Evidence of Egg Diversity in Squamate Evolution from Cretaceous Anguimorph Embryos.

Lizards are remarkable amongst amniotes, for they display a unique mosaic of reproduction modes ranging from egg-laying to live-bearing. Within this patchwork, geckoes are believed to represent the only group to ever have produced fully calcified rigid-shelled eggs, contrasting with the ubiquitous parchment shelled-eggs observed in other lineages. However, this hypothesis relies only on observations of modern taxa and fossilised gecko-like eggshells which have never been found in association with any embryonic or parental remains. We report here the first attested fossil eggs of lizards from the Early Cretaceous of Thailand, combining hard eggshells with exquisitely preserved embryos of anguimoph (e.g. Komodo dragons, mosasaurs). These fossils shed light on an apparently rare reproduction strategy of squamates, demonstrate that the evolution of rigid-shelled eggs are not an exclusive specialization of geckoes, and suggest a high plasticity in the reproductive organs mineralizing eggshells.

Ovum transmigration after salpingectomy for ectopic pregnancy.

STUDY QUESTION: What proportion of pregnancies are a result of ovum transmigration after salpingectomy for ectopic pregnancy? SUMMARY ANSWER: Approximately one-third of spontaneously conceived pregnancies are a result of pick-up of the ovum from the ovary contralateral to the remaining tube in women with a history of salpingectomy. WHAT IS KNOWN ALREADY: The corpus luteum has been found contralateral to tubal ectopic pregnancies in 32% of reported cases. The rate of contralateral ovum pick-up in intrauterine pregnancies is not known. STUDY DESIGN, SIZE, DURATION: We conducted a retrospective cohort study of clinical and ultrasound records collected over a 12-year period 1999-2010. Ten per cent of cases identified were excluded from the final analysis due to incomplete data or bilateral corpora lutea. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Included were 842 pregnancies in 707 women with a history of unilateral salpingectomy for ectopic pregnancy and subsequent spontaneous pregnancy. The study was set in the Early Pregnancy Unit of a large UK inner city teaching hospital. The outcome measure was the side of the corpus luteum in relation to the side of the remaining tube. MAIN RESULTS AND THE ROLE OF CHANCE: The corpus luteum was located in the ovary contralateral to the remaining tube in 266/842 pregnancies (31.6%; 95% CI 28.5-34.8%). There was no significant difference in this proportion between intrauterine and ectopic pregnancies [246/769 (32.0%) versus 21/73 (28.8%), P = 0.60]. LIMITATIONS, REASONS FOR CAUTION: This was a retrospective study and so did not address the conception rate according to the laterality of ovulation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings were very similar to the frequency of ectopic pregnancies found contralateral to the corpus luteum described in previous studies. Ovum pick-up from the cul-de-sac probably occurs reasonably frequently and is unlikely to have a causative role in the pathogenesis of ectopic pregnancy. It is not known how often this phenomenon occurs in women with intact Fallopian tubes. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was obtained. The authors have no conflicts of interest to declare.

Insights into bone metabolism of avian embryos in ovo via 3D and 4D 18F-fluoride positron emission tomography.

PURPOSE: The chick embryo is a well-known economical in vivo model system and is widely applied in preclinical research, e.g., bone development studies. It is therefore surprising that no studies concerning the application of (18)F-fluoride microPET to bone metabolism have been reported so far. This may be due to motion artifacts or the lack of convenient tracer injection sites. METHODS: We resolved the above problems using a combination of sample preparation, anesthesia, microPET imaging, and computational processing, and describe a convenient way of visualizing three- and four- dimensional features of bone metabolism in living chick embryos. RESULTS: The application of (18)F-fluoride microPET facilitates repeat measurements, highly reproducible and motion-artifact-free skeletal imaging, and provides quantitative measurements of in ovo metabolic activities in the bones of developing chick. During microPET measurement, radio tracer was injected intravascularly using a custom-made catheter system, allowing us to additionally investigate early time points in tracer kinetics and uptake. CONCLUSIONS: Our results show that bone metabolism in living chick embryos can be reproducibly studied and quantified in ovo, even for multiple tracer injections over a longer time period. The use of dynamic (18)F-fluoride microPET imaging made it possible to visualize and analyze even small bone structures with excellent quality. Moreover, as our data are comparable to data from corresponding rodent experiments, the use of embryonated chicken eggs is a convenient and economical alternative to other animal models.

Ovum pick-up and in vitro embryo production (OPU-IVEP) in Mediterranean Italian buffalo performed in different seasons.

This study was designed to evaluate the effect of season on in vivo oocyte recovery and embryo production in Mediterranean Italian buffalo (Bubalus bubalis). For this purpose repeated transvaginal ultrasound-guided ovum pick up (OPU) was conducted twice a week throughout autumn, mid-winter (transitional period) and spring-summer. The number and size of follicles was determined before puncture. The recovered oocytes were first classified in morphological categories and then used for in vitro embryo production (IVEP) according to standard procedures. The mean number of total follicles observed per session did not differ among the three periods we examined (on average 4.6). Although season did not considerably affect the number of oocytes recovered (on average 2.3/buffalo/session), the number of degenerated and abnormally expanded oocytes increased during autumn. Furthermore, the percentage of abnormally expanded oocytes significantly increased during autumn (6.1%) compared with both the transitional period and spring-summer (1.9 and 2.3%, respectively). Interestingly, the embryo output we recorded at day 7, in terms of tight morulae-blastocysts was higher in autumn (30.9%) compared to the other two periods (13.3% and 10.3%, respectively, in spring-summer and in the transitional period; P<0.01). The results of this trial demonstrated that the morphological features of the oocytes did not vary substantially among the considered periods, with the exception of degenerated and abnormally expanded oocytes. On the other hand, the oocyte developmental competence improved in autumn compared to spring-summer and the transitional period. This datum reflects buffalo reproductive pattern expressed in vivo at Italian latitudes.

Ultrasonography of reproductive structures and hormonal correlates of follicular development in female American alligators, Alligator mississippiensis, in southwest Louisiana.

Ultrasonography has been used effectively to study reproduction in a variety of reptile species, but its application to crocodilians has been relatively limited. We present results from a study testing the efficacy of using ultrasonography to monitor reproduction in the American alligator, Alligator mississippiensis. Ultrasound results were then compared with plasma hormone levels. A total of 124 females were examined during March, April, May, and early June (2001-2003). Ultrasound results were validated on a series of reproductive females (n=14) necropsied for other studies. Previtellogenic follicles, vitellogenic follicles, recently shelled eggs, fully developed well-calcified eggs, and atretic follicles were readily discernible with ultrasound in mature females. Reproductive structures were observed in 57 females of which 43 were actively reproductive, while 14 were non-reproductive, but contained large atretic follicles from prior years. Oviducts were discernible in females with eggs. Ovarian state was also correlated with hormone levels. These results are in agreement with previous studies that showed that 50% or less of the adult female alligator population is reproductively active in a given year. Ultrasonography can be used to make an accurate assessment of reproductive condition in wild alligator populations.

The relation between ultrasonographic observations in the oviduct and plasma progesterone, luteinizing hormone and estradiol during the egg laying cycle in ostriches.

In this study we investigated the temporal relationship between ovulation, egg formation, oviposition and the changes in plasma concentrations of progesterone, luteinizing hormone and estradiol-17beta during the egg laying cycle in farmed ostriches. In 10 egg-producing birds, transcutaneous ultrasound scanning was performed at 3h intervals and blood sampling at hourly intervals during a period of at least 48h (one egg laying cycle). In hens (n=8) that ovulated during the observational period, the ovulated egg was first detected 2h after oviposition; thus, ovulation occurred shortly after oviposition in all birds. During the period between two consecutive ovipositions, the developing egg remained for 9h in the proximal part (infundibulum, magnum or isthmus) and for 39h in the distal part of the oviduct (uterus). In ovulating hens, plasma progesterone concentrations showed a characteristic and consistent profile: from basal levels of around 0.1ng/ml concentrations started to increase 12h before oviposition, reached an average maximum of 3.5ng/ml at 3h before oviposition and returned to basal levels 3h and 30min after oviposition. Changes in plasma luteinizing hormone and estradiol-17beta concentrations showed comparable patterns of elevation and decline relative to the timing of oviposition and ovulation. However, variation in their individual basal concentrations was generally larger and peak values were less conspicuous than those of progesterone. In non-ovulating hens (n=2) neither progesterone, nor luteinizing hormone nor estradiol-17beta showed elevations to peak concentrations before oviposition. These data demonstrate that during the egg laying cycle of ostriches, events such as ovulation, egg development and oviposition evolve according to a rather strict time schedule, and that progesterone, luteinizing hormone and estradiol-17beta reach peak concentrations shortly before ovulation. Additionally, our findings also show that on-farm ultrasound scanning is a useful technique to discriminate between ovulating and non-ovulating hens.

Inactivation of single-celled Ascaris suum eggs by low-pressure UV radiation.

Intact and decorticated single-celled Ascaris suum eggs were exposed to UV radiation from low-pressure, germicidal lamps at fluences (doses) ranging from 0 to 8,000 J/m2 for intact eggs and from 0 to 500 J/m2 for decorticated eggs. With a UV fluence of 500 J/m2, 0.44-+/-0.20-log inactivation (mean+/-95% confidence interval) (63.7%) of intact eggs was observed, while a fluence of 4,000 J/m2 resulted in 2.23-+/-0.49-log inactivation (99.4%). (The maximum quantifiable inactivation was 2.5 log units.) Thus, according to the methods used here, Ascaris eggs are the most UV-resistant water-related pathogen identified to date. For the range of fluences recommended for disinfecting drinking water and wastewater (200 to 2,000 J/m2), from 0- to 1.5-log inactivation can be expected, although at typical fluences (less than 1,000 J/m2), the inactivation may be less than 1 log. When the eggs were decorticated (the outer egg shell layers were removed with sodium hypochlorite, leaving only the lipoprotein ascaroside layer) before exposure to UV, 1.80-+/-0.32-log reduction (98.4%) was achieved with a fluence of 500 J/m2, suggesting that the outer eggshell layers protected A. suum eggs from inactivation by UV radiation. This protection may have been due to UV absorption by proteins in the outer layers of the 3- to 4-microm-thick eggshell. Stirring alone (without UV exposure) also inactivated some of the Ascaris eggs (approximately 20% after 75 min), which complicated determination of the inactivation caused by UV radiation alone.

True hermaphroditism-the importance of ultrasonic assessment.

Intersex states are associated with rare syndromes within a wide clinical spectrum, true hermaphroditism being the rarest of them all. It is a condition that is characterized by ovotestis, i.e. the presence of ovarian and testicular tissues in the same individual, either in separate gonads or in the same gonad. Diagnostic investigation of hermaphroditism involves ultrasound examination of the gonads, karyotype analysis and histological studies. We report the case of a 21-year-old individual with true hermaphroditism, in whom ovotestis was diagnosed on ultrasonography and which was later confirmed by anatomopathological study of the gonads. The role of ultrasound in the initial diagnostic investigation of intersex states, especially true hermaphroditism, is discussed.

Crystalline calcium phosphate and magnetic mineral content of Daphnia resting eggs.

Daphnia is a key crustacean zooplankton of freshwater food chains. One factor that ensures successful propagation is the Daphnia resting eggs, which are able to retain structural integrity under extreme conditions. Until recently little was known about the chemical composition, microanatomy, and physical properties of the egg itself. The current study demonstrates that the resting eggs: (1) have shells that are made up of crystalline calcium phosphate and include a honeycombed structure, and (2) contain magnetic material having properties consistent with magnetite. These properties of the resting eggs may ensure Daphnia survival in harsh environments.

The effects of twisting and type of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up in cattle.

The effects of twisting and type (single- or double-lumen) of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up (US-guided OPU) were investigated in cattle. The first study using slaughterhouse ovaries revealed that twisting of the needle during follicle aspiration improved the oocyte recovery rate without deleterious effects on the attachment of cumulus layers. Vacuum pressure affected the oocyte recovery and cumulus attachment, regardless of the needle type. The needle type did not affect the oocyte recovery or cumulus attachment with an optimized vacuum pressure. In the second study, US-guided OPU was performed in live cows using two types of needles with a vacuum pressure of 75 mmHg. The needle type did not affect the oocyte recovery or cumulus attachment of the recovered oocytes. The results revealed that twisting of the needle is effective in follicle aspiration, and suggested that a single-lumen needle is as useful as a double-lumen needle for US-guided OPU in cattle.

Ultrasonography of the female reproductive organs in farmed ostriches (Struthio camelus spp.).

The main objective of this study was to determine whether transcutaneous ultrasonography could be used as a diagnostic tool to monitor the reproductive organs of female breeding ostriches. An additional aim was to investigate whether the use of this technique could facilitate prediction of the start or end of the egg production season. A technique for on-farm ultrasound scanning is described and examples of ultrasonographic images of different ovarian structures, developing ova and eggs within the genital tract are presented. These data were obtained by scanning mature female breeding ostriches (n=8). In vivo scanning took place a day prior to slaughter, and immediately after slaughter the reproductive organs were scanned in vitro in a water bath. By comparing ultrasonographic images with post mortem ovarian morphology, it appeared that the following morphological structures can be identified using ultrasonography: ovarian follicles of different sizes (diameter (phi) 1-9 cm), atretic follicles, post-ovulatory follicles (POFs), ova at different stages of development and eggs within the genital tract. Of the number of follicles counted during post mortem investigation, 58% (95% confidence interval 0.41-0.79) had been detected during previous in vivo examination. In the second part of our study, ultrasonographic scans were made at weekly intervals in two farmed female ostriches (n=2) during the breeding season in order to determine the predictive value of the technique. By comparing the images of ovarian activity with individual egg production of these hens, preliminary evidence was obtained that scanning might be of value in predicting egg production, especially at the start and the end of the breeding season. It is concluded that transcutaneous ultrasound scanning in mature female breeding ostriches is an easy, noninvasive technique for the monitoring of ovarian (in)activity, for visualization of different functional ovarian structures, for following the development of individual ova and for visualization of an egg with calciferous shell within the oviduct, and that this technique will be a valuable tool in future research on reproductive physiology and pathology, and the development of more advanced reproductive technologies, such as artificial insemination.

Fine structure of the eggs of Anopheles (Anopheles) punctimacula.

The egg of Anopheles (Anopheles) punctimacula is described from scanning electron micrographs. Eggs of An. punctimacula are boat shaped, with lateral floats extending 70% of the length of the egg. Plastronlike polyhedral chorionic cells with distinctive boundaries and round tubercles in the cell field cover the dorsal, lateral, and ventral surfaces. Narrow decks enclosing a field of irregular jagged tubercles and 2-4 lobed tubercles are present at both egg poles.

Experimental imaging of the acoustic nonlinearity parameter B/A for biological tissues via a parametric array.

The acoustic nonlinearity parameter B/A is an important parameter in nonlinear acoustics and has been suggested to be a novel parameter for ultrasound tissue characterization. In this paper, the principle and experimental results for B/A imaging by using a parametric array are presented. As two primary waves with different frequencies are radiated simultaneously from a circular piston source, a secondary wave at the difference frequency is generated due to the nonlinear interaction of the two primary waves. The axial sound pressure amplitude curve for the generated difference frequency wave in the near field is measured and agrees well with the theoretical calculation. Unlike the second harmonic wave, the difference frequency component of the parametric array grows almost linearly with distance from the piston source and goes through fewer oscillations in the near field. It therefore provides a better source for acoustic nonlinearity parameter tomography. On the basis of the finite amplitude insert substitution method and the conventional CT technology, an experimental imaging system for acoustic nonlinearity parameter B/A via a parametric array is set up. B/A images for several biological specimens, including normal and pathological tissues, are obtained.

Computed tomographic evaluation of dinosaur egg shell integrity.

The purpose of this study was to determine whether computed tomography (CT) could be used to identify hatching holes in partially embedded dinosaur eggs. One Faveololithus and two Dendroolithus eggs were examined using a fourth generation CT scanner. The eggs were partially embedded in a fossilized sediment matrix, with the exposed portion of the shell appearing intact. In CT images of all three eggs, the shells appeared hyperdense relative to the matrix. Hatching holes were visible as large gaps in the embedded portion of the shell, with inwardly displaced shell fragments. It was concluded that CT is an effective technique for nondestructively assessing dinosaur egg shell integrity.

Comparison of ultrasound-guided vs laparoscopic transvaginal ovum pick-up (OPU) in simmental heifers.

Transvaginal ovum pick-up (OPU) offers several advantages over standard embryo transfer procedures. For a systematic comparison of the ultrasound-guided (U-OPU) and the laparoscopic OPU (L-OPU) method, groups of Simmental heifers were subjected to 1 of the 2 OPU-methods for 8 wk (15 sessions) followed by a treatment-free interval of 11 wk and then another 8-wk period of OPU using the alternative method. Parameters that were evaluated included the number of follicles aspirated, number and morphology of recovered cumulus-oocyte complexes (COCs), and developmental capacity of oocytes after in vitro maturation (IVM) and fertilization (IVF). Blood samples were also taken from the donors twice a wk for progesterone measurement. To evaluate effects of long-term OPU on subsequent fertility of donors, all heifers (n = 14) were inseminated during the first natural estrus after the OPU experiment. The proportion of Class I oocytes was significantly (P < 0.001) higher after U-OPU than after L-OPU (38.7% vs 21.0%). Following IVM/IVF, this difference in oocyte quality was reflected by the cleavage rate (U-OPU: 58.1%; L-OPU: 52.1%; P < 0.05) and the rate of development to morulae and blastocysts (U-OPU: 27.1%; L-OPU: 13.9%; P < 0.001). Among other factors, the greater changes in vacuum pressure during L-OPU vs U-OPU might be responsible for the difference in oocyte quality. This problem may be overcome by a more flexible system for regulating the vacuum. Progesterone levels were higher during the L-OPU than the U-OPU periods. Seven donors (50%) were diagnosed pregnant by ultrasonography on Day 28 and by palpation per rectum on Day 42.

Observations on the female reproductive cycle of captive giant tortoises (Geochelone spp.) using ultrasound scanning.

The reproductive activities of one adult female Galápagos tortoise (Geochelone nigra) and three adult female Aldabra tortoises (Geochelone gigantea) were monitored over 2 yr using ultrasound scanning. A nonrestraining technique of tactile stimulation was used for all examinations. Developing, preovulatory, and atretic ovarian follicles, as well as eggs at various stages of shell deposition, were identified and measured. In G. nigra, follicles became preovulatory at a diameter of 40-42 mm and eggs were laid 34-84 days (mean = 55.6, n = 5) after the thin-shelled eggs were first detected in the oviducts. Geochelone nigra was capable of retaining eggs, with the shell already formed, until the next breeding season. No eggs have been produced by G. gigantea during their stay in Zürich Zoo although follicles of 38-40 mm have been observed frequently in two animals.

Patterns of follicular development during periods of anovulation in pasture-fed dairy cows after calving.

The ovaries of 17 Friesian cows aged two to seven years were examined daily by transrectal ultrasound from one week after calving. The position and size of all follicles more than 2 mm in diameter were recorded. A large (more than 9 mm in diameter) follicle was present on at least one ovary by six to 17 days after calving, and in two of the cows it ovulated; the remaining cows had from two to nine large follicles before they ovulated. The mean (SEM) interval from calving to the first ovulation was 43.4 (5.3) (range 13 to 93) days, after 4.2 (0.6) waves of follicles.

Ultrasonography as a tool for monitoring in ovo chicken development. 1. Technique and morphological findings.

Preliminary studies were performed to develop a method for using real-time, B-mode ultrasonography (US) to directly image the internal morphology of the chicken egg and developing embryo. Different soft tissue interfaces will reflect US waves differentially. These reflected waves, or echoes are then converted into a two-dimensional image of internal morphology. A major limitation of diagnostic US is its inability to penetrate through gas or hard tissue (bone, shell) interfaces. Methodology development to overcome the acoustic obstacle presented by the eggshell and air cell constituted the initial part of the preliminary study. An acoustical window was achieved by creating a 2-cm fenestration through the large end of the eggshell, then filling the air cell with sterile saline. Morphological features of the yolk and embryo were recorded at 0, 2, 6, 9, 14, and 17 days of incubation. The second part of the preliminary study explores whether the acoustic window, once created, could then be closed, and if closed, whether egg viability could be maintained. A second concurrent trial was conducted with 32 eggs that were fenestrated, imaged, recorded, reclosed, and incubated. Two methods of closure were attempted: one using dialysis membrane and tape; the other using an eggshell allograft. Hatchability was partially retained with both window closure methods.

Ultrasonography as a tool for monitoring in ovo chicken development. 2. Effects of eggshell alteration and ultrasonography on embryonic and posthatch development.

The influence of creating and closing acoustic windows on embryonic and posthatch development for the purpose of chicken embryogenesis monitoring by real-time ultrasonography (US) was evaluated at 2, 6, 9, 14, and 17 days of incubation. Acoustic windows were closed using either a porous dialysis film and tape (FM) or an eggshell allograft attached with collodion (CP). Results from eggs closed in each manner with and without concurrent nonsterile US examination were compared with two control groups. Window creation reduced hatchability. The hatchability reduction was caused primarily by bacterial contamination. Contamination was more common in the FM eggs than in the CP eggs and was greater in eggs that also underwent US. Hatchability increased and contamination decreased when US was performed closer to hatch. Egg weight loss was increased after Day 6 of incubation in FM + US eggs treated on Day 2 and after Day 9 in all eggs with windows except CP eggs treated on Day 9 and CP + US eggs treated on Days 6 and 9. Hatch weight decreased in chicks from eggs that had windows, particularly in FM eggs.

Investigation of abnormal first-trimester gestations by color Doppler imaging.

Color Doppler imaging was employed to assess the uteroplacental circulation in abnormal first-trimester gestations. Included in the study were 40 blighted ova, 35 missed abortions, 10 trophoblastic diseases, and 3 ectopic gestations. Normal gestations served as a control group. Flow was detected more frequently in the blighted ova than in the missed abortions. Flow was detected in all cases of trophoblastic disease and ectopic gestations. Resistance to flow was significantly lower in the trophoblastic group as compared to the other groups.