Female Bullfrogs Research Articles

Plasticity in the Functional Properties of NMDA Receptors Improves Network Stability during Severe Energy Stress.

Brain energy stress leads to neuronal hyperexcitability followed by a rapid loss of function and cell death. In contrast, the frog brainstem switches into a state of extreme metabolic resilience that allows them to maintain motor function during hypoxia as they emerge from hibernation. NMDA receptors (NMDARs) are Ca2+-permeable glutamate receptors that contribute to the loss of homeostasis during hypoxia. Therefore, we hypothesized that hibernation leads to plasticity that reduces the role of NMDARs within neural networks to improve function during hypoxia. To test this, we assessed a circuit with a large involvement of NMDAR synapses, the brainstem respiratory network of female bullfrogs, Lithobates catesbeianus Contrary to our expectations, hibernation did not alter the role of NMDARs in generating network output, nor did it affect the amplitude, kinetics, and hypoxia sensitivity of NMDAR currents. Instead, hibernation strongly reduced NMDAR Ca2+ permeability and enhanced desensitization during repetitive stimulation. Under severe hypoxia, the normal NMDAR profile caused network hyperexcitability within minutes, which was mitigated by blocking NMDARs. After hibernation, the modified complement of NMDARs protected against hyperexcitability, as disordered output did not occur for at least one hour in hypoxia. These findings uncover state-dependence in the plasticity of NMDARs, whereby multiple changes to receptor function improve neural performance during metabolic stress without interfering with their normal role during healthy conditions.

Presence of ascites in bullfrog breeders reared and kept on a frog farm

ABSTRACT Ascites is a pathology characterized by the extravasation of fluid from blood vessels and its accumulation in the abdominal cavity, caused by several associated factors. In this paper, we report the occurrence of this syndrome in breeding male and female Bullfrogs fed a commercial fish feed with 40% crude protein. Liver malformation was already observed due to lack of protein deposition in adult animals from the same spawning. The culture of ascitic fluid showed absence of bacterial growth. Kidney histopathology showed, in both sexes, a large number of tubular structures with strongly eosinophilic hyaline material suggesting colloid growth in both the cortical region and spinal cord. The analysis also revealed granulomas in various stages of development, many showing central necrotic material. The kidneys, glomerular, were mostly hypoplastic, with enlarged Bowman space, and many were hyalinized or hemorrhagic; the parenchyma showed dystrophic calcification, and many tubules containing fibrinoid material. The liver displayed a large amount of melanomacrophages in the parenchyma and foci of mono-lymphocytic hepatitis and marked cytoplasmic rarefaction, as well as several hepatocytes with pyknotic nuclei and necrotic cells and dissociation of the hepatic trabeculae. Ascites may be caused by the lesions observed in these organs.

How to Build a Fast and Highly Sensitive Sound Detector That Remains Robust to Temperature Shifts.

Frogs must have sharp hearing abilities during the warm summer months to successfully find mating partners. This study aims to understand how frog hair cell ribbon-type synapses preserve both sensitivity and temporal precision during temperature changes. Under room (∼24°C) and high (∼32°C) temperature, we performed in vitro patch-clamp recordings of hair cells and their afferent fibers in amphibian papillae of either male or female bullfrogs. Afferent fibers exhibited a wide heterogeneity in membrane input resistance (Rin) from 100 mΩ to 1000 mΩ, which may contribute to variations in spike threshold and firing frequency. At higher temperatures, most fibers increased their frequency of spike firing due to an increase in spontaneous EPSC frequencies. Hair cell resting membrane potential (Vrest) remained surprisingly stable during temperature increases, because Ca2+ influx and K+ outflux increased simultaneously. This increase in Ca2+ current likely enhanced spontaneous EPSC frequencies. These larger "leak currents" at Vrest also lowered Rin and produced higher electrical resonant frequencies. Lowering Rin will reduce the hair cells receptor potential and presumably moderate the systems sensitivity. Using membrane capacitance measurements, we suggest that hair cells can partially compensate for this reduced sensitivity by increasing exocytosis efficiency and the size of the readily releasable pool of synaptic vesicles. Furthermore, paired recordings of hair cells and their afferent fibers showed that synaptic delays shortened and multivesicular release becomes more synchronous at higher temperatures, which should improve temporal precision. Together, our results explain many previous in vivo observations on the temperature dependence of spikes in auditory nerves.SIGNIFICANCE STATEMENT The vertebrate inner ear detects and transmits auditory information over a broad dynamic range of sound frequency and intensity. It achieves remarkable sensitivity to soft sounds and precise frequency selectivity. How does the ear of cold-blooded vertebrates maintain its performance level as temperature changes? More specifically, how does the hair cell to afferent fiber synapse in bullfrog amphibian papilla adjust to a wide range of physiological temperatures without losing its sensitivity and temporal fidelity to sound signals? This study uses in vitro experiments to reveal the biophysical mechanisms that explain many observations made from in vivo auditory nerve fiber recordings. We find that higher temperature facilitates vesicle exocytosis and electrical tuning to higher sound frequencies, which benefits sensitivity and selectivity.

A tradeoff between mate-quality recognition and species recognition in male Bufo gargarizans that co-occur with Rana catesbeiana

Typically, anuran amphibians favor larger females as mates because larger females lay more eggs; thus, males in amplexus can increase the number of eggs fertilized, and fitness. However, males may also prefer those females that were closest to the norm for their population in overlapping populations, and these individuals do not receive the benefits of enhanced fertilization success. In this study, we test how male Bufo gargarizans choose mates in the presence and absence of an invasive species, bullfrogs (Rana catesbeiana). When given a simultaneous choice between a small male and a large female toad, males discriminated between the sexes and attempted to clasp a large female. However, when one male toad was presented with a large female bullfrog and a small female toad, the males chose both with equal frequency. Therefore, male B. gargarizans appears to trade-off between species and mate-quality recognition, such that those toads co-occurring with heterospecifics do not blindly prefer mate-quality to ensure conspecific matings.

Effects of testosterone on contractile properties of sexually dimorphic forelimb muscles in male bullfrogs (Rana catesbeiana, Shaw 1802)

SummaryThis study examined the effects of testosterone (T) on the contractile properties of two sexually dimorphic forelimb muscles and one non-dimorphic muscle in male bullfrogs (Rana catesbeiana, Shaw 1802). The dimorphic muscles in castrated males with testosterone replacement (T+) achieved higher forces and lower fatigability than did castrated males without replaced testosterone (T0 males), but the magnitude of the differences was low and many of the pair-wise comparisons of each muscle property were not statistically significant. However, when taken as a whole, the means of seven contractile properties varied in the directions expected of masculine values in T+ animals in the sexually dimorphic muscles. Moreover, these data, compared with previous data on male and female bullfrogs, show that values for T+ males are similar to normal males and are significantly different from females. The T0 males tended to be intermediate in character between T+ males and females, generally retaining masculine values. This suggests that the exposure of young males to T in their first breeding season produces a masculinizing effect on the sexually dimorphic muscles that is not reversed between breeding seasons when T levels are low. The relatively minor differences in contractile properties between T+ and T0 males may indicate that as circulating T levels rise during breeding season in normal males, contractile properties can be enhanced rapidly to maximal functional levels for breeding success.

Time interval from ovulation to extrusion in female bullfrog in different photoperiods

It was analyzed in this work the influence of photoperiod on time interval from ovulation induction period to extrusion of ovocits in female bullfrogs (Lithobates catesbeianus). It was used 54 females reared from metamorphosis to 9 months of age under three photoperiods: dark time (DL 0:24), 16 hours of daylight (DL 16:8) and 12 hours of daylight (DL 12:12). Ovulation was induced by intramuscular application of two doses of LHRHa with 12 hours of interval between the injections. After 10, 25, 28, 31, 34 and 37 hours from the first hormone injection, 10-gram samples (3,000 eggs) were extracted from each female at each time interval and fertilized. Egg hatching rate was checked in each sample 72 hours after fertilization. Analysis of variance showed a significant effect of extrusion delay and the interaction between photoperiod and this delay. Extrusion should be carried out 33, 24 and 26 hours after the first hormone dosage in females reared in environments without light, with 12 hours of daylight and with 16 hours of daylight, respectively, to obtain the maximum fertilization rate.

Geographical variation in body size and sexual size dimorphism of introduced American bullfrogs in southwestern China

Invasive species often exhibit geographical variations in life history traits that may allow them to successfully invade different environments. We investigated geographical variation in body size and sexual size dimorphism (SSD) of invasive bullfrogs in southwestern China, by sampling two breeding populations (descendants of a single source population) inhabiting sites at low (1,412 m, Shiping) and high (2,692 m, Luguhu) altitudes. Both populations exhibited significant SSD, with females larger than males. At high altitude, mean body size of both sexes and the degree of SSD were significantly reduced; the reduction in mean body size with increasing altitude was more pronounced in females, although not significantly so. Female bullfrogs also showed a significant decrease in average age at high altitude that may be a major factor related to this pattern; average age of male bullfrogs did not vary significantly with altitude. Growth rate of both sexes was also lower at high altitude. Our results provide the first evidence that introduced bullfrog’s exhibit geographical variation in morphology in invaded areas in response to different environments, likely due to changes in climate. Additional research is required to determine the mechanism of this variation (i.e., physiological or developmental plasticity, mortality rate, selective pressure) and most importantly, to evaluate the potential for variation in the impacts of introduced bullfrogs on native ecosystems in China.

Diet of Introduced Bullfrogs (Rana catesbeiana): Predation on and Diet Overlap with Native Frogs on Daishan Island, China

We examined diet of introduced Bullfrogs (Rana catesbeiana) and three native frog species (Rana limnocharis, Rana nigromaculata, and Bufo bufo gargarizans) co-occurring at a group of ponds on Daishan Island, east of China, to gain insight into the nature of potential interactions between Bullfrogs and native frog species. For postmetamorphic Bullfrogs, aquatic prey items dominated volumetrically. Prey size, diet volume and volumetric percentage of native frogs in diet increased with Bullfrog body size. The number and volumetric percentage of native frogs in the diet were not different for female and male Bullfrogs, and both were higher for adults than for juveniles. Diet overlap between males and juveniles was higher than that between males and females and between females and juveniles. Diet overlap with each native frog species of male Bullfrogs was lower than that of female Bullfrogs and juvenile Bullfrogs. We did not exam effects of Bullfrogs on native frogs but our results suggest that the primary threat posed by juvenile Bullfrogs to native frogs on Daishan Island is competition for food, whereas the primary threat posed by male Bullfrogs is direct predation. Female Bullfrogs may threaten native frogs by both competition and predation. These differences among Bullfrog groups may be attributed to differences in body size and microhabitat use.

Renal Failure and Bilateral Thymoma in an American Bullfrog, Rana catesbiana

ABSTRACT An adult female American bullfrog, Rana catesbiana, from a zoological collection, was examined because of a slowly progressive distension of the coelomic cavity. Approximately 750 cc of clear fluid was removed by celiocentesis. Using cardiocentesis, a blood sample was obtained and plasma was removed for biochemical analyses. The frog was kept in a isotonic solution of saline that contained calcium gluconate, and enrofloxacin was administered orally for 17 d. The frog was found dead in its cage at 17 d following removal of the coelomic fluid and was submitted for necropsy. Major gross findings were cystic kidneys and bilateral cystic masses in the cervical region. Using light microscopy, glomeruli were swollen and were surrounded by thick rims of dense fibrous connective tissues; some also were sclerotic. Additionally there was chronic, diffuse, severe tubulointerstitial nephritis. The bilaterally symmetrical cystic masses in the cervical region had microscopic features most consistent with those ...

Androgen Receptors and Sexual Dimorphisms in the Larynx of the Bullfrog

As in most anuran amphibians, both male and female bullfrogs (Rana catesbeiana) vocalize. Sex differences in vocalizations in the bullfrog may be due to sex differences in the larynx. We examined the laryngeal muscle to determine whether it possessed androgen receptors and whether there were morphological sexual dimorphisms in the larynx. Using a polyclonal antibody and immunocytochemistry, we found androgen receptors in the laryngeal dilator muscle of both sexes. Males possessed approximately 13% more receptor-positive muscle nuclei than females. We also stained the dilator muscle for the presence of succinate dehydrogenase. Density of staining for the enzyme was significantly greater in male muscle than in female muscle, indicating greater oxidative capacity of muscle in males. This procedure also showed both a significantly greater cross-sectional area for the dilator muscle in males and a greater area for individual fibers. Male muscle consisted almost entirely of fast-twitch oxidative/glycolytic fibers. Female muscle contained a mixture of fast-twitch glycolytic fibers and two subclasses of fast-twitch oxidative/glycolytic fibers. Finally, both the length and width of the entire laryngeal complex and the length and width of the dilator were significantly greater in males than in females. In summary, laryngeal muscle of bullfrogs possessed androgen receptors and is thus likely to be androgen sensitive. Androgens, during development or at adulthood, may be responsible for the anatomic and enzymatic sexual dimorphisms in the larynx.

Effects of bombesin on behavioral thermoregulation in the bullfrog.

Bombesin is a member of a class of neuroactive chemicals that have potent thermoregulatory effects in ectothermic and endothermic vertebrate species. Bombesin-like peptides are found in the brains of ectothermic and endothermic vertebrates and have been implicated in the central nervous system modulation of behavioral thermoregulation. Amphibians rely on behavioral thermoregulation to maintain their body temperature within developmental stage-dependent critical limits. To investigate the influence of bombesin on behavioral thermoregulation, we examined the effects of central injections of bombesin on thermal habitat selection at different stages of bullfrog development. Tadpoles and adult male and female frogs were allowed to select a preferred temperature, within an aquatic thermal gradient, before and after receiving an intracerebroventricular injection of bombesin. In larval and adult female bullfrogs, bombesin administration caused a decrease in preferred temperature values. This effect was clearly dose-dependent in tadpoles. Bombesin effects were variable in adult males, probably due to an overriding stress response to handling exhibited by males. The bombesin-induced hypothermia was blocked by [D-Phe6, Des-Met14]-bombesin (6-14), ethyl amide, a bombesin/gastrin-releasing peptide receptor antagonist. These data suggest that bombesin/gastrin-releasing peptide receptors are functional in the central nervous system of larval and adult amphibians and that receptor binding can modulate thermoregulation. They raise the question: under what natural conditions is endogenous bombesin/gastrin-releasing peptide released in the brain to activate thermoregulatory behavior?

Large-Scale Preparation of a Ribonuclease fromRana catesbeiana(Bullfrog) Oocytes and Characterization of Its Specific Cytotoxic Activity against Tumor Cells

Rana catesbeianaribonuclease (RC-RNase) is a pyrimidine–guanine sequence-specific ribonuclease found only inR. catesbeiana(bullfrog) oocytes, but not in other organs. The protein is localized in the yolk granules of oocytes but not in other organelles, as detected by immunohistochemistry. More than 99% of RC-RNase was found in the yolk granule pellet when a mild separation method was employed under physiological conditions. The ribonuclease was purified by precipitation of yolk granules, extraction of RC-RNase with 0.09MNaCl, selective removal of impurities by Hepes buffer, and chromatographies on phosphocellulose and carboxymethyl cellulose columns. Three milligrams of RC-RNase was purified from a 1-g pellet of yolk granules prepared from 2 g of ovary tissue. Therefore, 150 milligrams of RC-RNase could be obtained from a mature female bullfrog (600 g in weight) which had 100 g of ovary tissue. The properties of RC-RNase isolated from yolk granules tested so far are identical to those of RC-RNase isolated from the cytosolic fraction and similar to those of a sialic acid-binding lectin from bullfrog oocytes. To investigate the possible role of RC-RNase in the regulation of cell growth and differentiation during embryogenesis, its cytotoxic activity against various cell lines was examined. The degradation of ribosomal RNA was found in RC-RNase-treated HeLa cells. However, both events were not found in RNase A-treated HeLa cells. Therefore, RC-RNase is proposed to have both ribonucleolytic and cytotoxic activity and a specific receptor on the tumor cell surface is suspected to be involved in the recognition and binding, and possibly entry of RC-RNase.

Arginine Vasotocin Facilitation of Advertisement Calling and Call Phonotaxis in Bullfrogs

Although the neuropeptide arginine vasotocin (AVT) is found in several auditory and vocalization regions of the bullfrog ( Rana catesbeiana) brain, its functions in these areas are unknown. We examined the effects of AVT injection on two auditory-evoked reproductive behaviors: advertisement calling in male bullfrogs and call phonotaxis in female bullfrogs. AVT (5011 μg; intraperitoneal) significantly increased advertisement call frequency and decreased calling latency in male bullfrogs, compared to saline injection. This dose of AVT also significantly decreased the time required for female bullfrogs to reach a call source as well as the latency of females to leave the starling position during call playback. In both males and females, these effects were significant at 0.5 hr and persisted until at least 2 hr after injection. AVT thus facilitated display of two sexually-dimorphic reproductive behaviors in bullfrogs. These effects may be due to direct effects of AVT on auditory processing regions in the bullfrog brain.

Sexual differences in the tympanic frequency responses of the American bullfrog (Rana catesbeiana)

Adult male and female American bullfrogs (Rana catesbeiana) have sexually dimorphic middle ears. The most conspicuous feature is the relatively large tympanum of adult males. The acoustic responsiveness of the tympana of adult male and female bullfrogs was studied with a laser vibrometer. Clear sex-related differences in middle ear frequency responses were observed between the sexes. The middle ear of adult males was generally more sensitive, but, more significantly, it showed a distinct peak in sensitivity at very low frequencies around 200 Hz that was entirely lacking in the female middle ear. The male middle ear therefore is especially responsive to the distinct low-frequency peak in the male mating call centered around 200–300 Hz. The enlarged middle ear of male bullfrogs therefore may have evolved as a result of selective pressure to improve detection of the mating vocalizations of other males. Female bullfrogs lack tympanic sensitivity to the low-frequency peak in the male mating call, and presumably respond more to the higher frequency components of call.

Gonadal steroid modulation of vasotocin concentrations in the bullfrog brain.

Concentrations of the neuropeptide arginine vasotocin (AVT) are sexually dimorphic in some regions of the bullfrog brain. Since these differences in AVT content may be due to sexual differences in plasma steroid concentrations, we performed a gonadectomy-steroid replacement experiment (30-day treatment) to assess the effects of specific steroids on AVT concentrations (determined by RIA) in adult male and female bullfrogs (Rana catesbeiana). These treatments had significant effects in six brain areas. Gonad removal decreased AVT concentrations in the amygdala pars lateralis, septal nucleus and habenula of both sexes. Gonadectomy decreased AVT content in optic tectum, torus semicircularis and pretrigeminal nucleus of males only. Dihydrotestosterone treatment resulted in AVT concentrations at or above sham levels in all six areas, whether or not gonadectomy decreased content. Estradiol fully restored AVT concentrations only in the septal nucleus and habenula (of both sexes) and in the amygdala of females. Estradiol partially restored AVT levels in the amygdala of males. These results indicate that the gonads maintain AVT concentrations in several brain areas and that steroids can modulate levels of AVT in bullfrog brain. Effects of each steroid vary depending upon the region and sex of the frog. AVT cells are present in three of these regions--the amygdala pars lateralis, septal nucleus and pretrigeminal nucleus. Steroids may be directly affecting AVT synthesis in these cells and these populations may be the source of AVT fibers in the other steroid-sensitive areas.

Influence of Androgen on the GnRH-Stimulated Secretion and Biosynthesis of Gonadotropins in the Pituitary of Juvenile Female Bullfrogs, Rana catesbeiana

The influence of androgen on pituitary sensitivity to gonadotropin-releasing hormone (GnRH) was investigated in juvenile female bullfrogs. Newly metamorphosed bullfrogs were treated in vivo for 7 days or their pituitaries were treated in vitro for 24 hr with 5α-dihydrotestosterone (DHT). Pituitary sensitivity to GnRH was assessed by incubating glands with 100 ng/ml GnRH. The secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was quantified by separate radioimmunoassays. Gth biosynthesis was quantified using immunoprecipitation to measure the incorporation of [ 35S]methionine. Prior exposure to DHT, in vitro or in vivo, significantly elevated the GnRH-induced secretion of LH and FSH. However, DHT had a differential influence on the two gonadotropins; both the basal and the GnRH-induced secretion of LH was enhanced, whereas only the GnRH-induced secretion of FSH was elevated. DHT did not significantly alter Gth biosynthesis. Treating older frogs (5 months postmetamorphic) in vivo with DHT (for 7 days) combined with a GnRH agonist (GnRHa) for 3 days enhanced the GnRH-stimulated Gth biosynthesis compared to treatment with either DHT or GnRHa alone. Therefore, while DHT may act on the pituitary to enhance gonadotropin secretion in response to GnRH, this action does not result in a concomitant increase in Gth biosynthesis. Juvenile female bullfrogs may require an increase in both GnRH and DHT in order to stimulate Gth biosynthesis.

Sexual differences in hormonal control of release calls in bullfrogs

Release calls in anuran amphibians are given when animals are inappropriately clasped by others. Since other call types, such as mate calls, are sexually dimorphic in frogs and toads, sonagram analysis was used to determine whether release call characteristics might also be sexually dimorphic in bullfrogs ( Rana catesbeiana). Only intercall intervals differed significantly between males and females. Call duration, dominant frequency characteristics, and the display of secondary or tertiary harmonic frequencies were similar in both sexes. In the spring, but not the fall, calling rates were significantly lower in female bullfrogs, compared to males. Females also had significantly lower plasma androgen concentrations and higher plasma estrogen in the spring, compared to males. In both sexes, plasma androgen and estrogen were significantly higher in the spring, compared to the fall. The neuropeptide arginine vasotocin significantly decreased release call rates in females in the spring while it significantly increased rates in males. Vasotocin had no significant effect in the fall. Prostaglandin E 2 significantly inhibited release calling in both males and females. On the other hand, prolactin significantly inhibited calling in female bullfrogs, but had no affect in males. Thus, although acoustic characteristics of release calls were similar in male and female bullfrogs, hormonal control of call rates was sexually dimorphic and seasonally variable.

Effect of vasotocin on locomotor activity in bullfrogs varies with developmental stage and sex

Although neurohypophysial peptides are present in many regions of the developing and adult bullfrog ( Rana catesbeiana) brain, the function of these peptides remains unclear. To investigate possible behavioral actions, we examined locomotor activity following peptide injection in bullfrogs at various developmental stages. An intraperitoneal (ip) injection of arginine vasotocin (AVT) in tadpoles (stages V, X, or XVII) produced an immediate and dose-dependent inhibition of locomotor activity. On the other hand, AVT stimulated activity when administered ip to juvenile or adult female bullfrogs, but did not influence activity in juvenile or adult males. The minimum effective dose of AVT, when injected directly into the brain of tadpoles, was 100-fold less than that observed when injected ip, suggesting a central nervous system site of action for this peptide. A vasopressin receptor antagonist (d(CH 2) 5[Tyr(Me) 2]AVP administered ip or icv) significantly increased locomotor activity in tadpoles, compared to controls. Oxytocin, vasopressin, and AVP 4–9 inhibited activity in tadpoles while mesotocin, des Gly(NH 2)AVP, and pressinoic acid had no significant effect. Injection of PGF 2α also significantly decreased activity levels in tadpoles. However, pretreatment of tadpoles with indomethacin, a prostaglandin synthesis inhibitor, did not prevent the behavioral effects of AVT, suggesting that prostaglandin synthesis is not required for this response. In summary, AVT influenced locomotor activity in bullfrog tadpoles and female frogs. This effect shifted during development from an inhibitory action in tadpoles to a stimulatory effect in metamorphosed female frogs. The effect of AVT on juvenile and adult frog locomotion was sexually dimorphic, as this peptide altered female behavior but not male behavior.

Improved method for purification of Xenopus laevis vitellogenin and demonstration of cross-reactivity among wide-range species by radioimmunoassay.

This study was performed to improve the purification of Xenopus vitellogenin and establish the radioimmunoassay. The procedure of purification consisted of ammonium precipitation, DEAE-Sephadex chromatography and Sephadex G-200 gel chromatography. Using this procedure, 934 mg vitellogenin was purified from 49 ml of estradiol treated female Xenopus plasma (about 19 mg/ml). Vitellogenins purified from male and female plasma after a single injection of estradiol showed good correspondence in electrophoretic patterns and amino acid compositions, indicating that vitellogenin synthesis in the male occurs in four different genes as in the female. The radioimmunoassay for vitellogenin was established using an antibody in the plasma obtained from rabbits injected with purified Xenopus female vitellogenin. The titer was 20,000 times dilution of the plasma, and the minimum detectable amount of vitellogenin was 0.1 microgram. The cross-reactivity of this antibody with newt vitellogenin was about 65% and that of chick 6%. The cross-reaction was also observed in female bullfrog plasma. Vitellogenin content was increased gradually during the first 6 days after injection of estradiol in female and the elevated level of vitellogenin dropped afterward.

The effect of multiple laparotomies, biopsies and feeding on the oocytes of Rana catesbeiana

The ovaries of adult female bullfrogs with immature (stages I to III) and mature (stages IV and V) oocytes were not affected by repeated removal of ovarian samples over a period of 25 days. The surgical procedure proved to be an effective way to ascertain and follow ovarian development.