Lisosomal storage disease caused by ingestion of Astragalus spp in llamas: an emergent concern.

Lysosomal storage diseases are inherited or acquired disorders characterized by dysfunctional lysosomes that lead to intracytoplasmic accumulation of undegraded substrates, causing impaired cellular function and death. Many acquired lysosomal storage diseases are produced by toxic plants, which have indolizidine alkaloids, including swainsonine, that inhibits lysosomal α-mannosidase and Golgi α-mannosidase II. Swainsonine-induced nervous disease associated with various plants has been reported, including species of the genus Astragalus, Sida, Oxitropis, Swainsona, and Ipomoea. Two species of Astragalus (i.e. Astragalus garbancillo and Astragalus punae) have been found to cause neurologic disease in llamas. In addition, A. garbancillo was also associated with malformations in the offspring, and possibly abortions and neonatal mortality in llamas. The diagnosis of Astragalus spp. intoxication is established based on clinical signs, microscopic and ultrastructural findings, lectin histochemistry, abundance of these plants in the grazing area and determination of swainsonine in plant specimens.

Dynamics of changes in broiler spatial distribution induced by a robot with autonomous navigation along the growing cycle.

Welfare problems in broiler chickens are associated with accelerated growth in high density and barren environments. Encouraging broiler movement yields benefits by increasing locomotion, foraging, and environmental exploration. Robot sensors with autonomous navigation capabilities developed to collect husbandry information could collaterally induce movement of birds while traversing the chicken houses. This study examines the short-time dynamic of changes in broiler spatial distribution within the robot's zone of influence throughout the growing cycle. Two batches of mixed-sex Cobb-500 were raised in a commercial broiler farm until 42 d of age, in 2 houses divided into 4 equally sized sectors. In half of the sectors an AviSense robot sustained 2-h per day of autonomous navigation. The minute prior and the 4 min following the robot entering the zone of influence were video recorded weekly. Control sectors without a robot were analyzed equivalently. Number of individuals within the zone of influence of the robot were obtained at 1-s intervals and relative density (%) was estimated. Physical interactions between broilers and the robot, as well as interactions with the environment were also recorded. The entrance of the robot triggers within seconds a strong depopulation of the zone with birds walking to neighboring areas (P < 0.03, in all ages). The decreases in relative density induced by the robot appears more pronounced, and repopulation of the zone was slower, in younger than in older birds (P < 0.05). Broilers´ showed physical interactions towards the robot and were also touched and/or slightly pushed by the robots (19 and 84% of videos recorded, respectively). They were also found scratching and/or pecking the ground after the robot passed (64% of videos). Findings strongly suggest that robots, beyond their specific capabilities as environmental sensors, were effective in promoting increased movement in broilers along the growing cycle and could also favor additional exploratory behaviors. Thus, these robots could be considered as environmental enrichment elements that contribute to welfare improvements during intensive rearing.

Hepatic and renal lesions in sheep intoxicated with Urochloa hybrid Mulato II in Argentina.

A flock of 48 sheep in Argentina grazing on a pasture of hybrid Urochloa (formerly Brachiaria) Mulato II (Urochloa ruziziensis × Urochloa decumbens × Urochloa brizantha) developed facial dermatitis, severe jaundice, and weakness after brief physical activity. Blood biochemistry of 3 animals revealed azotemia, elevated aspartate aminotransferase activity, and increased direct, indirect, and total bilirubin concentrations. The urine was markedly turbid and contained large concentrations of bile pigments and protein. At autopsy of 2 animals, there was severe jaundice and subcutaneous submandibular edema. The livers were enlarged, intensely yellow, and had a marked acinar pattern. Gallbladders were distended, and the kidneys were diffusely dark in one animal and yellow-green in the other. Microscopically, there was lymphoplasmacytic and histiocytic cholangiohepatitis with abundant crystals in the lumen of bile ducts and in the cytoplasm of macrophages. The proximal and distal convoluted renal tubules had protein casts in their lumens, and crystals were observed in the lumen and epithelial cells. Lectin histochemistry showed strong affinity for Arachis hypogaea agglutinin in hepatic macrophages. In the one sheep that was tested for heavy metals, copper concentrations in the liver and kidney were within the RIs. Despite the immediate change of pasture, morbidity and mortality were 100% within 3 mo. The association between the consumption of this pasture, and the clinical, biochemical, pathology, and lectin histochemistry findings confirmed intoxication with Urochloa hybrid Mulato II. To our knowledge, intoxication by this hybrid of Urochloa has not been reported previously.

High-resolution ethograms, accelerometer recordings, and behavioral time series of Japanese quail.

Although many small vertebrates are capable of performing high-speed behaviors, most studies continue to focus on low-resolution temporal scales (>>1 s). Herein, we present video-recordings, behavior time series, and the computer software for video-analysis of Japanese quail within social groups. Home-boxes were monitored using both top and side video-cameras. High-resolution ethograms were developed for analyses. Pairs of females were assigned as either controls or using one of two methods for attachment of an accelerometer (patch or backpack). Behavior was recorded during 1 h on the first 2-days, sampled at 1 s intervals (days 1 and 2). On day 8, an unfamiliar male was placed in the home-box and its behavior was recorded during the first 10 min, sampled every 1/15 s. Male accelerometer recordings were also obtained. Video-recordings and resulting detailed high-resolution behavioral time series are valuable for reuse in comparative studies regarding the temporal dynamics of behavior within social environments. In addition, they are necessary for the assessment of novel machine learning algorithms that could be used for deciphering the output of accelerometer recordings.

Transcriptional analysis of landmark molecular pathways in lung adenocarcinoma results in a clinically relevant classification with potential therapeutic implications.

Lung adenocarcinoma (LUAD) is a molecularly heterogeneous disease. In addition to genomic alterations, cancer transcriptional profiling can be helpful to tailor cancer treatment and to estimate each patient's outcome. Transcriptional activity levels of 50 molecular pathways were inferred in 4573 LUAD patients using Gene Set Variation Analysis (GSVA) method. Seven LUAD subtypes were defined and independently validated based on the combined behavior of the studied pathways: AD (adenocarcinoma subtype) 1-7. AD1, AD4, and AD5 subtypes were associated with better overall survival. AD1 and AD4 subtypes were enriched in epidermal growth factor receptor (EGFR) mutations, whereas AD2 and AD6 showed higher tumor protein p53 (TP53) alteration frequencies. AD2 and AD6 subtypes correlated with higher genome instability, proliferation-related pathway expression, and specific sensitivity to chemotherapy, based on data from LUAD cell lines. LUAD subtypes were able to predict immunotherapy response in addition to CD274 (PD-L1) gene expression and tumor mutational burden (TMB). AD2 and AD4 subtypes were associated with potential resistance and response to immunotherapy, respectively. Thus, analysis of transcriptomic data could improve patient stratification beyond genomics and single biomarkers (i.e., PD-L1 and TMB) and may lay the foundation for more personalized treatment avenues, especially in driver-negative LUAD.

Pascalia glauca poisoning in ruminants: Report of 12 outbreaks in northwestern Argentina.

Pascalia glauca, a perennial herb known for its toxicity in ruminants, is one of the most important toxic plants in northwestern Argentina. This work describes several outbreaks of natural intoxication with P. glauca from different sources affecting cattle, sheep and goats in different production systems, with morbidity varying between 1% and 100%. Clinical signs included tremors in the hindquarters, recumbency and death. The liver showed swelling, congestion and a mottled appearance. Histopathologic findings included centrilobular hepatocellular necrosis with hemorrhage.

Age matters: Differential effects of the exposure to elevated environmental temperatures on representative variables of the immune system in juvenile and adult female Japanese quail.

Several days under the influence of high environmental temperature negatively impact on some immune-related variables of poultry species. However, it is little known whether this exposure affects differentially the immune response on different life stages. This study was aimed at characterizing the immune responses of juvenile and adult female Japanese quail (Coturnix coturnix) when exposed to a cyclic chronic heat stressor. Independent sets of juvenile (24) and adult (21) female Japanese quail were bred under thermoneutral conditions or exposed to an increasing temperature from 24 °C to 34 °C, for 8 h along nine consecutive days (chronic heat stressor). The inflammatory response against a mitogen, the antibody production against a non-pathogenic antigen and the heterophil to lymphocyte (H/L) ratio were measured after the ninth day of chronic heat exposure. Adult females had a more robust inflammatory response compared to their juvenile counterpart. The inflammatory response, the production of antibodies, and the H/L ratio were suppressed by the chronic heat stressor in the juvenile females; while in their adult counterpart, the production of antibodies was not affected by the chronic heat stress. Under the exposure of high environmental temperatures, juvenile females would be more susceptible to potentially harmful pathogens that require a robust immune response, while their adult counterparts would be able to mount an optimal acquired immune response against pathogens requiring this type of response. Our findings suggest that while heat stress during breeding should be avoided in both juvenile and adult females, more precautions should be taken during early life stages.

Intoxication of llamas by Astragalus punae in Argentina.

Several plants that contain indolizidine alkaloids, including swainsonine, are toxic to livestock, causing dysfunctional lysosomes and storage disease. Swainsonine induces a neurovisceral disease, known as locoism, in sheep, goats, and cattle, which occurs in several parts of the world, including, but not limited to, the western United States, China, and parts of Australia. In South America, locoism has been described in the Andean region of Argentina affecting sheep, cattle, and llamas. Intoxication by consumption of Astragalus punae was suspected in 4 llamas in Jujuy Province, northwestern Argentina. The grazing area contained abundant specimens of A. punae. The clinical course was ~15 d, and included moderate ataxia, incoordination of hindlimbs, and progressive loss of body condition. Microscopically, fine cytoplasmic microvacuolation was observed in the proximal convoluted renal tubules. Ultrastructurally, these changes consisted of severely dilated lysosomes. Swainsonine was detected in stem and leaf samples of A. punae at a concentration of 0.06%. Based on clinical history and signs, histologic and ultrastructural changes, and plant analysis, a diagnosis of swainsonine toxicosis caused by consumption of A. punae was made, which has not been reported previously, to our knowledge.

Yolk corticosterone and progesterone levels in Greater Rhea (Rhea americana) eggs vary in a changing social environment.

Maternal hormones in avian egg yolks may signal and prepare offspring for the prevailing conditions. However, this adjustment requires some degree of flexibility in regulating yolk hormone deposition. The Greater Rhea (Rhea americana) has a particular mating system that combines mixed polygyny and polyandry, communal nesting, and exclusive paternal care of chicks. In this species, we previously found that yolk hormone deposition varies among eggs of different captive populations and could influence chicks' physiology and behavior. However, it is still unknown whether females can modify yolk hormone deposition in a changing social environment. Using a captive population of Greater Rheas, in this study, we quantified yolk hormone levels before and after a reduction in the number of females present in the population. We found that females deposited on average higher yolk corticosterone and lower yolk progesterone after the change in their social environment. Since corticosterone deposited into the yolk comes exclusively from the female's plasma, our results suggest that females had, on average, higher plasma corticosterone levels. The change in the number of females may increase the events of male-male competitions, courtships, and matings, leading to an increase of corticosterone in the females' plasma and then into their eggs. Since we previously found that higher yolk corticosterone and lower yolk progesterone were associated with the production of chicks that have an attenuated stress response, the present study results suggest that yolk hormone deposition is mediated by flexible mechanisms that could adjust development to the prevailing conditions.

Gene Expression Profiling as a Potential Tool for Precision Oncology in Non-Small Cell Lung Cancer.

Recent technological advances and the application of high-throughput mutation and transcriptome analyses have improved our understanding of cancer diseases, including non-small cell lung cancer. For instance, genomic profiling has allowed the identification of mutational events which can be treated with specific agents. However, detection of DNA alterations does not fully recapitulate the complexity of the disease and it does not allow selection of patients that benefit from chemo- or immunotherapy. In this context, transcriptional profiling has emerged as a promising tool for patient stratification and treatment guidance. For instance, transcriptional profiling has proven to be especially useful in the context of acquired resistance to targeted therapies and patients lacking targetable genomic alterations. Moreover, the comprehensive characterization of the expression level of the different pathways and genes involved in tumor progression is likely to better predict clinical benefit from different treatments than single biomarkers such as PD-L1 or tumor mutational burden in the case of immunotherapy. However, intrinsic technical and analytical limitations have hindered the use of these expression signatures in the clinical setting. In this review, we will focus on the data reported on molecular classification of non-small cell lung cancer and discuss the potential of transcriptional profiling as a predictor of survival and as a patient stratification tool to further personalize treatments.

Short- and long-term dynamics of the physiological and behavioral response to heat stress and thymol supplementation in Japanese quail.

Organisms have evolved endogenous timing systems that enable them to predict temporal changes and to coordinate complex internal processes. However, temporal dynamics of biological responses are most often ignored in fields such as dietary supplementation of farm animals exposed to artificial environmental challenges. Herein, we hypothesized that the potential for thymol (2-isopropyl-5-methylphenol) to alleviate physiological and behavioral consequences of heat stress is time-dependent on both long-term (i.e. weeks) and short-term (i.e. within day) time scales. First, during 3-weeks adult female Japanese quail (Coturnix japonica) were exposed daily to 9h of increased environmental temperature (34.2 ± 0.1 °C). Controls remained at standard temperatures (23.6 ± 0.1 °C). Simultaneously, half received thymol dietary supplementation and the other half a control basal diet. On day 4, both thymol and heat stress decreased body weight and feed intake respect to controls (basal, standard temperature). After three weeks, feed intake recovered for thymol groups. Therefore, we performed a second experiment focused on the critical first week of treatment, sampling variables three times a day. The beneficial effects of thymol supplementation were mainly observed during the morning, including prevention of high respiratory rates and reduction in the weight of droppings induced by heat stress, and increased walking under both temperatures. In summary, thymol's potential for alleviating heat stress consequences is time-dependent, and can be conceived as an emergent property resulting from the complex interplay between the dynamics of the biological response to thymol and heat stress. Findings highlight the importance of considering time-related factors when developing supplementation protocols to mitigate environmental challenges.

Integrative transcriptome analysis of malignant pleural mesothelioma reveals a clinically relevant immune-based classification.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasia affecting the lung mesothelium. Immune checkpoint inhibitors (ICI) in MPM have not been extremely successful, likely due to poor identification of suitable candidate patients for the therapy. We aimed to identify cellular immune fractions associated with clinical outcome and classify patients with MPM based on their immune contexture. For each defined group, we sought for molecular specificities that could help further define our MPM classification at the genomic and transcriptomic level, as well as identify differential therapeutic strategies based on transcriptional signatures predictive of drug response. METHODS: The abundance of 20 immune cell fractions in 516 MPM samples from 7 gene expression datasets was inferred using gene set variation analysis. Identification of clinically relevant fractions was performed with Cox proportional-hazards models adjusted for age, stage, sex, and tumor histology. Immune-based groups were defined based on the identified fractions. RESULTS: T-helper 2 (TH2) and cytotoxic T (TC) cells were found to be consistently associated with overall survival. Three immune clusters (IG) were subsequently defined based on TH2 and TC immune infiltration levels: IG1 (54.5%) was characterized by high TH2 and low TC levels, IG2 (37%) had either low or high levels of both fractions, and IG3 (8.5%) was defined by low TH2 and high TC levels. IG1 and IG3 groups were associated with worse and better overall survival, respectively. While no differential genomic alterations were identified among immune groups, at the transcriptional level, IG1 samples showed upregulation of proliferation signatures, while IG3 samples presented upregulation of immune and inflammation-related pathways. Finally, the integration of gene expression with functional signatures of drug response showed that IG3 patients might be more likely to respond to ICI. CONCLUSIONS: This study identifies a novel immune-based signature with potential clinical relevance based on TH2 and TC levels, unveiling a fraction of patients with MPM with better prognosis and who might benefit from immune-based therapies. Molecular specificities of the different groups might be used to tailor specific potential therapies in the future.

Modulating offspring responses: concerted effects of stress and immunogenic challenge in the parental generation.

The perception, processing and response to environmental challenges involves the activation of the immuno-neuroendocrine (INE) interplay. Concerted environmental challenges might induce trade-off when resource allocation to one trait occurs at the expense of another, also producing potential transgenerational effects in the offspring. We evaluated whether concerted challenges, in the form of an immune inoculum against inactivated Salmonella enteritidis (immune challenge, ICH) and a chronic heat stress (CHS) exposure on adult Japanese quail, modulate the INE responses of the parental generation and their offspring. Adults were inoculated and later exposed to a CHS along nine consecutive days. For the last 5 days of the CHS, eggs were collected for incubation. Chicks were identified according to their parental treatments and remained undisturbed. Induced inflammatory response, heterophil/lymphocyte (H/L) ratio and specific humoral response against sheep red blood cells (SRBC) were evaluated in both generations. Regardless of the ICH, stressed adults showed a reduced inflammatory response and an elevated H/L ratio compared with controls. In offspring, the inflammatory response was elevated and the specific SRBC antibody titres were diminished in those chicks prenatally exposed to CHS, regardless of the ICH. No differences were found in the H/L ratio of the offspring. Together, our results suggest that CHS exposure influences the INE interplay of adult quail, establishing trade-offs within their immune system. Moreover, CHS not only affected parental INE responses but also modulated their offspring INE responses, probably affecting their potential to respond to future challenges. The adaptability of the developmental programming of offspring would depend on the environment encountered.

Intoxication by Astragalus garbancillo var. garbancillo in llamas.

Lysosomal storage diseases are inherited and acquired disorders characterized by dysfunctional lysosomes. Intracytoplasmic accumulation of undegraded substrates leads to impaired cellular function and death. Several plant species are toxic to livestock because of the presence of indolizidine alkaloids, including swainsonine, which cause a storage disease. Swainsonine-induced nervous disease (i.e., locoism) of sheep and cattle is well recognized in several parts of the world, particularly in the western United States and in parts of Australia. Spontaneous intoxication by Astragalus garbancillo var. garbancillo was suspected in a group of 70 llamas (Lama glama) in Jujuy Province, northwestern Argentina. The animals grazed an area dominated by stands of A. garbancillo var. garbancillo. Clinical signs were staggering, ataxia, hypermetria, and progressive weight loss. The clinical course in individual animals was ~50 d. The main microscopic changes were Purkinje cell degeneration, necrosis, and loss, associated with intracytoplasmic vacuolation, meganeurite formation, and Wallerian degeneration. Specific positive labeling for ubiquitin was observed in axonal spheroids. Composite leaf and stem samples of A. garbancillo var. garbancillo analyzed by high-performance liquid chromatography contained 0.03% swainsonine. Based on the microscopic lesions, clinical history, and plant analysis, a diagnosis was made of storage disease caused by consumption of swainsonine-containing A. garbancillo var. garbancillo.

Aggressive dominance can decrease behavioral complexity on subordinates through synchronization of locomotor activities.

Social environments are known to influence behavior. Moreover, within small social groups, dominant/subordinate relationships frequently emerge. Dominants can display aggressive behaviors towards subordinates and sustain priority access to resources. Herein, Japanese quail (Coturnix japonica) were used, given that they establish hierarchies through frequent aggressive interactions. We apply a combination of different mathematical tools to provide a precise quantification of the effect of social environments and the consequence of dominance at an individual level on the temporal dynamics of behavior. Main results show that subordinates performed locomotion dynamics with stronger long-range positive correlations in comparison to birds that receive few or no aggressions from conspecifics (more random dynamics). Dominant birds and their subordinates also showed a high level of synchronization in the locomotor pattern, likely emerging from the lack of environmental opportunities to engage in independent behavior. Findings suggest that dominance can potentially modulate behavioral dynamics through synchronization of locomotor activities.

High-resolution behavioral time series of Japanese quail within their social environment.

The behavioral dynamics within a social group not only could depend on individual traits and social-experience of each member, but more importantly, emerges from inter-individual interactions over time. Herein, we first present a dataset, as well as the corresponding original video recordings, of the results of 4 behavioral tests associated with fear and aggressive response performed on 106 Japanese quail. In a second stage, birds were housed with conspecifics that performed similarly in the behavioral tests in groups of 2 females and 1 male. By continuously monitoring each bird in these small social groups, we obtained time series of social and reproductive behavior, and high-resolution locomotor time series. This approach provides the opportunity to perform precise quantification of the temporal dynamics of behavior at an individual level within different social scenarios including when an individual showing continued aggressive behaviors is present. These unique datasets and videos are publicly available in Figshare and can be used in further analysis, or for comparison with existing or future data sets or mathematical models across different taxa.

Dynamics of thymol dietary supplementation in quail (Coturnix japonica): Linking bioavailability, effects on egg yolk total fatty acids and performance traits.

Phytogenic additives such as thymol are encountering growing interest in the poultry industry. However, there are still questions concerning dynamics of their bioavailability, biological action, optimal dosage and duration of supplementation needed to achieve meaningful effects, as well as persistence of induced changes after supplement withdrawal. We studied the link between the dynamics of free thymol concentration and the changes in fatty acids composition in quail egg yolk, both during a month-long chronic dietary supplementation and after 3 weeks of supplement withdrawal (post-supplementation). Fifty, 85 days-old, female quail of homogeneous body weights (251±1g) in egg-laying peak were used. To evaluate potential dose-dependent effects, three increasing doses 2, 4, and 6.25 g of thymol/kg of feed (THY2, THY4 and THY6, respectively) and two controls were evaluated (n = 10). In parallel, we assessed the concomitant changes in free thymol excretion, potential liver histopathological changes, and birds´ performance traits. Egg yolk and droppings show a dose-dependent increase in THY concentration after 9 days of supplementation and a decrease after post-supplementation. In egg yolk, these changes were accompanied by reduced saturated fatty acid concentrations achieved by 28 days of supplementation in THY2 and 14 days of supplementation in THY4 and THY6. However, after post-supplementation the aforementioned effect disappeared in THY2 but not in THY4 and THY6. While THY2 failed to increase polyunsaturated fatty acids, THY4 and THY6 increased polyunsaturated fatty acids by day 14 of supplementation and remained increased after post-supplementation. Fatty acids changes induced by thymol are consistent with improved nutritional quality of eggs. No treatment effects were observed in liver histopathology and female performance. Findings suggest that both dose of thymol and duration of supplementation modulate thymol and fatty acids concentrations in egg yolk and thymol concentration in droppings. Furthermore, the persistence of those effects after post-supplementation period is also a dose-dependent phenomenon.

Effect of maternal environment on yolk immunoreactive corticosterone and its influence on adrenocortical and behavioral activity in chicks of Greater Rhea (Rhea americana).

Maternal corticosterone in avian eggs may modify offspring phenotype in order to increase survival in poor environments. In the Greater Rhea (Rhea americana), we previously found that yolk immunoreactive corticosterone is influenced by the quality of the maternal environment: eggs laid by females of the intensive rearing system (IRS), living in poor captive conditions, had higher yolk immunoreactive corticosterone than those produced by females of the semi-extensive rearing system (SRS), living in better conditions. Here, we evaluate if these different hormone levels are associated with the production of different phenotypes. We collected eggs from the IRS and SRS for hormonal quantification and artificial incubation. Then, half of the chicks selected from each environment were exposed to a capture and restraint protocol, and the rest remained undisturbed and were used as controls. In the IRS, we found that higher yolk immunoreactive corticosterone was associated with the production of chicks that had reduced hatchability, lower hatchling mass and higher baseline fecal glucocorticoid metabolites (FGM) than those produced by SRS females. Moreover, after capture and restraint, IRS chicks did not modify their FGM nor their behaviors compared to their controls, while SRS chicks increased their FGM and spent more time ambulating and less time pecking, compared to their controls. These results indicate that yolk immunoreactive corticosterone could modify offspring phenotype. Although future studies are needed to elucidate their implications for fitness, our results suggest that yolk corticosterone could be mediating an adaptive maternal effect that allows individuals to better cope with poor conditions.

Expression of aggressiveness modulates mesencephalic c-fos activation during a social interaction test in Japanese quail (Coturnix japonica).

It is well known that during a social conflict, interactions are dependent on the animal's propensity to behave aggressively as well as the behavior of the opponent. However, discriminating between these two confounding factors was difficult. Recently, a Social Interaction (SI) test using photocastrated males as non-aggressive stimuli was proposed as a useful tool to evaluate aggressiveness. The avian Intercollicular- Griseum centralis complex (comparable to mammalian periaqueductal gray) has been reported as a crucial node in the descending pathways that organize behavioral and autonomic aspects of defensive responses and aggressiveness. Herein, using the SI test, we evaluated whether mesencephalic areas are activated (expressed c-fos) when photostimulated adult males are confronted with non-responsive (non-aggressive) opponents. Furthermore, we also examined whether mesencephalic activation is related to male performance during the SI test (i.e., aggressive vs. non-aggressive males) in birds reared in enriched or in standard environments. Five mesencephalic areas at two anatomic levels (intermediate and rostral) and locomotion during SI testing were studied. Aggressive males showed increased c-fos expression in all areas studied, and moved at faster speeds in comparison to their non-aggressive and control counterparts. Non-aggressive males and the test controls showed similar c-fos labeling. In general, rearing condition did not appear to influence c-fos expression nor behavior during the SI test. Findings suggest that mesencephalic activation is involved when males are actively expressing aggressive behaviors. This overall phenomenon is shown regardless of both the environmental stimuli provided during the birds´ rearing and the potentially stressful stimuli during the SI trial.