Rana Rugulosa Research Articles

Effects of Dietary Star Apple (Chrysophyllum cainito L.) Peel Extract on Growth Performance, Intestinal Histology, Hematology, and Non-Specific Immune Parameters in Common Lowland Frog (Rana rugulosa Wiegmann)

Star apple (Chrysophyllum cainito L.) has been used to treat several diseases. However, using this plant as a feed additive in aquaculture production is rare. Therefore, this work aimed to investigate the effects of dietary star apple peel extract (SAPE) on growth performance, intestinal histology, hematology, and non-specific immune parameters in common lowland frog (Rana rugulosa Wiegmann). Peel samples from star apple fruits were collected, dried, and extracted with methanol. Alkaloids, flavonoids, phenolics, saponins, tannins, and steroids were detected in SAPE at varied concentrations. Frogs of average weight 8.50 ± 0.10 g were fed diets incorporating 0, 10, 15, and 20 g SAPE/kg diet. After six weeks of feeding, the frogs' growth and physiological parameters were evaluated. The results revealed that the growth and intestinal histology features of frogs fed SAPE diets were significantly increased compared with the control (P <0.05). No significant differences among the treatments were observed in the survival rate, red blood cells, hematocrit, and hemoglobin (P >0.05). Serum albumin was significantly enhanced in frogs fed a 10 g SAPE/kg diet (P <0.05). Dietary SAPE significantly decreased serum alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, triglyceride, and cholesterol compared with the control (P <0.05). Furthermore, frogs fed SAPE diets had significantly elevated lysozyme activity, leukocyte respiratory burst activity, complement activity, and immunoglobulin M compared with the control (P <0.05). In conclusion, these results showed the beneficial effects of SAPE on the growth and health status of frogs with the appropriate level of 17.75 to 20.00 g/kg diet. Keywords: Medicinal plants, Common lowland frog, Growth performance, Immunity, Hematology

Morphology and cytochemical patterns of peripheral blood cells of tiger frog (Rana rugulosa).

Tiger frog (Rana rugulosa) is a national second-class protected amphibian species in China with an important ecological and economic value. In recent years, due to excessive human hunting, pollution and habitat loss, the wild population of tiger frog has declined sharply. To protect wildlife resources, the artificial breeding of tiger frogs has rapidly developed in China. Diseases are increasing and spreading among tiger frogs due to the increasing scale of artificial farming. The blood examination is the most straightforward and less invasive technique to evaluate the animal health condition. Thus, it is essential to obtain the normal hematological indicators of tiger frogs. The objective of this study was to investigate the morphometry, microstructure and cytochemical patterns of peripheral blood cells in tiger frogs. The number of blood cells in tiger frogs was counted on a blood count board, and the cell sizes were measured by a micrometer under light microscope. The morphology and classification of blood cells were studied by Wright-Giemsa staining, and the cytochemical pateerns was investigated by various cytochemical staining including periodic acid-Schiff (PAS), Sudan black B (SBB), peroxidase (POX), alkaline phosphatase (AKP), acid phosphatase (ACP), chloroacetic acid AS-D naphthol esterase (CAE) and α-naphthol acetate esterase (ANAE) staining. Besides erythrocytes and thrombocytes, five types of leukocytes were identified in tiger frogs: neutrophils, eosinophils, basophils, lymphocytes and monocytes. The mean erythrocyte, leukocyte and thrombocyte counts were 1.33 ± 0.15 million/mm3, 3.73 ± 0.04 × 104/mm3 and 1.7 ± 0.01 × 104/mm3, respectively. Small lymphocytes were the most abundant leukocytes, followed by large lymphocytes, Neutrophils, eosinophils and monocytes, basophils were the fewest. Eosinophils were strongly positive for PAS, positive for SBB, POX, ACP, CAE, ANAE, while weakly positive for AKP staining; basophils were strongly positive for PAS, ACP, positive for SBB, CAE, weakly positive for ANAE, negative for AKP, POX staining; neutrophils were strongly positive for ACP, SBB, positive for PAS, POX, weakly positive for AKP, CAE and ANAE staining; monocytes were positive for PAS, SBB, ANAE, weakly positive for ACP, AKP, POX, CAE staining; large lymphocytes and thrombocytes were positive for PAS, ACP, weakly positive for ANAE, while negative for SBB, POX, AKP, CAE; small lymphocytes were similar to large lymphocytes, except for strongly positive for PAS and ACP staining. The blood cell types and morphology of tiger frogs were generally similar to those of other amphibians, while their cytochemical patterns had some notable species specificity.Our study could enrich the knowledge of peripheral blood cell morphology and cytochemistry in amphibians, and provide baseline data for health condition evaluation and disease diagnosis of tiger frogs.

Genetic and Morphologic Identification of Spirometra ranarum in Myanmar.

In the present study, we identified a Spirometra species of Myanmar origin (plerocercoid) by molecular analysis using mitochondrial cox1 and nad1 genes, as well as by morphological observations of an adult tapeworm. Spargana specimens were collected from a paddy-field in Taik Kyi Township Tarkwa Village, Yangon, Myanmar in December 2017. A total of 5 spargana were obtained from 20 frogs Hoplobatrachus rugulosus; syn: Rana rugulosa (Wiegmann, 1834) or R. tigrina (Steindachner, 1867). The plerocercoids were used for experimental infection of a dog. After 4 weeks of infection, an adult tapeworm was recovered from the intestine of the dog. Morphologically, the distinct features of Spirometra sp. (Myanmar origin) relative to S. erinaceieuropaei and S. decipiens include a uterine morphology comprising posterior uterine coils that larger than the terminal uterine ball and coiling of the uteri diagonally (swirling) rather than spirally. The cox1 sequences (1,566 bp) of the Myanmar-origin Spirometra species showed 97.9% similarity to a reference sequence of S. decipiens (GenBank no. KJ599679) and 90.5% similarity to a reference sequence of S. erinaceieuropaei (GenBank no. KJ599680). Phylogenetic tree topologies were identical and presented high confidence level of values for the 3 major branches of the 3 Spirometra species in cox1 and nad1 genes. These results indicated that Myanmar-origin Spirometra species coincided with those of S. ranarum and may be considered as a valid species.

An investigation of sudden death in farmed infantSiamesecrocodiles during winter and spring in Hainan, China

Sudden deaths without any specific clinical sign occurred among one year old young ones on Siamese crocodile (Crocodylus siamensis) farms in Hainan province, China, during winter and spring in 2012 - 2013. The autopsy showed that the internal organs seemed normal except full of clear ascites in abdominal cavity. There were total six bacterial species isolated, Edwardsiella tarda, Proteus penneri, Proteus vulgaris, Enterobacter asburiae, Citrobacter freundii and Micrococcus luteus from either the heart bloods or the spleens and livers. The virulence of isolated strains was tested by intraperitoneal inoculation in female Chinese edible frogs (Rana rugulosa or Hoplobatrachus rugulosus). The six-day mortality revealed that E. tarda was the most pathogenic, whereas P. penneri and E. asburiae exhibited no infection at all, and the rest demonstrated similar inconspicuous toxicity. All these findings suggest that E. tarda would be the principal suspect causing death of Siamese crocodile. The sensitivities of the E. tarda isolates were tested against 20 kinds of antimicrobial drugs. The results showed that ceftazidime, norfloxacin and amikacin were highly sensitive to the isolates of E.tarda.

Growth performance and intestinal morphology of common lowland frog (Rana rugulosa) fed diets supplemented with lotus (Nelumbo nuc

The aims of this research were to study the effect of Nelumbo nucifera Gaertn. stamen extract (NNSE) as a natural feed additive on growth performance and intestinal morphology of common lowland frog (Rana rugulosa). Frogs with an average of 16.35±0.68 g were fed with diets containing 0, 1, 3 and 5% of NNSE for 11 weeks. The results showed that final weight, weight gain, average daily gain and specific growth rate of frogs fed the diets incorporated with NNSE were significantly higher than those of frogs fed the basal diet (P 0.05). Villi heights, villi widths, the thicknesses of villi, longitudinal muscularis and circular muscularis in the intestines of frogs fed the diets containing NNSE had significantly higher than frogs fed the control diet (P<0.05). Feeding behavior, feed acceptability and health of frogs in the experimental groups were similar to the control group, indicating that NNSE did not have any toxic effect on frog. The optimal concentration of NNSE observed in this investigation was 5%. Thus, our research supports the use of NNSE as natural feed additive in the diet to improve growth and intestinal morphology of frog.

Effects of dietary waterlily (Nymphaea pubescens) stamen extract on growth performance and intestinal morphology of common lowland frog (Rana rugulosa)

The effects of diets containing various levels (0, 1, 3 and 5%) of waterlily (Nymphaea pubescens) stamen extract (NPSE) on growth performance and intestinal morphology were investigated in common lowland frog (Rana rugulosa) with an initial weight of 16.09±0.50 g. After 11 weeks of feeding, growth parameters were evaluated. The results showed that the frogs fed with diets supplemented with 3 and 5% of NPSE exhibited significantly higher weight gain, specific growth rate and feed conversion ratio than frogs fed diets supplemented with 0 and 1% of NPSE (P 0.05). Villi heights, villi widths and the thickness of intestinal muscle layers in posterior intestine were significantly increased in frogs fed the diets containing NPSE (P 0.05). Feeding behavior and feed acceptability of the experimental groups were the same as the control group. The optimal levels of NPSE observed in this present study were ranged between 3-5%. Our findings suggest that NPSE can be applied in the diets as a growth promoter in common lowland frog.

Growth Performance and Feed Utilization of Common Lowland Frog (Rana rugulosa Wiegmann) Fed with Supplementation by Bromelain Extracted from Pineapple Feed

Growth Performance and Feed Utilization of Common Lowland Frog (<i>Rana rugulosa</i> Wiegmann) Fed with Supplementation by Bromelain Extracted from Pineapple Feed

The Characterization and Activity of Digestive Enzymes in Different Sizes of Common Lowland Frog (Rana rugulosa Wiegmann)

The Characterization and Activity of Digestive Enzymes in Different Sizes of Common Lowland Frog (<i>Rana rugulosa</i> Wiegmann)

Origin of Secretin Receptor Precedes the Advent of Tetrapoda: Evidence on the Separated Origins of Secretin and Orexin

At present, secretin and its receptor have only been identified in mammals, and the origin of this ligand-receptor pair in early vertebrates is unclear. In addition, the elusive similarities of secretin and orexin in terms of both structures and functions suggest a common ancestral origin early in the vertebrate lineage. In this article, with the cloning and functional characterization of secretin receptors from lungfish and X. laevis as well as frog (X. laevis and Rana rugulosa) secretins, we provide evidence that the secretin ligand-receptor pair has already diverged and become highly specific by the emergence of tetrapods. The secretin receptor-like sequence cloned from lungfish indicates that the secretin receptor was descended from a VPAC-like receptor prior the advent of sarcopterygians. To clarify the controversial relationship of secretin and orexin, orexin type-2 receptor was cloned from X. laevis. We demonstrated that, in frog, secretin and orexin could activate their mutual receptors, indicating their coordinated complementary role in mediating physiological processes in non-mammalian vertebrates. However, among the peptides in the secretin/glucagon superfamily, secretin was found to be the only peptide that could activate the orexin receptor. We therefore hypothesize that secretin and orexin are of different ancestral origins early in the vertebrate lineage.

Ovine thyroid stimulating hormone (TSH) heterologously stimulates production of thyroid hormones from Chinese soft-shell turtle ( Pelodiscus sinensis) and bullfrog ( Rana catesbeiana and Rana rugulosa) thyroids in vitro

Thyroid hormones are important for regulating a variety of developmental processes in vertebrates, including growth, differentiation, metamorphosis, and oxidative metabolism. In particular, this study focused on the in vitro production of thyroxine (T 4) and triiodothyronine (T 3) from thyroids in American bullfrogs ( Rana catesbeiana), Chinese bullfrogs ( Rana rugulosa Wiegmann), and Chinese soft-shell turtles ( Pelodiscus sinensis) treated with ovine thyroid stimulating hormone (TSH) at different culture intervals (2, 4, 8, and 12 h) and dosages (1, 10, 50 or 100 ng). The levels of T 4 and T 3 in the tested animals were elevated upon stimulation in a time- and dose-dependent manner, indicating de novo synthesis of T 4 and T 3. Significantly higher hormone levels were observed in the Chinese bullfrog compared to the other two species, for both the time–course and dose–response experiments. Although the bullfrog secreted significantly higher levels of T 4 and T 3, a higher T 4-conversion capacity was found in the Chinese soft-shell turtle. The highest ratios of T 3 to T 4 were observed in the American bullfrog and Chinese soft-shell turtle for the time–course and dose–response experiments, respectively. These findings suggest that the Chinese soft-shell turtle and bullfrog thyroids can accept ovine TSH for T 4- and T 3-formation in a time- and dose-dependent manner, supporting the hypothesis that the binding interactions between TSHs and thyroidal receptors are conserved in vertebrates.

Aeromonas hydrophila subsp. ranae subsp. nov., isolated from septicaemic farmed frogs in Thailand.

A group of seven sucrose-negative Aeromonas strains (referred to as group Au) isolated from the internal organs of septicaemic farmed frogs (Rana rugulosa) in Thailand was subjected to a polyphasic taxonomic study including fluorescent amplified fragment length polymorphism (FAFLP) and ERIC-PCR fingerprinting, 16S rDNA sequencing, microplate DNA-DNA hybridizations and extensive phenotypic characterization. Comparison of FAFLP and ERIC-PCR fingerprints indicated that the group Au isolates belonged to the species Aeromonas hydrophila DNA hybridization group (HG) 1 in which they represent a genotypic subgroup closely affiliated to A. hydrophila subsp. hydrophila and subsp. dhakensis. One representative of the Au group exhibited > or = 99.0% 16S rDNA sequence similarity with the type strains of the two A. hydrophila subspecies. DNA-DNA hybridization with type and reference strains of all known Aeromonas taxa revealed that the Au group represented a homogeneous taxon that exhibited the highest relatedness with members of the two A. hydrophila subspecies, ranging from 75 to 93%. Phenotypic characterization on the basis of 152 features further revealed that the Au group isolates differed from A. hydrophila subsp. hydrophila or subsp. dhakensis in a total of 13 biochemical properties. Of these, assimilation of L-glycine and isobutyrate as sole carbon source, acid production from salicin and D-sucrose, and aesculin hydrolysis were of diagnostic value. From the results of this study, it can be concluded that the Aeromonas frog isolates of the Au group represent a new subspecies of A. hydrophila, for which the name Aeromonas hydrophila subsp. ranae subsp. nov. is proposed. Its type strain is Au-1D12(T) (=LMG 19707(T) = CCUG 46211(T)).

Optimum protein and energy levels for the Thai native frog, Rana rugulosa Weigmann

This study determined the effect of dietary protein and energy levels on growth and feed utilization of the Thai native frog (Rana rugulosa Weigmann). The frogs were fed four isocaloric diets (5300 kcal kg−1) with protein levels 30, 35, 40 and 45% in experiment 1 and with four isonitrogenous (37%) diets with gross energy levels of 4500, 4900, 5300 and 5700 kcal kg−1 in experiment 2. The frogs were fed to apparent satiation twice daily for 12 weeks in three replicate concrete ponds (30 frogs per pond) with an average frog weight of 3.5 and 7.8 g for experiments 1 and 2, respectively. Average weights but not survival rates were significantly different at the end of the study. Feed intake, energy intake and protein efficiency ratio of frogs fed the 30–45% protein diets were not significantly different. Feed conversion ratios of frogs fed the 30% protein diet were significantly higher than for those fed 35, 40 and 45% protein diet. The amount of protein consumed by frogs fed the 30% protein diet was significantly lower than frogs fed the 35, 40 and 45% protein diets. Energy retention in frogs fed the 35% protein diet was significantly higher than other treatments. Protein retention in frogs fed diets with 30 and 35% protein was significantly higher than the frogs fed the 40 and 45% protein diet. The optimum protein level was determined as 36.72%. In experiment 2, frogs were fed with four levels of energy in a diet with a constant protein level of 37%. The average final weight and average daily gain of frogs fed with the 4900 kcal kg−1 diet were significantly higher than the other treatments. Frogs fed with the 4500 kcal kg−1 diet had significantly higher feed intake, feed conversion and lower protein efficiency ratio than frogs fed with the 4900–5700 kcal kg−1 diet. The amount of protein consumed by frogs fed the 4900–5700 kcal kg−1 diet was significantly less than frogs fed diet with 4000 kcal kg−1, but energy consumed by frogs fed the 5300–5700 kcal kg−1 diet was significantly greater than those fed the 4500–4900 kcal kg−1 diet. On the other hand, protein and energy retention in frogs fed the 4900 kcal kg−1 diet were not significantly higher than other treatments. Survival rate and body composition were similar in all treatments. The studies indicate optimum dietary protein, energy and protein to energy ratio were 36.7%, 4900 kcal kg−1 and 74.89 mg kcal−1, respectively.

Differences in mGnRH and cGnRH-II contents in pituitaries and discrete brain areas of Rana rugulosa W. according to age and stage of maturity

(1) In tadpoles, chicken-II gonadotropin-releasing hormone (cGnRH-II) could be measured in the brains before metamorphosis, but mammalian gonadotropin-releasing hormone (mGnRH) did not appear until the stage of metamorphosis, i.e. cGnRH-II appeared earlier than mGnRH during ontogenesis. (2) During the metamorphic climax, mGnRH content increased more rapidly than cGnRH-II; the content of mGnRH was about two times of that of cGnRH-II. (3) In juveniles and adults, the content of mGnRH and cGnRH-II, and the distribution pattern of mGnRH (but not cGnRH-II) in the brains and pituitaries changed with age and stages of gonadal development. mGnRH mainly distributed in the rostral brain areas, whereas cGnRH-II had a widespread distribution in the brain. (4) Both mGnRH and cGnRH-II were present in the pituitaries at each stage of maturity. The gonadotropin-releasing hormone (GnRH) content at sexually maturity was significantly higher than that at other stages of gonadal development, and the content of mGnRH was about 15–18 times of that of cGnRH-II. (5) These results suggest that both mGnRH and cGnRH-II are potentially involved in the direct regulation of pituitary gonadotropes, and mGnRH may be the major active form, cGnRH-II may also serve as a neurotransmitter or neuromodulator in the brain.

Improved disease resistance in Rana rugulosa (Daudin) after β-glucan administration

There is an increasing problem with bacterial disease associated with stress in farmed frogs. In general, little is known about the immune system in farmed tropical ranid species and, in particular, about its functional ability. As part of a study on the non-specific immune system, immunostimulation by β-glucan was evaluated in preventing bacterial disease in cultured ranid frogs. Fifty frogs were grown to a mean weight of 40 g and separated into three experimental groups. These were exposed to a motile aeromonad bacterial challenge alone (Au), bacterial challenge with β-glucan (Au + glucan) or saline only (controls) to serve as controls. There were four replicate tanks containing five frogs per tank in each experimental group. After bacterial challenge, all the animals were observed twice daily, and the number of mortalities per tank was recorded. The liver was sampled in order to recover bacteria, and any colonies grown were identified. The survival of the β-glucan-treated frogs was significantly higher than that of the bacterial challenge group alone.

Seasonal variations in production in vitro of corticosteroids by the frogs (Rana rugulosa) adrenal.

SUMMARY Cyclical changes in the activity of adrenocortical tissue are a wellestablished phenomenon in many amphibian species but up to now they have been studied by observing changes in cytology or enzyme activity, e.g. steroid-3β-ol dehydrogenase. The present study was designed to provide a measure of corticosteroid secretion throughout the seasons using the in-vitro approach. Concomitant histological changes are also recorded and together provide evidence that the adrenal tissue of Rana rugulosa shows maximum activity in the spring spawning season and reaches a minimum in winter.

Pathways for the biosynthesis of corticosteroids from pregnenolone by adrenal tissue of the frog, Rana rugulosa

The formation of corticosteroids from pairs of differently labeled precursors was studied using adrenal tissue from the frog, Rana rugulosa. The precursors chosen were [16- 3H]-pregnenolone and [4- 14C]-progesterone; [4- 14C]-progesterone and [1,2- 3H]-deoxycorticosterone; [1,2- 3H]-deoxycorticosterone and [4- 14C]-corticosterone. Products were isolated after different periods of time, and product yield time curves were constructed. From pregnenolone, one compound was formed which was not produced from the other precursors. Its identity is unknown, but it does not seem likely that this played a part as an intermediate. Otherwise, all of the products isolated during the experiments were doubly labeled, and produced in a similar manner from both precursors. Furthermore, the maximal incorporation of radioactive label into the different products showed a clearly defined sequence. From these results it appears that in the frog adrenal, the main pathway for the formation of corticosteroids from pregnenolone is: pregnenolone→progesterone→deoxycorticosterone ▪ Other pathways, particularly those which envisage 11β-hydroxyprogesterone or 18-hydroxycorticosterone as intermediates, appear to be relatively unimportant. It is particularly noteworthy that progesterone and pregnenolone were equally good precursors of the corticosteroids. In this respect the adrenal of the frog resembles that of other submammalian tetrapods, but shows a marked contrast with those from certain mammalian species.