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.     

Detection of bovine immunodeficiency virus DNA in the blood and semen of experimentally infected bulls

Five 18- to 24-month-old bulls were inoculated with either a cell suspension containing bovine immunodeficiency virus (BIV-FL112; 3 bulls) or a BIV-free cell suspension (2 bulls). Blood and semen specimens were collected once a week for 14 weeks, and seroconversion was confirmed by indirect immunofluorescent antibody (IFA) testing. The presence of BIV in blood and semen was determined by virus isolation and/or polymerase chain reaction (PCR) assays. Antibodies to BIV were detected in the 3 experimentally infected bulls as early as day post inoculation (DPI) 17, and levels peaked at DPI 37-58. BIV was isolated from the peripheral blood mononuclear cells (MNCs) of the infected bulls at DPI 9 (2 bulls) and DPI 23 (1 bull), and could be isolated from one animal up to DPI 65. PCR analysis of MNC DNA, using BIV pol gene primers, detected virus in all three of the experimentally infected bulls from DPI 9 until the termination of the experiment at DPI 98. Efforts to isolate a significant number of non-spermatozoal cells (NSC) by gradient separation from the semen of the experimentally infected bulls were unsuccessful. Two methods for the extraction of total NSC DNA from up to 2 ml of non-extended semen were employed; however, no BIV pol fragment was amplified from these DNA preparations. Additionally, 30 bulls from artificial insemination (AI) centers were evaluated for BIV infection by PCR. No amplification products were obtained from MNC DNA from the AI submissions using primer sets for both the BIV pol and env genes.     

Isolation of a subgroup two adenovirus from calf with weak calf syndrome.

A viral agent, designated Id-1, was isolated from the buffy coat of a calf suffering from weak calf syndrome. The virus replicated on bovine salivary gland cells and caused cytopathic effect within four days after infection-Cytopathic effect was characterized by rounding and clumping of cells. Stained preparations of infected monolayers revealed multiple intranuclear inclusions. The agent was found to be resistant to chloroform, ether, trypsin, sodium desoxycholate, oxytetracycline and a pH range of three to nine. The virus was sensitive to 5-iodo-2'-deoxyuridine and to a temperature of 70 degrees C. Cross neutralization tests with Id-1 antiserum and bovine adenovirus type 7 (strain Fujuroi) antiserum resulted in complete neutralilation of both viruses with four or less antibody units of homologous or heterologous antiserum.     

Bovine pneumonic pasteurellosis: chemiluminescent response of bovine peripheral blood leukocytes to living and killed Pasteurella haemolytica, Pasteurella multocida, and Escherichia coli

A luminol-dependent chemiluminescence (LDCL) assay was used to evaluate the response of bovine polymorphonuclear leukocytes; (neutrophils [PMN]) to living and heat-killed Escherichia coli, Pasteurella multocida (type A, serotype 3), and P haemolytica (biotype A, serotype 1), and to heat-killed P haemolytica and sterile culture supernatant from living P haemolytica. Control cultures containing PMN that had not been phagocytically stimulated with bacteria had a modest increase in LDCL during the initial 10 minutes of incubation, followed by a gradual decline throughout the 120-minute incubation period. Bovine PMN emitted LDCL more efficiently when the cells were exposed to living E coli or P multocida than when they were exposed to the same bacteria killed by heat. The mean LDCL values for reaction mixtures containing living E coli or P multocida peaked at 30 minutes of incubation and remained above values for mixtures containing the same heat-killed bacteria. Kinetics of the LDCL response of bovine PMN to heat-killed P haemolytica were similar (although reduced in amplitude) to that observed with killed E coli or P multocida. The LDCL response of bovine PMN to living P haemolytica was not like that for E coli or P multocida, and was characterized by the development of a peak response at 10 minutes followed by a precipitous decrease in responsiveness and a subsequent complete cessation of LDCL. Addition of sterile culture supernatant from living P haemolytica to test samples containing heat-killed P haemolytica induced a response similar to that obtained with the living microorganism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Corynebacterium pseudotuberculosis exotoxin: fatal hemolytic anemia induced in gnotobiotic neonatal small ruminants by parenteral administration of preparations containing exotoxin

Inoculation of live Corynebacterium pseudotuberculosis, culture supernatant, ammonium sulfate-fractionated crude exotoxin, or chromatographically purified exotoxin preparations into gnotobiotic small ruminants (n = 13) caused death of the ruminants within 48 hours. Characteristic changes observed in animals living greater than or equal to 2 hours after inoculation included hemorrhage and edema at the site of injection, severe hemolytic anemia and hemoglobinuria, dark red fluid in body cavities, lung edema, and icterus. The crude exotoxin preparation caused a syndrome of acute shock in 2 lambs that died within 15 minutes after inoculation. Clinical and pathologic responses of animals inoculated with culture supernatant and purified toxin were similar. Histopathologic evidence indicated that the exotoxin caused necrotic changes in the proximal convoluted tubules of the kidneys. Inoculation with live organisms caused multiple foci of suppurative inflammation in skeletal muscle and adjacent adipose tissue, whereas such changes were not observed in animals administered exotoxin preparations. Although C pseudotuberculosis exotoxin induced a hemolytic anemia in the experimental animals, it did not lead to in vitro lysis of ovine, caprine, or bovine erythrocytes, unless they had been sensitized with Rhodococcus (Corynebacterium) equi filtrate. The toxic sphingomyelin-specific phospholipase D from C pseudotuberculosis had a molecular weight of 31,000 daltons and an isoelectric point of approximately 9.6. The elution profile of exotoxin on a carboxymethyl Sephadex column was studied and the majority of the enzymatic activity was eluted by a NaCl gradient (0.25M to 0.7M) with a maximum at 0.35M NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 physiological tolerance of the grey carpet shark (<Emphasis Type="Italic">Chiloscyllium punctatum</Emphasis>) and the epaulette shark (<Emphasis Type="Italic">Hemiscyllium ocellatum</Emphasis>) to anoxic exposure at three seasonal temperatures

The epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) are commonly found in periodically hypoxic environments. The ecophysiological time available for these animals to safely exploit these niches during different seasonal temperatures was examined. The time to loss of righting reflex (T _LRR) was examined in response to an open ended anoxic challenge at three seasonal temperatures (23, 25 and 27°C). Ventilation rates were measured in an open ended anoxic challenge at 23°C and during 1.5 h of anoxia followed by 2 h of re-oxygenation at 23 and 25°C. The mean T _LRR of epaulette and grey carpet sharks was inversely proportional to temperature. The T _LRR was similar between species at 23°C; however, grey carpet sharks had significantly reduced T _LRR at higher temperatures. During the standardised anoxic challenge, epaulette sharks entered into ventilatory depression significantly earlier at 25°C. During re-oxygenation, epaulette sharks exposed to anoxia at 23°C had no significant increase in ventilation rates. However, after anoxic challenge and re-oxygenation at 25°C, epaulette sharks showed a significant increase in ventilation rates during re-oxygenation. Grey carpet sharks displayed no evidence of ventilatory depression during anoxia. However, during re-oxygenation, grey carpet sharks had significantly elevated ventilation rates above pre-experimental levels and control animals. These data demonstrate that the anoxia tolerance times of both species were temperature dependent, with a significant reduction in the T _LRR occurring at higher temperatures. Epaulette sharks had a significantly greater T _LRR at higher temperatures than grey carpet sharks, which did not enter into a ventilatory depression.