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.     

Evaluation of semen from individual male domestic fowl by assessment of sperm perivitelline interaction in vitro and in vivo

1. Spermatozoa in semen samples from 8 individual male domestic fowls were shown to have a differential and characteristic ability to hydrolyse holes in the inner perivitelline layer from laid eggs in an in vitro assay. 2. The number of holes produced by samples of spermatozoa per unit area of inner perivitelline layer in vitro was linearly correlated with sperm ATP content ( r = 0.85) and motility (r=0.76). 3. The number of holes formed in the inner perivitelline layer in vitro was also linearly correlated with the numbers of holes formed in the inner perivitelline layer of eggs fertilised in vivo , in inseminated hens (r=0.90); and was correlated logarithmically with the proportion of fertile eggs laid by these hens.     

Cellular immune response in rainbow trout Salmo gairdneri Richardson to Yersinia ruckeri O-antigen monitored by the passive haemolytic plaque assay test

Abstract. The specificity and kinetics of the immune response of rainbow trout ( Salmo gairdneri ) to single injections of an O-antigen extracted from the bacterial pathogen Yersinia ruckeri , which causes enteric redmouth in fish, were investigated by the passive haemolytic plaque assay and serum antibody quantitation. Doses ranging from 5 ng to 500 mg in 10-fold increments were injected intraperitoneally into groups of trout held at 17 × 1°5°C. The occurrence of plaque forming cells (PFC) and humoral antibody was followed for 35 days after injection. Trout gave an immune response to doses of 500 ng and above. Seven days after injection no humoral antibody was detected, but PFC were found in the spleen. The maximum PFC numbers occurred 11 days after injection. On day 21, few PFC were found, whereas serum antibody titres were highest. The antibody from immunized trout showed little or no cross-reactions with sheep red blood cells passively labelled With antigens from other fish pathogens.     

Some aspects of the absorption of picloram by gorse (Ulex europaeus L.)

Plants of Ulex europaeus were grown from cuttings and studies made of the absorption of 14C-picloram applied with unlabelled 2,4,5-T. In vivo experiments in a growth chamber showed that absorption ceased after 10 h, but was resumed on wetting. In in vitro experiments the Q10 and the activation energy for uptake decreased with increasing temperature. Absorption was increased by addition of non-ionic surfactants, by lowering the pH and by removal of cuticular wax. Gorse shoots were shown to have a high wax content but scanning electron microscopy revealed no obvious wax structures and the contact angle of spray droplets was less than 900, indicating that the surfaces were not difficult to wet.     

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.     

Influence of brown trout, Salmo trutta L., predation on the benthic fauna of earthen ponds

Abstract. Sections of the bottom of three earthen fish ponds in central Scotland were enclosed to prevent access by juvenile brown trout, Salmo trutta L., stocked at different densities from July to October. Monthly benthic samples were taken inside and outside the enclosures with a suction sampler. The benthos was dominated by Oligochaeta, whose density and biomass were significantly lower outside the enclosures; production was also lower outside but the P:B ratio was much higher. Total Chironomidae were more abundant outside, but not the larger predatory species which appeared to control the inside population. Species diversity of Oligochaeta and Chironomidae was greater outside while mean size was reduced, presumably as a result of size-selective predation by trout. Numbers of Mollusca and Asellidae were lower in the presence of fish, but Hirudinea and Sialidae were unaffected. Increasing fish density boosted chironomid numbers and biomass, indicating a predominant response to organic enrichment, but reduced the numbers of most other benthic groups presumably as a result of predation.