Seasonal changes in plasma steroid levels in relation to ovarian steroidogenic ultrastructural features and progesterone receptors in the house gecko, Hemidactylus flaviviridis, in Oman.

A comprehensive study of the annual ovarian cycle in the house gecko, Hemidactylus flaviviridis, was conducted in Oman. Circulating estradiol (E(2)), progesterone (P), and testosterone (T) were measured during the active and inactive phases of the cycle. The steroid levels were related to the steroidogenic ultrastructural features such as the abundance of smooth endoplasmic reticulum (SER), the presence of cisternal whorls, and close association of lipid droplets with swollen vesiculated mitochondria and SER. The steroids were measured using a sensitive detection technique HPLC-MS/MS. E(2) levels began to rise in January at the onset of vitellogenesis and continued to rise between February and May relative to ovulation, postovulation, gravidity and oviposition. Afterwards, E(2) remained low during the inactive phase (June-December). P levels increased significantly in March and peaked in April, which coincided with luteinization. P levels began to decline relative to luteolysis (May-June). Afterwards, it remained low throughout the inactive phase. T levels rose significantly in March-April coinciding with vitellogenesis, but decreased rapidly and significantly in May and remained low during the inactive phase. Progesterone receptors (PR), identified using immunohistochemistry, were strongly expressed during the breeding period, but were absent during the non-breeding period. The appearance of the steroidogenic ultrastructural features in the preovulatory and lutein granulosa cells was correlated with the significant rise in the three steroid levels and the PR. As the steroid levels declined, the granulosa cells underwent a general degeneration and disruption of the associated steroidogenic features.

Opportunistic pathogens relative to physicochemical factors in water storage tanks.

Household water in Oman, as well as in other countries in the region, is stored in tanks placed on house roofs that can be subjected to physicochemical factors which can promote microbial growth, including pathogens and opportunistic pathogens which pose health risks. Water samples were collected from 30 houses in a heavily populated suburb of Muscat. The tanks used were either glass reinforced plastic (GRP), polyethylene or galvanised iron (GI). Heterotrophic bacteria, coliforms, faecal coliforms and iron sulphur bacteria varied significantly in the three tanks. Yeast and mould count showed significant variations. Isolation of Aeromonas spp., fluorogenic and pathogenic Pseudomonas, Pasteurella, Salmonella, Serratia and Tatumella, and Yersinia and Legionella in biofilms varied in the three tanks. The fungi isolates in the three tanks were Penicillium, Cladosporium and Aspergillus. Nephelometric turbidity unit, threshold odour number and free chlorine varied significantly in the three tanks. True colour unit values did not show a significant difference; however, GRP tanks had algae, autotrophic and pigmented microorganisms. In addition, GI tanks had sediments and corrosion. The results of this investigation are important to evaluate the status of the present household water tanks in countries with high annual temperatures, which may affect public health.

Analysis of elemental composition of the eggshell before and after incubation in the loggerhead turtle ( Caretta caretta) in Oman.

To date, there are limited studies on loggerhead turtle (Caretta caretta) eggshell ultrastructure and its elemental composition. Eggs were collected from turtle nests immediately after oviposition and post hatching. Three eggshell layers were recognized. The outer calcareous layer consists of loose nodular units of different shapes and sizes with loose attachment between the units, resulting in numerous spaces and openings. Each unit consists of CaCO3 crystals in aragonite (99%) and calcite (1%). The middle layer has several strata with numerous openings connecting the calcareous and the inner shell membrane. Crystallites of the middle layer are a mix of amorphous material with aragonite (62%) and calcite (38%). The inner shell membrane has numerous reticular fibers mixed predominantly with halite (NaCl) and small amounts of sylvite. Thermogravimetry analysis of the calcareous showed a low exothermic peak at 425°C, which corresponds to a transitional phase from aragonite to calcite. A high endothermic peak at 814°C corresponds to decomposition of calcite CaCO3 to CaO and CO2. Electron diffraction confirmed the presence of NaCl halite crystal. A significant difference was found in the percentage of elements and crystal configurations in the three layers. This study has value in assessing the emergence success in this endangered species.

Viability of multiple antibiotic resistant bacteria in distribution lines of treated sewage effluent used for irrigation.

Viability of multiple antibiotic resistant bacteria (MARB) in tertiary treated sewage effluent (TTSE) used for irrigation, was investigated at the Sultan Qaboos University sewage treatment plant (STP). This water recycle system is used here as a model for the systems commonly used throughout Oman and the Gulf region. Samples of TTSE were collected weekly from four sites, 1.5 km from each other. Chlorine levels declined gradually at the three sites with increasing distance from the STP. Viable bacteria, coliforms and nitrate concentrations increased significantly while biological oxygen demand (BOD) declined after STP chlorination. Mean values of turbidity changed slightly. Trace elements values were insignificant. A total of 336 bacteria from 8 genera revealed that the dominant isolates were Enterobacter spp., Pseudomonas spp., and Aeromonas spp. Among the isolates 59.8% were multiply resistant to several antibiotics. Resistance was higher to ampicillin followed by sulphamethoxazole, carbenicillin, streptomycine and minocycline. Frequency of resistance to the 14 antibiotics varied among the isolates. The present system related to the viability of MARB in TTSE used for irrigation may have serious implications for public health and wildlife. Results of this investigation will be of value in modifying current STPs systems and thus avoiding serious health issues.

Plasma hormone levels in the green turtles Chelonia mydas during peak period of nesting at Ras Al-Hadd-Oman.

Circulating estradiol (E(2)), progesterone (Pro), testosterone, and corticosterone (B) levels were monitored in the green turtles Chelonia mydas during different nesting phases. Successful nesting includes emergence from sea, chamber and nest excavation, oviposition, burying the nest, and returning to sea. Unsuccessful nesting includes chamber and nest excavations but without oviposition. Blood samples were taken from the cervical sinus and collected within 5-min of capture to minimize stress. The samples were collected between 2000 and 0100 h during the peak season (May-October). High-performance liquid chromatography using a u.v. detection system coupled with tandem quadrupole mass spectrometry was used to measure B. Plasma B levels were significantly higher in successful and unsuccessful phases over emergence and excavation phases. However, B levels in successful versus unsuccessful or emergence versus excavation phases were not significantly different. Plasma steroid levels were measured by the Coat-A-Count RIA technique. Pro levels were significantly higher (P<0.005) in successful over unsuccessful turtles and also successful turtles over turtles in the other phases (P<0.01). The Pro levels immediately after nesting were found to be higher than that reported previously. Plasma testosterone values were higher in successful turtles but not significantly different from the turtles in other phases. Estrogen levels were undetected in all phases. Overall, the hormone values during different phases of nesting may play a major role in formulating the nesting behavior and physiology of the nesting activities in the green turtle.

Partial development of the steroidogenic ultrastructural features in degenerative corpora lutea after a single injection of pituitary extract in the Western painted turtle (Chrysemys picta).

Pituitary glands were removed from sexually mature female turtles (Chrysemys picta) and they were injected intraperitoneally (i.p.) into other mature females of the same species (experimental). In addition mature females of the same species received saline injection only (controls). Initially all the turtles used in this study were steroidogenically inactive with corpora lutea already undergoing luteolysis (degeneration) as these turtles had ovioposited their eggs approximately 2 weeks earlier. Forty-eight hour post injection the corpora lutea were removed from the control and experimental turtles. In the experimental turtles, the lutein granulosa cells developed ultrastructural features such as tubular and cisternal smooth endoplasmic reticulum (SER) and mitochondria with tubular cristae associated with lipid droplets. However, the controls maintained degenerative corpora lutea without steroidogenic ultrastructural features. The circulating progesterone (Pro) levels in the experimental turtles were significantly higher than the controls (P<0.049). Although the 48h development of steroidogenic ultrastructural features in the lutein granulosa cells was only partial in development, the effect of the pituitary taken from the inactive donor triggered an activating process within a short period, clear evidence of gonadotropic effect on the inactive corpora lutea. The present data offer interesting information on the short-term effect of gonadotropins during the non-reproductive period. This information may have useful implication under natural conditions particularly during the onset of a new reproductive cycle where the ovary is still inactive.

Oviductal morphology in relation to hormonal levels in the snapping turtle, Chelydra serpentina.

Microscopic and in situ visual observations were used to relate circulating hormone levels to morphological changes in the oviduct of the snapping turtle Chelydra serpentina throughout the ovarian cycle. Increase in levels of progesterone (P), estradiol (E2) and testosterone (T) levels coincide with an increase in number and growth of endometrial glands, luminal epithelial cells and secretory droplets throughout the oviduct. Testosterone and estradiol levels rose significantly (P < 0.05) after the May-June period and remained high throughout the rest of the summer. Progesterone levels remained stable throughout the summer, with a brief decline in July due to luteolysis. Hormonal values declined significantly (P < 0.001) at the end of the ovarian cycle in the fall. In situ visual observation of fresh oviducts at different stages of gravidity in recently ovulated turtles revealed that proteinaceous like components from the endometrial glands were released into the lumen to form fibers. The morphological features of the oviduct remained active throughout the summer months even though the snapping turtle is a monoclutch species which deposits all the eggs in late-May to mid-June. The high steroid levels correlate with and may be responsible for the secretory activity present throughout the summer and their decline correlates with change to low secretory activity in the fall. Calcium deposition accompanied by morphological changes in luminal cells are suggestive of secretory activity. In the egg-bearing turtles, uterine Ca2+ concentrations measured by flame atomic absorption spectrophotometry revealed significantly higher Ca2+ concentrations (P < 0.001) in eggs with soft shell than eggs without shell. There was a significant increase in calcium granules and proteinaceous fibers in luminal surface of the uterus during the period of eggshelling. This supports the fact that in the snapping turtle like in other reptiles, eggshelling process occurs in the uterus.

Seasonal fluctuations in the plasma concentrations of progesterone and oestradiol-17 beta in the female snapping turtle (Chelydra serpentina).

Plasma concentrations of progesterone and oestradiol-17 beta were determined by radioimmunoassay procedures in the sexually mature female snapping turtle at four different stages of the annual ovarian cycle. The highest level of progesterone in the peripheral plasma was detected during the luteal stage (June), whereas the concentration of oestradiol was highest (280 pg/ml) in May, immediately before ovulation.

Cytoplasmic progesterone receptors in uterine tissue of the snapping turtle (Chelydra serpentina).

A high affinity progesterone-binding component was detected in the cytosol of the uterus of the snapping turtle, Chelydra serpentina. Density gradient centrifugation indicated that binding of [3H]progesterone and [3H]promegestone (R5020) was to a fraction with a heavier sedimentation coefficient than bovine serum albumin (BSA) appearing as a broader peak in the 6-7 S region; it was not affected by excess cortisol. Another binding peak, lighter than BSA and appearing with [3H]R5020 and [3H]progesterone near the 4 S region, was affected by excess cortisol. Excess progesterone decreased both the heavier and lighter peaks. Analysis of steroid specificity revealed that, of the natural steroids, progesterone had the highest affinity for the uterine cytosol. This was followed by deoxycorticosterone, 5 alpha-pregnanedione, testosterone, oestradiol-17 beta, corticosterone, 5 alpha-dihydrotestosterone and cortisol. Non-linear regression analysis of saturation data indicated the presence of two classes of high affinity binding sites: progesterone-binding sites (R-sites) with equilibrium association constants (Ka) of 2.9 +/- 0.28 litres/nmol (mean +/- 95% confidence limit) for [3H]R5020 and 0.34 +/- 0.20 litres/nmol for [3H]progesterone, and corticosteroid-binding globulin-like sites (G-sites) with Ka of 4.5 +/- 1.6 litres/nmol for progesterone. The concentration of R-sites was between 0.66 +/- 0.10 and 2.6 +/- 0.55 pmol/mg protein while that of G-sites was between 0.73 +/- 0.05 and 5.0 +/- 0.27 pmol/mg protein. DEAE-cellulose filtration assay also confirmed the presence of R-sites and G-sites in the cytosol. R-sites were detectable without oestrogen priming during the preovulatory and vitellogenic phases (low progesterone, high oestrogen concentrations) when the ovarian follicles are mature (18-22 mm diameter).(ABSTRACT TRUNCATED AT 250 WORDS)

Biomonitoring marine habitats in reference to antibiotic resistant bacteria and ampicillin resistance determinants from oviductal fluid of the nesting green sea turtle, Chelonia mydas.

During the egg-laying process, oviductal fluid was collected using a non-invasive procedure from the cloacal vent of the green turtles. Forty-two independent isolates of antibiotic-resistant bacteria from 11 genera were obtained from 20 turtles during nesting. The dominant isolate was Citrobacter (52.4%), followed by Pseudomonas, Proteus, Enterobacter, Salmonella, Escherichia coli, Shigella, Edwardsiella, Morganella, Providencia and Arcomobacter. Most of the isolates were resistant to ampicillin. Ampicillin-resistant isolates showed variations in their resistance for the following classes of ß-lactamases: extended-spectrum ß-lactamases (EBSLs), AmpC type ß-lactamases C (AmpC), and screen-positive ß-lactamase. None of the isolates produced metallo ß-lactamase. Some ampicillin-resistant genes were detected by multiplex polymerase chain reaction (PCR) only. Inhibitor based test (IBT) categorized some isolates as AmpC ß-lactamase producers. ß-Lactamase genes were detected from a few strains. The sequencing of those genes revealed the presence of cephamycinase (CMY) and AmpC ß-lactamases. The oviductal fluid was used in this study as a source of bacterial antibiotic-resistant determinants for biomonitoring marine turtles exposed to contaminated effluents. This data can be of value in understanding the decline of this endangered species as a result of exposure to marine pollution which is threatening their survival.

Detection of low plasma estradiol concentrations in nesting green turtles (Chelonia mydas) by HPLC/Ms-Ms.

In previous studies on nesting green turtles under natural conditions from different geographical regions, 17-ß-estradiol (E(2) ) was either undetectable or detected at very low levels. RIA and other related techniques were not sensitive enough to measure low E(2) values in the green turtles. In this study, a sensitive method was used in detecting low hormone concentrations: high performance liquid chromatography with tandem quadruple mass spectrometry (HPLC-MS/MS). Using this technique, estradiol for the first time was detected in nesting green turtles during the peak season (June-October) at Ras Al-Hadd Reserve, Oman. The E(2) values recorded from this study were the highest ever recorded from nesting green turtles in any geographical region, but the levels did not vary significantly throughout different phases of nesting. The presence of E(2) during nesting presumably plays a role in the physiology and behavior of this species. Ras Al-Hadd hosts one of the largest nesting populations of green turtles in the world, and an understanding of their nesting patterns may be of value in conservation and management programs for this endangered species.

Ultrastructural features and elemental distribution in eggshell during pre and post hatching periods in the green turtle, Chelonia mydas at Ras Al-Hadd, Oman.

Eggshells were randomly collected from turtle nests immediately after oviposition and at the end of incubation to examine the ultrastructural features using scanning JSM-5600LV microscopy. Three layers were recognized; an outer calcareous, a middle multistrata and an inner membrane. The calcareous layer had loose nodular units varying in shape and size without interlocking attachments. In freshly laid eggs, each nodular unit had spicules arranged in folded stacks. The spicules became unfolded during incubation, to form radiating configurations. Elemental composition and mapping of the layers were analyzed using energy dispersive spectroscopy (EDS). The elements were unevenly distributed throughout the eggshell and Ca(2+) decreased significantly after hatching. X-ray diffraction was used to identify the crystals of the eggshells. It revealed that nodular units of the calcareous were made up of CaCO(3), as aragonite (91%), calcite (6%) and vaterite (3%). The middle layer consisted of organic amorphous material with aragonite (89%) and calcite (11%). The shell membrane consisted of reticular fibers with crystals predominantly of NaCl halite. Thermogravimetry analysis of the calcareous layer indicated a complete evaporation of bonded H(2)O at 480 degrees C and CO(2) at 830 degrees C. Using the differential thermal analysis (DTA), aragonite was transformed to stable calcite at 425 degrees C.

Coastal sewage discharge and its impact on fish with reference to antibiotic resistant enteric bacteria and enteric pathogens as bio-indicators of pollution.

Presence of antibiotic resistant bacteria as bio-indicators of pollution was monitored in fish (colons and gills) and in sewage treated effluent (STE) in two marine sites in the Gulf of Oman, Muscat. One site was polluted as the sewage effluent STE was directly discharged into the sea while the unpolluted site was 10 km away. A total of 42 water samples (250 mL each) were collected from both sites. In the polluted site, 14 samples were taken directly from the STE discharge point and 14 from the surrounding area near the STE discharging point (7-10 m). Water and fish samples were collected from polluted and unpolluted sites. Chlorine and nitrate levels were significantly higher in STE samples. A significant difference (P<0.005) was found in total bacterial count of fish colon and gills in the two sites. Most probable number (MPN) was significantly higher in STE and polluted seawater over unpolluted. Coliforms were detected in the polluted site only. A total of 320 target enteric normal flora of animals and pathogenic species were isolated from 10 genera taken from water and fish samples. The isolates were exposed to 14 antibiotics. Most of the isolates were resistant to at least one antibiotic with ampicillin was the dominant antibiotic. This is a clear indication that sewage effluent causes contamination of marine wildlife along coastal lines.

Changes in plasma levels of adrenaline, noradrenaline, glucose, lactate and CO2 in the green turtle, Chelonia mydas, during peak period of nesting.

Plasma concentrations of stress hormones [adrenaline (ADR), noradrenaline (NR)], lactate, glucose and CO2 were monitored during peak nesting period (May-October) at different phases of nesting in the green turtle, Chelonia mydas. These include, emergence from sea, excavating body and nest chambers, oviposition, covering and camouflaging the nest and then returning to sea. Turtles that completed all phases of nesting including oviposition before returning to sea were considered "successful" turtles, while those that completed all phases but failed to lay their eggs were "unsuccessful". Blood samples were taken from the cervical sinus within 5min of capture to avoid stress due to handling. The turtles were usually sampled for blood between 20:00 and 1:00h of nesting time to ensure uniformity in the sampling. Plasma ADR and NR values were highly significant (P<0.001) in successful turtles over emergence, excavating and unsuccessful turtles. Plasma glucose levels remained stable throughout the nesting phases while lactate levels were significantly higher in successful turtles over the other phases (P<0.05) which signifies anaerobic metabolism during nesting. Plasma CO2 values were negatively correlated with ADR and NR (r=-0.258, P=0.03; r=-0.304, P=0.010), respectively. Hematocrit was significantly higher in successful phase (P<0.05) compared to other phases, and this may signify a higher degree of stress in successful turtles. Body temperature were significantly lower (P<0.005) in the excavating phase compared to the other three phases. Overall, body temperatures were lower than sand temperatures around the nest, which may indicate a behavioral thermoregulation used by the turtles during nesting. This information will be of value to the ongoing conservation program at Ras Al-Hadd Reserve in the Sultanate of Oman.

Seasonal changes in gonadal activity and the effects of stress on reproductive hormones in the common snapping turtle, Chelydra serpentina.

The seasonal gonadal cycle (including gonadal histology, sex steroids, and gonadotropins) was studied in freshly captured common snapping turtles, Chelydra serpentina, from Wisconsin, and the effects of capture stress were evaluated. The ovarian and testicular cycles are shorter than those reported in other freshwater turtles; the cycles commence in mid-May and terminate in early September, immediately after the completion of gonadal growth and maturation. In the female, testosterone (T), 17beta-estradiol (E2), and progesterone (Pro) were highly correlated with follicular growth and vitellogenesis. Ovulation in captivity and under natural conditions occurred after mid-May. In captivity, ovulation was a rapid process (24-48 hr); as the follicles descended into the uterine horns there was a significant increase in E2 and Pro and eggs were retained in the uterine horns for about 2 weeks before oviposition. In the male, T was significantly correlated with testicular growth and spermiation. Follicle-stimulating hormone (FSH) reached significantly higher levels (P < 0.01) in males (8.99 +/- 0.38 ng/ml) than in females (2.66 +/- 0.22 ng/ml), but luteinizing hormone was undetectable in both sexes. FSH was not correlated with the steroids in either sex. Sex steroids and FSH began to rise before spermiation and vitellogenesis and remained elevated until completion of gonadal growth and maturation. Leydig cells, the main source of plasma androgen in this species, became active shortly after emergence from hibernation and remained steroidogenically active for the rest of the cycle. Sertoli cells became active only after spermatogenesis was under way but also stayed active for the rest of the summer. Courtship and mating behaviors were observed in spring, summer, and fall. The snapping turtle is strictly aquatic with no basking behavior and limited behavioral thermoregulation so there is little daily fluctuation in body temperature. Environmental correlates indicate that the snapping turtle is temperature dependent: recrudesence occurs with a slight increase in water temperature during spring and early summer, while a dramatic drop in gonadal activity accompanies a slight decrease in temperature in fall. Changes in temperature may underlie changes in gonadal activity in the face of relatively stable FSH. Male turtles subjected to captivity and periodic blood sampling show a significant decline in T. The hormonal levels continued to decline whether the turtles are exposed to optimum or extreme temperatures. However, there is more rapid decline in T values in animals with regressed testes (June) than in those with well-developed testes (July). Male and female turtles kept in captivity at different phases of the cycle exhibit different patterns and degrees of response to stress, possibly related to the hormonal levels and the condition of the gonads.

The role of arginine vasotocin and prostaglandin F2 alpha on oviposition and luteolysis in the common snapping turtle Chelydra serpentina.

The administration of arginine vasotocin (AVT) to gravid snapping turtles with steroidogenically active corpora lutea and high plasma progesterone concentration (1480 +/- 155 pg/ml) did not trigger oviposition, whereas 12 days after ovulation when luteolysis occurred and plasma progesterone concentration was low (570 +/- 78 pg/ml), treatment with AVT caused oviposition. Controls with high plasma progesterone concentration (1605 +/- 185 pg/ml) oviposited 15-23 days after ovulation when plasma progesterone concentration dropped to 201 +/- 35 pg/ml. Deluteinization-induced oviposition was initiated 15 hr after surgery and was completed by 30 hr. Oviposition of complete clutches occurred and was correlated with a significant drop in plasma progesterone. Sham-operated turtles did not exhibit oviposition and no significant change in progesterone concentration was observed. A single injection of prostaglandin F2 alpha (PGF) in recently ovulated turtles induced early luteolysis and a significant decrease in plasma progesterone concentration after 24-30 hr. A single administration of PGF caused the disappearance of steroidogenic features such as the smooth endoplasmic reticulum and mitochondria with tubular cristae 48 hr later. Also PGF triggered the invasion of a hyaline-like material from the luteal theca into the luteal cell mass which eventually induced luteolysis. The role of AVT, PGF, and progesterone in relation to egg retention and oviposition is discussed.

Development of the ovarian follicles in relation to changes in plasma parameters and 5 delta 3 beta HSD in snapping turtle, Chelydra serpentina.

Follicular development in snapping turtle, Chelydra serpentina, was studied in relation to changes in plasma levels of estradiol-17 beta, total protein, calcium, inorganic phosphate and cholesterol. Histochemical reaction for 3 beta-HSD in granulosa and theca cells of the follicles is strong during the preovulatory and vitellogenic phases when the follicles attain their maximum size (18-22 mm) and estradiol is at a high level. The reaction for 3 beta-HSD is weal during the postovulatory phase when the developing follicles are at their minimum size (5-7 mm) and estradiol is low. Administration of estradiol-17 beta causes a rise in plasma calcium, total protein, inorganic phosphate and cholesterol.

Secretory proteins in the reproductive tract of the snapping turtle, Chelhydra serpentina.

SDS-polyacrylamide gel electrophoresis was used to separate the secretory proteins produced by the epithelial and endometrial glands of the uterine tube and uterus in the snapping turtle Chelydra serpentina. The proteins were analyzed throughout the phases of the reproductive cycle from May to August, including preovulatory, ovulatory, postovulatory or luteal, and vitellogenic phases. The pattern of secretory proteins is quite uniform along the length of the uterine tube, and the same is true of the uterus, but the patterns for uterine tube and uterus are clearly different. We identify 13 major proteins in C. serpentina egg albumen. Bands co-migrating with 11 of these are found in the uterine tube, but at most 4 are found in the uterus, suggesting that the majority of the albumen proteins are most likely secreted in the uterine tube, not in the uterus. Although some of the egg albumen proteins are present in the uterine tube only at the time of ovulation, most of the bands corresponding to albumen proteins are present throughout the breeding season even though the snapping turtle is a monoclutch species. These results suggest that the glandular secretory phase in the uterine tube is active and quite homogeneous in function regardless of location or phase of the reproductive cycle.