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There is a research gap in the evaluation of blood decomposition processes and the use of potential blood decomposition odor markers in establishing time of death ranges. On-set of coagulation in terms of blood decomposition can potentially display vital signs in determining how long the blood has been drying. Research exploiting the use of canines for cadaver detection has associated challenges in regard to the age of the blood used as a training aid, and ultimately a lack of scientific foundation as to blood odor signatures with respect to decomposition time. This novel research is an evaluation of specific volatile organic compounds (VOCs) within three substrates at various stages of decomposition. This study simultaneously evaluated blood decomposition profiles on two target substrates and its effect on Bluestar’s intensity. The purpose of this research further intends to increase the knowledge of detection windows of the presumptive reagent, Bluestar, by introducing a parallel understanding of distinctive volatile odor profiles in relation to the number of resulting false negatives and the observable intensity based on apparent luminescence. This study analyzed blood at three distinctive time frames: fresh (0-72 hours), intermediate (96-144 hours), and late (168-216 hours) potentially revealing different VOCs specific to each phase. Instrumental evaluation utilized DVB/CAR/ PDMS coated SPME fibers that were injected into a GC-MS for the identification of extracted blood decomposition odor profiles at each of the three decomposition time frames. Within the three-phases, two substrates were analyzed: metal nails (zinc and stainless steel), to determine if the number of detected VOCs present in natural blood decomposition is affected by the substrate’s composition. The odor profile resulted in a total of twelve detected VOCs detected above the headspace of the human donated pathogen free blood. These compounds were detected with the blood-contaminated nails in lower abundance, thereby showing the effect of substrate interactions. As for the compounds detected in metal nails a total of three compounds were unique to the stainless steel and four compounds were found above the headspace of the yellow zinc nail substrate. This is also one of the first studies to evaluate commonly used chemical reagents such as Bluestar and correlate the field response with the specific odor signature for that blood sample time frame. Based on the scale ranking results, Bluestar’s luminescence did not highlight a correlation between intensity and aging of blood on the substrates. This research presents a bridge to the knowledge gap of blood odor profile composition and presumptive reagents.

Bobwhite populations have been declining across their historic range for several decades. A number of hypotheses have been posited regarding factors contributing the observed decline. One such factor that that has recently gained attention from wildlife professionals is the contribution of disease and parasitism to bobwhite population regulation. Despite limited evidence implicating infectious agents in bobwhite population declines, researchers appear to largely disregard the role of the immune system in controlling said infectious agents. The immune system is the most important component of any organism’s defense against infectious agents. Although the immune system is well studied in a number of gallinaceous species (e.g. chickens and other poultry) there is a paucity of information regarding the immune system of bobwhites. Furthermore, the lack of information also pertains to the effects of varying factors encountered in the natural environment that may impact the immune system, susceptibility to infectious agents, and potentially mortality. Nutrition plays an important role in supporting the immune system with evidence of tradeoffs between other physiologically important processes and the immune system resulting from nutritional or resource limitation commonly encountered in natural systems. Other factors inherent to natural systems cannot typically be controlled by land managers (i.e. weather, disease processes, predation, etc.) with the exception of habitat management. Even then, responses of vegetative communities must yield to abiotic factors such as the weather (e.g. drought). However, nutritional pathways may be altered through the provision of supplemental food resources by land managers. Hence, nutritional pathways may be utilized to alter the immune function of bobwhites and consequently may buffer bobwhite populations against the effects of infectious agents if these processes are limiting bobwhite survival. The goals of my study were to assess whether measures of immune function from wild captured bobwhites were important parameters in bobwhite survival. To assess the effects of immune function on the survival of bobwhites, I utilized two immune assays; a bacteria killing assay (BKA) and an enzyme linked immunosorbent assay (ELISA) to assess the innate and adaptive immune function. I used the Nest Survival data type in Program Mark to asses survival of bobwhites captured over the years of 2010 – 2011, 2011 – 2012, and 2013 – 2014. Immune function measures (BKA and ELISA – denoted as IgY in survival models) as well as covariates related to year of monitoring, age (juvenile/adult), initial capture mass and feed supplementation status (supplemented/control) were utilized as covariates within the analysis to determine their relative importance in explaining survival. Only bobwhites with relevant immune data (BKA/IgY) were included to achieve a sample size of 124 individuals. The top models for survival through the analysis interval included the models containing the parameters, Year, Feed_Stat, and IgY. The top model (Year+Feed_Stat+IgY) contained the additive effects of all three aforementioned parameters (Wi = 0.501). The parameter Year was determined to be the most important parameter and appeared to “wash-out” the effects of Feed_Stat and IgY. A post-hoc analysis excluding Year indicated that Feed_Stat and IgY were the most important parameters. The model averaged probability of a bobwhite collected over a 3-year period surviving during the study was ~22% The role of nutrition on the bobwhite immune system was evaluated by providing a captive population of all male bobwhites with feeds of varying nutrient concentrations and compositions (i.e. vitamin, trace mineral, and antioxidant content.). The adaptive and innate immune systems were assessed via a battery of laboratory assays as well as in vivo assays. Diet did not influence innate immune function as measured by bactericidal killing capacity of serum, adaptive immune function measured as circulating immunoglobulin Y (IgY), and cell-mediated immune function as measured via subcutaneous swelling following phytohemagglutinin injection into the patagium. A significant time effect was observed for the adaptive and innate immune function parameters, circulating IgY and bactericidal killing capacity between time points (Day #1, Day #14, and Day #28). Through what nutritional pathways (e.g. nutritional component) the immune parameters were elevated is unknown. Whether nutritional quality is of consequence to the immune system of wild birds is yet to be seen and studies implicating nutritional profiles, available resources, and condition of bobwhites during immune compromising events warrants further study. The role of nutritionally supplemented diets consisting of varying nutritional qualities and components on muscle damage following extreme exertion was evaluated by providing a captive population of all male bobwhites with feeds of varying nutrient concentrations and compositions (i.e. vitamin, trace mineral, and antioxidant content.). Bobwhites were then exposed to a trial of muscular exertion for 15 minutes. Muscle damage resultant from exertion was assessed via determination of the concentration of enzymes indicative of muscle damage, creatine kinase (CK) and aspartate aminotransferase (AST) at 4 hours and 24 hours post exertion. Pathological examination of muscle tissues from the breast and thigh muscles were also utilized to determine effect of diet on incidence of muscle damage. A difference in CK concentrations between the NRC diet and NATURAL diet at 4 hours was observed. Four hours post exertion, CK concentrations in the NRC diet were an average of 8,690 +/- 3,572 U/L. No other differences between diets on CK and AST activity were observed at either 4 hours or 24 hours post exertion. I found that there was a significant difference in incidence of muscle damage as indicated by muscle damage lesion identification. It appears that nutritional quality did indeed affect muscle damage in bobwhites following strenuous muscular exertion, with bobwhites on the HVTM+A diet having the highest incidence of muscle damage as indicated by muscle damage lesions. The specific pathways regulating the occurrence of muscle damage in bobwhites fed diets with elevated nutrients is not known. However, the potential for reduction of muscle damage through increased nutritional supplementation does not appear in this specific study to be beneficial. A better understanding of the implications of specific nutrients, vitamins, antioxidants, etc. is necessary and additional research warranted.

This research project will impact the forensic field by providing an insightful scientific perspective on the chemical composition of decomposition odors of human analogues emitted into the environment. By studying the chemical makeup of decomposition odor or the “scent of death”, enhanced understanding of stage-specific volatile organic compounds (VOCs) can provide better understanding for better detection methods to be developed. This study also achieved a preliminary understanding of when a specific area can be cleared of any remaining decomposition odor. Not only will this study aid in the potential location of holding sites or transportation methods, it will also aid in optimal training of human remains detection dogs. There has been limited scientific research into how decomposition residual volatile organic compounds change over time, with even less scientific research into how the compounds change in a dry, arid environment such as west Texas. Emerging research has begun to look into the compounds present in soil during decomposition, but limited studies are specifically looking at the change of compounds as a longitudinal analysis over time, after the removal of the decomposing item. The purpose of this study was to monitor and provide abundances of the target decomposition odor vapors emanating from the soil after removal of decomposing human analogues (sus Scrofa). Pig meat samples and carcasses were placed in an outdoor study site to model the decomposition process and VOCs were collected intermittently over a one-month period. The odor collection process consisted of allowing pigs to decompose for a period of 14 days, 17 days and 21 days, then removing the pigs and sampling the soil once per week for a period of four weeks. Instrumental analysis utilized Divinylbenzene/Carbon/Polydimethylsiloxane (DVB/CAR/PDMS) coated Solid Phase-Microextraction (SPME) fibers that were injected into a Gas Chromatography-mass Spectrometry (GC-MS) system for the identification of extracted soil headspace odor profiles of target decomposition odors. The soil samples were sampled individually in 15mL glass vials for a period of 24 hours to allow for headspace extraction time optimization. Weather conditions such as temperature and humidity were recorded during each soil sample collection, as well as soil pH and moisture content. During each sample extraction, at each of the three intervals, both abundance and type of target volatiles were analyzed. The purpose was to document the change in both the type of volatiles present and their abundance at each of the set intervals. VOCs of interest were identified over the duration of the study, showing distinctive trends in compound abundance and disappearance. The benefit produced by the study has heightened the understanding of how decomposition odor changes over a set period of time. This research will ultimately aid in the knowledge and understanding of odor composition and concentration levels of residual decomposition odor for given decomposition times, with the generated information supporting search and recovery of holding sites and possible modes of transportation at various stages of decomposition.