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.     

Host Plant-Based Artificial Diets Enhance Development,Survival and Fecundity of the Edible Long-Horned Grasshopper Ruspolia differens (Orthoptera: Tettigoniidae)

Wild swarms of the long-horned grasshoppers Ruspolia differens (Serville) which are widely harvested for consumption and sale in Africa are seasonal and unsustainable, hence the need for innovative ways of artificially producing the insects. We investigated the development, survival, and reproduction of R. differens in the laboratory on diets mixed with host plants [Digitaria gayana Kunth, Cynodon dactylon (L.) and Megathyrsus maximus Jacq (Poales: Poaceae); Ageratum conyzoides L. (Asterales: Asteraceae)] identified from guts of their wild conspecifics with a view to developing a suitable diet for artificial mass rearing of the edible insect. A standard diet comprising ground black soldier fly, Hermetia illucens L. (Diptera: Startiomyidae) larvae, soybean flour, maize flour, vitamin premix, and ground bones was tested for rearing R. differens as a control against the same ingredients incorporated with individual powders of the different host plants. Whereas R. differens developed more slowly in the diet mixed with D. gayana than in the control diet; its development was faster in the diet mixed with C. dactylon. Mortalities of R. differens in host plant-based diets were 42.5–52.5%, far lower than in the control diet with 71% mortality. The insects raised on the diet mixed with M. maximus laid approximately twice more eggs compared to R. differens fecundities from the rest of the diets. However, inclusion of host plants in the diets had no detectable influence on R. differens adult weight and longevity. These findings support inclusion of specific host plants in artificial diets used for mass rearing of R. differens to enhance its survival, development, and fecundity.     

Monitoring biochemical changes in bacterial spore during thermal and pressure-assisted thermal processing using FT-IR spectroscopy

Pressure-assisted thermal processing (PATP) is being widely investigated for processing low acid foods. However, its microbial safety has not been well established and the mechanism of inactivation of pathogens and spores is not well understood. Fourier transform infrared (FT-IR) spectroscopy was used to study some of the biochemical changes in bacterial spores occurring during PATP and thermal processing (TP). Spore suspensions (approximately 10(9) CFU/mL of water) of Clostridium tyrobutyricum, Bacillus sphaericus, and three strains of Bacillus amyloliquefaciens were treated by PATP (121 degrees C and 700 MPa) for 0, 10, 20, and 30 s and TP (121 degrees C) for 0, 10, 20, and 30 s. Treated and untreated spore suspensions were analyzed using FT-IR in the mid-infrared region (4000-800 cm(-1)). Multivariate classification models based on soft independent modeling of class analogy (SIMCA) were developed using second derivative-transformed spectra. The spores could be differentiated up to the strain level due to differences in their biochemical composition, especially dipicolinic acid (DPA) and secondary structure of proteins. During PATP changes in alpha-helix and beta-sheets of secondary protein were evident in the spectral regions 1655 and 1626 cm(-1), respectively. Infrared absorption bands from DPA (1281, 1378, 1440, and 1568 cm(-1)) decreased significantly during the initial stages of PATP, indicating release of DPA. During TP changes were evident in the bands associated with secondary proteins. DPA bands showed little or no change during TP. A correlation was found between the spore's Ca-DPA content and its resistance to PATP. FT-IR spectroscopy could classify different strains of bacterial spores and determine some of the changes occurring during spore inactivation by PATP and TP. Furthermore, this technique shows great promise for rapid screening PATP-resistant bacterial spores.     

Responses of field grown tomato plants to arbuscular mycorrhizal fungal colonization under varying intensities of drought stress

The effects of root colonization by the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck and Smith on growth, flower and fruit production, and fruit quality were studied in field-grown tomato plants exposed to varying intensities of drought stress. Inoculated (M+) and non-inoculated (M−) tomato seedlings were exposed to varying intensities of drought stress by adjusting irrigation intervals. Mycorrhizal plants had significantly higher uptake of N and P in both roots and shoots regardless of intensities of drought stress. AM inoculation also significantly increased shoot dry matter and the number of flowers and fruits. The fruit yields of M+ plants under severe, moderate, mild drought-stressed conditions were higher than M− plants by 24.7%, 23.1%, 16.2% and 12.3%, respectively. Furthermore, M+ plants produced tomato fruits that contain significantly higher quantities of ascorbic acid and total soluble solids (TSS) than M− plants. Mycorrhizal effects increased with increasing intensity of drought. The overall results suggest that mycorrhizal colonization affects host plant nutritional status, water stratus and growth under field conditions and thereby alters reproductive behaviour, fruit production and quality of fruits under both well-watered and drought-stressed conditions.     

Changes in protein synthesis during drought conditioning in roots of jack pine seedlings (Pinus banksiana Lamb.)

The impact of drought conditioning on the ability of eight-week-old jack pine (Pinus banksiana Lamb.) seedlings to withstand drought was assessed. Two progressive cycles of drought conditioning significantly increased the survival of seedlings subjected to a subsequent prolonged drought. The in vivo accumulation of several root membrane proteins during drought conditioning was correlated with an increase in seedling survival. A group of root proteins, ranging in molecular mass from 43 to 47 kDa, increased accumulation during one cycle of drought conditioning and to a lesser extent during two cycles of drought conditioning. The accumulation of several low molecular mass membrane and soluble proteins also increased during drought conditioning, suggesting that these proteins may play an important role in the enhancement of drought tolerance. In vitro translation studies showed a general increase in the abundance of protein products encoded by mRNAs from drought-conditioned seedlings. Although the majority of the in vitro translation products appeared in both control and drought-conditioned seedlings, one mRNA encoding a 15 kDA translated protein was more prominent during the second cycle of drought conditioning.     

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.     

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.