Immunolocalization of Melatonin and Follicle‐Stimulating Hormone Receptors in Caprine Ovaries and their Effects During in vitro Development of Isolated Pre‐Antral Follicles

The expression of melatonin type 1 (MT1) and FSH (FSHR) receptors in caprine ovaries and the effects of these hormones on the in vitro development of isolated pre‐antral follicles were evaluated. Follicles (≤200 μm) were cultured for 12 days in α‐MEM (control) or melatonin (100 or 1000 pg/ml) or sequential melatonin medium (100 pg/ml: from day 0 to day 6; 1000 pg/ml: from day 6 to day 12; experiment 1) and in control or sequential FSH (100 ng/ml from day 0 to day 6; 500 ng/ml from day 6 to day 12) or sequential melatonin or this latter plus sequential FSH (experiment 2). MT1 and FSHR expressions were observed in granulosa cells from secondary and antral follicles. The oocytes from primordial and primary follicles also express FSHR. Sequential melatonin increased the percentage of normal follicles and oocyte recovery compared with the control or melatonin (1000 pg/ml) at day 12. In experiment 2, all the treatments increased the normal follicles and growth compared with the control. In conclusion, this study demonstrated the presence of MT1 and FSHR in caprine ovaries. The addition of increased concentrations of melatonin (sequential medium) or FSH can be used to promote the in vitro development of caprine pre‐antral follicles.     

Transform-limited, narrow-linewidth lasing action in organic semiconductor microcavities

Lasing action in organic vertical-cavity surface-emitting laser (OVCSEL) structures is demonstrated. Optically pumped OVCSELs with an active layer composed of a thin-film organic semiconductor tris-(8-hydroxyquinoline) aluminum (Alq3) doped with DCM laser dye produced very narrow linewidth (0.2 +/- 0.1 angstrom), high-power (3 watts) emission that could be varied in different devices from orange to red. The efficient energy transfer from Alq3 to DCM results in a threshold input energy of 300 microjoules per square centimeter. An operational lifetime >10(6) laser pulses was achieved for a device operated well above threshold in atmosphere. The linewidths above threshold are Fourier transform-limited and could potentially be narrowed further.     

Variations in Pectoral Girdle Muscles in Dogs

Muscle variations take the form of additional muscle bundle or belly, unusual muscle origin or termination, as well as complete muscle absence. Knowledge of such variations not only has clinical importance for guiding surgery, but also can help reveal phylogenetic relationships. To improve our understanding of muscle variations in dogs, 57 medium‐sized, cross‐breed male and female adult animals were dissected as part of a gross anatomy course between 2005 and 2011. Variations in pectoral girdle muscles were observed in 7 (12.3%) dogs and took the form of an additional muscle bundle in the brachiocephalicus muscle (n = 2), in the omotransversarius muscle (n = 2), in the cervical part of the rhomboideus muscle (n = 2) and in the cervical part of the serratus ventralis muscle (n = 1). Muscle variation was bilateral in only one dog; it involved the omotransversarius muscle. The variations did not seem to be sex dependent. Such variations can appear regularly in dogs and should be taken into consideration during anatomical dissection.     

Immunohistochemical Localization of Fibroblast Growth Factor‐2 in the Sheep Ovary and its Effects on Pre‐antral Follicle Apoptosis and Development In Vitro

Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF‐2 protein expression in ovine ovaries and to verify the effect of FGF‐2 on the morphology, apoptosis and growth of ovine pre‐antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α‐MEM⁺) alone or supplemented with FGF‐2 at different concentrations (1, 10, 50, 100 or 200 ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF‐2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10 ng/ml FGF‐2, and significantly higher than those observed in 50, 100 or 200 ng/ml FGF‐2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200 ng/ml FGF‐2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200 ng/ml FGF‐2 (p < 0.05). However, the percentage of apoptotic cells only decreased (p < 0.05) in ovarian tissues cultured in 1 or 10 ng/ml FGF‐2 compared with the control medium and other FGF‐2 treatments. In conclusion, this study demonstrated the presence of FGF‐2 in ovine ovaries. Furthermore, 10 ng/ml FGF‐2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis.     

Morphological Investigations of the Glandulae Profundae Plicae Semilunares Conjuctivae in the Domestic Swine (Sus scrofa domesticus) and the Wild Hog (Sus scrofa ferus)

Samples of glandulae profundae plicae semilunares (Harderian glands) from five domestic swine and five wild hogs were used for this research. The gland samples were fixed in Bouin solution and mounted in paraffin. The paraffin slices were stained with haematoxylin and eosin, according to the periodic-acid-Schiff (PAS) method, with alcian blue (pH 2.5), with toluidine blue (pH 4.0), and applying a combination of staining with alcian blue (pH 2.5) and the PAS method. In domestic swine and wild hogs, these glands are tubular-alveolar with wide glandular lumina. A great deal of acid mucopolysaccharides and PAS-positive substances were noted within swines’ glandular cells, while, in wild hogs, PAS-positive substances were not frequent, acid mucopolysaccharides being noted in only a few glandular-acini cells. The appearance of the metachromatic phenomenon was not noted either in domestic swine or in wild hogs. In domestic swine, the level of acid mucopolysaccharides is probably due either to the housing method or to the influence of the alkaline substances that may appear on an eye's mucous conjunctive membrane during intensive breeding, such that the Harderian glands protect the mucous membrane by extracting acid mucopolysaccharides.     

Use of protocatechuic acid as the sole antioxidant in the base medium for in vitro culture of ovine isolated secondary follicles

This study evaluated the effect of the protocatechuic acid (PCA) as the sole antioxidant in the base medium for in vitro culture of ovine secondary follicles. Secondary follicles (200‐230 μm) were isolated and cultured in α‐minimal essential medium supplemented with BSA, insulin, glutamine and hypoxanthine (α‐MEM: antioxidant‐free medium) or α‐MEM also added by transferrin, selenium and ascorbic acid (α‐MEM+: with antioxidant) or α‐MEM added by PCA (56.25; 112.5; 225; 450; or 900 μg/ml). Moreover, after culture, oocytes were matured and the chromatin configuration and DNA fragmentation were evaluated. After 12 days, the treatment containing 56.25 μg/ml PCA showed higher percentage of normal follicles than control medium or the other treatments (p < .05), except for 900 μg/ml PCA (p > .05). The antrum formation was significantly higher in treatments containing 56.25, 112.5 or 900 μg/ml PCA, compared to the α‐MEM and similar (p > .05) to the other treatments. The rates of fully grown oocytes (≥110 μm) were similar (p > .05) among all treatments containing PCA and α‐MEM+, and those were superior (p < .05) than α‐MEM, except for 450 μg/ml PCA (p > .05). GSH levels and mitochondrial activity were higher (p < .05) in α‐MEM+ than in α‐MEM and similar (p > .05) to all PCA treatments. The rates of meiotic resumption and DNA fragmentation were similar (p > .05) among α‐MEM+ and 56.25 μg/ml PCA. In conclusion, PCA at 56.25 μg/ml as the sole antioxidant added to the medium for ovine isolated secondary follicle culture maintains follicular survival, GSH and active mitochondria levels, meiotic developmental competence and DNA integrity of cultured oocytes.     

Carbon structures with three-dimensional periodicity at optical wavelengths

Porous carbons that are three-dimensionally periodic on the scale of optical wavelengths were made by a synthesis route resembling the geological formation of natural opal. Porous silica opal crystals were sintered to form an intersphere interface through which the silica was removed after infiltration with carbon or a carbon precursor. The resulting porous carbons had different structures depending on synthesis conditions. Both diamond and glassy carbon inverse opals resulted from volume filling. Graphite inverse opals, comprising 40-angstrom-thick layers of graphite sheets tiled on spherical surfaces, were produced by surface templating. The carbon inverse opals provide examples of both dielectric and metallic optical photonic crystals. They strongly diffract light and may provide a route toward photonic band-gap materials.     

Growth Dynamics of White Muscle Fibres in Relation to Somatic Growth of Larvae of European Sea Bass (Dicentrarchus labrax, L.)

The body length and diameter of muscle fibres from the right hypaxial tail myomeres were measured in 100 sea bass larvae (Dicentrarchus labrax, L.) at ages of 40 to 90 days, from a fish farm in Croatia. The larvae were kept at a temperature of 19-20 degrees C and fed with standard feed for their respective ages. Body lengths of larvae 40-90 days old varied from 8 to 42 mm, increasing significantly in 10-day intervals (p < 0.001), except between days 50 and 60 when there was no increase in the body length. The measured diameters of muscle fibres ranged from 10 to 80 microm, the mean value increasing except between days 50 and 60. The fibre diameter in larvae aged up to 60 days was 10-40 microm, while that in larvae older than 70 days was 51-80 microm. In 90-day-old larvae there were no fibres of 10-20 microm in diameter; however, the number of fibers of 61-80 microm diameter increased. A statistically significant concordance was determined between the body length and fibre diameter of larvae 70 to 90 days old. Between days 50 and 60, arrested growth of the larval body and corresponding decrease in fibre diameter was observed. From day 45 until days 59, larvae were fed with Artemia nauplii and microparticles of formulated diets. From the day 60, minced fish was added to formulated diets. This change in diet probably resulted in the observed growth arrest.     

13,14‐Dihydro‐15‐Keto Prostaglandin F2α Release in Response to Oxytocin Challenge Early Post‐Partum in Anoestrous Nelore Cows Submitted to Temporary Calf Removal and Progesterone Priming

The study evaluated, in early post‐partum anoestrous Nelore cows, if the increase in plasma oestradiol (E2) concentrations in the pre‐ovulatory period and/or progesterone priming (P4 priming) preceding ovulation, induced by hormonal treatment, reduces the endogenous release of prostaglandin PGF₂αand prevents premature lysis of the corpus luteum (CL). Nelore cows were subjected to temporary calf removal for 48 h and divided into two groups: GPE/eCG group (n = 10) and GPG/eCG group (n = 10). Animals of the GPE/eCG group were treated with a GnRH agonist. Seven days later, they received 400 IU of eCG, immediately after PGF₂α treatment, and on day 0, 1.0 mg of oestradiol benzoate (EB). Cows of the GPG/eCG group were similarly treated as those of the GPE/eCG group, except that EB was replaced with a second dose of GnRH. All animals were challenged with oxytocin (OT) 9, 12, 15 and 18 days after EB or GnRH administration and blood samples were collected before and 30 min after OT. Irrespective of the treatments, a decline in P4 concentration on day 18 was observed for cows without P4 priming. However, animals exposed to P4 priming, treated with EB maintained high P4 concentrations (8.8 ± 1.2 ng/ml), whereas there was a decline in P4 on day 18 (2.1 ± 1.0 ng/ml) for cows that received GnRH to induce ovulation (p < 0.01). Production of 13,14‐dihydro‐15‐keto prostaglandin F₂α (PGFM) in response to OT increased between days 9 and 18 (p < 0.01), and this increase tended to be more evident in animals not exposed to P4 priming (p < 0.06). In conclusion, the increase in E2 during the pre‐ovulatory period was not effective in inhibiting PGFM release, which was lower in P4‐primed than in non‐primed animals. Treatment with EB promoted the maintenance of elevated P4 concentrations 18 days after ovulation in P4‐primed animals, indicating a possible beneficial effect of hormone protocols containing EB in animals with P4 priming.