Nuclei and tracts in the thalamus of crocodiles.

The thalamus is one of the most important divisions of the forebrain because it serves as the major hub for transmission of information between the brainstem and telencephalon. While many studies have investigated the thalamus in mammals, comparable analyses in reptiles are incomplete. To fill this gap in knowledge, the thalamus was investigated in crocodiles using a variety of morphological techniques. The thalamus consists of two parts: a dorsal and a ventral division. The dorsal thalamus was defined by its projections to the telencephalon, whereas the ventral thalamus lacked this circuit. The complement of nuclei in each part of the thalamus was identified and characterized. Alar and basal components of both the dorsal and ventral thalamus were distinguished. Although some alar-derived nuclei in the dorsal thalamus shared certain features, no grouping could account for all of the known nuclei. However, immunohistochemical observations suggested a subdivision of alar-derived ventral thalamic nuclei. In view of this, a different approach to the organization of the dorsal thalamus should be considered. Development of the dorsal thalamus is suggested to be one way to provide a fresh perspective on its organization.

Body condition as a descriptor of American alligator (Alligator mississippiensis) health status in the Greater Everglades, Florida, United States.

Body condition is used as an indicator of the degree of body fat in an animal but evidence of its actual relationship with health diagnostics (e.g., blood parameters) is usually lacking across species. In American alligators (Alligator mississippiensis), body condition has been used as a performance metric within the Greater Everglades ecosystem to provide insight on hydrological and landscape changes on alligator populations. However, there is no clear evidence that spatial body condition changes relate to different health conditions (low food intake vs sickness) and whether this link can be made when relating body condition values with blood parameters. We assessed the relationship between alligator body condition and 36 hematological and biochemistry (blood) parameters in four areas across two physiographic regions (Everglades and Big Cypress) of the Greater Everglades (sample size = 120). We found very strong to weak evidence of linearity between 7 (Big Cypress) and 19 (Everglades) blood parameters and relative condition factor index, from which cholesterol (38%) and uric acid (41%) for the former and phosphorus (up to 52%) and cholesterol (up to 45%) for the latter (mean absolute error MAE = 0.18 each) were the predictors that individually explain most of the body condition variation. The best combination of blood parameters for the Everglades were cholesterol, phosphorus, osmolality, total protein, albumin, alpha 2, beta, and gamma globulins, and corticosterone accounting for 40% (37 ± 21%, MAE = 0.16) of the variation found in alligator body condition for this region. We found better predictability power in models when analyzed at smaller rather than larger scales showing a potential habitat effect on the body condition-blood parameters relationship. Overall, Everglades alligators in poorer condition are likely dehydrated or have an inadequate diet and the spatial differences found between physiographic regions suggest that these areas differ in prey availability/quality.

Caiman's fat enriched with n-3 fatty acids: potential food supplement.

Broad-snouted caiman (Caiman latirostris) products (meat, fat and oil) are currently beginning to be valued as a food of special interest due to its high content of n-3 fatty acids. Thus, the objective of this study was to characterize the fats of caiman fed with diets enriched with flaxseeds (Linus usitatissimun) rich in n-3 fatty acids, lignans and antioxidants. Caimans were fed six days a week with: a control diet (C), and a diet enriched with ground flaxseed = 90% C + 10% flaxseed ground (FS), during 30 (FS30) and 60 (FS60) days. Animals fed the flaxseed-enriched diets increased linolenic acid content and reduced the n-6/n-3 ratio of fats relative to controls, and this improvement increased over time. The proportion of eicosapentaenoic acid also increased, but there was no difference at the time the enriched diets were offered. Caiman fat of the FS30 and FS60, showed a decrease in lipoperoxidation (24% and 40%) and reactive oxygen species (44% and 76%) accompanied by an increase in antioxidant systems. Consumption of a flax-enriched diet by caimans increases the content of essential fatty acids and improves the lipoperoxidative status of fat. This provides an enriched fat with potential for the development products for human consumption.

The latitudinal and longitudinal allelopathic patterns of an invasive alligator weed (Alternanthera philoxeroides) in China.

Allelopathy has been considered a good explanation for the successful invasion of some invasive plants. However, the real latitudinal and longitudinal allelopathic effects on native species have rarely been documented since many exotics have spread widely. We conducted a Petri dish experiment to determine the latitudinal and longitudinal allelopathic patterns of an invasive alligator weed (Alternanthera philoxeroides) on a common crop (Lactuca sativa) in China, and find what determines the allelopathic intensity. The results showed that the allelopathic effects of A. philoxeroides increased with the latitude while decreased with the longitude. This indicated that A. philoxeroides used its allelopathy to gain competitive advantages more in its recent invaded communities than that in its early invaded ones as A. philoxeroides is expanding from southeast China to northwest China. Furthermore, we found that the allelopathic intensity of A. philoxeroide was negatively correlated to the leaf contents of soluble carbohydrate (SC), carbon (C) and nitrogen (N), but that was positively correlated to the leaf contents of soluble protein (SP), free amino acids (FAA), plant polyphenol (PP), phosphorus (P) and potassium (K). These results suggested that the allelopathic intensity of A. philoxeroide was more determined by the limited P and K nutrients as well as the intermediate allelochemicals (SP, FAA, PP) rather than the unlimited C, N and SC. Thus, we can speculate that the negative or positive effects of plant aqueous extracts are a function of not only the extract concentrations but also the trade-offs between inhibition and promotion of all components in the extracts. Then we could reduce the allelopathic effects of A. philoxeroide by controlling the component contents in the plant tissues, by fertilization or other managements, especially in the plant recent invaded communities.

Anti-Melanogenesis Activity of Crocodile (Crocodylus siamensis) White Blood Cell Extract on Ultraviolet B-Irradiated Melanocytes.

Ultraviolet (UV) radiation generates a range of biological effects in the skin, which includes premature skin aging, hyperpigmentation, and cancer. Therefore, the development of new effective agents for UV-related skin damage remains a challenge in the pharmaceutical industry. This study aims to test the inhibitory effect of crocodile white blood cell (cWBC) extract, a rich source of bioactive peptides, on ultraviolet B (UVB)-induced melanocyte pigmentation. The results showed that cWBC (6.25-400 µg/mL) could inhibit tyrosinase without adduct formation by 12.97 ± 4.20% on average. cWBC pretreatment (25-100 µg/mL) had no cytotoxicity and reduced intracellular melanin to 111.17 ± 5.20% compared with 124.87 ± 7.43 for UVB condition. The protective role of cWBC pretreatment against UVB was exhibited by the promotion of cell proliferation and the prevention of UVB-induced morphological change as observed from F actin staining. The decrease of microphthalmia-associated transcription factor expression levels after cWBC pretreatment might be a mechanism by which cWBC suppresses UVB-induced pigmentation. These results suggest that cWBC could be beneficial for the prevention of UVB-induced skin pigmentation.

Crocodile Oil Disrupts Mitochondrial Homeostasis and Exacerbates Diabetic Kidney Injury in Spontaneously Diabetic Torii Rats.

Diabetic nephropathy is currently the leading cause of end-stage renal disease (ESRD) in type 2 diabetes. Studies have suggested that supplementation with some fatty acids might reduce the risk and delay the progression to ESRD in patient with chronic kidney disease. Crocodile oil (CO) contains a variety of fatty acids, especially omega-3, -6 and -9, that have been reported to be beneficial to human health. This study examined the impact of long-term CO supplementation on the development of diabetic nephropathy in spontaneously diabetic Torii (SDT) rats. After diabetic verification, SDT rats were assigned to receive vehicle or CO at 500 and 1000 mg/kg BW, respectively, by oral gavage. Age-matched nondiabetic Sprague-Dawley rats were given vehicle or high-dose CO. After 28 weeks of intervention, CO failed to improve hyperglycemia and pancreatic histopathological changes in SDT rats. Unexpectedly, CO dose-dependently exacerbated the impairment of kidney and mitochondrial functions caused by diabetes. CO also disturbed the expressions of proteins involved in mitochondrial biogenesis, dynamics, and mitophagy. However, no significant alterations were observed in nondiabetic rats receiving high-dose CO. The findings reveal that CO has deleterious effects that aggravate diabetic kidney injury via disrupting mitochondrial homeostasis, possibly due to its improper omega-6: omega-3 ratio.

Ghrelin and ghrelin receptor (GHSR) in Chinese alligator, alligator sinensis: Molecular characterization, tissue distribution and mRNA expression changes during the active and hibernating periods.

The Chinese alligator (Alligator sinensis) is a freshwater crocodilian endemic to China. So far, the endocrine regulation of feeding and growth in Chinese alligator is poorly understood. In this study, the molecular structure and tissue expression profiles of ghrelin and its receptor GHSR in the Chinese alligator were characterized for the first time. The full-length cDNA of ghrelin was 1770 bp, including a 37 bp 5 '-UTR (untranslated region), a 435 bp ORF (open reading frame) and a 1298 bp 3 '-UTR. The ORF encodes a ghrelin precursor, which consists of 145 amino acid residues, including a signal peptide with 52 amino acid residues at the N-terminus, a mature peptide with 28 amino acid residues, and a possibly obestain at the C-terminus. The full-length cDNA of GHSR was 3961 bp, including a 5'-UTR of 375-bp, an ORF of 1059-bp and a 3' -UTR of 2527-bp. The ORF encodes a protein of 352 amino acid residues containing seven transmembrane domains, with multiple N glycosylation modification sites and conserved cysteine residue sites. The active core "GSSF" of Chinese alligator ghrelin was identical to that of mammals and birds, and the ghrelin binding site of GHSR was similar to that of mammals. The amino acid sequences of both ghrelin and GHSR share high identity with American alligator (Alligator mississippiensis) and birds. Ghrelin was highly expressed in cerebrum, mesencephalon, hypothalamus and multiple peripheral tissues, including lung, stomach and intestine, suggesting that it could play functions in paracrine and/or autocrine manners in addition to endocrine manner. GHSR expression level was higher in hypothalamus, epencephalon and medulla oblongata, and moderate in multiple peripheral tissues including lung, kindey, stomach and oviduct, implicating that ghrelin/GHSR system may participate in the regulation of energy balance, food intake, water and mineral balance, gastrointestinal motility, gastric acid secretion and reproduction. During hibernation, the expression of ghrelin and GHSR in the brain was significantly increased, while ghrelin was significantly decreased in heart, liver, lung, stomach, pancreas and ovary, and GHSR was significantly decreased in heart, liver, spleen, lung, kindey, stomach, ovary and oviduct. These temporal changes in ghrelin and GHSR expression could facilitate the physiological adaption to the hibernation of Chinese alligator. Our study could provide basic data for further studies on the regulation of feeding, physiological metabolism and reproduction of Chinese alligator, which could also be useful for the improvement of artificial breeding of this endangered species.

Effect of flaxseed and flaxseed oil supplemented in caiman diet on meat fatty acids.

Increasing polyunsaturated or long-chain fatty acids in meat for human consumption improves both nutritional quality and consumer perception. The increase could occur through the addition of rich sources of omega-3 fatty acids (such as flaxseed or flaxseed oil) to the animal diet. The aim of this study was to evaluate the effects of dietary supplement with two presentations of flax (crushed seeds or oil) on the change of FA content in two cuts of caiman meat (tail and neck). We measured fatty profile in two different caiman meat cuts (neck and tail) from 30 animals (total length 96.7 ± 4.9 cm, snout-vent length 47.8 ± 3 cm, weight 4.2 ± 0.6 kg), raised in individual enclosures, fed three a week for 50 days with crushed chicken head and a dry food formulated for these reptiles in a 70/30 ratio (C, n = 10), control diet with 10% crushed flaxseed (FS, n = 10), and control diet with 10% flaxseed oil (FO, n = 10), while the remaining days animals were fed the control diet. Meats from animals fed both enrichment diet (FS and FO) showed an increase of C18:3n-3 and ΣUFA with respect to control diet. Although both enriched diets raised the levels of C18:3n-3, the neck showed higher values than the tail. We observed that the neck is more susceptible than the tail to be improved by FO, which could suggest that it is more beneficial to consume neck meat. In order to be implemented in caiman farms, flaxseed oil is more expensive than seed, but more effective, easier to manage, and is practical for application on caiman farms.

The Effects of Crocodile Blood Supplementation on Delayed-Onset Muscle Soreness.

Delayed-onset muscle soreness (DOMS) is associated with increases in acute inflammatory and biochemical markers, muscle swelling, pain, and reduced functional performance. This study aimed to investigate the preventative effects of crocodile blood supplementation on DOMS induced by eccentric exercise. Sixteen healthy males were randomly allocated to either a crocodile blood (CB, n = 8) or a placebo (PL, n = 8) treatment. Participants receiving the CB treatment consumed four capsules of freeze-dried CB powder (1 g day-1) over 18 days. Participants receiving the other treatment were administered a placebo over the same period. An eccentric exercise protocol was performed, and functional performance, visual analogue scale (VAS)-measured pain, knee range of movement (ROM), thigh circumference (swelling), and cytokines, enzymes, and biochemical parameters were assessed immediately after exercise as well as after 24 h, 48 h, and 72 h. CB supplementation could significantly maintain maximum voluntary isometric contraction (MVIC) at 24 h (p = 0.001) and 48 h after exercise (p = 0.001) when comparing values at different times for the CB group. In the CB group, thigh circumference decreased only immediately after eccentric exercise (p = 0.031) in comparison with pre-eccentric exercise values. An 18-day supplementation (1 g day-1) of crocodile blood does aid in the maintenance of functional performance and muscle swelling after eccentric exercise. Our data indicate that 1 g day-1 of crocodile blood supplementation should be safe for human consumption.

Lead, mercury, and selenium alter physiological functions in wild caimans (Caiman crocodilus).

Environmental contaminants affect ecosystems worldwide and have deleterious effects on biota. Non-essential mercury (Hg) and lead (Pb) concentrations are well documented in some taxa and are described to cause multiple detrimental effects on human and wildlife. Additionally, essential selenium (Se) is known to be toxic at high concentrations but, at lower concentrations, Se can protect organisms against Hg toxicity. Crocodilians are known to bioaccumulate contaminants. However, the effects of these contaminants on physiological processes remain poorly studied. In the present study, we quantified Hg, Pb and Se concentrations in spectacled caimans (Caiman crocodilus) and investigated the effects of these contaminants on several physiological processes linked to osmoregulatory, hepatic, endocrine and renal functions measured through blood parameters in 23 individuals. Mercury was related to disruption of osmoregulation (sodium levels), hepatic function (alkaline phosphatase levels) and endocrine processes (corticosterone levels). Lead was related to disruption of hepatic functions (glucose and alanine aminotransferase levels). Selenium was not related to any parameters, but the Se:Hg molar ratio was positively related to the Na+ and corticosterone concentrations, suggesting a potential protective effect against Hg toxicity. Overall, our results suggest that Hg and Pb alter physiological mechanisms in wild caimans and highlight the need to thoroughly investigate the consequences of trace element contamination in crocodilians.

Importance of the postcranial skeleton in eusuchian phylogeny: Reassessing the systematics of allodaposuchid crocodylians.

Our current knowledge on the crocodyliform evolution is strongly biased towards the skull morphology, and the postcranial skeleton is usually neglected in many taxonomic descriptions. However, it is logical to expect that it can contribute with its own phylogenetic signal. In this paper, the changes in the tree topology caused by the addition of the postcranial information are analysed for the family Allodaposuchidae, the most representative eusuchians in the latest Cretaceous of Europe. At present, different phylogenetic hypotheses have been proposed for this group without reaching a consensus. The results of this paper evidence a shift in the phylogenetic position when the postcranium is included in the dataset, pointing to a relevant phylogenetic signal in the postcranial elements. Finally, the phylogenetic relationships of allodaposuchids within Eusuchia are reassessed; and the internal relationships within Allodaposuchidae are also reconsidered after an exhaustive revision of the morphological data. New and improved diagnoses for each species are here provided.

Potential of crocodile blood as a medication and dietary supplement: A systemic review.

Crocodile blood has long been used as a traditional medicine in many Asian countries to treat diseases such as asthma, allergies, and many others. Yet, only recently has the safety and effectiveness of using crocodile blood as a medicine been examined using modern scientific methods; with both conserved and novel active components identified from crocodile blood. Further in vitro and in vivo investigations found that crocodile blood can have a wide range of beneficial effects, including antimicrobial, antiviral, anti-oxidative, anti-inflammatory, antitumour effects, anti-anaemia, and enhancement of wound healing. A systematic research of literature published in English-language journals up to April 2020 was conducted in PubMed, Google Scholar, and Web of Science. Based on the biological and chemical knowledge of crocodile immunity and crocodile blood, this article aims to: provide a critical review on the proposed properties of crocodile blood, identify the knowledge gap and offer some insights for future investigations regarding the use of crocodile blood as a medication or dietary supplement.

Antioxidant effect of acerola fruit powder, rosemary and licorice extract in caiman meat nuggets containing mechanically separated caiman meat.

The aim of this work was to evaluate the antioxidant effect of acerola fruit powder, rosemary and licorice extract in caiman meat nuggets containing high amounts of mechanically separated caiman meat (MSCM) during 120 days of frozen storage. Five treatments were prepared: a control without antioxidant (CON), sodium erythorbate at 500 mg/kg (ERY), acerola fruit powder at 500 mg/kg (AFP), with licorice extract at 500 mg/kg (LIE) and rosemary extract at 500 mg/kg (ROE). The addition of natural antioxidants increased color, cohesiveness, and decreased TBARS values. The redness values were higher for LIE and these values were different to the CON. Nuggets with licorice extract had significantly lower TBARS values at the end of the storage period compared to the control. All the treatments were similar in sensory analysis, with good acceptability. These results indicate licorice extract used in caiman meat nuggets containing a high amount of MSCM caiman has the potential to improve quality during frozen storage.

Molecular cloning, characterization, and gene expression behavior of glucocorticoid and mineralocorticoid receptors from the Chinese alligator (Alligator sinensis).

The Chinese alligator is an endemic crocodilian species in China. We isolated and obtained the glucocorticoid and mineralocorticoid receptor genes coding from the kidney of Alligator sinensis by nested polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). The glucocorticoid receptor (GR) gene has 2343 base pairs encoding 780 amino acids, while the mineralocorticoid receptor (MR) gene is 2958 bp in length encoding 985 amino acids. Quantitative real-time PCR was used to detect the distribution of messenger RNA (mRNA) levels. The maximum mRNA expressions were observed in the ovary and kidney, suggesting that these receptors may be involved in basic cellular functions or stress response of alligators. Besides this, RT-qPCR was performed to analyze the abundance of GR and MR mRNA transcripts in early embryonic development of the Chinese alligator in the kidney, liver, and heart. The mRNA levels of GR and MR at earlier stages in kidney, liver, and heart indicates that they might involve in the transcriptional regulation of early embryos and activate many precise developmental effects in fetal tissues. We also measured the protein expression in the liver embryonic developmental stages and found that the GR and MR proteins were restricted to both the nuclei and cytoplasm. The protein expression levels in the liver at different embryonic developmental stages have extremely prominent differences. Taken together, our results showed the full coding regions of GR and MR, their characteristics, and embryonic developmental mRNA and protein expressions of both genes in A. sinensis. This study could provide the necessary information for further investigating the diverse functions of GR and MR in A. sinensis.

Repair function of essential oil from Crocodylus Siamensis (Schneider, 1801) on the burn wound healing via up-regulated growth factor expression and anti-inflammatory effect.

ETHNOPHARMACOLOGICAL RELEVANCE: Crocodile oil has been used by traditional physicians around the world to treat wound healing and inflammation. However, the scientific rationale and mechanism behind its use in vivo has not been fully researched. AIMS OF THE STUDY: We mainly investigated the mechanism during crocodile oil treatment of up-regulated growth factor expression and anti-inflammatory on burn wound healing in rats. MATERIALS AND METHODS: The moisture and nitric oxide (NO) levels in the skin of rats were analyzed in the first 14 days after burn and the changes of the structure of the skin tissues in the wound healing were studied by hematoxylin-eosin (H.E.) staining within 21 days after scald. The inflammatory factor on burn wound healing in rats was dected by ELISA kits and Q-PCR. the expression of a variety of growth factors (TGF-ß1, VEGE-α, EGF) and PCNA in the skin tissue after burns was evaluated using immunohistochemistry. The down-regulated phosphorylation of p38 MAPK in the wound healing was confirmed by Western-blot analysis. In addition, TEM was used to observe the ultrastructure of scalded skin. RESULTS: This study showed that crocodile oil could significantly reduce the protein and mRNA levels of TNF-α, IL-1ß and IL-6. And it was found that the phosphorylation of p38 MAPK was down-regulated in the wound healing (p < 0.05). Meanwhile, crocodile oil can promote the expression of a variety of growth factors (TGF-ß1, VEGE-α, EGF) and PCNA in the skin tissue after burns, and promote the repair of collagen fibers in the dermis, preventing the production of melanin and maintain the appearance of repaired skin.

Microbiota of Four Tissue Types in American Alligators (Alligator mississippiensis) Following Extended Dietary Selenomethionine Exposure.

Selenium represents an essential trace nutrient that is necessary for biological functions. Deficiencies can induce disease, but excess can induce toxicity. Selenium deficiency is a major concern in underdeveloped countries, while also posing as a toxic pollutant in waterways surrounding landfills, agricultural areas, and fossil fuel production sites. We examined the microbiome of selenomethionine (SeMet) fed American alligators (Alligator mississippiensis) at the beginning and end of a 7-week exposure experiment. Alligators were randomly divided into three groups: control and 1000 or 2000 ppm SeMet. DNA from before exposure (oral and cloaca swabs) and post-exposure (oral, cloaca, small & large intestines) sampling were extracted and amplified for bacterial 16 s rRNA. While treatment did not seem to have much effect, we observed a predominance of Fusobacteriaceae and Porpyromonodaceae across all tissue types. Cetobacterium and Clostridium are the most abundant genera as potential indicators of the aquatic and carrion feeding lifestyle of alligators.

Magnetic resonance diffusion tensor tractography of a midbrain auditory circuit in Alligator.

Knowledge of brain circuitry is critical for understanding the organization, function, and evolution of central nervous systems. Most commonly, brain connections have been elucidated using histological and experimental methods that require animal sacrifice. On the other hand, magnetic resonance diffusion tensor imaging and associated tractography have emerged as a preferred method to noninvasively visualize brain white matter tracts. However, existing studies have primarily examined large, heavily myelinated fiber tracts. Whether tractography can visualize fiber bundles that contain thin and poorly myelinated axons is uncertain. To address this question, the midbrain auditory pathway to the thalamus was investigated in Alligator. This species was chosen because of its evolutionary importance as it is the reptilian group most closely related to birds and because its brain contains many thin and poorly myelinated tracts. Furthermore, this auditory pathway is well documented in other reptiles, including a related crocodilian. Histological observations and experimental determination of anterograde connections confirmed this path in Alligator. Tractography identified these tracts in Alligator and provided a 3-dimensional picture that accurately identified the neural elements of this circuit. In addition, tractography identified one possible unrecognized pathway. These results demonstrate that tractography can visualize circuits containing thin, poorly myelinated fibers. These findings open the door for future studies to examine these types of pathways in other vertebrates.

Cloning, characterisation and expression profile of kisspeptin1 and the kisspeptin1 receptor in the hypothalamic-pituitary-ovarian axis of Chinese alligator Alligator sinensis during the reproductive cycle.

Kisspeptin1 (Kiss1), a product of the Kiss1 gene, plays an important role in the regulation of reproduction in vertebrates by activating the Kiss1 receptor (Kiss1R) and its coexpression with gonadotrophin-releasing hormone (GnRH) in GnRH neurons. The purpose of this study was to clone the Kiss1 and Kiss1R genes found in the brain of Alligator sinensis and to explore their relationship with reproduction. The full-length cDNA of Kiss1 is 816bp, the open reading frame (ORF) is 417bp and the gene encodes a 138-amino acid precursor protein. The full-length cDNA of Kiss1R is 2348bp, the ORF is 1086bp and the gene encodes a 361-amino acid protein. Quantitative polymerase chain reaction showed that, except for Kiss1R expression in the hypothalamus, the expression of Kiss1 and Kiss1Rduring the reproductive period of A. sinensis was higher than that in the hypothalamus, pituitary gland and ovary during the hibernation period. The changes in GnRH2 mRNA in the hypothalamus were similar to those of GnRH1 and peaked during the reproductive period. This study confirms the existence of Kiss1 and Kiss1R in A. sinensis and the findings strongly suggest that Kiss1 and Kiss1R may participate in the regulation of GnRH secretion in the hypothalamus of alligators during the reproductive period. Furthermore, this is the first report of the full-length cDNA sequences of Kiss1 and Kiss1R in reptiles.

Examining the Effects of Chronic Selenium Exposure on Traditionally Used Stress Parameters in Juvenile American Alligators (Alligator mississippiensis).

Environmental contaminants, such as the trace element selenium (Se), are a continuing concern to species worldwide due to their potential pathophysiological effects, including their influence on the stress response mediated through glucocorticoids (GCs; stress hormones). Environmental concentrations of Se are increasing due to anthropogenic activities, including the incomplete combustion of coal and subsequent disposal of coal combustion wastes. However, most studies examining how Se affects GCs have been focused on lower trophic organisms. The objectives of this study were to investigate the effects of long-term Se exposure on traditionally used stress parameters and to identify which of these parameters best indicate Se accumulation in liver and kidney of the American alligator (Alligator mississippiensis), a top trophic carnivore found in the southeastern United States and known to inhabit Se-containing areas. Alligators were divided into three dietary treatments and fed prey spiked with 1000 or 2000 ppm of selenomethionine (SeMet) or deionized water (control treatment) for 7 weeks. Following the 7-week treatment protocol, blood and tissue samples were obtained to measure plasma corticosterone (CORT; the main crocodilian GC), tail scute CORT, the ratio of peripheral blood heterophils (H) to lymphocytes (L) as H/L ratio, and body condition. To evaluate which parameter best indicated Se accumulation in the liver and kidney, principal component and discriminant analyses were performed. The only parameter significantly correlated with liver and kidney Se concentrations was scute CORT. Our results suggest that measurement of CORT in tail scutes compared with plasma CORT, H/L ratios, and body condition is the best indicator of Se-exposure and accumulation in crocodilians.

Quantitative duplex real-time polymerase chain reaction assay with TaqMan probe detects and quantifies Crocodylus porosus in food chain and traditional medicines.

Consumption and exploitation of crocodiles have been rampant for their exotic, nutritive and medicinal attributes. These depredations are alarming and although they have continued to be monitored by wildlife and conservation agencies, unlawful trading of crocodiles shows an increasing trend worldwide. Recently, conventional polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) assays for crocodile have been documented but they are only suitable for identification and cannot quantify adulterations. We described here a quantitative duplex real-time PCR assay with probes to quantify contributions from Crocodylus porosus materials simultaneously. A very short amplicon size of 127bp was used because longer targets could have been broken down in samples, bringing considerable uncertainty in molecular analysis. We have validated a TaqMan probe-based duplex real-time PCR (qPCR) assay for the detection of 0.004 ng DNA in pure state and 0.1% target meat in model chicken meatball. False negative detection was eliminated through an endogenous control (141-bp site of eukaryotic 18S rRNA). Analysis of 12 model chicken meatballs adulterated with C. porosus reflected 96.3-120.2% target recovery at 0.1-10% adulterations. A validation test of 21 commercial food and traditional medicine (TM) crocodile-based products showed 100% effectiveness. Short amplicon sizes, alternative complementary target, exceptional stability and superior sensitivity suggested the assay could be used for the identification and quantitative determination of C. porosus in any food or TM samples even under degraded conditions.