HEMATOLOGY, PLASMA BIOCHEMISTRY, AND PLASMA PROTEIN ELECTROPHORESIS REFERENCE INTERVALS FOR BLUE IGUANAS (CYCLURA LEWISI) FROM GRAND CAYMAN ISLAND.

The blue iguana (Cyclura lewisi) is an endangered rock iguana species native to Grand Cayman, in the Cayman Islands. Health assessments were conducted on captive and free-roaming iguanas in 2001 and 2003-2014 and were performed in the summer wet season (June-July) of 2003-2004 and 2010-2014 and in the winter dry season (November-December) of 2001 and 2005-2009. Morphometric data were recorded from iguanas when blood samples were collected: 903 samples were collected and data from 890 samples from 775 iguanas were included. Samples were analyzed for hematology, plasma biochemistry, protein electrophoresis, mineral panels, 25-hydroxyvitamin D, and testosterone. Reference intervals were created for captive subadults, captive adults, and free-roaming adults when data were sufficient. Significant differences among these groups were described, as were differences on the basis of sex, season, and origin (captive vs free-roaming). In captive iguanas, most analytes were significantly different between subadults and adults, mature heterophils and copper were significantly higher in the dry season, zinc levels were significantly higher in the wet season, and cholesterol and triglycerides were significantly higher in adult females than adult males. Testosterone in adult males was significantly higher in the dry season. These results will aid in future health assessments and disease investigations in wild and captive populations of blue iguanas and are of comparative value for other Cyclura species that are free-roaming, captive, and, especially, in similar conservation release programs.

Influence of light/dark cycle and orexins on breathing control in green iguanas (Iguana iguana).

Light/dark cycle affects the physiology of vertebrates and hypothalamic orexin neurons (ORX) are involved in this function. The breathing pattern of the green iguana changes from continuous to episodic across the light/dark phases. Since the stimulatory actions of ORX on breathing are most important during arousal, we hypothesized that ORX regulates changes of breathing pattern in iguanas. Thus, we: (1) Localized ORX neurons with immunohistochemistry; (2) Quantified cyclic changes in plasma orexin-A levels by ELISA; (3) Compared breathing pattern at rest and during hypoxia and hypercarbia; (4) Evaluated the participation of the ORX receptors in ventilation with intracerebroventricular microinjections of ORX antagonists during light and dark phases. We show that the ORX neurons of I. iguana are located in the periventricular hypothalamic nucleus. Orexin-A peaks during the light/active phase and breathing parallels these cyclic changes: ventilation is higher during the light phase than during the dark phase. However, inactivation of ORX-receptors does not affect the breathing pattern. Iguanas increase ventilation during hypoxia only during the light phase. Conversely, CO2 promotes post-hypercarbic hyperpnea during both phases. We conclude that ORXs potentiate the post-hypercarbic (but not the hypoxic)-drive to breathe and are not involved in light/dark changes in the breathing pattern.

Living on the edge: Glucocorticoid physiology in desert iguanas (Dipsosaurus dorsalis) is predicted by distance from an anthropogenic disturbance, body condition, and population density.

Ecological factors, such as habitat quality, influence the survival and reproductive success of free-living organisms. Urbanization, including roads, alters native habitat and likely influences physiology, behavior, and ultimately Darwinian fitness. Some effects of roads are clearly negative, such as increased habitat fragmentation and mortality from vehicle collision. However, roads can also have positive effects, such as decreasing predator density and increased vegetation cover, particularly in xeric habitats due to increased water run-off. Glucocorticoids are metabolic hormones that reflect baseline metabolic needs, increase in response to acute challenges, and may mediate endogenous resource trade-offs between survival and reproduction. Here we examined circulating concentrations of corticosterone (baseline and stress-induced) in desert iguanas (Dipsosaurus dorsalis) in relation to the distance from a major anthropogenic disturbance, a high-traffic road in Palm Springs, CA. Additionally, we analyzed body condition and population density as additional predictors of glucocorticoid physiology. Surprisingly, we found lower baseline CORT levels closer to the road, but no effect of distance from road on stress-induced CORT or stress responsiveness (difference between baseline and stress-induced concentrations). Both population density and body condition were negative predictors of baseline CORT, stress-induced CORT, and stress responsiveness. Given the known effect of roads to increase run-off and vegetation density, increased water availability may improve available forage and shade, which may then increase the carrying capacity of the habitat and minimize metabolic challenges for this herbivorous lizard. However, it is important to recognize that surfaces covered by asphalt are not usable habitat for iguanas, likely resulting in a net habitat loss.

Validation of an enzyme immunoassay for the quantification of testosterone in green iguana males (Iguana iguana).

The endocrinological study by immunological methods allows elucidating mechanisms of response to environmental challenges and reproductive regulatory mechanisms in animals. However, it is often overlooked that immunological assays for the detection and quantification of steroid hormones require prior validation tests. In this study, the efficacy of a commercial enzyme immunoassays (EIA) was evaluated for the quantification of plasma testosterone (T) in males from a population of green iguanas (Iguana iguana) in semi-captivity. The enzyme immunoassay was validated for specificity, accuracy and precision. Testosterone concentrations obtained by EIA were compared to estimates obtained on the same samples by high-performance liquid chromatography (HPLC). The proposed protocol has shown linearity and parallelism, T recovery was found to be within 80-110% accuracy, and precision variation was <10%. The EIA method allowed the differentiation of the plasma T concentration of male iguanas during the reproductive season (29.7 ± 14.4 ng mL-1, n = 4) and outside the reproductive season (6.8 ± 2.0 ng mL-1, n = 4). The HPLC method has been able to detect concentrations of T only for those individuals during the reproductive season. The T concentrations obtained by the two methods were not statistically different (p > 0.05) indicating that the commercial EIA kit analyzed can be employed in the laboratory routine to quantify plasma T concentration and consequently differentiate the reproductive status of green iguana males.

Obtaining plasma to measure baseline corticosterone concentrations in reptiles: How quick is quick enough?

There is growing interest in the use of glucocorticoid (GC) hormones to understand how wild animals respond to environmental challenges. Blood is the best medium for obtaining information about recent GC levels; however, obtaining blood requires restraint and can therefore be stressful and affect GC levels. There is a delay in GCs entering blood, and it is assumed that blood obtained within 3 min of first disturbing an animal reflects a baseline level of GCs, based largely on studies of birds and mammals. Here we present data on the timing of changes in the principle reptile GC, corticosterone (CORT), in four reptile species for which blood was taken within a range of times 11 min or less after first disturbance. Changes in CORT were observed in cottonmouths (Agkistrodon piscivorus; 4 min after first disturbance), rattlesnakes (Crotalus oreganus; 2 min 30 s), and rock iguanas (Cyclura cychlura; 2 min 44 s), but fence lizards (Sceloporus undulatus) did not exhibit a change within their 10-min sampling period. In both snake species, samples taken up to 3-7 min after CORT began to increase still had lower CORT concentrations than after exposure to a standard restraint stressor. The "3-min rule" appears broadly applicable as a guide for avoiding increases in plasma CORT due to handling and sampling in reptiles, but the time period in which to obtain true baseline CORT may need to be shorter in some species (rattlesnakes, rock iguanas), and may be unnecessarily limiting for others (cottonmouths, fence lizards).

Unraveling avian and reptilian hematology: An optical and electron microscopic study of the buffy coat.

BACKGROUND: Blood centrifugation and buffy coats are at the cornerstone of hematology. In mammals, the buffy coat has a layered disposition (from bottom to top) with neutrophils on top of erythrocytes, followed by monocytes/lymphocytes, and platelets. In nonmammals, this distribution is unknown. Recently, the cell tube block (CTB) technique was developed to study the buffy coat, but it was never applied to nonmammal buffy coats. OBJECTIVES: This study aimed to evaluate using the CTB technique to study reptilian and avian buffy coats and to propose its use for clinical applications. METHODS: Blood from five birds and eight reptiles of different species was obtained to make CTBs that were processed for optical/electron microscopy. H&E, Sirius red, and immunohistochemistry staining against CD3 (to label T lymphocytes) were applied to the CTBs. RESULTS: In birds, the buffy coat had a layered appearance with the granulocyte layer containing granulocytes (heterophils and eosinophils) and nucleated erythrocytes followed by a mononuclear cell layer containing lymphocytes, monocytes, and thrombocytes. In some animals, a nucleated erythrocyte layer was observed admixed with the granulocyte/mononuclear cell layer. A small clot within the buffy coat was seen in seven reptiles, and less definition of layers occurred in reptiles, with only one or two layers. Lymphocytes appeared toward the top of the buffy coat. CONCLUSIONS: From a comparative hematology perspective, the buffy coat of mammals differs from that of birds and more from that of reptiles. The CTB technique can be used to study these differences in avian and reptilian hematology, especially to study atypical circulating cells, hemoparasites, or blood cell proportions in health and disease.

PHARMACOKINETICS OF CEFTIOFUR CRYSTALLINE FREE ACID STERILE SUSPENSION IN GREEN IGUANAS ( IGUANA IGUANA) AFTER SINGLE INTRAMUSCULAR ADMINISTRATION.

The objective of this study was to establish the pharmacokinetic parameters of ceftiofur crystalline free acid (CCFA) for a single intramuscular injection in green iguanas ( Iguana iguana). Six green iguanas received an injection of 5 mg/kg CCFA into the triceps muscle. Using high-performance liquid chromatography, concentrations of ceftiofur free acid equivalents in plasma samples collected at predetermined time points were evaluated up to 21 days following drug administration. Noncompartmental pharmacokinetic analysis was applied to the data. The observed maximum plasma concentration (Cmax obs) was 2.765 ± 0.864 µg/mL, and the time of observed maximum concentration (Tmax obs) was 6.1 ± 9.2 hr. The area under the curve (0 to infinity) was 239.3 ± 121.1 µg·hr/mL. No significant adverse drug reactions were clinically observed, and no visible injection site reactions were noted. Minimum inhibitory concentrations of bacterial isolates from iguanas were used to establish a target plasma concentration of 2.0 µg/mL. Based on the results from this study, a potential dosing interval for ceftiofur crystalline free acid administered at 5 mg/kg intramuscularly for iguanas maintained at a temperature of 30°C would be 24 hr based on a target plasma concentration of 2 µg/mL; however, multidose studies still need to be performed.

Differential responses of the somatotropic and thyroid axes to environmental temperature changes in the green iguana.

Growth hormone (GH), together with thyroid hormones (TH), regulates growth and development, and has critical effects on vertebrate metabolism. In ectotherms, these physiological processes are strongly influenced by environmental temperature. In reptiles, however, little is known about the direct influences of this factor on the somatotropic and thyroid axes. Therefore, the aim of this study was to describe the effects of both acute (48h) and chronic (2weeks) exposure to sub-optimal temperatures (25 and 18°C) upon somatotropic and thyroid axis function of the green iguana, in comparison to the control temperature (30-35°C). We found a significant increase in GH release (2.0-fold at 25°C and 1.9-fold at 18°C) and GH mRNA expression (up to 3.7-fold), mainly under chronic exposure conditions. The serum concentration of insulin-like growth factor-I (IGF-I) was significantly greater after chronic exposure (18.5±2.3 at 25°C; 15.92±3.4 at 18°C; vs. 9.3±1.21ng/ml at 35°C), while hepatic IGF-I mRNA expression increased up to 6.8-fold. Somatotropic axis may be regulated, under acute conditions, by thyrotropin-releasing hormone (TRH) that significantly increased its hypothalamic concentration (1.45 times) and mRNA expression (0.9-fold above control), respectively; and somatostatin (mRNA expression increased 1.0-1.2 times above control); and under chronic treatment, by pituitary adenylate cyclase-activating peptide (PACAP mRNA expression was increased from 0.4 to 0.6 times). Also, it was shown that, under control conditions, injection of TRH stimulated a significant increase in circulating GH. On the other hand, while there was a significant rise in the hypothalamic content of TRH and its mRNA expression, this hormone did not appear to influence the thyroid axis activity, which showed a severe diminution in all conditions of cold exposure, as indicated by the decreases in thyrotropin (TSH) mRNA expression (up to one-eight of the control), serum T4 (from 11.6±1.09 to 5.3±0.58ng/ml, after 2weeks at 18°C) and T3 (from 0.87±0.09 to 0.05±0.01ng/ml, under chronic conditions at 25°C), and Type-2 deiodinase (D2) activity (from 992.5±224 to 213.6±26.4fmolI(125)T4/mgh). The reduction in thyroid activity correlates with the down-regulation of metabolism as suggested by the decrease in the serum glucose and free fatty acid levels. These changes apparently were independent of a possible stress response, at least under acute exposure to both temperatures and in chronic treatment to 25°C, since serum corticosterone had no significant changes in these conditions, while at chronic 18°C exposure, a slight increase (0.38 times above control) was found. Thus, these data suggest that the reptilian somatotropic and thyroid axes have differential responses to cold exposure, and that GH and TRH may play important roles associated to adaptation mechanisms that support temperature acclimation in the green iguana.

Stress responsiveness predicts individual variation in mate selectivity.

Steroid hormones, including glucocorticoids, mediate a variety of behavioral and physiological processes. Circulating hormone concentrations vary substantially within populations, and although hormone titers predict reproductive success in several species, little is known about how individual variation in circulating hormone concentrations is linked with most reproductive behaviors in free-living organisms. Mate choice is an important and often costly component of reproduction that also varies substantially within populations. We examined whether energetically costly mate selection behavior in female Galápagos marine iguanas (Amblyrhynchus cristatus) was associated with individual variation in the concentrations of hormones previously shown to differ between reproductive and non-reproductive females during the breeding season (corticosterone and testosterone). Stress-induced corticosterone levels - which are suppressed in female marine iguanas during reproduction - were individually repeatable throughout the seven-week breeding period. Mate selectivity was strongly predicted by individual variation in stress-induced corticosterone: reproductive females that secreted less corticosterone in response to a standardized stressor assessed more displaying males. Neither baseline corticosterone nor testosterone predicted variation in mate selectivity. Scaled body mass was not significantly associated with mate selectivity, but females that began the breeding period in lower body condition showed a trend towards being less selective about potential mates. These results provide the first evidence that individual variation in the corticosterone stress response is associated with how selective females are in their choice of a mate, an important contributor to fitness in many species. Future research is needed to determine the functional basis of this association, and whether transient acute increases in circulating corticosterone directly mediate mate choice behaviors.

Genotoxic effects in blood cells of Mus musculus and Iguana iguana living near coal mining areas in Colombia.

Coal is a mixture of chemicals with the capacity of promoting biochemical changes that may lead to DNA damage. In this study, the comet assay in peripheral blood cells, and the micronucleus test in blood smears were used to evaluate potential genotoxic effects derived from exposure to coal mining activities on wild populations of Mus musculus and Iguana iguana. Four locations from Colombia were evaluated: La Loma and La Jagua de Ibirico, two municipalities located near coal mining fields at the Department of Cesar; and Valledupar and Arjona, cities used as reference sites, both localized at least 100 and 200km far from the mines, respectively. Compared to Valledupar and Arjona, animals collected in close proximity to coal mining areas showed highest percentages of DNA damage for both species, evidencing that living around coal mining fields may result in an increase of DNA lesions in blood cells of rodents and reptiles. The results for micronucleus test were conflicting. Mice from Arjona had greater number of cells with micronucleus than those from the other studied locations, probably as a result of infection found by blood parasites. In summary, it was demonstrated that animals living around coal mining areas have a greater chance of having DNA damage, as measured by the comet assay, than those from sites far from the coal dust source.

Cannabis intoxication in three Green iguanas (Iguana iguana).

This report describes clinical signs and plasma biochemical changes associated with significant cannabis consumption in three Green iguanas (Iguana iguana) which resulted in seizures, cardiovascular and digestive tract aberrations, elevated hepatic enzymes and bile acid concentrations for a number of weeks post recovery. One case required extensive antiseizuring therapy to recover. All Green iguanas eventually made a full recovery.

Human disturbance alters endocrine and immune responses in the Galapagos marine iguana (Amblyrhynchus cristatus).

Anthropogenic disturbance is a relevant and widespread facilitator of environmental change and there is clear evidence that it impacts natural populations. While population-level responses to major anthropogenic changes have been well studied, individual physiological responses to mild disturbance can be equally critical to the long-term survival of a species, yet they remain largely unexamined. The current study investigated the impact of seemingly low-level anthropogenic disturbance (ecotourism) on stress responsiveness and specific fitness-related immune measures in different breeding stages of the marine iguana (Amblyrhynchus cristatus). Specifically, we found stress-induced elevations in plasma corticosterone among tourist-exposed populations relative to undisturbed populations. We also found changes in multiple immunological responses associated with stress-related effects of human disturbance, including bacterial killing ability, cutaneous wound healing, and hemolytic complement activity, and the responses varied according to reproductive state. By identifying health-related consequences of human disturbance, this study provides critical insight into the conservation of a well-known species that has a very distinct ecology. The study also broadens the foundation of knowledge needed to understand the global significance of various levels of human disturbance.

Plasma protein electrophoresis of Trachemys scripta and Iguana iguana.

BACKGROUND: Protein electrophoresis is widely applied in veterinary medicine, but is not used often in reptiles, in part because of lack of reference values. OBJECTIVE: The goals of this study were to compare plasma protein profiles obtained by cellulose acetate electrophoresis (CAE) and agarose gel electrophoresis (AGE), measure precision and examine interference by sample hemolysis, and establish preliminary reference intervals for 2 reptile species. METHODS: Heparinized plasma samples from healthy and diseased adult female Iguana iguana (n=40) and Trachemys scripta (n=60) were analyzed by CAE and AGE. Total protein concentration was measured by the biuret method. Electrophoresis results were compared using Bland-Altman plots and Passing-Bablok regression analysis. Precision and the effects of sample hemolysis were determined. Results from clinically healthy animals were used to determine reference intervals. RESULTS: Five protein fractions were identified in both species, with bisalbuminemia observed in 23/40 iguanas. High correlation was observed between the 2 methods for all fractions, with few proportional and systematic errors. Coefficients of variation were lower using AGE vs CAE and for I. iguana vs T. scripta. Two additional bands were observed in hemolyzed samples from T. scripta; 1 additional band was observed for I. iguana. Minimum and maximum values were reported for healthy I. iguana (n=14) and T. scripta (n=22). CONCLUSIONS: Although both methods are acceptable, the performance of AGE was slightly better than that of CAE for analysis of plasma from reptiles. Furthermore, reptile electrophoretic patterns should be interpreted based on the method used, the species analyzed, and the quality of the plasma sample.

Determination of lead and zinc concentrations in the blood and liver of the captive common green iguana (Iguana iguana).

Heavy metal toxicosis is a well-known phenomenon in wild, captive-animal, and domestic animal medicine. However, the occurrence among reptiles is not well documented. One reason for this is the lack of information regarding reference blood and tissue levels of heavy metals in reptiles. To determine normal blood lead, plasma zinc, and liver lead and zinc concentrations, blood and liver samples were collected from 4 adult and 16 juvenile, healthy green iguanas (Iguana iguana). Lead and zinc levels were measured using atomic absorption spectroscopy. Using the mean +/- two SD as the normal reference range, the present study suggests the following for captive common green iguana: 1) whole blood lead level: 0.06 +/- 0.06 microg/ml; 2) plasma zinc level: 2.68 +/- 1.66 microg/ml; 3) liver lead level (wet-weight basis): <1.0 +/- 0.0 microg/g; 4) liver lead level (dry-weight basis): <3.0 +/- 0.0 microg/g; 5) liver zinc level (wet-weight basis): 24.9 +/- 11.6 microg/g; and 6) liver zinc level (dry-weight basis): 83.4 +/- 44.6 microg/g. These values are fairly consistent with published reference levels in other mammalian and avian species.

Biochemical reference intervals and intestinal microflora of free-ranging Ricord's iguanas (Cyclura ricordii).

Caribbean iguanas, including the Ricord's iguana (Cyclura ricordii) are ranked as critically endangered on the International Union of the Conservation of Nature (IUCN) Red List. The taxon is endemic to Hispaniola, where it inhabits xeric lowlands and is found in a very limited range in two subpopulations in the southwestern area of the island. The goals of this study were to obtain and analyze plasma biochemical and intestinal microbiological data from Ricord's iguanas in the subpopulation of the Neiba Valley, on Cabritos Island National Park, Dominican Republic, and to establish biochemical reference intervals. A total of 23 blood samples from clinically healthy iguanas (16 males, 7 females) and 14 fecal samples from clinically healthy iguanas were analyzed. Adult female iguanas had higher concentrations of cholesterol, phosphorus, triglycerides, urea, uric acid, and aspartate transaminase activity compared with male iguanas, although the difference was not statistically significant. Seventeen bacterial isolates were obtained, including: Salmonella sp. (serogroup B; serogroup D negative) Escherichia coli, Brevibacterium sp., Enterobacter cloacae, and Enterococcus durans. These data will help contribute to the management and recovery of Ricord's iguana populations.

Tameness and stress physiology in a predator-naive island species confronted with novel predation threat.

Tame behaviour, i.e. low wariness, in terrestrial island species is often attributed to low predation pressure. However, we know little about its physiological control and its flexibility in the face of predator introductions. Marine iguanas (Amblyrhynchus cristatus) on the Galapagos Islands are a good model to study the physiological correlates of low wariness. They have lived virtually without predation for 5-15 Myr until some populations were first confronted with feral cats and dogs some 150 years ago. We tested whether and to what extent marine iguanas can adjust their behaviour and endocrine stress response to novel predation threats. Here, we show that a corticosterone stress response to experimental chasing is absent in naive animals, but is quickly restored with experience. Initially, low wariness also increases with experience, but remains an order of magnitude too low to allow successful escape from introduced predators. Our data suggest that the ability of marine iguanas to cope with predator introductions is limited by narrow reaction norms for behavioural wariness rather than by constraints in the underlying physiological stress system. In general, we predict that island endemics show flexible physiological stress responses but are restricted by narrow behavioural plasticity.

Pharmacokinetics of ketoprofen in the green iguana (Iguana iguana) following single intravenous and intramuscular injections.

The nonsteroidal antiinflammatory drug ketoprofen (KTP) is a commonly used antiinflammatory and analgesic agent in reptile medicine, but no studies documenting its pharmacokinetics in this species have been published. Ketoprofen was administered as a racemic mixture to green iguanas (Iguana iguana) intravenously (i.v.) and intramuscularly (i.m.) at 2 mg/kg. Pharmacokinetic analyses were performed and indicated that ketoprofen in iguanas administered by the intravenous route has a classical two-compartmental distribution pattern, a slow clearance (67 ml/ kg/hr) and a long terminal half-life (31 hr) compared to ketoprofen studies reported in mammals. When delivered by the intramuscular route, bioavailability was 78%. These data indicate the daily dosing that is generally recommended for reptile patients, as an extrapolation from mammalian data, may be more frequent than necessary.

Inter-population variation of carotenoids in Galápagos land iguanas (Conolophus subcristatus).

Carotenoids have received much attention from biologists because of their ecological and evolutionary implications in vertebrate biology. We sampled Galápagos land iguanas (Conolophus subcristatus) to investigate the types and levels of blood carotenoids and the possible factors affecting inter-population variation. Blood samples were collected from populations from three islands within the species natural range (Santa Cruz, Isabela, and Fernandina) and one translocated population (Venecia). Lutein and zeaxanthin were the predominant carotenoids found in the serum. In addition, two metabolically modified carotenoids (anhydrolutein and 3'-dehydrolutein) were also identified. Differences in the carotenoid types were not related to sex or locality. Instead, carotenoid concentration varied across the localities, it was higher in females, and it was positively correlated to an index of body condition. Our results suggest a possible sex-related physiological role of xanthophylls in land iguanas. The variation in the overall carotenoid concentration between populations seems to be related to the differences in local abundance and type of food within and between islands.

Steroid hormones and aggression in female Galápagos marine iguanas.

We studied steroid hormone patterns and aggression during breeding in female Galápagos marine iguanas (Amblyrhynchus cristatus). Females display vigorously towards courting males after copulating (female-male aggression), as well as fight for and defend nest sites against other females (female-female aggression). To understand the neuroendocrine basis of this aggressive behavior, we examined changes in testosterone (T), estradiol (E2), corticosterone (CORT), and progesterone (P4) during the mating and nesting periods, and then measured levels in nesting females captured during aggressive interactions. Testosterone reached maximal levels during the mating stage when female-male aggression was most common, and increased slightly, but significantly, during the nesting stage when female-female aggression was most common. However, fighting females had significantly lower T, but higher E2 and P4, than non-fighting females. It remains unclear whether these changes in hormone levels during aggressive interactions are a cause or a consequence of a change in behavior. Our results support the "challenge hypothesis", but suggest that E2 and/or P4 may increase in response to aggressive challenges in females just as T does in males. Females may be rapidly aromatizing T to elevate circulating levels of E2 during aggressive interactions. This hypothesis could explain why non-fighting females had slightly elevated baseline T, but extremely low E2, during stages when aggressive interactions were most common. Although P4 increased rapidly during aggressive encounters, it is unclear whether it acts directly to affect behavior, or indirectly via conversion to E2. The rapid production and conversion of E2 and P4 may be an important mechanism underlying female aggression in vertebrates.