Cause and Effect Analysis between Influencing Factors Related to Environmental Conditions, Hunting and Handling Practices and the Initial Microbial Load of Game Carcasses

Environmental, hunting and handling factors affect the microbial load of hunted game and the resulting meat products. The aim of this study was to systematically investigate the influence of several factors on the initial microbial load (IML) of game carcasses during the early hunting chain. Eviscerated roe deer body cavities (n = 24) were investigated in terms of total viable count and the levels of Pseudomonas spp., Lactobacillus spp., Enterobacteriaceae and Escherichia coli (E. coli). Furthermore, a risk analysis based on the obtained original IML data, literature search and a Failure Mode and Effects Analysis (FMEA) was performed. The IML could be explained in a regression model by factors including the higher body weight (BW), damaged gastrointestinal tract by the shot, ambient temperature or rain. The levels of Lactobacillus spp. (p = 0.0472), Enterobacteriaceae (p = 0.0070) and E. coli (p = 0.0015) were lower on the belly flap surface when gloves were used during evisceration. The literature search revealed that studies examining influencing factors (IF) on the IML of game carcasses found contradictory effects of the comparable IF on IML. Potential handling failures may lead to a higher IML of game carcasses during the early hunting chain ranked by FMEA. Several handling practices for game carcasses are recommended, such as ensuring efficient cooling of heavier BW carcasses to limit bacterial growth or eviscerating heavier carcasses before lighter ones

Influence of a high fat diet during adolescence on the reinforcing effects of cocaine and alcohol

RESUMEN La adolescencia es un período de especial vulnerabilidad en el desarrollo del ser humano, en el que se dan grandes cambios a nivel estructural y funcional (Spear, 2000; Chambers et al., 2003). En esta etapa de gran vulnerabilidad, los jóvenes muestran trastornos de la alimentación, consumo de sustancias, búsqueda de sensaciones y conductas de riesgo (Bava and Tapert, 2010). Actualmente, el consumo excesivo de comida especialmente rica en azúcares y grasas, se ha convertido en un serio problema para nuestra sociedad, habiéndose producido un incremento en las tasas de obesidad a nivel mundial que afecta particularmente a la población adolescente. La alimentación está regulada tanto por mecanismos homeostáticos como hedónicos. Un malfuncionamiento de cualquiera de estos sistemas puede provocar una alteración en la alimentación y producir obesidad (Kenny et al., 2011). El sistema de refuerzo, constituido esencialmente por el sistema dopaminérgico mesocorticolímbico, regula la motivación para buscar o consumir estímulos reforzantes como las drogas o la comida altamente palatable. Por lo tanto, el consumo de drogas o la alimentación por razones hedónicas activan las mismas vías del refuerzo. Inicialmente nuestro organismo se desarrolló en un contexto caracterizado por la escasez nutricional y conseguir alimentos era el principal objetivo a alcanzar en el día a día. Ya que nuestro cuerpo procesa los alimentos de forma rápida, mostramos una preferencia innata por las comidas con alto contenido calórico y energético. Este tipo de alimentos, al igual que la cocaína o el alcohol, producen liberación de dopamina en el sistema de recompensa cerebral. Hoy en día, en la sociedad de la abundancia y la variedad de alimentos, comemos no sólo por hambre, sino también por placer (Gold, 2011). Por lo tanto, comer por placer afecta a los mecanismos neurales relacionados con el refuerzo y promueve el mantenimiento de esta conducta (ver revisión Avena et al., 2008; Volkow et al., 2013). En resumen, tanto los reforzadores naturales como las drogas estimulan el circuito cerebral de recompensa, produciendo placer y euforia. El estado nutricional es un importante factor modulador en el desarrollo de la adicción, y algunos estudios han mostrado similitudes psicológicas y biológicas entre la ingesta de comida rápida y la adicción a las drogas, (Garber and Lustig, 2011; Avena et al., 2012). Por ejemplo, tanto la adicción a las drogas como la obesidad pueden ser definidas como trastornos en los que el valor de determinado refuerzo (droga o comida) está anormalmente incrementado en relación y a expensas de otros refuerzos. La adicción a drogas y los atracones de comida se caracterizan por una pérdida de control sobre la conducta consumatoria, la escalada, la dependencia y el ansia por consumir (craving), mostrando ambos trastornos una elevada comorbilidad (Swanson et al., 2011). Numerosos estudios han puesto de manifiesto que el consumo de drogas durante la adolescencia incrementa la probabilidad de desarrollar dependencia y problemas relacionados con estas sustancias en la edad adulta (Arteaga et al., 2010; Merline et al., 2004). Se ha propuesto igualmente una teoría de puerta de entrada también para la comida y el abuso de sustancias (Degenhardt y cols., 2008). Esta teoría postula que la ingesta compulsiva de comida puede facilitar el desarrollo de otros comportamientos desadaptativos, como es el consumo de drogas. Por el momento, los estudios preclínicos indican que el atracón de comida rica en azúcar provoca a una sensibilización conductual a la anfetamina (Avena y Hoebel, 2003) y aumenta la autoadministración de alcohol (Avena y cols., 2004). Se han realizado estudios utilizando comida rica en azúcar, sin embargo, los estudios con comida rica en grasa son escasos. Hasta la fecha todavía no se han evaluado las consecuencias de realizar atracones de grasa durante la adolescencia sobre el consumo de drogas como la cocaína y el alcohol. Tampoco conocemos los efectos de suspender el consumo de una dieta alta en grasas, sobre los efectos gratificantes de la cocaína o el alcohol. Así pues, el objetivo de la presente tesis doctoral ha sido evaluar cómo una dieta alta en grasa consumida durante la adolescencia, puede modificar los efectos reforzantes de la cocaína y el alcohol. Estudiamos dos tipos diferentes de consumo de la dieta alta en grasa, el acceso continuado a la dieta rica en grasa (los animales consumen la dieta durante todo el periodo de tiempo correspondiente, sin limitar su acceso) y el atracón, que es un acceso limitado e intermitente. Este último patrón se ha demostrado que induce un consumo de grasa en forma de atracones (Puhl y cols., 2011; Bocarsly y cols., 2011). En el protocolo de administración intermitente los ratones tienen acceso a la dieta rica en grasa solo durante 2 horas los lunes, miércoles y viernes con un acceso ilimitado a la dieta estándar. Como metodología principal para evaluar la respuesta a la cocaína y el alcohol utilizamos los paradigmas de Condicionamiento de la Preferencia de Lugar y el procedimiento de Autoadministración operante. El Condicionamiento de la Preferencia de lugar es el paradigma más utilizado para medir el efecto de las claves ambientadas asociadas con los efectos reforzantes de las drogas (Aguilar et al., 2013). Por otro lado, el modelo de autoadministración operante evalúa directamente la motivación del animal por conseguir la sustancia. Ambos modelos proporcionan una evaluación completa de los efectos reforzantes de las drogas, ya que nos permiten medir tanto las claves individuales como las externas. También hemos estudiado los efectos metabólicos que estos tipos de dieta grasa producían en los niveles plasmáticos de leptina, grelina y corticosterona. Igualmente, hemos estudiado mediante la reacción en cadena de la polimerasa (PCR) en tiempo real, la expresión de genes pertenecientes a los sistemas dopaminérgico, cannabinoide y opioide, debido a su implicación tanto en el refuerzo inducido por las drogas como por la comida. En cuanto a los resultados obtenidos, en el primer estudio hemos observado que, aunque no hay cambios en ninguna de las conductas estudiadas mientras se mantiene el consumo de grasa, el cese de esta dieta produce un incremento en el nivel de ansiedad, aumenta la respuesta aguda a la cocaína y produce condicionamiento de la preferencia de lugar con dosis subumbrales de cocaína. Todo esto se acompaña por cambios a nivel de expresión del gen para el receptor opioide mu, el cannabinoide CB1 y GHSR. Por otro lado, observamos el poder de la dieta rica en grasa como refuerzo alternativo, ya que cuando la administramos tras condicionar a los animales con una dosis efectiva, éstos tardan menos en extinguir la preferencia y no presentan reinstauración con dosis con las que el grupo control sí reinstaura la preferencia de lugar. En el segundo estudio hemos evaluado el efecto de realizar atracones de una dieta rica en grasa y hemos observado que provocan un aumento en la sensibilidad a dosis subumbrales de cocaína en el condicionamiento de la preferencia de lugar y se autoadministran más cocaína que el grupo con dieta estándar. Por otro lado, observamos también cambios en la expresión de los genes responsables de los receptores opioide mu, cannabinoide CB1 y de la grelina GHSR. En el tercer estudio realizamos el mismo procedimiento que en el estudio anterior, pero con el fin de evaluar si este tipo de administración de dieta rica en grasa también incrementaba la sensibilidad al alcohol. De nuevo encontramos que los animales que realizan atracones son más sensibles a dosis subumbrales de alcohol y se autoadministran más que el grupo con dieta estándar. Además, presentan cambios a nivel de expresión de genes tras la autoadministración, diferentes a los obtenidos con la simple administración de la grasa en estudios anteriores. En el cuarto estudio evaluamos si estos efectos sobre la ingesta de alcohol tenían un efecto a largo plazo, incluso habiendo cesado el consumo de grasa. Observamos cómo los animales que realizaron atracones durante la adolescencia y llevaban 15 días sin acceso a la grasa seguían presentando un incremento en la autoadministración de alcohol, pero ya no eran más sensibles a los efectos reforzantes de una dosis subumbral de alcohol en el condicionamiento de la preferencia de lugar. En el quinto estudio evaluamos el papel que tiene un estrés crónico, como es el aislamiento, en la modulación que produce la dieta rica en grasa en atracón sobre los efectos reforzantes de la cocaína. Observamos resultados opuestos a los obtenidos con animales agrupados, ya que los animales aislados con una dieta estándar fueron más sensibles a dosis subumbrales de cocaína que los que realizaron atracones de dieta rica en grasa. Además, los grupos que desarrollaron la preferencia (aislados dieta estándar y agrupados dieta atracón grasa) también presentaron niveles incrementados de leptina. En el sexto y último estudio trazamos un perfil conductual con todos los grupos que hemos utilizado en la presente tesis doctoral, observando que los animales que realizan atracones de grasa presentan pocas alteraciones conductuales. Únicamente exhiben un incremento en la actividad locomotora y conductas agresivas en la interacción social con un oponente. Sí que observamos déficits a nivel de aprendizaje espacial con los animales que comen comida rica en grasa de forma continuada y además de un comportamiento agresivo. En ambos tipos de dieta, el cese del consumo produce un incremento en el nivel de ansiedad. Los resultados obtenidos nos indican el consumo de una dieta alta en grasa de forma continuada produce efectos muy diferentes a los producidos por el consumo intermitente en atracón. Mientras que el consumo en forma de atracón no produce grandes efectos metabólicos, incrementa la sensibilidad a los efectos reforzantes del alcohol y la cocaína. Sin embargo, el consumo de grasa de forma continuada produce grandes cambios metabólicos, como hiperleptinemia y aumento de peso, pero no afecta la respuesta a la cocaína. Sin embargo, tras el cese en el consumo continuado de una dieta alta en grasa, aparece un incremento en la respuesta a los efectos condicionados reforzantes de la cocaína, confirmando así que el sistema de refuerzo ha quedado sensibilizado tras el consumo de grasa. Ambos tipos de dieta alta en grasa produce cambios en la expresión génica de los receptores opioide mu, cannabinoide CB1 y del receptor de grelina GHSR, indicando que ambos patrones de consumo de grasa modifican de forma diferente la función del sistema de refuerzo cerebral, produciendo consecuencias a largo plazo, incluso cuando la comida rica en grasa ya no está disponible. Nuestro trabajo demuestra que los hábitos nutricionales no solo producen alteraciones metabólicas en nuestro organismo o modifican nuestro peso corporal. También modifican nuestro SNC y alteran la forma en que respondemos a las drogas de abuso. Una de las principales aportaciones de este trabajo es la demostración de que la dieta grasa consumida de forma continuada, que induce grandes cambios metabólicos y obesidad, así como alteraciones conductuales, no parece modular la respuesta a las drogas hasta que esta deja de consumirse, momento en el que observamos una respuesta incrementada. Sin embargo, los atracones de grasa, que no alteran metabólicamente al sujeto, ni modifican su peso corporal o su conducta, sí que incrementan la respuesta a drogas como la cocaína o el etanol, incluso cuando ya ha dejado de consumirse. Por lo tanto, esperamos que los artículos que componen la presente Tesis Doctoral ayuden a incrementar el conocimiento y la conciencia sobre la importancia de los hábitos nutricionales en el abordaje de un problema multifactorial como es la adicción a las drogas.  ABSTRACT Adolescence is a highly vulnerable developmental period in which numerous structural and functional maturational changes occur (Spear, 2000; Chambers et al., 2003). In this early stage of vulnerability, adolescents exhibit eating disorders, substance abuse, novelty-seeking and risk-taking behaviors (Bava and Tapert, 2010). Regarding these environmental hazards, the rise in obesity rates worldwide has encouraged extensive research to improve understanding of this problem, in which the excessive intake of food, especially sugar-rich and high-fat food has become a serious problem for society. As we know, addiction is a chronic and multifactorial relapsing disorder resulting from an interaction of biological and environmental aspects, characterized by a loss of control in the use of the substance and relapse (Koob and Volkow, 2010). Developmental factors are important components of vulnerability, with strong evidence supporting that exposure to drugs of abuse during adolescence leads to a significantly higher likelihood of drug dependence and drug-related problems in adulthood. Feeding is regulated by homeostatic and hedonic mechanisms. Abnormalities in these functions can cause several feeding alterations and produce obesity (Kenny et al., 2011). The reward system, which is essentially constituted by the dopamine mesocorticolimbic system, regulates motivation to seek and take rewarding stimuli, such as drugs or highly palatable food. Therefore, drug abuse or hedonic eating activate the same reward pathways. Our body was initially developed in a context characterized by nutritional deficiencies and getting food was the daily main objective to achieve. It processes foods quickly and we have innate preference for high energy meals, especially those high in fat and sugar, because in a situation of scarcity those would be the energy stores that would help us to survive. This type of food, like drugs of abuse, produces a dopamine release in the brain reward system, which explains its pleasurable effects. Nowadays, there is such abundance and variety of food, that this survival-oriented evolutionary adaptation has lost its sense, since we eat not only for hunger, but also for pleasure (Gold, 2011). Thus, hedonic eating affects neural mechanisms connected with reward and maintains this behavior (For reviews: Avena et al., 2008; Volkow et al., 2013). In summary, both natural reinforcers and drugs stimulate the brain reward circuit, producing pleasure and euphoria. The nutritional status is an important factor in the development of addiction, since some studies indicate psychological and biological similarities between fast food intake and addiction to drugs, sharing common reward mechanisms (Garber and Lustig, 2011; Avena et al., 2012). For example, both drug addiction and obesity can be defined as disorders in which the value of the type of reinforcement (drug or food, respectively) is abnormally increased in relationship to other reinforcements (Avena et al., 2012; Volkow et al., 2013). Drug addiction and binge eating are characterized by a loss of control over consummatory behaviors, exhibiting escalation, dependence and craving when the reward is not available, and both present a high comorbidity (Swanson et al., 2011). Several studies report that drug use during adolescence often predicts an increased likelihood of continued use into adulthood (Arteaga et al., 2010; Merline et al., 2004). Based on these relationships, a Theory of Gateway has also been proposed for eating disorders and substance abuse (Degenhardt et al., 2008), in which it is postulated that eating disorders, such as binge eating, can lead to the development of another desadaptive behavior, such as drug abuse. At the moment, preclinical studies indicate, for example, that intake of certain types of sugar leads to a sensitization to amphetamine (Avena and Hoebel, 2003) and to an increase in alcohol self-administration (Avena et al., 2004). Studies with sugar are abundant, unlike studies with high-fat food and drug vulnerability. For example, there are no studies reporting the effects of bingeing on fat during adolescence and vulnerability to drug abuse, like cocaine or alcohol, nor are there studies examining the effects of interrupting fat consumption on the subsequent vulnerability to drugs like cocaine or alcohol. Thus, the objective of the present work was to evaluate how a high-fat diet exposure during adolescence modulates the reinforcing effects of cocaine and alcohol. We studied two different types of fat diet consumption: continuous access (animals have ad libitum access to high-fat food without a limit) and the intermittent and limited access. The latter has been shown to induce a binge eating pattern (Puhl and cols., 2011; Bocarsly et al., 2011), and our protocol is based on that used by Corwin et al. (1998). Here, mice had access to the high-fat diet only for 2 hours every Monday, Wednesday and Friday, with ad libitum access to the standard diet. The main methodology employed to assess the vulnerability to cocaine and alcohol rewarding effects was the Conditioned Place Preference paradigm and the operant Self-Administration procedure. The conditioned place preference is the most commonly used test to evaluate the environmental cues conditioned to the rewarding effects of the drug (Aguilar et al., 2013), while the self-administration procedure evaluates directly the animals’ motivation to obtain the substance of abuse. Both models provide a complete scenario to evaluate vulnerability to drug abuse, since they allow us to measure both the external and the individual cues. We have also studied some metabolic effects on circulating leptin, ghrelin and corticosterone levels and examined changes in gene expression with real time polymerase chain reaction (rt-PCR) related to dopaminergic, opioid and endocannabinoid systems, because of their involvement in the rewarding properties of drugs and food. Regarding our results, in the first study we observed no effects on the behavioral response to drugs while fat is available. However, following fat discontinuation, mice exhibited increased anxiety, augmented locomotor response to cocaine, and developed preference for subthreshold doses of cocaine. In addition, there were changes in MOr, CB1 and GHSR gene expression. On the other hand, we observed how high-fat diets act as an alternative reinforcer, as its administration after conditioned place preference reduces the number of sessions required to extinguish the preference and decreased sensitivity to drug priming-induced reinstatement. In the second study, we observed that animals that binged on fat were more sensitive to the reinforcing effects of a subthreshold dose of cocaine in the conditioned place preference and enhanced self-administration. We also observed changes in MOr, CB1 and GHSR gene expression. In the third study, we employed the same procedure as in the former study, but with the aim of evaluating if the increased sensitivity also arises with alcohol. Animals in the high-fat binge group presented more sensitivity to the rewarding effects of subthreshold doses of ethanol and greater ethanol consumption with a higher motivation to obtain the drug. In addition, they presented changes in gene expression after the self-administration procedure which are different from those found with the fat administration alone. In the fourth study, we evaluated if these effects of bingeing on fat and alcohol intake would have a long term consequences, even when fat consumption is interrupted. Our results showed how after 15 days from the last binge session, animals still presented a greater ethanol consumption, but they were not sensitive to subthreshold doses in the conditioned place preference anymore. The fifth study aimed to evaluate the role of chronic stress, such as isolation, on the modulatory effects of fat on the rewarding properties of cocaine. We observed opposite effects as those found in grouped animals, as isolated mice with a standard diet access were more sensitive to subthreshold doses of cocaine than those fed with a fat binge eating pattern. In addition, the groups that developed preference (isolated with standard diet and grouped with high-fat binge) were those who presented increased circulating leptin levels. In the sixth and last study, we drew a behavioral profile with the groups used in the present doctoral thesis, and found a few alterations in animals that binge on fat, such as hyperlocomotion and aggressive behaviors. On the other hand, we observed marked spatial learning deficits in animals eating fat continuously as well as increased attack behaviors with conspecifics. In both diet patterns, discontinuation of fat led to an increase in anxiety levels. Our results show that bingeing on fat is quite different from eating fat continuously. While the former does not produce great metabolic effects, it produces significant changes in the sensitivity to drugs such as alcohol and cocaine. On the other hand, continuously eating fat produces big metabolic changes like hyperleptinemia and increased bodyweight and but has no effect regarding vulnerability to drug use. However, when access to fat is interrupted (withdrawal period), the increased response to the rewarding effects of drugs arises, confirming that the reward system has become sensitized. Both fat consumption patterns produce several changes in gene expression of mu opioid receptor, of cannabinoid receptor CB1 and of ghrelin receptor GHSR, indicating that both patterns of fat consumption changed the reward system function in different manners, having long-lasting effects, even when fat is no longer available. Our work shows that the nutritional habits not only produce metabolic alterations in our organism or modify our body weight, they also modify our CNS and change the way w

Performance of Lead-Free versus Lead-Based Hunting Ammunition in Ballistic Soap

BACKGROUND: Lead-free hunting bullets are an alternative to lead-containing bullets which cause health risks for humans and endangered scavenging raptors through lead ingestion. However, doubts concerning the effectiveness of lead-free hunting bullets hinder the wide-spread acceptance in the hunting and wildlife management community. METHODS: We performed terminal ballistic experiments under standardized conditions with ballistic soap as surrogate for game animal tissue to characterize dimensionally stable, partially fragmenting, and deforming lead-free bullets and one commonly used lead-containing bullet. The permanent cavities created in soap blocks are used as a measure for the potential wound damage. The soap blocks were imaged using computed tomography to assess the volume and shape of the cavity and the number of fragments. Shots were performed at different impact speeds, covering a realistic shooting range. Using 3D image segmentation, cavity volume, metal fragment count, deflection angle, and depth of maximum damage were determined. Shots were repeated to investigate the reproducibility of ballistic soap experiments. RESULTS: All bullets showed an increasing cavity volume with increasing deposited energy. The dimensionally stable and fragmenting lead-free bullets achieved a constant conversion ratio while the deforming copper and lead-containing bullets showed a ratio, which increases linearly with the total deposited energy. The lead-containing bullet created hundreds of fragments and significantly more fragments than the lead-free bullets. The deflection angle was significantly higher for the dimensionally stable bullet due to its tumbling behavior and was similarly low for the other bullets. The deforming bullets achieved higher reproducibility than the fragmenting and dimensionally stable bullets. CONCLUSION: The deforming lead-free bullet closely resembled the deforming lead-containing bullet in terms of energy conversion, deflection angle, cavity shape, and reproducibility, showing that similar terminal ballistic behavior can be achieved. Furthermore, the volumetric image processing allowed superior analysis compared to methods that involve cutting of the soap blocks

The transition to non-lead rifle ammunition in Denmark: National obligations and policy considerations

The issue of Denmark regulating use of lead-free rifle ammunition because of potential risks of lead exposure in wildlife and humans was examined from a scientific and objective policy perspective. The consequences of adopting or rejecting such regulation were identified. Denmark is obliged to examine this topic because of its national policy on lead reduction, its being a Party to the UN Bonn Convention on Migratory Species, and its role in protecting White-tailed Sea Eagles (Haliaeetus albicilla), a species prone to lead poisoning from lead ingestion. Lead-free bullets suited for deer hunting are available at comparable cost to lead bullets, and have been demonstrated to be as effective. National adoption of lead-free bullets would complete the Danish transition to lead-free ammunition use. It would reduce the risk of lead exposure to scavenging wildlife, and humans who might eat lead-contaminated wild game meat. Opposition from hunting organizations would be expected

Reproducibility.

<p>Reproducibility was assessed by repeating shots. (A, B) Two cavities for the TAG bullet at 900 m/s appear similar except for small parts created by unpredictable fragments. (C) A strong correlation of the volumes between all first and second shots is found (n = 16), showing high reproducibility. (D) The correlation, i.e. reproducibility is highest for NVU and TSX.</p

Bullet types.

<p>Four commercially available bullets were used for the experiments.</p

Deforming copper bullet (TSX).

<p>(A–D) Cavities at increasing energies. (E) Cavity volume plotted over deposited energy with higher for the quadratic regression than for linear regression. (F) Ratio of volume and deposited energy increases with deposited energy (). Small fragments are indicated by yellow arrows.</p

Partially fragmenting copper bullet (TAG).

<p>(A–D) Cavities at increasing energies. The aluminum tip which supports the bullet expansion can be seen at similar depths. (E) Cavity volume plotted over deposited energy with linear and quadratic regression curves showing similar . (F) Ratio of volume and deposited energy is constant over the deposited energy.</p

Cutting vs. CT-Method.

<p>(A) The cutting method results in piecewise truncated cones which cannot accurately represent elliptical cross-sections. (B) Using the CT images, an accurate model of the cavity is created. (C) For both methods, the cross-sectional area is plotted over the depth. (D) The cavity volumes of 32 blocks measured by both methods show a strong correlation.</p