Enzyme Immunoassays for the Determination of Ovine LH and FSH

The development of competitive enzyme immunoassays for ovine plasma LH (oLH) and FSH (oFSH) is described. Standards and plasma samples were preincubated with diluted antiserum to oLH or oFSH and the reacted solution (100 μl per well) was transferred to plates previously coated with oLH or oFSH, respectively. The second antibody used was anti‐rabbit IgG horseradish peroxidase. The measuring range was 0.39–50 ng/ml for each hormone and the 50% relative binding sensitivity was 9 ng/ml for oLH. The respective value for oFSH was 3.5 or 34 ng/ml with different hormone and antibody preparations used for the assay. The enzyme immunoassays were used to determine oLH and oFSH levels in plasma from ewes of two breeds during the oestrous cycle. The assays detected the first FSH surge coincident with the LH surge, the second FSH surge about 24 h later and the periodic fluctuations of FSH concentrations during the luteal phase of the oestrous cycle. These enzyme immunoassays are an efficient and economic alternative to the established radioimmunoassays (RIA) for oLH and oFSH.     

The use of midday leaf water potential for scheduling deficit irrigation in vineyards

Midday leaf water potential (Ψ_md) was monitored for 3 years at a commercial vineyard (cv. Pinot Noir) under four irrigation strategies. Three treatments were established based on irrigating vines with 4–6 mm/day, when daily measured Ψ_md was more negative than the pre-defined threshold. After the first experimental year, thresholds were adjusted for each treatment as: (1) Control (C), irrigated when Ψ_md was less than −0.6 MPa at the beginning of the season and gradually fell to −0.8 MPa at about mid-June, after which the threshold was maintained at −0.8 MPa until harvest. (2) Control–Deficit (CD), irrigated as C from bud-break to mid-June (around the middle of Stage II of fruit growth), and from then until harvest when Ψ_md decreased below −1.2 MPa. (3) Deficit–Deficit (DD), irrigated when Ψ_md was less than −1.0 from bud break to mid-May (about the middle of fruit growth Stage I), and after that time the Ψ_md threshold became −1.2 MPa until harvest. A fourth treatment was applied following a soil water budget approach (WB). All treatments were replicated five times but irrigation in the Ψ_md-based treatments were independently applied to each of the replicate plots, whereas irrigation for WB was applied equally to all replications. The more site-specific information obtained from Ψ_md thresholds in C provided substantial advantages for yield homogeneity and repeatability of results with respect to WB, thus demonstrating the method’s greater ability to account for spatial variability. Average applied water for the 3 years in C, CD, and DD was 374, 250, and 178 mm, respectively, while the yields were 11.8, 9.2, and 6.1 kg/vine, respectively. The CD treatment produced better juice quality than C, and was superior in other quality parameters to both C and DD. However, over the study period, an important carryover effect was observed in the yields and the grape size of CD, which tended to diminish from year to year relative to C.     

Relationship between root growth potential and field performance in Aleppo pine

Commercial stocks of Aleppo pine in Spain vary in quality, but there is no accepted standard for evaluating quality. A RGP test was applied to six commercial seedling stocklots at two dates (November, February) and under two test conditions (growth-chamber, greenhouse). The RGP’s predictive ability was evaluated on two contrasting sites. There was a considerable variation in the RGP depending on application date, test conditions and stock factors. The RGP results for November were correlated with each other but they did not explain outplanting performance. The February results in the growth-chamber correlated well with survival at both sites. Regression models explained survival both in the lower (R ² = 97%) and in the higher (R ² = 92%) quality sites. RGP has a valid predictive ability for this species although it is sensitive to the test conditions. In this sense, a shorter and more intensive test performed right before planting may be more reliable.     

The Control of Reactive Oxygen Species Influences Porcine Oocyte In Vitro Maturation

The aim of this study was to examine the effect of varying intracellular reactive oxygen species (ROS) levels during oocyte in vitro maturation with enzymatic ROS production systems (xanthine + xanthine oxidase or xanthine + xanthine oxidase + catalase), scavenger systems (catalase or superoxide dismutase + catalase) or cysteine on porcine oocyte maturation. Oocyte ROS levels showed an increase when H₂O₂ or O₂?^‐ production systems were added to the culture medium (p < 0.05). On the other hand, the presence of ROS scavengers in the maturation medium did not modify oocyte ROS levels compared with the control after 48 h of maturation, but the addition of cysteine induced a decrease in oocyte ROS levels (p < 0.05). The ROS production systems used in this work did not modified the percentage of oocyte nuclear maturation, but increased the decondensation of sperm head (p < 0.05) and decreased the pronuclear formation (p < 0.05). In turn, the addition of O₂?^‐ and H₂O₂ scavenging systems during in vitro maturation did not modify the percentage of oocytes reaching metaphase II nor the oocytes with decondensed sperm head or pronuclei after fertilization. However, both parameters increased in the presence of cysteine (p < 0.05). The exogenous generation of O₂?^‐ and H₂O₂ during oocyte in vitro maturation would not affect nuclear maturation or later sperm penetration, but most of the spermatozoa cannot progress to form the pronuclei after fusion with the oocyte. The decrease in endogenous ROS levels by the addition of cysteine would improve pronuclear formation after sperm penetration.     

Thinning decreased soil respiration differently in two dryland Mediterranean forests with contrasted soil temperature and humidity regimes

The effects of a thinning treatment on soil respiration (Rs) were analysed in two dryland forest types with a Mediterranean climate in east Spain: a dry subhumid holm oak forest (Quercus ilex subsp. ballota) in La Hunde; a semiarid postfire regenerated Aleppo pine (Pinus halepensis) forest in Sierra Calderona. Two twin plots were established at each site: one was thinned and the other was the control. Rs, soil humidity and temperature were measured regularly in the field at nine points per plot distributed into three blocks along the slope for 3 years at HU and for 2 years at CA after forest treatment. Soil heterotrophic activity was measured in laboratory on soil samples obtained bimonthly from December 2012 to June 2013 at the HU site. Seasonal Rs distribution gave low values in winter, began to increase in spring before lowering as soil dried in summer. This scenario indicates that with a semiarid climate, soil respiration is controlled by both soil humidity and soil temperature. Throughout the study period, the mean Rs value in the HU C plot was 13% higher than at HU T, and was 26% higher at CA C than the corresponding CA T plot value, being the differences significantly higher in control plots during active growing periods. Soil microclimatic variables explain the biggest proportion of variability for Rs: soil temperature explained 24.1% of total variability for Rs in the dry subhumid forest; soil humidity accounted for 24.6% of total variability for Rs in the semiarid forest. As Mediterranean climates are characterised by wide interannual variability, Rs showed considerable variability over the years, which can mask the effect caused by thinning treatment.

     

Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10^?5 and (2.54 ± 0.32) 10^?5, and for MDH, the U were (4.72 ± 0.42) 10^?5 and (4.38 ± 0.25) 10^?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10^?3 and (0.94 ± 0.12) 10^?3, and for MDH (9.08 ± 0.93) 10^?3 and (1.89 ± 0.10) 10^?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.     

Hydrology-oriented (adaptive) silviculture in a semiarid pine plantation: How much can be modified the water cycle through forest management?

Hydrology-oriented silviculture might adapt Mediterranean forests to climatic changes, although its implementation demands a better understanding and quantification on the water fluxes. The influence of thinning intensity (high, medium, low and a control) and its effect on the mid-term (thinned plots in 1998 and 2008) on the water cycle (transpiration, soil water and interception) and growth [basal area increment (BAI)] were investigated in 55-year-old Aleppo pine trees. Thinning enhanced a lower dependence of growth on climate fluctuations. The high-intensity treatment showed significant increases in the mean annual BAI (from 4.1 to 17.3 cm²) that was maintained in the mid-term. Thinning intensity progressively increased the sap flow velocity (v _s) in all cases with respect to the control. In the mid-term, an increased functionality of the inner sapwood was also observed. Mean daily tree water use ranged from 5 (control) to 18 (high intensity) l tree^?1. However, when expressed on an area basis, daily transpiration ranged from 0.18 (medium) to 0.30 mm (control), meaning that in spite of the higher transpiration rates in the remaining trees, stand transpiration was reduced with thinning. Deep infiltration of water was also enhanced with thinning (about 30 % of rainfall) and did not compete with transpiration, as both presented opposite seasonal patterns. The changes in the stand water relationships after 10 years were well explained by the forest cover metric. The blue to green water ratio changed from 0.15 in the control to 0.72 in the high-intensity treatment, with the remaining treatments in the 0.34–0.48 range.