HPLC quantification of coumarin in bastard balm (Melittis melissophyllum L., Lamiaceae)

Coumarin occurs in many plants used as flavoring and is known to possess hepatotoxic effects. Despite in the EFSA ‘Compendium of botanicals containing toxic substances’ coumarin is reported to be present in Melittis melissophyllum (bastard balm), a plant traditionally used as beverage in Italy and Serbia, to the best of our knowledge quantitative data has never been reported. Thus, the amount of coumarin in bastard balm leaves and its variation during the annual phenological cycle were determined. The subsp. melissophyllum resulted to contain high levels of coumarin (14,392 mg/kg), mainly in the early stages of the plant cycle, suggesting prudence in its use as beverage. Furthermore, coumarin was found to be useful as marker compound to differentiate the bastard balm subspecies occurring in Italy, since the subsp. albida contained a much lower content of this molecule (19–34 mg/kg).     

Colocalization of DNA fragmentation and caspase-3 activation during atresia in pig antral follicles

Apoptosis is the cellular mechanism of ovarian follicular atresia. The major downstream effector of this phenomenon in many tissues is caspase-3 but little is known about its role in pig ovarian apoptosis. In the present study, we detected the localization of caspase-3 in parallel with nuclear fragmentation (TUNEL) on healthy and early atretic antral follicles. In healthy antral follicles caspase-3 and TUNEL positivity were occasionally recorded within theca layer. The incidence of DNA fragmentation, as indicated also by the biochemical detection, increased mainly in the granulosa layer of early atretic follicles. Quantitative analysis revealed, besides, that atresia was accompanied by a higher incidence of caspase-3 (57.20 +/- 20.05 versus 3.64 +/- 0.61 positive cells in atretic versus healthy follicles, respectively; P < 0.05), of TUNEL positivity (20.13 +/- 9.33 versus 0.42 +/- 0.12; P < 0.05) and simultaneous immunostaining for caspase-3 and TUNEL (15.02 +/- 6.95 versus 0.31 +/- 0.05; P < 0.05) in the granulosa layer. In detached granulosa cells isolated from the follicular fluid of early atretic follicles a further significantly increase was recorded in the percentage of TUNEL positivity and in the incidence of cells that showed colocalization of caspase-3 activity and DNA fragmentation. Granulosa cells of early atretic follicles exhibited a higher positivity for caspase-3 localized in the cytoplasm and occasionally in the nucleus area of granulosa cells. These results indicate that capsase-3 was involved and precociously activated during the process of atresia. Finally, the progressively higher incidence of TUNEL positivity and of double immunostaining in atretic cells collected within the follicular fluid seems to indicate that proteases activity leads only tardily in a detectable DNA fragmentation.     

Genotyping,pedigree reconstruction and endocrinological characterization of Acipenser naccarii (Bonaparte, 1836) using microsatellite markers and plasma steroid levels

This study aimed to set up a method for the long‐term management of Adriatic sturgeon, through the analysis of sex steroids and the genetic profiling of individuals in order to maximize the already reduced genetic variability of this species. Ten A. naccarii adults (nine of captive origin, one captured in the Ticino river and then moved into captivity) and eight subadults located in a semi‐natural land‐locked pond in Abbiategrasso (Milan, Italy) were analysed. Plasma testosterone differed significantly in the eight analysed subadults allowing their differentiation into two groups: the first group with an average testosterone concentration of 5.42 ± 1.31 ng/ml (probably female) and the second group with an average of 423.14 ± 75.97 ng/ml (probably male), as subsequently confirmed by artificial stripping. The plasma testosterone level was also significantly different between adult males and females (371.37 ± 43.58 vs. 95.34 ± 51.10 ng/ml), while the E2 levels showed no significant differences. Animals were genotyped on the basis of 10 microsatellite loci and their parental relationships were defined: four adults, two females and two males, generated the eight subadults. On the basis of pedigree analyses and genetic distances, 15 unrelated couples were identified for the future breeding seasons. Finally, the adult female captured in the Ticino River showed an interesting genetic profile, widely different from all of the other 17 specimens analysed, and represents a valuable source of genetic diversity.     

Lipidic microdomain reorganization during the in vitro capacitation of boar spermatozoa

Veterinary Research Communications -