Rana Tigerina Research Articles

Effects of Salinity on the Egg Fertilization, Hatching, and Tadpole Growth and Survival Rates of the Thailand Frog (Rana tigerina Dubois, 1981)

We studied the effects of salinity on the egg fertilization, hatching, and tadpole growth and survival rates of the Thailand frog (Rana tigerina Dubois, 1981), an introduced species that is commonly cultured in the Mekong Delta, Vietnam. Salinity levels of 2, 6, and 4% significantly affected the fertilization and hatching rates of the eggs, as well as the tadpole survival rates from the newly hatched to yolk sac exhaustion stages, respectively p<0.05. Notably, during the stage from yolk sac exhaustion to juvenile tadpole, a salinity of 3% resulted in an increase in the final weight of the tadpoles throughout the stage and a significant increase in Phase 1 (the tadpole stage from yolk sac exhaustion to the onset of foreleg emergence) and Phase 2 (the tadpole stage from the onset of foreleg emergence to the onset of hind leg emergence); however, it did not show a significant effect on tadpole survival rates in Phases 1 and 2 of this stage p>0.05. A salinity level of up to 5% showed no significant effect on the growth performance but caused a significant reduction in the survival rates of the tadpoles during this stage. We recommend that the artificial reproduction process of Thai frogs should be performed using salinities of <2%, with salinities of ≤2 and 3% being suitable for rearing tadpoles from the newly hatched to yolk sac exhaustion stage and from the yolk sac exhaustion to juvenile stage, respectively.

The effects of different stocking densities and feed types on frogs’ growth and survival rates (Rana tigerina Dubois, 1981) reared in composite tanks

We performed two independent experiments, each with a completely random design in three repetitions and lasting 80 days, on the growth and survival of the artificially reared Thailand frog (Rana tigerina Dubois, 1981). Experiment 1 compared three stocking densities: 75 (D75), 100 (D100), and 125 (D125) individuals m−2. Experiment 2 used a stocking density of 100 individuals m−2 to study the effects of different feed types: frog feed, snakehead fish feed, catfish feed, and tilapia feed. Daily weight gain (DWG) and specific growth rate (SGR) did not differ between D75, D100, and D125, but these parameters tended to be higher at D100. At the end of the experiment, the average final weight and survival rate in D75 (204.59 ± 35.4 g and 79.67 ± 4.04%) and D100 (215.05 ± 38.65 g and 79.00 ± 2.75%) were not different, but both were significantly higher than those in D125 (184.64 ± 37.75 g and 57.00 ± 6.56%). In addition, frogs fed snakehead fish feed showed the highest DWG, SGR, and yield and the lowest feed conversion ratio (FCR), followed by frogs given frog feed, tilapia feed, and catfish feed, which showed decreasing yields and increasing FCR. In conclusion, a stocking density of 100 individuals m−2 using snakehead fish feed is suitable for rearing the Thailand frog under our experimental conditions.

Impact of extraction condition on the yield and molecular characteristics of collagen from Asian bullfrog (Rana tigerina) skin

The present study is the first report on collagen extraction as acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC), which could maximize the exploitation of Asian bullfrog (Rana tigerina) skin as an alternative collagen material in food and pharmaceutical fields. Extraction methods for ASC and PSC from Asian bullfrog skin were developed using different concentrations of acetic acid (0.50, 0.75, and 1.00 M) and pepsin (1.00, 2.00, and 3.00% [w/w]). PSCs exhibited a higher extraction yield (22.59%–28.30%) and recovery (22.48%–31.12%) than ASCs. An electrophoretic study revealed that collagens possessed type I collagen. ASC with 0.75 M acetic acid (ASC-0.75) and PSC with 3.00% (w/w) pepsin (PSC-3.00) were selected and analysed on their solubility, molecular characteristics, and amino acid composition. A better solubility with a higher isoelectric point was observed for PSC-3.00 than ASC-0.75. Fourier transform infrared- and circular dichroism-spectra revealed a high-interacted compact structure with a triple helical-structure of the collagen peptides, respectively. A similar amino acid profile with a high imino acid content was observed. Therefore, collagens from Asian bullfrog skin were successfully extracted, where the optimized conditions provided a high yield with less adverse effects on collagen properties and molecular characteristics.

Asian bullfrog (Rana tigerina) skin gelatin extracted by ultrasound-assisted process: Characteristics and in-vitro cytotoxicity

Impact of ultrasound-assisted process (UAP) on yield and characteristics of Asian bullfrog (Rana tigerina) skin gelatin was studied and the in-vitro cytotoxicity of the resulting frog skin gelatin was evaluated using CaCo-2, Raw264.7 and L929 cell lines. Different ultrasonication modes (5 min on-time with 25 min off-time (5/25) and 10 min on-time with 50 min off-times (10/50)) were implemented for different total ultrasonication times (10–60 min) during gelatin extraction at 55 °C. UAP could effectively augment yield and recovery of frog skin gelatin, compared with the typical process (without UAP). Different UAPs used affected characteristics and properties of gelatin differently. Increased total ultrasonication time increased color changes and turbidity of resulting gelatin and induced protein fragmentation, regardless of modes used. At the same total ultrasonication time, mode of 10/50 provided gelatin with higher hydroxyproline content and higher gel strength than 5/25 mode. Gelatin extracted by UAP with 10/50 mode for 60 min (FU) possessed high yield and good gel property. Gelling and melting temperatures and FTIR-spectra of FU were similar to those of gel from typical process. Based on in-vitro cytotoxicity toward different cell lines, frog skin gelatin could be used as biocompatible material, which could be safe for human consumption.

Oil from Asian Bullfrong (Rana tigerina) Skin: Antimicrobial Activity and Its Application in Emulsion Gelatin-based Film

Oil from Asian Bullfrong (Rana tigerina) Skin: Antimicrobial Activity and Its Application in Emulsion Gelatin-based Film

Characteristics and Gel Properties of Gelatin from Skin of Asian Bullfrog (Rana tigerina)

Characteristics and gel properties of gelatin from frog skin as influenced by extraction temperatures (45–75 °C) were investigated. Yield of gelatin increased as the extraction temperature increased (P 0.05). Gelling and melting temperatures of frog skin gelatin were 23.47–24.87 and 33.22–34.66 °C, respectively. Gels became more yellowish with increasing extraction temperatures (P < 0.05). All gelatin gels were sponge or coral-like in structure but varied in patterns as visualized by scanning electron microscopy (SEM). Gelatin from frog skin could be used as a replacement for land animal counterpart.

Modulation of delayed type hypersensitivity response by skin peptide from Rana tigerina

Modulation of delayed type hypersensitivity response by skin peptide from Rana tigerina

On the action and mechanism of withaferin-A from Withania somnifera, a novel and potent melanin dispersing agent in frog melanophores

The present work was carried out to determine the effects of lyophilized root extracts of Withania somnifera along with pure withaferin-A, on the isolated skin melanophores of frog, Rana tigerina which are disguised type of smooth muscle cells and offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The lyophilized extract of W. somnifera and its active ingredient withaferin-A induced powerful dose-dependent physiologically significant melanin dispersal effects in the isolated skin melanophores of R. tigerina, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of W. somnifera and its active ingredient withaferin-A got highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of W. somnifera and withaferin-A is mediated by cholino-muscarinic like receptors having similar properties.

Mediation of cholino-piperine like receptors by extracts of Piper nigrum induces melanin dispersion in Rana tigerina tadpole melanophores

Aim: The present study was carried out to determine the effects of lyophilized dried fruit extracts of Piper nigrum and pure piperine on the tadpole melanophores of frog Rana tigerina which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The nature of specific cellular receptors present on the neuro-melanophore junction and their involvement in pigmentary responses has been explored.Material: Effects of lyophilized extracts of P. nigrum and pure piperine were studied on the isolated tail melanophores of tadpoles of the frog R. tigerina as per the modified method.Results: The extract of P. nigrum and its active ingredient piperine caused significant melanin dispersal responses leading to darkening of the tail melanophores, which were completely antagonized by atropine and hyoscine. These per se melanin dispersal effects were also found to be markedly potentiated by neostigmine an anticholinesterase agent.Conclusion: It appears that the melanin dispersal effects of the extracts of P. nigrum and pure piperine leading to skin darkening are mediated by cholinergic muscarinic or piperine-like receptors having similar properties.

Efficacy of frog skin lipids in wound healing.

BackgroundFrog skin has been sequentially and scientifically evaluated by our group for its wound healing efficiency. Owing to the complex structure of skin, attempts were being made to analyse the role of individual constituents in different phases of healing. Our earlier papers have shown the significance of frog skin not only in wound healing but also enhancing the proliferating activity of the epidermal and dermal cells which are instrumental for normal healing process. We also have identified for the first time novel antimicrobial peptides from the skin of Rana tigerina and thereby reduce the complications involved in the sepsis.Purpose of the study and ResultsThe current study envisages the role of frog skin lipids in the inflammatory phase of wound healing. The lipid moiety of the frog skin dominated by phospholipids exhibited a dose dependent acceleration of healing irrespective of the mode of application. The efficiency of the extract is attributed partially to the anti-inflammatory activity as observed by the histochemical and immunostimulatory together with plethysmographic studies.ConclusionsThus, frog skin for the first time has been demonstrated to possess lipid components with pharmaceutical and therapeutic potential. The identification and characterization of such natural healing molecules and evaluating their mechanism of action would therefore provide basis for understanding the cues of Nature and hence can be used for application in medicine.

First isolation and identification of Elizabethkingia meningoseptica from cultured tiger frog, Rana tigerina rugulosa

Elizabethkingia meningoseptica has been recognised as an occasional but serious opportunistic bacterial pathogen to human beings. Recently, it was frequently isolated from tiger frog, Rana tigerina rugulosa, with cataract disease, which is the most common disease of unknown aetiology of frogs in Hainan, China. The purpose of this study was to identify and characterise the bacterial strains isolated from the recent outbreaks of cataract disease in farmed tiger frog in Hainan, China, and to evaluate their pathogenicity to the frog and their sensitivity to 20 chemotherapeutic agents. The 16S rRNA gene sequences of strains W0701 (1478 bp), W0702 (1477 bp) and W0703 (1478 bp) showed 98.6–98.7% similarity with the sequence of E. meningoseptica type strain (ATCC 13253) and 99.9–100% similarity with that of E. meningoseptica NTU 870424-IL. Six strains (W0701–W0706) were selected to represent 24 isolates retrieved from six moribund frogs. The morphological, physiological and biochemical characteristics of the six representative isolates were consistent with those of E. meningoseptica strains. The organisms were only susceptible to vancomycin and moderately susceptible to cefoperazone among the 20 investigated chemotherapeutic agents. Virulence test with strain W0702 was conducted and pathogenicity (by intramuscular injection) was demonstrated in the tiger frog. In conclusion, 24 isolates obtained from frogs with cataract disease were the E. meningoseptica strains highly pathogenic to tiger frog, and this is the first report of E. meningoseptica as a pathogen for tiger frog.

Chromatin organization and basic nuclear proteins in the male germ cells of Rana tigerina

The process of chromatin condensation during spermiogenesis in Rana tigerina is similar to the heterochromatization in somatic cells, where 30 nm fibers are coalesced together into a dense mass in spermatozoa without changing their initial size and nucleosomal organization. This conclusion was supported by the finding that the full set of core histones (H2A, H2B, H3, H4) are still present in sperm chromatin, but histone H1 is replaced by its variant, H1V. Rabbit anti-sera were raised against histone H3, H1, H1V, and H5 (H1 variant in chick erythrocyte). Anti-histone H1 antiserum cross-reacted with histone H1V, which implied the presence of a common epitope. Anti-histone H1V and H5 also showed cross-reaction with each other but not with histone H1, which implied the presence of a common epitope not shared by histone H1. Immunocytochemical studies, using the above antibodies as probes, showed that histones H3 is present in all steps of spermatogenic and spermiogenic cells, and somatic cells including red blood cells, Sertoli cells, and Leydig cells, while histone H1 is present in all of the cells mentioned except in spermatozoa where it is replaced by histone H1V. Histone H1V appears in the early spermatids starting from spermatid 1 (St1), and it persists throughout the course of spermatid differentiation into spermatozoa. Histone H1V is also found in chromosomes of metaphase spermatocyte and red blood cells. Thus histone H1V may cause the final and complete condensation of chromatin in Rana spermatozoa, a process which is similar to the heterochromatization occurring in somatic cells such as metaphase chromosome and chick erythrocyte nucleus.

Application of frog (Rana tigerina Daudin) skin collagen as a novel substrate in cell culture.

Collagen and collagen-based materials have extensive application in biomedical devices and tissue engineering. The current paper pertains to the application of frog (Rana tigerina Daudin) skin collagen as a novel substrate in cell culture. The study deals with the behavior, morphology, and physiology of keratinocytes and fibroblasts over dry and reconstituted collagen substratum, which are the key cells involved in wound repair. The advantage of using frog skin collagen as a substratum lies in the ease with which the reconstituted gel can be formed. Further, frog skin collagen is highly hydrophilic, which may be attributed to the fact that amphibians, as the first vertebrates connecting water and land, must have evolved certain physiologic specializations. These studies also contribute to the hypothesis that part of the healing efficacy of frog skin may be due to the collagen since proliferation, migration, and differentiation of epithelial cells are prime requisites for a normal healing mechanism.

Studies on Rana tigerina skin collagen

The current paper pertains to the study of frog skin, more specifically Rana tigerina skin collagen, which is a major extracellular matrix protein known to play an important role in the wound-healing process. This study revealed interesting differences in the frog skin collagen when compared to the hitherto known vertebrate collagens. This could probably be attributed to the position of the amphibians in the vertebrate hierarchy. Therefore, detailed investigations on the various physico-chemical properties, such as reconstitution, redissolution, viscosity and denaturation were carried out. The study confirms the structural relationship of collagen to habitat and function.

Tigerinins: Novel Antimicrobial Peptides from the Indian FrogRana tigerina

Four broad-spectrum, 11 and 12 residue, novel antimicrobial peptides have been isolated from the adrenaline-stimulated skin secretions of the Indian frog Rana tigerina. Sequences of these peptides have been determined by automated Edman degradation, by mass spectral analysis and confirmed by chemical synthesis. These peptides, which we have named as tigerinins, are characterized by an intramolecular disulfide bridge between two cysteine residues forming a nonapeptide ring. This feature is not found in other amphibian peptides. Conformational analysis indicate that the peptides tend to form beta-turn structures. The peptides are cationic and exert their activity by permeabilizing bacterial membranes. Tigerinins represent the smallest, nonhelical, cationic antimicrobial peptides from amphibians.

Virulence properties of motile aeromonads isolated from farmed frogs Rana tigerina and R. rugulosa.

Virulence factors were compared in Aeromonas species isolated from clinically normal and septicaemic farmed frogs from Thailand. Haemolysin activities against frog erythrocytes were significantly different within the collection of aeromonads. Groups of high haemolytic activity (unspeciated Aeromonas, Au), moderate haemolytic activity (A. hydrophila), and low haemolytic activity (A. veronii biovar sobria, A. veronii biovar veronii, A. caviae, A. schubertii) were noted. DNA colony hybridisation studies revealed that Au isolates possessed a haemolysin gene (ASH1) which was not present in any of the other Thai aeromonads or type strains tested. Elastinolytic activity was demonstrated in 90% of the Au isolates, 60% of the A. hydrophila isolates and in none of the other motile aeromonads. The cytotoxic activity of the Aeromonas isolates varied according to the source of cells used in the assays. Cells from rainbow trout were extremely sensitive to Au toxins but less so to toxins produced by other species. In contrast mammalian cells showed very little sensitivity to Au toxins but were more sensitive to toxins produced by A. hydrophila. Selection of suitable assay substrates is therefore important.

Histamine receptors in the skin melanophores of Indian bullfrog Rana tigerina

Histamine and 2-methyl histamine caused dose-dependent aggregation of the integumental melanophores of Rana tigerina both in vitro and in vivo. The aggregating effects were antagonised by mepyramine and metiamide, specific H 1 and H 2 receptor blockers, respectively. Compound 48/80 and EDTA augmented the melanin-aggregating effects of exogenously applied histamine and 2-methyl histamine in in vivo experiments. 4-Methyl histamine, a specific H 2 receptor agonist, dispersed the frog melanophores in in vitro studies, the dispersing effects were blocked by metiamide.

Growth potential of Rana tigerina skin lipids in cell cultures.

This study involves the investigation of the lipid composition of the skin of Rana tigerina which has a significant healing capacity. The results indicated that the lipid extract enhanced keratinocyte and fibroblast cell proliferation progressively and were found to be much more efficient in comparison to agents known to cause cell proliferation and to be anti-inflammatory such as hydrocortisone. Cell proliferation was dose dependent and suppression occurred only at very high doses. [3H]thymidine incorporation studies produced the same results. Because proliferation, migration, and differentiation of the basal cells is essential for initiation and progression of wound healing, any agent enhancing their proliferation would hasten the healing process. This paper therefore aims at elucidating the effect of composition of the total lipid extract confirming the efficacy of frog skin in wound healing and thereby providing an understanding of the natural mechanism of healing.

Active Acoustic Gleaning from the Water Surface by the Indian False Vampire Bat, Megaderma lyra

Abstract1 The method employed by the Indian false vampire bat Megaderma lyra (body mass about 40 g) to detect and capture prey from the surface of an artificial pond in an outdoor enclosure was studied. Frogs (Rana tigerina) (body mass about 10 g) were used as prey. The bats exhibited searching flights at the surface of the water for a few minutes in darkness, detected a stationary frog with its head jutting out of the water, and with a dart captured the frog in the mouth after hovering over it for about 2 s. The detected frogs did not show any movement when the bats hovered over them.2 When a freshly killed frog alone was available and pinned with its head protruding out of the surface of the water, the bats captured it after making fewer numbers of attempts in a shorter period of time compared with the capture of live frogs. The number of attempts and time taken to capture a frog increased when the frog's eyeballs alone were exposed above the water surface, and the bats failed to detect frogs when their entire head was submerged just below the water.3 Sound and video recordings showed that M. lyra continuously emitted faint ultrasounds while making searching flights at the pond. Apparently the bats made ‘early’ and ‘late’ detections, with higher repetition rates of sound emission in the case of the latter. A final buzz before capturing prey was not recorded and the change in the duration of emitted sounds during approach and after capturing the frog was marginal. The second harmonic at 43.6 kHz (SD 3.5 kHz) was more dominant and the duration was 1.0 ms (SD 0.2 ms).4 It is clear that M. lyra uses echolocation to detect and capture prey from the water surface. A comparison is made with an earlier report on M. lyra catching prey from the ground by a passive acoustic location.

Effect of catecholamines on the isolated melanophores of the Indian frog, Rana tigerina

Effect of catecholamines on the isolated melanophores of the Indian frog, Rana tigerina