Volatile compounds of bovine milk as related to the stage of the estrous cycle.

Previous reports on behavioral assays with trained dogs suggested that milk samples from cows at diestrus, proestrus, and estrus had different odors. To identify the odor differences, volatile compounds in milk were isolated and analyzed by gas chromatography and mass spectrometry. About 80 peaks were detected in each chromatogram, of which 59 were present in all samples, and 23 were tentatively identified. The major identified compounds included the following six structurally distinct classes: ester, aldehyde, ketone, alcohol, fatty acid, and lactone. Although no unique peaks were found to be specific to samples taken at diestrus, proestrus, or estrus, 36 compounds exhibited significant differences in concentration among the three reproductive stages. These quantitative differences may account for the variation of milk odors during the estrous cycle. In order to investigate the quantitative differences systematically, multivariate discriminant techniques were used to relate the gas chromatographic profiles with the three stages of the estrous cycle. Stepwise discriminant analysis indicated that 15 of the 59 peaks in each chromatogram could best be used to reveal the differences among milk samples taken at diestrus, proestrus, and estrus stages. The discriminant function based on the 15 key peaks could classify all of the samples into their original categories at a total accuracy of 97.9%. Canonical analysis indicated that milk samples from different stages were clearly separated from each other in a two-dimensional space.

Neonatal pain: from concept to research questions and a role for the advanced practice nurse.

The assessment and treatment of neonatal pain remain inconsistent in spite of scientific evidence and agreement among practitioners that neonates do experience pain. In fact, pain forms the basis for many subsequent maturational events and may mediate future responses to pain. The article outlines the concept of pain in the neonate, provides suggestions for future research, and discusses the instrumental role of the advanced practice nurse in the recognition and treatment of neonatal pain.

Cyclic changes in volatile constituents of bovine vaginal secretions.

Headspace gas chromatography was used to examine the volatile components of bovine vaginal secretions in three consecutive estrous cycles from each of four cows. Results indicated that there was an interesting peak with a retention of 2.7 min. It showed a successive rise and fall zero to three days before estrus. Gas chromatographic evidence and mass spectral data confirmed that this peak was acetaldehyde. Another unidentified compound with a retention time of 27 min was found to be unique to proestrus. It occurred two to three days prior to estrus, but was absent at any other time of the cycle.

Development and evaluation of a minicolumn assay for the detection of aflatoxin M1 in milk.

A practical field method for the chemiselective immobilization and detection of aflatoxin M1 in milk has been developed and is being marketed. In this new method, aflatoxin M1 is selectively adsorbed at the interface of a layer of neutral sand and a band of magnesium silicate (Florisil) packed in a glass minicolumn. Aflatoxin M1, at > or = .5 ppb in contaminated milk, can be easily visualized as a band of bright blue fluorescence. Briefly, raw or homogenized and pasteurized milk is diluted with water (1: 1, vol/vol) and passed through a C18 cartridge. Aflatoxin M1 is then partitioned by polarity, eluted with acetone-methylene chloride, and added to the minicolumn. The minicolumn is washed and viewed under long wave UV light. The limit of detection for this assay was .2 ppb, which was similar to the .3 ppb obtained using an immunoaffinity column, followed by minicolumn detection. The assay was accurate, rapid, easy to perform, and stable.

Effect of hydrated sodium calcium aluminosilicates on aflatoxicosis in broiler chicks.

In three experiments, three different hydrated sodium calcium aluminosilicates (HSCAS) were incorporated into chick diets (.5%) containing either 0 or 5.0 (Experiments 1 and 2) or 0 or 2.5 (Experiment 3) mg/kg aflatoxin (AF). Male broiler chicks consumed their respective diets and water ad libitum from 1 to 21 days of age. When compared with controls, body weights in chicks receiving 5.0 mg AF/kg were reduced by 214 g in Experiment 1 and 220 g in Experiment 2. The addition of .5% of the HSCAS compounds significantly diminished the growth inhibitory effects caused by AF by 39 to 68% in Experiment 1, by 46 to 88% in Experiment 2, and by 38 to 90% in Experiment 3. The increases in relative organ weights and the decreases in serum biochemical values caused by AF were significantly diminished to differing degrees by all three of the HSCAS compounds. These data demonstrate that these specific HSCAS compounds can be protective against the effects of AF in young growing broilers and further emphasizes the fact that all silicate-type sorbents are not equal in their ability to protect against aflatoxicosis. It also seems possible to specially process compounds to increase their efficacy for protection against the toxicity of AF.

Immunotoxicity of ochratoxin A to growing gilts.

Ochratoxin A (OA) was incorporated in the diets of growing gilts (mean body weight, 20.1 kg) at a concentration of 2.5 mg of OA/kg of feed and was fed continuously for 35 days. Humoral and cell-mediated immunologic measurements were evaluated to determine the effects of OA on immune function in swine. Cutaneous basophil hypersensitivity to phytohemagglutinin (PHA), delayed hypersensitivity to tuberculin, PHA-induced lymphocyte blastogenesis, interleukin-2 production, total and isotype immunoglobulin concentrations, antibody response to chicken RBC, and macrophage activation were used to evaluate immune function. Gilts treated with OA had reduced cutaneous basophil hypersensitivity response to PHA, reduced delayed hypersensitivity to tuberculin, decreased stimulation index for lymphoblastogenesis, decreased interleukin-2 production when lymphocytes were stimulated with concanavalin A, and decreased number and phagocytic activity of macrophages. Differences were not observed for total and isotype immunoglobulin concentrations, or humoral hemagglutination (chicken RBC) titer. These data indicate that OA may suppress cell-mediated immune response in growing swine.

Patulin-induced cellular toxicity: a vital fluorescence study.

The mechanisms of patulin-induced cellular toxicity in an immortalized rat granulosa cell line were examined using several vital fluorescence bioassays. Monochlorobimane and 5-chloromethylfluorescein diacetate were used to monitor cellular glutathione (GSH) levels and revealed dose- and time-dependent depletion of GSH by patulin. A significant reduction in the fluorescence of the monochlorobimane-GSH conjugate by 0.1 microM patulin was observed between 1 and 2 hr. Similar GSH depletion by the mycotoxin was also observed in parallel studies on a liver (Clone 9) and a renal (LLC-PK1) cell line, although reduction of fluorescence occurred within 1 hr at the same dosage. Analysis of the electrical potential-dependent partitioning of rhodamine 123 into mitochondria also revealed significant effects of patulin within 1 hr at 0.1 microM. An initial dose-dependent reduction in mitochondrial fluorescence was followed by loss of selective partitioning of the fluorophore into mitochondria at higher doses and/or a longer exposure of cells to patulin. The reduction in mitochondrial fluorescence was paralleled by a dose-dependent decrease in intracellular pH detected with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Analysis of [Ca2+]i with indo-1 and fluo-3 revealed a significant dose-dependent influx of Ca2+ at 10 microM and an alteration of the pattern of ionomycin-induced Ca2+ influx at 1.0 microM following patulin treatment. A carboxyfluorescein fluorescence photobleaching assay was used to examine the effects of patulin on gap junction-mediated intercellular communication. Dose-dependent reduction in intercellular communication was observed within 2 hr with 1.0 microM patulin. These observations indicate that the fluorescence assays used in this study provide a sensitive index of toxicity caused by exposure to patulin. Further, the toxic effects of patulin may involve direct effects on cellular glutathione levels and mitochondrial function in addition to direct effects on the plasma membrane.

Fate of [(14)C]xanthotoxin (8-methoxypsoralen) in laying hens and a lactating goat.

The metabolism of xanthotoxin, a naturally occurring furanocoumarin photosensitizer, was studied in laying hens and a lactating goat treated with single oral doses equivalent to 10 mg xanthotoxin/kg of body weight. Within 48 h, essentially all of the administered radiocarbon was eliminated in the excreta of the laying hens, while in the goat 92% and 3% were excreted in the urine and feces, respectively. Radiocarbon residues in the milk, egg white, and egg yolk were low. Xanthotoxin, 8-hydroxypsoralen (xanthotoxol), 6-(7-hydroxy-8-methoxycoumaryl)-acetic acid (HCA) and 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyacetic acid (HCHA) were identified in the excreta of laying hens. In the goat, xanthotoxin was metabolized to HCA, HCHA, xanthotoxol, 5,8-dihydroxypsoralen, psoralenquinone, 5-hydroxy-8-methoxy-psoralen and 3[5-(6-hydroxy-7-methoxybenzofuryl)]-propanoic acid. Thus, identified metabolites in one or both of these species arose throughO-demethylation, oxidative cleavage of the furan ring, hydroxylation, reduction, oxidation, and hydrolysis of the lactone ring.

Evaluation of the developmental toxicity of chlorinated phenols utilizing Hydra attenuata and postimplantation rat embryos in culture.

Chlorinated phenols (CPs) represent a major component of hazardous oily and wood-preserving wastes that are widely distributed in chemical dumpsites throughout the United States. Pentachlorophenol (C5P) has been reported to be highly embryolethal and embryotoxic in rats. However, data pertaining to the developmental toxicities of other important CPs are limited. In this study, the toxicities of phenol, CP homologues and their isomers, selected phenyl acetates, anisoles, sodium phenates, and tetrachlorobenzoquinones (a total of 38 chemicals) were evaluated using cultures of Hydra attenuata (HA). Developmental hazard index (A/D ratio) was determined for selected test chemicals (i.e., those chemicals which resulted in an early toxic endpoint at the lowest whole-log concentration in the adult hydra assay). These same chemicals were evaluated at equimolar concentration in postimplantation rat whole embryo culture (WEC). HA and WEC studies demonstrated a linear relationship between toxicity and the degree of chlorine substitution with C5P greater than 2,3,4,5-C4P greater than 2,3,5-C3P greater than 3,5-C2P greater than 4-CP greater than phenol. The A/D ratios from the HA assay were approximately 1 for all of the chemicals tested. Findings from the WEC assay indicated similar results based on growth, gross morphology, and DNA and protein content of embryos. The results obtained in the HA and WEC assays suggest that the chlorinated phenols are not potent teratogens. The combination of HA and WEC may facilitate the rapid detection and ranking of hazardous chemicals associated with complex mixtures of chemical wastes.

Cocontamination of swine diets by aflatoxin and diacetoxyscirpenol.

The effects of dietary aflatoxin (AF) and diacetoxyscirpenol (DAS), singly and in combination, were evaluated in growing crossbred barrows. The experimental design consisted of 4 treatments of 9 barrows each fed diets containing 1) 0 mg AF and 0 mg DAS/kg feed (control), 2) 2.5 mg AF/kg feed, 3) 2.0 mg DAS/kg feed, or 4) 2.5 mg AF + 2.0 mg DAS/kg feed for 28 days (10-14 weeks of age). Production performance, serum biochemical, hematologic, and pathologic measurements were made. Body weight and body weight gain were significantly decreased by each toxin but more so by the combination treatment. The effects were additive in nature. Liver and spleen weights, as percentages of body weight, were increased by the AF and AF + DAS treatments, and AF or AF + DAS treatments induced diffuse hepatocellular vacuolar change, early portal fibrosis, and early bile duct hyperplasia. Aflatoxin increased serum values of creatinine and gamma glutamyl transferase, cholinesterase, and alkaline phosphatase activities; increased packed cell volume and hemoglobin; and decreased urea nitrogen and total iron binding capacity. DAS reduced serum iron binding capacity. The AF + DAS treatment increased serum gamma glutamyl transferase and alkaline phosphatase activities, increased hemoglobin, and decreased serum iron binding capacity. Generally, the combination treatment could be described as additive or less than additive, with most of the effects attributable to AF. Under the conditions and parameters monitored in this study, AF and DAS had no synergistic toxic effects when incorporated into diets of growing barrows.

Detection and detoxification of aflatoxins: prevention of aflatoxicosis and aflatoxin residues with hydrated sodium calcium aluminosilicate.

Our recent findings demonstrate that HSCAS can prevent aflatoxicosis in chickens and swine and significantly decreases the level of aflatoxin M1 residues in the milk of lactating dairy cattle. The basic mechanism for this action appears to involve sequestration of aflatoxin in the gastrointestinal tract and chemisorption (i.e., tight binding) to HSCAS which results in a reduction in aflatoxin bioavailability. Research is in progress to elucidate the specificity of HSCAS action and to construct a series of selective chemisorbents for mycotoxin control in livestock and poultry.

Liquid chromatographic determination of aflatoxins, ochratoxin A, and zearalenone in grains, oilseeds, and animal feeds by post-column derivatization and on-line sample cleanup.

A liquid chromatographic method using on-line sample cleanup, reverse flow analytical column loading, gradient elution, and postcolumn derivatization with iodine permits direct, rapid determination of aflatoxins B1, B2, G1, and G2, as well as ochratoxin A and zearalenone. Limits of quantitation are 5 ppb for the aflatoxins and ochratoxin A and 30 ppb for zearalenone. This procedure performs well as a multimycotoxin screen for cereal grains and oilseeds, with more limited success in complete animal feeds.

Developmental toxicity of diacetoxyscirpenol in the mouse.

Mycotoxins (frequently referred to as secondary metabolites of toxigenic fungi) are commonly found in foodstuffs and are important because of their association with disease. The mycotoxin diacetoxyscirpenol, or 3-hydroxy-4,15-diacetoxy-12,13-epoxytrichothec-9-ene (DAS), is produced by numerous species of Fusarium and is reportedly toxic to humans and animals. The teratogenic potential of DAS was determined in time-mated ICR mice. DAS (dissolved in a 1:9 mixture of propylene glycol/saline) was administered intraperitoneally to pregnant mice at levels of 1.0, 1.5, 2.0, 3.0 and 6.0 mg/kg body weight in a single dose on one of gestation days 7-11 during the period of organogenesis. Term fetuses were examined for anomalies by routine teratologic procedures. Reabsorption frequency was dose-related and occurred as follows: 100% at 6.0 mg/kg on all gestation days tested; 90-99% at 3.0 mg/kg on days 7-9 and 100% on days 10 and 11; 26-51% at 2.0 mg/kg on days 7-9 and 100% on days 10 and 11; 9-77% at 1.5 mg/kg on days 7-10 and 100% on day 11; 7-34% at 1.0 mg/kg on days 7-11. A significant reduction in mean fetal body weight and a variety of fetal malformations (i.e. external and skeletal) were observed following maternal exposure to DAS. This is the first report to implicate this mycotoxin as a teratogen.

Determination of deoxynivalenol (vomitoxin) by high-performance liquid chromatography with electrochemical detection.

A rapid method for the analysis of deoxynivalenol (DON) was developed using high-performance liquid chromatography (HPLC) with reductive electrochemical detection (ED). Deoxynivalenol produced by Fusarium roseum growing on solid cornmeal and rice substrates and from naturally contaminated wheat was extracted and quantitated via ED. DON levels in wheat were verified by gas chromatography and structurally confirmed by mass spectrometry. DON was optimally resolved by HPLC employing a radially compressed octadecylsilane column and a mobile phase of deoxygenated methanol-40 mM borate buffer (35:65) at a flow-rate of 1.0 ml/min. Under these conditions DON exhibited an average retention time of 3.6 min. Reductive ED (-1.4 V) allowed a 12-fold increase in sensitivity and greater selectivity than classical UV absorption at 224 nm. A detection limit for DON of 25 pg/microliter was achieved under these conditions. The determination of DON in crude grain extracts was hindered by extractable interfering substances, whereas ED was more functional-group selective (i.e. reduction of the carbonyl moiety). ED permits a direct quantitation of DON from crude grain extracts and may facilitate the determination of this agent and associated metabolites in biological samples.