the early patterns of temperature change at the two positions. For the first half-hour after death temperature at the thoracic position changed very slowly or not at all; it then began to change more quickly, reaching its maximum rate near one hour post-mortem. In contrast, temperature at the cloacal position decreased rapidly almost immediately after death; no stationary period was evident. As a result, thoracic temperature was several degrees higher than cloacal temperature in the early hours after death. At death, how- ever, 108°F. was characteristic of both positions, although substant- ial variation occurred. This difference gradually decreased until the two temperatures were the same. This situation accounts for the noticeably higher regression coefficients found for the thoracic temperature data. It also indicates that linear regression is un- suitable for showing the relation between the data. During the 1966 study period, more intensive efforts were made to learn how mallard body temperature changed after death. An experiment was designed to determine the effects on post-mortem body temperature of the following factors: ambient temperature, bird weight, and a period of soaking in water after death as opposed to remaining dry. All observations of the influence of ambient temperature were made in controlled-temperature rooms. Different groups of birds were main- tained at 30°, 40°, 50°and 60°F. To study the effect of bird weight, two distinct weight groupings were recognized. Birds weighing 1090 grams (2.4 pounds or more) were compared with birds weighing less than 1090 grams, this critical point being the mean weight of all the birds treated. All birds were weighed to the nearest 10 grams. An attempt was made to take males and females in equal numbers throughout the study. The weight classification used also resulted in an effective separation of males from females. To study the effect of a period of soaking in water, immediately after death and weighing, approximately half of the birds were placed in a tub of water of the same temperature as the surrounding air for a three-minute period. During this time the plumage was ruffled slightly to allow water penetration and increase wetting. After removal the bird was shaken free of loosely clinging water. This treatment was envisioned as typical of conditions prevailing under hunting situations. The other half of the birds in the sample were kept dry. Body temperatures were measured and recorded by means of copper- constantan thermocouples (24 AWG, duplex, nylon-coated wire) used in -3- conjunction with a General Electric multipoint strip-chart recorder (Type HG; range, 0° to 140°F.). Thermocouple junctions were twisted and soft-soldered. Finally they were coated with a thin protective layer of dental acrylic. Each thermocouple was sheathed in a six- inch length of medical polyethylene tubing to facilitate its use as a probe. As each bird was killed it was hung in a breast-out tail-down position on a large pljrwood panel. Thermocouple probes were inserted at a position just anterior to the sternum and then posteriorly into the thoracic cavity, passing dorsal of the heart. Total penetration of the measuring junctions was three inches. Once in position the junctions were exposed by sliding the tubing back slightly. Tempera- ture measurements for each bird were made periodically and recorded on a moving chart. Measurements from each bird were continued for 20 hours. Consecutively numbered tags were attached to each bird with the following information: time of death, sex, weight (to nearest 10 grams), ambient temperature, wet or dry plumage, manner of killing, date, and number of the thermocouple used to measure temperature. It was expected that each of the variables studied would cause differences in cooling rates of the dead birds. So the primary aim was to define representative cooling curves for mallards on the basis of each variable condition. This procedure was expected to yield results that would permit estimates of time since death. Using estimates of variance from data taken during 1965 it was decided to include eight birds (replications) in each of the experi- mental groups. An analysis of variance was used to examine group differences (Table 4). For this purpose hours after death (1, 2, 3, . . . .20) was considered to be another variable condition, and individual temperature observations constituted the data. The analysis demonstrated significant differences in the variables: body weight, wet vs. dry condition, ambient temperature, and time since death. These differences indicate that each of the variables tested should be used in time since death estimation. Records of body temperature from death to 20 hours post-mortem were available for 113 mallards (five of these were records of birds killed during the 1965 phase of the study). In this total were 13 different groups, each representing one of the possible combinations of experimental conditions. The data in each of these groups were combined and plot- ted graphically. A curve was hand-fitted to each group. These curves represent the average cooling rates for mallards that existed under each of the 13 sets of laboratory conditions (Figures 1-4). In using these curves to estimate time since death under actual hunting conditions pertinent factors must be evaluated carefully. Even under the best of conditions and with the most tedious care, however, a certain amount of error occurs. The extent of this error will be learned after thorough field testing. The following points aid in using the curves. The Fahrenheit scale was used for all temperature measurements. In measuring body temperature, the sensitive part of the thermometer should penetrate 3 inches into the body cavity of the bird and insertion should be made just anterior to the sternum with the thermometer passing dorsal to the heart. A thermometer should be used which can be read while in position. A single, most representative ambient temperature that has existed since the death of the bird must be chosen. This may be relatively simple or very difficult depending on the circumstances. A crude interpretation of the bird's body temperature is useful. To use the information presented, an officer must keep detailed records of the changing air temperature and time. Separate cooling rates are distinguished for "heavy" (greater than 2.4 pounds) and "light" (less than 2.4 pounds) mallards. The average weight of birds in each group is given with each body tempera- ture curve (Figures 1-4, Appendix). Heavy and light correspond to male and female in the experimental birds. Both males and females should be significantly heavier in the fall, however. Weight of birds in the field should be determined as accurately as possible. It is necessary for the investigating officer to know if a bird falls into water when it is killed. The period in water and the degree of wetting is important. The primary effect is an increased cooling rate while in the water because initial body temp- eratures are depressed rapidly in proportion to the period of wetting. These known effects should aid in estimating the probable influences of different time periods of soaking that have occurred. Also, the "wet' curves are the result of conditions of identical air and water temp- eratures. There are special circumstances that may have pronounced effects on the cooling rate of a dead bird such as wind velocity, insulation of the bird from the open air, and field dressing or picking. No investigation was made of these variables. Electrical resistance of muscle tissue. A post-mortem change in the resistance offered by muscle tissues to the passage of an electrical current was considered a potential means of estimating time since death in mallards. Changes in conducti- vity are believed to be the result of chemical variations in cytoplasm and tissue fluids and structural alterations of cellular membranes. Resistance measurements were made in the tissue of the pectoral flexor muscles (Figure 5, Appendix). Two terminals or electrodes were inserted into the muscle and a potential created across the gap. This resistance was made the unknown leg of a Wheatstone bridge, and its value was found by comparison with standard internal resistances of the instrument. The instrument used was a Heathkit Impedence Bridge. Stainless steel hypodermic needles, number 15, were used as electrodes. Moulded into a small polyester block, the electrodes were uninsulated for 1.0 cm of their terminal length and spaced 1.0 cm apart between their parallel shafts. Their points were rounded. Beginning at death, hourly readings were made at each of three positions along the muscle. At each position two determinations were made; one parallel to the alignment of the fibers and another perpendicular to this alignment. The skin was lifted back from the muscle for each reading but the muscle remained covered between readings. Direct current was used to make the readings with the first two birds. Polarization of the electrodes caused single readings to be unstable and the instrument showed a steady rise in resistance. Despite an attempt to compensate for this by reading the instrument quickly, later plotting of the data revealed an erratic scattering of values. Nevertheless, a marked decrease in resistance was apparent during the first 5 hours after death, from above 2500 ohms at death to about 1500 ohms after 5 hours. A slight downward trend could be noted through about 30 hours post-mortem from several measurement positions. Polari- zation of the electrodes seemed to prevent reliable measurements from being made with direct current. Later the instrument was operated using alternating current, and the identical measurement procedure was applied to two birds. Polari- zation of the electrodes was prevented resulting in an improved correlation between muscle resistance and time after death. In Figure 6 (Appendix) resistance measurements from three positions of each of two birds have been averaged and plotted again at post-mortem interval. The pattern was similar to that found using direct current, but actual ohm values at corresponding periods after death were much smaller using alternating current. Resistance measurements made perpendicular to the alignment of muscle fibers were higher than the corresponding readings made parallel to the fibers for about 10 hours post-mortem. During this period the difference in resistance steadily decreased. Parallel readings had reache'd a near base level after about 7 hours post-mortem (800 ohms at death to 350 ohms), while perpendicular read- ings had reached a near base level after about 11 hours post-mortem (1200 ohms at death to 400 ohms). Later changes in resistance were slight. Resistance measurements using alternating current were attempt- ed on five additional birds, but the results were unsatisfactory. A new electrode assembly was used that was designed to be implanted in the muscle through feathers and skin and remain in place for the duration of measurements. The resistance values obtained were in- consistent and showed an erratic pattern of change. The initial values were lower and the final values higher than comparable A. C. readings made earlier. These results were attributed to problems in design and use of the new electrodes. Appearance of the eyes. The eyes of dead mallards were found to change in appearance in a definite pattern. The characters most useful were color and shape. Poor illumination prevents accurate observation of eye color so a flashlight was used to illuminate the eyes of all but the first four birds. The eyes were described as to general shape and turgor, shape of pupil and iris areas, color of pupil, and color of iris. The eyes of the birds were first observed and described one hour after death and at hourly intervals. Although an overall pattern was evident, variation occurred. Corresponding changes happened at different post-mortem intervals, and the eye conditions observed were inconsistent in duration. Even so, useful time of death estimates can be made on the basis of eye appearance . The following description of post-mortem eye changes is a com- posite presentation of all observations of eyes found to be of value in estimating time of death and the intervals at which they are most likely to occur. The range of occurrence and characteristics of eye changes are presented in Figure 7 (Appendix). Color terms follow the Villalobos system (Palmer, 1962). At death and for commonly 2 hours thereafter mallard eyes maintain their normal appearance as in the living bird. The pupil is ultramarine and slightly cloudy, the iris is chestnut and the whole eye has a smooth, moist, and turgid appearance. As moisture evaporates from the eye turgor is lost, small wrinkles appear, and indentations begin to form in the eye. These conditions typically prevail through about 9 hours post-mortem. A change occurs in pupil color near 5 to 8 hours post- mortem; the pupil becomes turquoise-cobalt, with a reflective or glossy appearance from inside the eye; no further changes in pupil color occur. With further moisture loss the corneal surface over the pupil generally becomes sunken and flat. The cornea remains elevated over the iris as a circular ridge around the sunken pupil. The iris becomes blackish- brown to ultramarine-violet at the same time and has a clear, watery look due to the fluid between 7 and 12 hours after death. After an indefinite period the cornea over the iris sinks to the level of the pupil and the entire eye appears flattened. This seems most character- istic of the period 14 to 24 hours after death. The iris loses its watery appearance at this time and becomes dull or opaque ultramarine- violet; it may appear medium gray to blackish-brown. However, the trend is usually toward darker colors as time since death increases. From the flattened condition of the eye, the pupil becomes increas- ingly sunken as moisture is lost. The pupil remains generally flat causing the iris to be pulled into an increasingly concave shape as it slopes upward to the white sclerotic ring outside the iris. This commonly begins near 22 hours post-mortem and gradually becomes more pronounced. After about 50 hours post-mortem the pupil area around the edges begins to assume the concave shape of the rest of the eye. The flattened area in the center of the eye gradually shrinks until the iris and pupil areas are uniformly and deeply concave. One or both eyes of a bird normally have reached this condition after about 60 hours post-mortem. Many eye conditions were not as distinct as those described, because they were complicated by irregular wrinkling. Since most of the changes described are the result of moisture loss from the eye, evaporation rate has an important effect on the time intervals involved. Muscle stiffening. The pattern and sequence of muscular stiffening is an important, established aid in human forensic pathology (Bendall, 1960 and Forster, 1963). It is most reliable for the relatively short period when rigor is becoming established. The final breaking of rigor seems more indistinct and variable. The onset of rigor in different muscle groups of mallards was observed by "feel". That is, pressure was carefully applied to a muscle area to test its flexibility. Care was taken to disturb the normal course of rigor as little as possible. Muscle groups in the bill, neck, legs, and breast were observed. For the purpose of comparison the degree of stiffening was described as none, slight, moderate, or rigid. After the first four birds were inspected, it became obvious that full rigor is established within 1 or 2 hours post-mortem. Thereafter, observations were concentrated in this general period. The pattern of stiffening observed among these muscle groups is variable because the time intervals required for the establishment of rigor were variable. Muscles controlling the bill often were observed to pass from initial stiffening to a condition of full rigor in 10 minutes or less, but in several instances stiffening proceeded over a much longer period. Stiffening of the bill began as early as 22 minutes and as late as 45 minutes post-mortem. Full rigor was reached as early as 27 minutes and as late as 75 minutes post-mortem. In one exceptional case, stiff- ening of the bill was observed to begin after 60 minutes post-mortem and terminate in full rigor only after 170 minutes post-mortem. In the muscles of the neck, stiffening began as early as 8 minutes and as late as 45 minutes post-mortem. Full rigor was achieved as early as 10 minutes and as late as 75 minutes post-mortem. In one bird, neck stiffening began at 70 minutes post-mortem and ended in full rigor after 140 minutes post-mortem. Muscles of the legs began to show stiffening as early as 8 minutes and as late as 40 minutes post-mortem. Full rigor was achieved as early as 10 minutes and as late as 70 minutes post-mortem. A period of 20 to 30 minutes was usually required in the stiffening process. Stiffening began in the flight muscles as early as 25 minutes and as late as 60 minutes post-mortem. Full rigor was reached as early as 40 minutes and as late as 90 post-mortem. Although time intervals required for stiffening and the sequences observed among parts are variable and conflicting, enough pattern existed to suggest that stress and handling from capture until killing might influence the results observed. Muscular activity was probably an important factor. Further investigations are planned under actual hunt- ing situations. pH of muscle tissue. Although post-mortem changes in muscle pH were expected, a suit- able means of measuring their magnitude needed to be investigated to determine their possible usefulness in a procedure for estimating time since death. Muscle samples were taken hourly from the left pectoral flexor muscles of two mallards, pulverized completely, and made to approximate 57o suspensions in distilled water. The pH of each sample was determined with three overlapping ranges of short-range pH paper. A change from pH 6.0 at death to 5.8 after 2 hours was observed. No later change seemed to occur. Later measurements were made on a single bird using a Beckman portable pH meter, model N. Immediately after death a combination pH electrode (Sargent, S-30070-10) was implanted in the pectoral flexor muscle. Readings were made thereafter at 5 minute intervals. At 10 minutes post-mortem, a pH of 6.70 was recorded; by 20 minutes post- mortem, 6.50 was recorded; by 30 minutes post-mortem, 6.40; by 40 minutes post-mortem, 6.30; and by 85 minutes post-mortem a final low of 6.20. Readings were discontinued after 115 minutes post-mortem. Activity of cilia on the tracheal lining. Ciliary activity of the tracheal lining of mallards was investi- gated as a possible indicator of time since death. The persistence of this function was studied by removing tracheal samples from 55 mallards at various post-mortem intervals (7-8, 12, 16, 25, 32 hours) and examining them for ciliary movement. A 4-inch segment of trachea was taken from each bird. The larynx was included in most instances. The samples were wrapped in a paper towel moistened with physiological saline solution and kept cool (30° to 40°F.) until examined. For examination, each sample was cut into a dorsal and ventral half and viewed under a dissecting microscope. Ciliary movement was apparent as a random waving motion on the inner lining of the trachea. Greater ciliary movement was present on the ventro-medial surface of the trachea and larynx than on the dorsal surface. The activity on the ventral surface was the criterion used in examining each trachea. This method of investigation revealed few sudden changes in intensity of ciliary movement after death. Gradual change appeared -9- to be the normal situation. Interpretation and description of ob- served conditions were rather subjective because considerable variation was seen between birds after the same post-mortem interval. For this reason it was difficult to define a clear pattern of change from the limited amount of data obtained. For 7 or 8 hours after death, ciliary movement remained uniformly strong over the ventral lining of the trachea. Activity became less vigorous in samples removed beyond 8 hours. Inactive areas were found interspersed with active areas in the sample. This effect became pro- nounced after 12 hours post-mortem, and inactive areas became larger and more common with increasing post-mortem interval. Occasional tracheal samples were found to be completely inactive, but this situa- tion seemed to be unrelated to the post-mortem interval. The greater portion of a sample was inactive after 32 hours, but small areas show- ed moderate to full activity. The strongest activity at this longest observed interval (and also at 25 hours post-mortem) was commonly observed on the floor of the larynx. Other criteria. An attempt was made to measure the force required to remove certain feathers from the mallard wing at hourly post-mortem intervals to see if a change occurred. The measuring device was inadequate for the job, how- ever, and should be investigated further. Another idea involved determining how long the muscles controlling movement of the iris were functional after death. Optical preparations used for dilating and contracting the pupils of human eyes, Neo- synephrine and Pilocarpine, respectively, were applied to the eyes of two birds before death and shortly after death, no appreciable change was noted in pupil size of any of the eyes so treated. -10- ACKNOWLEDGMENTS A great many people have given advice, assistance, made equip- ment available, and donated freely of their time during the two years this study was in progress. Without their help very little could have been accomplished. Administrative, research, and enforcement personnel of the Colo- rado Division of Game, Fish and Parks made the project possible. Controlled-temperature room facilities at the Surgical-Metabolic Laboratory and in the Anatomy-Physiology Building, Colorado State University were available to us through the courtesy of Drs, Kirvin Knox and Robert Phillips. Numerous people were helpful in a variety of ways from the following departments at Colorado State University: Animal Science, Chemistry, Engineering, Foods and Nutrition, Forest and Wood Sciences, Fishery and Wildlife Biology, Pathology, Physiology and Biophysics, Poultry Science, Veterinary Medicine, and Zoology. Drs. G. Ogura and J. Feegel, Denver General Hospital provided useful ideas, encouragement and references on the subject. Certain persons, because of their interests and contributions, warrant special thanks: John Gill, David O'Meara, Carrol Littlefield, David Anderson, John Criner, Carl Walters, John Oldemeyer, Dave Bowden, Dr. M. Morris, Dr. H. Steinhoff and Dr. R. Ryder. •11- LITERATURE CITED Bendall, J. R.. 1960. Post mortem changes in muscle. p. Ill -21k. In G. H. Bourne, The structure and function of muscle. Vol. 3: Pharmacology and disease. Academic Press, New York and London. Forster, B. 1963. The contractile deformation of skeletal muscle in rigor mortis. J. Forensic Med. 10:133-147. Gill, J. D. and David C. O'Meara. 1965. Estimating time of death in white-tailed deer. J. Wildl. Manage. 29(3) :471-486. July. Marshall, T. K. , and F. E. Hoare. 1962. Estimating the time of death; the rectal cooling after death and its mathematical expression. J. Forensic Sci. 7:56-81. Palmer, R. S. 1962. Handbook of North American birds. Vol. 1. New Haven, Conn. 567p. Snedecor, G. W. 1956. Statistical methods. 5th ed. The Iowa State College Press, Ames. xiii + 534p. ■12- APPENDIX List of Tables 1. Regression coefficients ^' and accompanying ambient temperatures for 13 adult drake mallards. 2. Pooled regression coefficients ^' by ambient temperature classes ' and points of temperature measurement. 3. Comparison of cooling rates of treatments based on ambient temperature classes and points of temperature measurement. 4. Analysis of variance showing group differences for variables: body weight (W) , wet vs dry condition (S), ambient temperature (A) and time since death (T) observed in studies of post-mortem changes in mallards. 5. Relative humidity in controlled-temperature rooms during post- mortem body temperature studies; measured with sling psychrometer at irregular intervals. List of Figures 1. Post-mortem aging curves as influenced by ambient temperature. Body temperature data were taken from dry mallards whose average weights were approximately 2.6 lbs. 2. Post-mortem aging curves as influenced by ambient temperature. Body temperature data were taken from dry mallards whose average weights were approximately 2.2 lbs. 3. Post-mortem aging curves as influenced by ambient temperature. Body temperature data were taken from mallards placed in water of ambient temperature for three minutes immediately following death and whose average weights were approximately 2.5 lbs. 4. Post-mortem aging curves as influenced by ambient temperature. Body temperature data were taken from mallards placed in water of ambient temperature for three minutes immediately following death and whose average weights were approximately 2.2 lbs. 5. Electrode unit used in measuring electrical resistance of muscle tissue and measurement positions (A, B, and C) along pectoralis flexor muscle. 6. Electrical resistance values, in ohms, obtained from right pectoral flexor muscles of birds No. 11 and 12 by hourly post- mortem intervals; each plotted value represents an average of three separate measurements made on each bird. •13- 7. Observation frequency ^' of certain post-mortem characteristics of mallard eyes "/ ; arrow indicates the general progression of change . •14- a/ Table 1. Regression coefficients and accompanying ambient temperatures for 13 adult drake mallards. Bird number Regression coefficients Ambient cloacal thoracic temp. ( F.) 1 -.129 -.145 38 2 -.130 -.144 37 3 -.121 -.134 37 4 -.137 -.148 38 7 -.149 -.156 40 8 -.140 -.137 39 9 -.161 -.153 39 10 -.147 -.157 39 13 -.163 -.165 30 14 -.163 -.171 30 15 -.153 -.145 30 16 -.152 -.164 32 17 -.158 -.160 33 a/ Regression coefficients describe the relationship between natural logarithmic values of differentials between body temperature and the average ambient temperature (the y variable) and hours after death (the x variable) . a/ Table 2. Pooled regression coefficients by ambient temperature classes °' and points of temperature measurement. Pooled regression coefficients, by Point of ambient temperature class measurement 31°F class 39°F class Cloacal -.157 -.132 Thoracic -.162 -.147 a/ Pooled regression coefficients derived from covariance analysis described by Snedecor (1956) . b/ 31°F class represented by birds numbered 13, 14, 15, 16, 17, an average of whose ambient temperatures is 31 degrees Fahrenheit. 39°Fclass represented by birds numbered 1, 2, 3, 4, 7, 8, 9, 10. •15- Table 3. Comparison of cooling rates of treatments based on ambient temperature classes and points of temperature measurement. * 31°F class, cloacal pos. (Birds no. 13,14,15,16,17) * 31°F class, thoracic pos. (Birds no. 13,14,15,16,17) 31 F class, cloacal pos. (Birds no. 13,14,15,16,17) * 39°F class, cloacal pos. (Birds no. 1,2,3,4,7,8,9,10) vs vs vs vs vs 39°F class, cloacal pos. (Birds no. 1,2,3,4,7,8,9,10) 39''f class, thoracic pos. (Birds no. 1,2,3,4,7,8,9,10) 31°F class, thoracic pos. (Birds no, 13,14,15,16,17) 39°F class, thoracic pos. (Birds no. 1,2,3,4,7,8,9,10) * A statistically significant difference exists between these two populations, as determined by covariance analysis (Snedecor, 1956). Table 4. Analysis of variance showing group differences for variables: body weight (W) , wet vs dry condition (S) , ambient temperature (A) and time since death (T) observed in studies of post-mortem changes in mallards. SOURCE DF SS MS s 1 w 1 A 2 T 19 SW 1 SA 2 ST 19 WA 2 WT 19 AT 38 SWA 2 SWT 19 SAT 38 WAT 38 SWAT 38 RESIDUAL 1680 TOTAL 1919 4338.70 2029.52 50308.08 532869.58 69.01 67.76 2008.14 476.45 231.07 6570.59 368.68 7.40 53.40 26.84 27.53 5882.75 605535.50 4538.70 2029.52 25154.04 28045.77 69.01 33.88 105.69 238.23 12.16 172.91 184.34 .39 1.41 .71 .72 3.50 42, .9 * 166, ,9 * 145, .5 * 8,013, ,1 * 176, .9 * 24, .0 * 30, ,2 * 335, .5 * 3, ,5 * 49, .4 * 256, ,0 * 0, .1 NS 0, ,4 NS 0, ,2 NS 0, .2 NS * - Significant at 99% level NS - Not significant -16- Table 5. Relative humidity in controlled-temperature rooms during post-mortem body temperature studies; measured with sling psychro- meter at Irregular intervals. Ambient Relative temperature humidity 30°F no information 40°F 65 - 70 % 50°F 55 - 60 % 60°F 25 - 30 % -17- (do) 3dniVa3dlM31 AQOa lAJ31dOI^-lSOd . 0) M U •H 0) •18- o \\ \\\ CVJ w f 4 m o» J I o / o / o ft -l ex Pu CO 0) 4-1 1-1 •U Q) r; 3 I 0) 1- •H n CO 4-1 < fi XI LlJ cfl 60 O >, 0) J3 IS III O •a 0) 00 T' o CO c (-1 0) (1) C/) 1 > ^ CO (O ^-( CK c •H CO -) o O W « 4-: 3 X cn CO (U X) > u )-i CO n ^ o CO an e c •H >, on (-1 n) T) e 6 0) u •u u u U-l o 1 c (1) 4-J ^ C/1 CO O 4J Oh 0) CNl 3 CO on 4-1 •H CO fxj TS •19- (1) 3 ■u n) (-1 +-l 3 O •H to n) to > u 3 o o ^1 p, & (0 0) u a) tn 1-1 _ a) ^ •U 60 •H di 60 tfl U XI O QJ (0 > CO 60 . •H tn 05 60 XI o CO ;j e (U ■u o B I 4-1 to O . CO CO 4-1 B to O 4-1 M CO 4-1 (U 60 •H ai -H •20- (Jo) 3aniva3diM3i xaoa i^3iaoN-isod u CO -a 3 (0 •H •a ^ B ^ C T) -H O E PQ (U dJ • M >-l 4-1 . =^ ^ <« g ^- ^ ti >^ ■U >^ 0) C en en 00 'O o (0 t-i CO 4J m c 6 fo (J en "+-1 01 CO 0) CO CO .^ ■ S o . 0) •H S M-l -21- o m UJ _i < o CO CM UJ I- < X o q: Q. Q. < pq CO C o •H (0 O ft (U CO n) (U 6 13 C CO 0) CO (fl •H 4J O CO 0) CJ c CO 4J CO •H CO (1) V4 cd u •H a (U LlI N CO _l < I- O < c •rH )-l . 3 (U to -H Cfl O Q) en 0 d a ^ •H V. O X) |>< 0) (U CO M 3 u-i en •H rH CO i-l O 4-1 u 0) a fd O 3 0) Xl o ■22- •H C a: iii OQ u. LjJ 1 (O (> ir CO LU 3 GQ S U. li. UJ O _l <> 1- CO z 3 UJ ^ CO o 1- _i 7^ < UJ o 2 H z cr o < _l _i < 3 o p Q _l rf' UJ UJ Q. UJ q: Q- q: f ' o o (NJ o o o 00 CD CVJ 00 < UJ Q (T. UJ I- U- < 01 CD J_ CVJ o O (M si^H0*30NvisiS3d 3nosnw o C 0) 4-1 en CO TJ Vl u to •H O. J3 0) M M-( O OJ U-l to ^ c CO to >-l o to 4-1 4-1 O C (U QJ ex tn QJ 4J U JZ o- 00 0) •H tJ 1-1 QJ e 3 O -H yi to y-4 > T3 -a 0) QJ C •1-' •M 4-1 CO O 4-1 -H ^ p. o ^ " o tn to e QJ J= O "■ tn C -H •H to > ^ u tn Q) 0) u 3 a -H -H "i ^ , > i T3 QJ )-l Q) 4-1 •H O >-i U3 C Q to e x; 4-1 1 a tn *-> CO •H tn Q) tn o QJ (X c S-i o >^ •-{ -H CO >-4 QJ O 3 ■r) •H O to S-i -^ E 4-1 O >^ tn QJ 43 4-1 c W CN QJ 6 .— 1 QJ ^ X! >-i ^ C 3 • to to 00 to •H ^ QJ [H -H 6 -23- Figure 7. Observation frequency^' of certain post-mortem character- istics of mallard eyes ' ; arrow indicates the general progression of change. Hours after death A B C D E F G H I 1 \ 13\ 8 20 2 2 \ \ A 12 20 2 1 1 3 \^ \10 16 6 5 3 4 ^6 ^ 8 15 7 6 3 5 3\ 13\ -.12 10 7 4 6 ^13 11^ 11 7 5 7 13"^ ^11 ^11 7 7 8 11 9^ ^13\ 8 7 9 9 8 ^14 \6 9 2 1 10 9 7 14 \ \ 6\ 9 3 1 11 8 7 14 5 \10 3 4 12 5 7 14 4\ \ ^^v 3 4 13 4 6 16 5 18\ 3 5 14 4 6 16 7 18. \5 6 15 2 6 16 8 20 \ \ ^ 5 16 2 4 20 8 20 \6. 5 17 2 1 21 8 20 \\ . 5 18 2 22 8 20 5\V 7 19 2 22 8 20 b> 7 20 2 22 5 20 '\ 7 21 1 22 5 20 6 \/ 9 22 1 22 4 20 5 11 23 1 22 4 20 4 12 24 1 22 4 20 4 12 27 22 22 2 9 30 22 22 2 9 33 22 22 1 10 36 22 22 1 10 39 22 22 10 42 22 22 10 45 22 22 13 48 22 22 13 54 22 22 13 60 22 22 10 3 66 22 22 7 5 72 22 22 2 5 78 22 22 2 5 -24- Figure 7. (continued) a/ Each frequency unit indicates that the characteristic (s) was descriptive of one mallard eye at the corresponding post-mortem interval. Observations for 11 birds are included. Only four were observed for 78 hours; two were observed through 58 hours; five were observed for 24 hours. Observation frequencies for characteristics D and F are extended past the 24 and 58 hour intervals (even though data are lacking) because these characteristics seem to persist unchanged for the full period. b/ Post-mortem characteristics of mallard eyes: A. Eyes as in life; moist, only slight loss in turgor; iris, chestnut. B. Turgor of eyes much reduced; indentations and wrinkling present, but not fully collapsed. C. Pupil ultramarine and slightly cloudy. D. Pupil turquoise-cobalt with a reflective or glassy appearance from inside the eye. E. Corneal surface over pupil sunken and largely flat; cornea elevated above iris as a circular ridge. F. Iris blackish brown to ultramarine violet; watery between cornea and iris in association with characteristic E, becom- ing an opaque violet-magenta to magenta with characteristics G and H. G. Cornea sunken flat over iris; whole eye is flat. H. Iris is pulled into an increasingly steep, concave position as the flat pupil area becomes more sunken. I. Area of iris and pupil a uniformly concave depression. As the Nation's principal conservation agency, the Department of the Interior has basic responsibilities for water, fish, wildlife, mineral, land, park, and recreational resources. Indian and Ter- ritorial affairs are other major concerns of this department of natural resources. The Department works to assure the wisest choice in managing all our resources so that each shall make its full contribution to a better United States now and in the future. UNITED STATES DEPARTMENT OF THE INTERIOR FISH AND WILDLIFE SERVICE BUREAU OF SPORT FISHERIES AND WILDLIFE WASHINGTON. D. C. 20240 POSTAGE AND FEES PAID DEPARTMENT OF THE INTERIOR