Indole-diterpenes are an important class of chemical compounds which can be unique to different fungal species. The highly complex lolitrem compounds are confined to Epichloë species, whilst penitrem production is confined to Penicillium spp. and Aspergillus spp. These fungal species are often present in association with pasture grasses, and the indole-diterpenes produced may cause toxicity in grazing animals. In this review, we highlight the unique structural variations of indole-diterpenes that are characterised into subgroups, including paspaline, paxilline, shearinines, paspalitrems, terpendoles, penitrems, lolitrems, janthitrems, and sulpinines. A detailed description of the unique biological activities has been documented where even structurally related compounds have displayed unique biological activities. Indole-diterpene production has been reported in two classes of ascomycete fungi, namely Eurotiomycetes (e.g., Aspergillus and Penicillium) and Sordariomycetes (e.g., Claviceps and Epichloë). These compounds all have a common structural core comprised of a cyclic diterpene skeleton derived from geranylgeranyl diphosphate (GGPP) and an indole moiety derived from tryptophan. Structure diversity is generated from the enzymatic conversion of different sites on the basic indole-diterpene structure. This review highlights the wide-ranging biological versatility presented by the indole-diterpene group of compounds and their role in an agricultural and pharmaceutical setting.

Perennial ryegrass (Lolium perenne L.), an economically important pasture and turf grass, is commonly infected with asexual Epichloë species endophytes. Endophytes provide enhanced bioprotection by producing alkaloids, and research often focusses on the negative impact on grazing animals. However, alkaloid distribution throughout the plant and their role in biocontrol of insect pests and diseases are less well understood. Additionally, intermediate compounds have not been investigated for their impacts on animal welfare and biological control in pasture-based scenarios. Here, a single liquid chromatography–mass spectrometry (LC-MS) method was used to measure seven alkaloids in different perennial ryegrass tissues infected with SE or NEA12 endophytes. High alkaloid recoveries and a clear plant matrix effect emphasize the importance of using matrix-matched standards for accurate quantitation. The method is sensitive, detecting alkaloids at low concentrations (nanogram levels), which is important for endophyte strains that produce compounds detrimental to livestock. Concentrations were generally highest in seeds, but distribution differed in the shoots/roots: peramine, terpendole E, terpendole C and lolitrem B were higher in shoots, whilst ergovaline, paxilline and epoxy-janthitrem I were more evenly distributed throughout the two tissues. Knowledge of alkaloid distribution may allow for concentrations to be predicted in roots based on concentrations in the shoots, thereby assisting future determinations of resistance to insects, especially subterranean root-feeding pests.

Background: The presence and concentration of lipids in serum of dairy cows have significant implications for both animal health and productivity and are potential biomarkers for several common diseases. However, information on serum lipid composition is rather fragmented, and lipid remodelling during the transition period is only partially understood. Methods: Using a combination of reversed-phase liquid chromatography-mass spectrometry (RP-LC-MS), hydrophilic interaction-mass spectrometry (HILIC-MS), and lipid annotation software, we performed a comprehensive identification and quantification of serum of dairy cows in pasture-based Holstein-Friesian cows. The lipid remodelling induced by negative energy balance was investigated by comparing the levels of all identified lipids between the fresh lactation (5–14 days in milk, DIM) and full lactation (65–80 DIM) stages. Results: We identified 535 lipid molecular species belonging to 19 classes. The most abundant lipid class was cholesteryl ester (CE), followed by phosphatidylcholine (PC), sphingomyelin (SM), and free fatty acid (FFA), whereas the least abundant lipids included phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), acylcarnitine (AcylCar), ceramide (Cer), glucosylceramide (GluCer), and lactosylceramide (LacCer). Conclusions: A remarkable increase in most lipids and a dramatic decrease in FFAs, AcylCar, and DHA-containing species were observed at the full lactation compared to fresh lactation stage. Several serum lipid biomarkers for detecting negative energy balance in cows were also identified.

Lolitrem B is the most potent indole-diterpene mycotoxin produced by Epichloë festucae var. lolii (termed LpTG-1), with severe intoxication cases reported in livestock. To date, there are no in vivo metabolism studies conducted for the mycotoxin. A mouse model assay established for assessing toxicity of indole-diterpenes was used to investigate metabolic products of lolitrem B. Mice were administered lolitrem B at 0.5 and 2.0 mg/kg body weight (b.wt) intraperitoneally before body and brain tissues were collected at 6 h and 24 h post-treatment. Samples were cryoground and subjected to a biphasic or monophasic extraction. The aqueous and lipophilic phases were analysed using liquid chromatography high-resolution mass spectrometry (LC–HRMS); data analysis was performed with Compound Discoverer™ software. A total of 10 novel phase I metabolic products were identified in the lipophilic phase and their distribution in the liver, kidney and various brain regions are described. The biotransformation products of lolitrem B were found to be present in low levels in the brain. Based on structure–activity postulations, six of these may contribute towards the protracted tremors exhibited by lolitrem B-exposed animals.

Identifying physiological determinants of dairy cow survival and their potential modulation by parity may reveal opportunities to improve herd health and longevity. This multisite, prospective, observational study investigated culling and mortality hazards using targeted lipidomic and standard metabolite assays. Blood samples, stratified by parity, were collected from 2 cow cohorts (1) dry and (2) peak-milk, from across 29 commercial Australian farms (14 pasture-based, 15 confinement-based). There were 717 nonlactating, late-pregnant, dry cows (∼27 d prepartum) and 794 peak-milk cows (∼58 DIM) sampled. A total of 186 lipid species (including glycerophospholipids, sphingomyelin, and triacylglycerols) and 11 routinely measured metabolites were evaluated. Sample cows were followed for an average of 693 d and exit reasons recorded. Competing risk survival models were used to estimate the cumulative incidence of culling and mortality by parity and cohort. Blood analytes were autoscaled within cohort and farm, controlling for farm-level effect on metabolites. Survival analysis was performed using an adaptive least absolute shrinkage and selection operator (LASSO) Cox full likelihood model that explored associations among blood analytes and hazards of removal, with a shared frailty of farm (accounting for farm-level baseline hazards), and 2 removal outcomes considered: culling (censoring: death, cull from farm accident, end of follow-up) and mortality (censoring: cull, death from farm accident, end of follow-up). Separate models were used to estimate survival outcome by cohort and parity groupings (first, second and third, and greater than third) or with parity as a categorical covariate. Due to high correlations, analyte data were reduced to 25 clusters using Ward's hierarchical clustering criterion. Bootstrapping of the LASSO variable selection procedure identified clusters with high selection frequency for use in the final model. The hazards and cumulative incidence of culling and mortality increased with parity. Glycerophospholipids with very-long-chain n-3 fatty acids were associated with reduced hazards of culling in parity 1 peak-milk cows (0.39 hazard ratio [HR]), whereas glycerophospholipids with n-3 α-linolenic acid were associated with reduced hazards of culling in parity >3 peak-milk cows (0.18 HR). Sphingomyelin with >C18 fatty acyl chains was associated with increased hazards of culling in parity >3 peak-milk cows (2.02 HR). Clusters containing the routinely evaluated analytes albumin, globulin, urea, magnesium, glucose, triglycerides, β-hydroxybutyric acid, bilirubin, and nonesterified fatty acid were associated with culling and mortality, consistent with their roles in health and reproduction. Lipids collected from dry cows were poor predictors of survival. Many novel plasma lipid targets for future research into survival of cattle were identified, with proinflammatory lipid profiles associated with increased risk of culling and mortality.

Dairy cow housing and management can be broadly described as either intensive confinement-based (CONFINE) or extensive pasture-based (PAST) systems. The diets between systems typically differ in their forage base, with CONFINE farms often utilizing maize silage in a TMR. Consequently, the lipid composition of diets differs between systems. The influence of housing system on blood lipidomics is currently unknown, but due to the bioactive role of lipids in influencing overall health and productivity, differences in diet may have consequences for reproduction, health, and aging of cows. The objective of this cross-sectional, multisite study was to investigate blood lipids and metabolites from cows in PAST and CONFINE systems, in the dry period (∼27 d prepartum) and at peak milk (∼58 DIM). After exclusions, blood samples from 303 PAST and 398 CONFINE dry-period cows and 350 PAST and 431 CONFINE peak-milk cows from 15 PAST and 15 CONFINE farms were analyzed. A total of 185 lipid species (including glycerophospholipids, sphingomyelins, and triacylglycerols) were evaluated using targeted liquid chromatography-MS, as were 11 routinely measured metabolites. Dry and peak-milk cohorts were analyzed separately throughout. Lipids and metabolites associated with housing system were selected using a variable stabilization approach that was achieved by calculating the frequency of inclusion in categorical (housing system) penalized models using bootstrapping. Variables were retained if inclusion frequency exceeded a false-positive threshold. Five different statistical models were used with variable stabilization. Dry cows in CONFINE systems had decreased globulin, urea, and glycerophospholipids associated with n-3 fatty acids. The highest total inclusion rates in the dry cohort were phosphatidylcholine (PC; 36:5), which mostly comprises palmitic acid (C16:0) and eicosapentaenoic acid (EPA; 20:5n-3), then phosphatidylethanolamine (PE; 38:5, 16:0/22:5n-3 or 18:0/EPA) and PC(34:3; 16:0/18:3 α-linolenic acid [ALA]). No lipids were increased in more than one stabilized model in CONFINE dry cows. Peak-milk CONFINE cows had increased glycerophospholipids associated with n-6 fatty acids. The highest total inclusion-rate lipids in the peak-milk cohort were phosphatidylinositol (PI; 38:3; 18:0/20:3n-6 dihomo-γ-linolenic acid), PC(34:2; 16:0/18:2 linoleic acid [LA]), PC(40:7; 18:2/22:5n-6), PC(34:1; 16:0/18:1), and PE(34:2; 16:0/LA). The CONFINE peak-milk cows also had decreased PC(34:3; 16:0/ALA). This study identified specific lipids that were strongly associated with housing systems, findings that have not been reported elsewhere. Given the important biological functions of omega fatty acids, the pattern of glycerophospholipids with increased n-6 and decreased n-3 in CONFINE cows may indicate housing systems create different risk profiles for reproduction, health, and aging.