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.     

The resistance of meat chickens vaccinated by aerosol with a live V4 Newcastle disease virus vaccine in the field to challenge with a velogenic Newcastle disease virus

Meat chickens housed on a commercial broiler farm in Australia were vaccinated once at 10 to 11 days-of-age by aerosol with live V4 Newcastle disease virus (NDV) vaccine. Groups of vaccinated and unvaccinated birds were flown to Malaysia, where they were challenged with a virulent strain of NDV. Survival rates in vaccinated chickens challenged 7, 14, 21 or 31 d after vaccination were 0.47, 0.77, 0.97 and 0.92, respectively. All unvaccinated chickens died due to Newcastle disease (ND) following challenge. Chickens in Australia and Malaysia were bled and the serums tested for haemagglutination-inhibiting (HI) antibody to NDV. Many vaccinated birds with no detectable antibody, and all birds with a log2 titre of 2 or greater, survived challenge. The results showed that this V4 vaccine induced protective immunity in a significant proportion of chickens within 7 d of mass aerosol vaccination. This early immunity occurred in the absence of detectable circulating HI antibody. Non-HI antibody mediated immunity continued to provide protection up to 31 d after vaccination. Almost all vaccinated birds were protected within 3 w of vaccination. It is concluded that the V4 vaccine is efficacious and could be useful during an outbreak of virulent ND in Australia.     

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.