A precision apparatus, with solid phase micro-extraction monitoring capability, for incorporation studies of gaseous precursors into insect-derived metabolites

An apparatus is described that facilitates the determination of incorporation levels of isotope labelled, gaseous precursors into volatile insect-derived metabolites. Atmospheres of varying gas compositions can be generated by evacuation of a working chamber followed by admission of the required levels of component gases, using a precision, digitised pressure read-out system. Insects such as fruit-flies are located initially in a small introduction chamber, from which migration can occur downwards into the working chamber. The level of incorporation of labelled precursors is continuously assayed by the Solid Phase Micro Extraction (SPME) technique and GC-MS analyses. Experiments with both Bactrocera species (fruit-flies) and a parasitoid wasp, Megarhyssa nortoni nortoni (Cresson) and oxygen-18 labelled dioxygen illustrate the utility of this system. The isotope effects of oxygen-18 on the carbon-13 NMR spectra of 1,7- dioxaspiro[5,5]undecane are also described.

Stakeholders' assessment of welfare indicators for sheep and cattle exported by sea from Australia

An adaptive conjoint analysis was use to evaluate stakeholders' opinion of welfare indicators for ship-transported sheep and cattle, both onboard and in pre-export depots. In consultations with two nominees of each identified stakeholder group (government officials, animal welfare representatives, animal scientists, stockpersons, producers/pre-export depot operators, exporters/ship owners and veterinarians), 18 potential indicators were identified Three levels were assigned to each using industry statistics and expert opinion, representing those observed on the best and worst 5% of voyages and an intermediate value. A computer-based questionnaire was completed by 135 stakeholders (48% of those invited). All indicators were ranked by respondents in the assigned order, except fodder intake, in which case providing the amount necessary to maintain bodyweight was rated better than over or underfeeding, and time in the pre-export assembly depot, in which case 5 days was rated better than 0 or 10 days. The respective Importance Values (a relative rating given by the respondent) for each indicator were, in order of declining importance: mortality (8.6%), clinical disease incidence (8.2%), respiration rate (6.8%), space allowance (6.2%), ammonia levels (6.1%), weight change (6.0%), wet bulb temperature (6.0%), time in assembly depot (5.4%), percentage of animals in hospital pen (5.4%), fodder intake (5.2%), stress-related metabolites (5.0%), percentage of feeding trough utilised (5.0%), injuries (4.8%), percentage of animals able to access food troughs at any one time (4.8%), percentage of animals lying down (4.7%), cortisol concentration (4.5Y.), noise (3.9y.), and photoperiod (3.4%). The different stakeholder groups were relatively consistent in their ranking of the indicators, with all groups nominating the some top two and at least five of the top seven indicators. Some of the top indicators, in particular mortality, disease incidence and temperature, are already recorded in the Australian industry, but the study identified potential new welfare indicators for exported livestock, such as space allowance and ammonia concentration, which could be used to improve welfare standards if validated by scientific data. The top indicators would also be useful worldwide for countries engaging in long distance sea transport of livestock.

Screening bacterial metabolites for inhibitory effects against Batrachochytrium dendrobatidis using a spectrophotometric assay

Certain bacteria present on frog skin can prevent infection by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), conferring disease resistance. Previous studies have used agar-based in vitro challenge assays to screen bacteria for Bd-inhibitory activity and to identify candidates for bacterial supplementation trials. However, agar-based assays can be difficult to set up and to replicate reliably. To overcome these difficulties, we developed a semi-quantitative spectrophotometric challenge assay technique. Cell-free supernatants were prepared from filtered bacterial cultures and added to 96-well plates in replicated wells containing Bd zoospores suspended in tryptone-gelatin hydrolysate-lactose (TGhL) broth medium. Plates were then read daily on a spectrophotometer until positive controls reached maximum growth in order to determine growth curves for Bd. We tested the technique by screening skin bacteria from the Australian green-eyed tree frog Litoria serrata. Of bacteria tested, 31% showed some degree of Bd inhibition, while some may have promoted Bd growth, a previously unknown effect. Our cell-free supernatant challenge assay technique is an effective in vitro method for screening bacterial isolates for strong Bd-inhibitory activity. It contributes to the expanding field of bioaugmentation research, which could play a significant role in mitigating the effects of chytridiomycosis on amphibians around the world.

Feeding low protein diets to meat chickens: Effects on emissions of toxic and odorous metabolites

Low protein diet and odour emissions in meat chickens

The Biosynthesis of Sesquiterpene Isocyanides and Isothiocyanates in the Marine Sponge Acanthella cavernosa (Dendy); Evidence for Dietary Transfer to the Dorid Nudibranch Phyllidiella pustulosa

The tropical marine sponge Acanthella cavernosa (Dendy) converts potassium [14C] cyanide to axisonitrile-3 (1); this precursor is also used for the synthesis of axisothiocyanate-3 (2) suggesting that isocyanides are precursors to isothiocyanates in A. cavernosa. Likewise, potassium [14C] thiocyanate is used for the synthesis of axisothiocyanate-3; unexpectedly this precursor also labelled axisonitrile-3. These results demonstrate either an interconversion between cyanide and thiocyanate prior to secondary metabolite formation or that the secondary metabolites can themselves be interconverted. Specimens of the dorid nudibranch Phyllidiellu pustulosa, preadapted to a diet of A. cavernosa, fed on 14C-labelled sponges and were subsequently found to contain the radioactive terpenes (1) and (2). Specimens of P. pustulosa, which had not expressed a dietary preference for A. cavernosa in the field, did not generally feed in aquarium tests with 14C-labelled sponges and, therefore, provided non-radioactive extracts. Since control experiments demonstrated the inability of P. pustulosa to synthesise the metabolites de novo, we therefore conclude that P. pustulosa acquires secondary metabolites by dietary transfer from A. cavernosa.

Fungal colonization of sago starch in Papua New Guinea

Sago starch is an important source of dietary carbohydrates in lowland Papua New Guinea. Over the past 30 years there have been sporadic reports of severe illness following consumption of sago starch. A common assumption is that fungal metabolites might be associated with the illness, leading to the need for a more thorough investigation of the mycoflora of sago starch. Sago starch was collected from areas of high sago consumption in Papua New Guinea for fungal analysis (69 samples). Storage methods and duration were recorded at the time of collection and pH on arrival at the laboratory. Yeasts were isolated from all samples except two, ranging from 1.2 × 103 to 8.3 × 107 cfu/g. Moulds were isolated from 65 of the 69 samples, ranging from 1.0 × 102 to 3.0 × 106 cfu/g. Of 44 samples tested for ergosterol content, 42 samples showed the presence of fungal biomass. Statistical analyses indicated that sago starch stored for greater than five weeks yielded significantly higher ergosterol content and higher numbers of moulds than sago stored for less than five weeks. The method of storage was also shown to influence mould numbers with storage in natural woven fibre containers returning significantly greater numbers than present in other storage methods tested. Potentially mycotoxigenic genera of moulds including Aspergillus and Penicillium were commonly isolated from sago starch, and as such storage factors that influence the growth of these and other filamentous fungi might contribute to the safety of traditional sago starch in PNG.

The efficacy of a vitamin D3 metabolite for improving the myofibrillar tenderness of meat from Bos indicus cattle

The influence of a once only administration of a metabolite of vitamin D3 (HY [middle dot] D(R)-25-hydroxy vitamin D3) on myofibrillar meat tenderness in Australian Brahman cattle was studied. Ninety-six Brahman steers of three phenotypes (Indo-Brazil, US and US/European) and with two previous hormonal growth promotant (HGP) histories (implanted or not implanted with Compudose(R)) were fed a standard feedlot ration for 70 d. Treatment groups of 24 steers were offered daily 10 g/head HY [middle dot] D(R) (125 mg 25-hydroxyvitamin D3) for 6, 4, or 2 d before slaughter. One other group of 24 steers was given the basal diet without HY [middle dot] D(R). Feed lot performance, blood and muscle samples and carcass quality data were collected at slaughter. Calcium, magnesium, potassium, sodium, iron and Vitamin D3 metabolites were measured in plasma and longissimus dorsi muscle. Warner-Bratzler (WB) shear force (peak force, initial yield) and other objective meat quality measurements were made on the longissimus dorsi muscle of each steer after ageing for 1, 7 and 14 d post-mortem at 0-2 [deg]C.There were no significant effects of HY [middle dot] D(R) supplements on average daily gain (ADG, 1.28-1.45 kg/d) over the experimental period. HY [middle dot] D(R) supplements given 6 d prior to slaughter resulted in significantly higher (P (R)) by phenotype/HGP interaction for peak force (P = 0.028), in which Indo-Brazil steers without previous HGP treatment responded positively (increased tenderness) to HY [middle dot] D(R) supplements at 2 d when compared with Indo-Brazil steers previously given HGP. There were no significant effects of treatment on other phenotypes. HY [middle dot] D(R) supplements did not affect muscle or plasma concentrations of calcium, potassium or sodium, but did significantly decrease plasma magnesium and iron concentrations when given 2 d before slaughter. There were no detectable amounts of 25-hydroxyvitamin D3 in the blood or muscle of any cattle at slaughter.

The environmental fate of diuron under a conventional production regime in a sugarcane farm during the plant cane phase

BACKGROUND: Field studies of diuron and its metabolites 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU), 3,4-dichlorophenylurea (DCPU) and 3,4-dichloroaniline (DCA) were conducted in a farm soil and in stream sediments in coastal Queensland, Australia. RESULTS: During a 38 week period after a 1.6 kg ha^-1 diuron application, 70-100% of detected compounds were within 0-15 cm of the farm soil, and 3-10% reached the 30-45 cm depth. First-order t1/2 degradation averaged 49 ± 0.9 days for the 0-15, 0-30 and 0-45 cm soil depths. Farm runoff was collected in the first 13-50 min of episodes lasting 55-90 min. Average concentrations of diuron, DCPU and DCPMU in runoff were 93, 30 and 83-825 µg L^-1 respectively. Their total loading in all runoff was >0.6% of applied diuron. Diuron and DCPMU concentrations in stream sediments were between 3-22 and 4-31 µg kg^-1 soil respectively. The DCPMU/diuron sediment ratio was >1. CONCLUSION: Retention of diuron and its metabolites in farm topsoil indicated their negligible potential for groundwater contamination. Minimal amounts of diuron and DCMPU escaped in farm runoff. This may entail a significant loading into the wider environment at annual amounts of application. The concentrations and ratio of diuron and DCPMU in stream sediments indicated that they had prolonged residence times and potential for accumulation in sediments. The higher ecotoxicity of DCPMU compared with diuron and the combined presence of both compounds in stream sediments suggest that together they would have a greater impact on sensitive aquatic species than as currently apportioned by assessments that are based upon diuron alone.

The physiological and metabolic impacts on sheep and cattle of feed and water deprivation before and during transport

Sheep and cattle are frequently subjected to feed and water deprivation (FWD) for about 12 h before, and then during, transport to reduce digesta load in the gastrointestinal tract. This FWD is marked by weight loss as urine and faeces mainly in the first 24 h but continuing at a reduced rate subsequently. The weight of rumen contents falls although water loss is to some extent masked by saliva inflow. FWD is associated with some stress, particularly when transportation is added. This is indicated by increased levels of plasma cortisol that may be partly responsible for an observed increase in the output of water and N in urine and faeces. Loss of body water induces dehydration that may induce feelings of thirst by effects on the hypothalamus structures through the renin-angiotensin-aldosterone system. There are suggestions that elevated cortisol levels depress angiotensin activity and prevent sensations of thirst in dehydrated animals, but further research in this area is needed. Dehydration coupled with the discharge of Na in urine challenges the maintenance of homeostasis. In FWD, Na excretion in urine is reduced and, with the reduction in digesta load, Na is gradually returned from the digestive tract to the extracellular fluid space. Control of enteropathogenic bacteria by normal rumen microbes is weakened by FWD and resulting infections may threaten animal health and meat safety. Recovery time is required after transport to restore full feed intake and to ensure that adequate glycogen is present in muscle pre-slaughter to maintain meat quality.

Regrouping unfamiliar animals in the weeks prior to slaughter has few effects on physiology and meat quality in Bos taurus feedlot steers

The response of cattle to alterations in social groupings can lead to physiological changes that affect meat quality. Feedlot practices frequently lead to a proportion of cattle in a pen being drafted for slaughter with the balance retained for a further period until they meet market specifications. An ability to regroup such retained cattle for short periods without consequences for meat quality would facilitate efficient use of feedlot pen space. The current experiment examined the impact on physiological variables and meat quality of regrouped British breed steers 4, 2 or 1 week before dispatch for slaughter. There was little effect of regrouping cattle on physiological variables associated with stress responses. Physical assessment of meat quality indicated that regrouping steers 1 week before slaughter led to higher compression and a tendency for higher peak force values in animals from one genotype than in their respective controls (1.89 v. 1.71 ± 0.05 kg, P = 0.017); however, these assessments were not matched by changes in sensory perception of meat quality. Average daily gain during feedlot finishing was negatively related to the temperament measure and flight time. It was also associated with breed, white cell count, plasma cortisol and haemoglobin at the midpoint of the 70-day finishing period. The results confirm the impact of flight time on growth rate during feedlot finishing and that regrouping cattle less than 2 weeks before slaughter may reduce meat quality.

Review: Near infrared spectroscopy of faeces to evaluate the nutrition and physiology of herbivores

Near infrared (NIR) spectroscopy, usually in reflectance mode, has been applied to the analysis of faeces to measure the concentrations of constituents such as total N, fibre, tannins and delta C-13. In addition, an unusual and exciting application of faecal NIR [F.NIR] analyses is to directly predict attributes of the diet of herbivores such as crude protein and fibre contents, proportions of plant species and morphological components, diet digestibility and voluntary DM intake. This is an unusual application of NIR spectroscopy insofar as the spectral measurements are made, not on the material of interest [i.e. the diet), but on a derived material (i.e. faeces). Predictions of diet attributes from faecal spectra clearly depend on there being sufficient NIR spectral information in the diet residues present in faeces to describe the diet, although endogenous components of faeces such as undigested debris of micro-organisms from the rumen and Large intestine and secretions into the gastrointestinal tract wilt also contribute spectral information. Spectra of forage and of faeces derived from the forage are generally similar and the observed differences are principally in the spectral regions associated with constituents of forages known to be of low, or of high, digestibility. Some diet components (for example, ureal which are likely to be entirely digested apparently cannot be predicted from faecal NIR spectra because they cannot contribute to faecal spectra except through modifying the microbial and endogenous components. The errors and robustness of F.NIR calibrations to predict the crude protein concentration and digestibility of the diet of herbivores are generally comparable with those to directly predict the same attributes in forage from NIR spectra of the forage. Some attributes of the animal, such as species, gender, pregnancy status and parasite burden have been successfully discriminated into classes based on their faecal NIR spectra. Such discrimination was likely associated with differences in the diet selected and/or differences in the metabolites excreted in the faeces. NIR spectroscopy of faeces has usually involved scanning dried and ground samples in monochromators in the 400-2500nm or 1100-2500nm ranges. Results satisfactory for the purpose have also been reported for dried and ground faeces scanned using a diode array instrument in the 800-1700nm range and for wet faeces and slurries of excreta scanned with monochromators. Chemometric analysis of faecal spectra has generally used the approaches established for forage analysis. The capacity to predict many attributes of the diet, and some aspects of animal physiology, from NIR spectra of faeces is particularly useful to study the quality and quantity of the diet selected by both domestic and feral grazing herbivores and to enhance production and management of both herbivores and their grazing environment.

Corymbia leaf oils, latitude, hybrids and herbivory: a test using common-garden field trials

Foliar oils, particularly monoterpenes, can influence the susceptibility of plants to herbivory. In plants, including eucalypts, monoterpenes are often associated with plant defence. A recent analysis revealed an increase in foliar oil content with increasing latitudinal endemism, and we tested this pattern using three eucalypt taxa comprising a latitudinal replacement cline. We also examined the relative concentrations of two monoterpenes (alpha-pinene and 1,8-cineole), for which meta-analyses also showed latitudinal variation, using hybrids of these three taxa with Corymbia torelliana. These, and pure C. torelliana, were then assessed in common-garden field plots for the abundance and distribution of herbivory by four distinct herbivore taxa. Differing feeding strategies among these herbivores allowed us to test hypotheses regarding heritability of susceptibility and relationships to alpha-pinene and 1,8-cineole. We found no support for an increase in foliar oil content with increasing latitude, nor did our analysis support predictions for consistent variation in alpha-pinene and 1,8-cineole contents with latitude. However, herbivore species showed differential responses to different taxa and monoterpene contents. For example, eriophyid mites, the most monophagous of our censused herbivores, avoided the pure species, but fed on hybrid taxa, supporting hypotheses on hybrid susceptibility. The most polyphagous herbivore (leaf blister sawfly Phylacteophaga froggatti) showed no evidence of response to plant secondary metabolites, while the distribution and abundance patterns of Paropsis atomaria showed some relationship to monoterpene yields.

Evaluation of the impacts of spaying by either the dropped ovary technique or ovariectomy via flank laparotomy on the welfare of Bos indicus beef heifers and cows

The welfare outcomes for Bos indicus cattle (100 heifers and 50 cows) spayed by either the dropped ovary technique (DOT) or ovariectomy via flank laparotomy (FL) were compared with cattle subjected to physical restraint (PR), restraint by electroimmobilization in conjunction with PR (EIM), and PR and mock AI (MAI). Welfare assessment used measures of morbidity, mortality, BW change, and behavior and physiology indicative of pain and stress. One FL heifer died at d 5 from peritonitis. In the 8-h period postprocedures, plasma bound cortisol concentrations of FL, DOT, and EIM cows were not different and were greater (P < 0.05) than PR and MAI. Similarly, FL and DOT heifers had greater (P < 0.05) concentrations than PR and MAI, with EIM intermediate. Creatine kinase and aspartate aminotransferase concentrations were greater (P < 0.05) in FL and EIM heifers compared with the other treatments, with a similar pattern seen in the cows. Haptoglobin concentrations were significantly (P < 0.05) increased in the FL heifers compared with other treatments in the 8- to 24-h and 24- to 96-h periods postprocedures, and in cows were significantly (P < 0.05) increased in the FL and DOT compared with PR in the 24- to 96-h period. Behavioral responses complemented the physiological responses; standing head down was shown by more (P < 0.05) FL cows and heifers to 3 d postprocedures compared with other treatments, although there was no difference between FL and DOT heifers at the end of the day of procedures. At this same time, fewer (P < 0.05) FL and DOT heifers and cows were observed feeding compared with other treatments, although in cows there was no difference between FL, DOT, and EIM. There were no significant differences (P > 0.05) between treatments in BW changes. For both heifers and cows, FL and DOT spaying caused similar levels of acute pain, but FL had longer-lasting adverse impacts on welfare. Electroimmobilization during FL contributed to the pain and stress of the procedure. We conclude that: i) FL and DOT spaying should not be conducted without measures to manage the associated pain and stress; ii) DOT spaying is preferable to FL spaying; iii) spaying heifers is preferable to spaying cows; and iv) electroimmobilization causes pain and stress and should not be routinely used as a method of restraint.

Earthy-muddy tainting of cultured barramundi linked to geosmin in tropical northern Australia

Tainting of outdoor pond-reared barramundi Lates calcarifer by muddy-earthy off-flavours is frequently reported across tropical Australia. To investigate the possible causes and effects of off-flavour tainting, we analysed water samples from outdoor rearing ponds for the presence of geosmin (GSM) and 2-methylisoborneol (2-MIB), 2 microbial metabolites often associated with tainting episodes. We then conducted controlled dose-effect experiments which measured the accumulation of tainting metabolites in the flesh, and the impact tainting had on taste and flavour attributes. GSM was deemed to be the compound most likely responsible for off-flavour tainting, persisting at moderate (similar to 1.00 mu g l(-1)) to extreme levels (similar to 14.36 mu g l(-1)), while 2-MIB was never detected during the study. Controlled experiments revealed that the accumulation of GSM in the flesh of market-sized barramundi was directly related to GSM levels of the holding water (0 to similar to 4 mu g l(-1)), with higher levels resulting in significant increases in undesirable taste and flavour attributes, particularly muddy-earthy flavour and weedy aftertaste. We identified the sensory detection threshold for GSM in farmed barramundi to be <= 0.74 mu g kg(-1), similar to estimates for GSM detection in rainbow trout Oncorhynchus mykiss (similar to 0.9 mu g kg(-1)) and for 2-MIB in channel catfish Ictalurus punctatus (0.7 mu g kg(-1)). Quantitative estimation of flesh-bound GSM using gas chromatography-mass spectrometry (GC-MS) agreed well with human sensory assessment scores and highlights the reliability of chemical analysis of GSM in barramundi flesh while also indicating the value of GC-MS analysis in predicting the impact of GSM on the sensory properties of farmed barramundi.

Consequences of Corymbia (Myrtaceae) hybridisation on leaf-oil profiles

The present study examines patterns of heritability of plant secondary metabolites following hybridisation among three genetically homogeneous taxa of spotted gum (Corymbia henryi (S.T.Blake) K.D.Hill & L.A.S.Johnson, C. citriodora subsp. variegata (F.Muell.) K.D.Hill & L.A.S.Johnson and C. citriodora (Hook.) K.D.Hill & L.A.S.Johnson subsp. citriodora (section Maculatae), and their congener C. torelliana (F.Muell.) K.D.Hill & L.A.S.Johnson (section Torellianae)). Hexane extracts of leaves of all four parent taxa were statistically distinguishable (ANOSIM: global R = 0.976, P = 0.008). Hybridisation patterns varied among the taxa studied, with the hybrid formed with C. citriodora subsp. variegata showing an intermediate extractive profile between its parents, whereas the profiles of the other two hybrids were dominated by that of C. torelliana. These different patterns in plant secondary-metabolite inheritance may have implications for a range of plant-insect interactions.