Post-mortem recovery,in vitro maturation and fertilization of fallow deer (Dama dama,Linnaeus 1758) oocytes collected during reproductive and no reproductive season

Habitat degradation leads to small and fragmented populations, lower genetic variability and fertility overtime. Assisted reproductive techniques represent important tools to cope with the dramatic loss of biodiversity. Fallow deer (Dama dama), beyond its high commercial value and wide distribution, may represent the most suitable model to study endangered cervids. In this study, oocytes were recovered post-mortem from fallow deer during the breeding and no breeding seasons and were in vitro matured (IVM). The ability of cryopreserved thawed sperm samples recovered by electroejaculation from four adult males was tested by in vitro fertilization of IVM oocytes. The number of oocytes collected per ovary did significantly vary across seasons from 6.2 ± 0.92 during breeding season to 10.4 ± 1.26 during no breeding season (p = .006). Oocytes collected during the breeding season showed higher in vitro fertilization rate compared to the no breeding season (p = .045). However, no embryos reached the blastocyst stage. Semen samples obtained by electroejaculation were successfully cryopreserved, although the cryopreservation process negatively affected most kinetic parameters, mainly at 2 hr post-thawing. Moreover, the percentage of rapid spermatozoa significantly decreased between fresh samples and at 2 hr post-thawing, whereas the percentage of slow spermatozoa increased across the same period (p < .05). Our study provides the logistic steps for the application of assisted reproductive techniques in fallow deer and might be of great interest for genetic resource bank planning.     

Influence of redox conditions and rice straw incorporation on nitrogen availability in fertilized paddy soils

Biology and Fertility of Soils - Temporal nitrogen (N) availability in fertilized rice paddies is the result of a balance of processes, mainly the gross rates of N mineralization, microbial and...     

Snow removal and its influence on temperature and N dynamics in alpine soils (Vallée d'Aoste,northwest Italy)

The effect of a lack of snow cover in winter was investigated in two soils, beneath larch and meadow, in NW Italy (Vallée d'Aoste Region). During the late 1980s and early 1990s and 2000s, this region experienced extreme climatic conditions including a low snow pack and lack of snow cover for extended periods with important effects on soil temperature and nutrient dynamics. In particular, the mountain belt in the Alps may be extremely sensitive to these phenomena, in relation to the rise in average snowline projected under a warmer global climate. The study area is located at an elevation of 1450 m asl in the Italian Alps (Mont Mars Natural Reserve). During the winter 2003/04, snow was continuously removed in a treatment plot while a reference plot was maintained undisturbed. Soil temperature was measured at 10 cm depth by data loggers (UTL‐1). Soil N transformations in the topsoil (10 cm depth) were determined by the buried‐bag technique. The removal of the snow cover caused a significant decrease in soil temperature, related to concurrent decreases in air temperature. The lowest soil temperatures recorded were –4.3°C and –4.5°C beneath larch and meadow, respectively, on January 31, 2004. Soil temperature in the undisturbed plots was maintained above the freezing point when the snow cover was present. The snow removal caused significant increases in net ammonification in both soils and net nitrification only under meadow, but did not affect microbial biomass N which decreased in both plots. Our results suggest that the lower temperature reached in the plot without snow favored the production of inorganic N by physical rather than microbial degradation of soil organic matter (SOM). Soil freezing could enhance soil‐aggregate disruption releasing physically protected SOM and fragmentation of OM itself.     

In vitro Developmental Competence of Adult Sheep Oocytes Treated with Roscovitine

The efficiency of in vitro sheep embryo production is still low compared to that observed in vivo and in other species. In this context, meiotic inhibition strategies emerged as a promising alternative to improve this biotechnology. So, this study aimed to evaluate, for the first time, the effects of roscovitine on in vitro maturation of sheep oocytes and their subsequent embryo development. For this, cumulus–oocyte complexes (COCs) were cultured for 6 h in the presence (Rosco) or absence (Control) of 75 μm roscovitine and, subsequently, in vitro matured (IVM) for 18 h with gonadotropins. At 0 (Immature), 6 and 24 h of culture, the nuclear status of oocytes was evaluated by Hoechst staining. Embryo cleavage and blastocyst formation were recorded 30 h after in vitro fertilization and on day 7 of culture, respectively. Blastocyst quality was evaluated by differential staining. At 6 h, the GV rate in the Rosco treatment (93.8%) was similar to that observed in the Immature oocytes (94.9%) and significantly higher compared to Control (41.3%). After IVM for 18 h, a high and similar proportion of oocytes from Rosco (93.6%) and Control (88.4%) reached the MII stage. In both treatments, approximately 70% of oocytes cleaved and 50% of them developed up to blastocyst. The mean percentage of blastocyst cells, embryoblast, trophoblast and pyknosis did also not differ between Control and Rosco. In conclusion, roscovitine, at the studied experimental conditions, was efficient to reversibly inhibit the meiosis of adult sheep oocytes without detrimental effect on development and quality of the in vitro produced embryos.     

Lipid droplet distribution of immature canine oocytes in relation to their size and the reproductive stage

This study investigated the distribution of lipid droplets (LD) in immature canine oocytes in relation to their size and the reproductive stage. Oocytes were collected from the ovaries of bitches at different estrous stages, divided according to their size (110‐120 µm; >120 µm), and stained with Nile Red to detect lipid droplet distribution. At the follicular phase most of the oocytes displayed a diffuse pattern of LD distribution, whereas at anestrus and luteal phase oocytes showed LD mainly in a peripheral/ perinuclear LD distribution. A significantly higher intensity of LD has been recorded in the oocytes > 120 µm compared to those of smaller size (110 ‐ 120 µm) at all stages of the estrous cycle. At follicular phase, oocytes > 120 µm displayed LD intensity similar to that of oocytes > 120 µm at luteal phase and higher compared to the oocytes of the other groups.     

Evolution of surface properties and organic matter stabilisation in podzolic B horizons as assessed by nitrogen and phosphate sorption

In podzolic B horizons illuviated Al, Fe and organic matter (OM) increase with the ongoing of the pedogenic process. Depending on OM load on mineral surfaces, modifications of the soil surface properties are expected and may influence OM stabilisation. The proportion of labile organic pools should thus vary depending on the type of podzolic horizon. In this work, we selected B horizons at increasing intensity of podzolisation, evaluated the labile OM pools through oxidation with 2 % NaClO and characterised surface properties with N₂ and phosphate sorption. Before and after oxidation, we assessed the NaOH-extractable OM fractions. Oxidation was more effective on the least polar organic compounds and led to an increase in the fulvic to humic acid ratio. Specific surface area (SSA) increased after oxidation only in the least podzolised horizons, while selectively preserved OM induced a decrease in SSA in the more developed Bs, Bsm and Bhs. Phosphate sorption induced a release of OM and always decreased after oxidation, although variations in P affinity for the surfaces were observed. The effect of oxidation on surface parameters pointed to a specific association between organics and minerals that changed during soil development. At the very beginning of podzolisation, the dominant forms seemed related to organo-metallic complexes with little interaction with surfaces. With Bs development, weak interactions between mineral surfaces and OM appeared, while at a later stage OM differentiated into bulky structures and tightly bound, rigid ones, with extremely low N₂ accessibility. The latter were not sensitive to low concentration NaClO while the former were easily oxidised.     

Interaction of phytases with minerals and availability of substrate affect the hydrolysis of inositol phosphates

The stability and activity of phytases in the soil environment may be affected by their sorption on soil particle surfaces and by substrate availability with important consequences for P cycling and nutrient bioavailability. This work evaluated the interaction of phytases with goethite, haematite, kaolinite, montmorillonite and two oxisol clays and investigated how this interaction is affected when myo-inositol hexakisphosphate (InsP₆) was sorbed on the mineral surfaces. phyA histidine acid phosphatases of fungal origin were used and their ability to release orthophosphate from the InsP₆-saturated minerals was evaluated.The phytases showed a high affinity for the mineral surfaces, with a loss of enzyme activity generally being observed over 24 h (up to 95% of the initially added activity). The loss of phytase activity was dependent on the type of mineral, with kaolinite and montmorillonite showing the greatest effect. Retention of enzyme activity was higher with the two oxisol clays, suggesting that the heterogeneous nature of clay surfaces and the presence of endogenous organic matter may limit the inhibition caused by interaction with minerals.In the presence of mineral surfaces saturated with InsP₆, the partitioning of enzyme activity between the solution and the solid phase was shifted more towards the solution phase, presumably due to the mineral surfaces being occupied by the substrate. However, phytases were not able to release any orthophosphate directly from InsP₆-saturated goethite and haematite, and hydrolysed InsP₆ that was desorbed from haematite. Conversely, in the case of kaolinite and of the oxisol clays, where desorption was limited, phytases appeared to be able to hydrolyse a small fraction of the InsP₆ adsorbed on the surfaces. These findings suggest that the bioavailability of P from inositol phosphates is governed to a large extent by the mineral composition of soil and by competitive effects for sorption on reactive surfaces among inositol phosphates and phytases.     

Relative Importance of Mineralogy and Organic Matter Characteristics on Macroaggregate and Colloid Dynamics in MG‐Silicate Dominated Soils

Soil aggregation and organic matter (OM) conservation are important in the prevention of land degradation. Aggregation processes and OM turnover influence each other and depend on the characteristics of both minerals and organic pools. We assessed the relative importance of the organic and mineral phases at the macroaggregate and colloidal scale in two soils (CHL and SRP, chlorite and serpentine‐rich, respectively) where Mg‐silicates dominated, by incubating them with a relatively degraded and oxidized organic fraction, that is the humic acids (HAs) extracted from the organic horizons of both CHL and SRP. The HA from SRP were more aromatic and richer in phenolic groups, whereas HA from CHL were N‐richer, more aliphatic and richer in carboxyl groups. The SRP soil formed larger amounts of macroaggregates, more stable than in CHL. At the colloidal scale, SRP was more flocculated and clay had a lower electrophoretic mobility than CHL. HA enhanced aggregate formation in both samples but improved aggregate stability only in CHL. In CHL, slight differences in electrophoretic mobility were visible, while in SRP, differences were more pronounced, with a point of zero charge at lower pH and larger hydrodynamic diameter. The abundance of Mg in SRP may have favoured the formation of weaker outer‐sphere interactions and the release of clay‐HA associations upon water dispersion, while in CHL Ca formed more stable bonds with HA. In SRP, ligand exchange reactions can be ruled out, conversely to the dominant bonding mechanism occurring in Al‐silicate dominated soils, with important consequences on the release of OM‐loaded clay particles. Copyright © 2016 John Wiley & Sons, Ltd.     

HPLC determination of fenoxaprop and fenoxaprop-ethyl in different soils

This paper describes a method for the determination of fenoxaprop ethyl and fenoxaprop residues in four soil types using two extraction procedures: extraction with 0.1 M hydrochloric acid/methanol, partitioning with dichloromethane and extraction with ethyl acetate. The extracts were purified on florisil or alumina cartridge. The analyses were performed by reverse phase HPLC with UV detection at 280 nm. The best results in terms of recovery, clean-up efficiency and independence of soil characteristics were obtained with the combination ethyl acetate extraction alumina clean-up. Under these conditions, the recoveries were higher than 70 % and the detection limit was 0.02 mg kg^?1 soil.     

Production and lambing rate of blastocysts derived from in vitro matured oocytes after gonadotropin treatment of prepubertal ewes.

The aim of this study was to evaluate the effect of gonadotropin treatment on the in vitro maturation, blastocyst production, and developmental potential to term of oocytes collected from Sardinian neonatal and prepubertal ewes at 4 to 6 wk of age. Cumulus-oocyte complexes were recovered at 24 h after withdrawal of a 1/6th size progestagenated pessary from the donors, of which each received 120 IU FSH/LH and 400 IU PMSG in a single dose 36 h before sponge removal. Treated donors produced a greater (P<.01) number of oocytes per animal (86.2 +/-7.9) compared with slaughterhouse (untreated) prepubertal ewes (55.5+/-6.1) of the same age or with treated neonatal ewes (6.1+/-0.7) 10 d old. During oocyte maturation, there were no differences in the percentage of germinal vesicle break-down (78.08 vs. 74.24), metaphase I (89.13 vs. 87.18), and metaphase II (77.91 vs. 76.38) when evaluated after 8, 14, and 24 h of maturation, respectively, between oocytes from treated and slaughterhouse (untreated) prepubertal ewes. The embryo cleavage (71.1 vs. 73.7) and blastocyst rates (22.2 vs. 19.8) were similar in the treated and the untreated prepubertal ewes after transfer of in vitro matured oocytes into ligated oviducts of temporary recipients. The in vitro viability rates of vitrified blastocysts (81.2 vs. 76.9) and the in vivo survival rates (46.1 vs. 50.0) of embryos derived from in vitro matured and in vivo fertilized oocytes showed no difference. The data suggest that gonadotropin treatment increases oocyte production per animal but has no effect on oocyte quality because embryo production and lambing rates of blastocysts derived from in vitro matured oocytes were not markedly different from those derived from untreated prepubertal ewes of the same age.