In vitro analysis of the anthelmintic activity of forage chicory (Cichorium intybus L.) sesquiterpene lactones against a predominantly Haemonchus contortus egg population.

The anthelmintic activity of chicory (Cichorium intybus L.) herbage has been attributed to sesquiterpene lactones. Chicory leaves contain significant amounts of lactucin (LAC), 8-deoxylactucin (DOL), and lactucopicrin (LPIC), but the proportions of these three sesquiterpene lactones vary among forage chicory cultivars. To determine whether the individual compounds differ in anthelmintic activity, we prepared sesquiterpene lactone-enriched extracts from leaves of two forage chicory cultivars, 'Grasslands Puna' (Puna) and 'Forage Feast', and tested their effects on the hatching of a predominantly Haemonchus contortus egg population. The dominant constituents in the Puna and Forage Feast extracts were DOL and LAC, respectively; LPIC concentrations in the two extracts were similar. Extracts from both cultivars inhibited egg hatching at all concentrations tested (P<0.001), but there were significant differences in egg responses to the two extracts (P<0.001). With Puna, egg hatching decreased sharply in a linear fashion when the combined LAC, DOL, and LPIC concentrations ranged from 0 to 5.0mg/ml. A biphasic effect on egg hatching occurred with the Forage Feast extract. The fraction of eggs that hatched decreased gradually to 65% as the sesquiterpene lactone concentrations increased from 0 to 6.7 mg/ml. Treatment with higher concentrations resulted in a sharp decline in egg hatchability. Concentrations of sesquiterpene lactones required for 50% lethality were determined by probit dose-effect analysis to be 2.6 mg/ml (95% confidence interval: 2.4-2.8 mg/ml) for the Puna extract and 6.4 mg/ml (95% confidence interval: 5.9-7.2mg/ml) for the Forage Feast extract (P<0.0001). These concentrations provided 1.3 and 1.5mg/ml of DOL and 0.8 and 3.9 mg/ml of LAC for Puna and Forage Feast extracts, respectively. Results suggest that LAC has minimal effect on egg hatching and that DOL or other constituent(s) in the extracts is inhibitory. Quantitative analyses of free sesquiterpene lactones in chicory leaf extracts suggest that Puna may be a better cultivar than Forage Feast for use in bioactive pastures for gastrointestinal parasite control in small ruminants.

Efficacy of an orange oil emulsion as an anthelmintic against Haemonchus contortus in gerbils (Meriones unguiculatus) and in sheep.

Haemonchus contortus is a blood-sucking abomasal parasite responsible for major losses to small ruminant producers worldwide. The recent increase in populations of anthelmintic resistant parasites has produced a demand for alternative control methods. An orange oil emulsion that has shown activity against plant parasitic nematodes and H. contortus in vitro was assessed for activity against H. contortus in a gerbil model and in the natural ovine host. In gerbil experiments, animals were infected with 600 infective third stage (L3) H. contortus larvae. In one experiment, gerbils were treated with 600 milligrams per kilogram bodyweight (mg/kg BW) orange oil once or daily for 5 days. In a second experiment, gerbils were treated with 1200 mg/kg BW orange oil once or daily for 5 days. On Day 9 post-infection, gerbils were killed, their stomachs removed, and the worms counted. The 600 mg/kg BW dosage caused 7% and 62.6% parasite reduction compared to a control group when given once or daily for 5 days, respectively. The 1200 mg/kg BW dosage of orange oil caused 25% and 87.8% parasite reduction compared to a control group when given once or daily for 5 days, respectively. The difference between the multiple treatment and control group were significant at both dosages (P<0.005). In the sheep trial, 18 lambs were orally inoculated with 10,000 L3 H. contortus. One month later, two groups of six lambs each were dosed with 600 mg/kg BW orange oil either once or daily for 3 days. Fecal egg counts were monitored daily starting on the first day of treatment (Day 0) and continuing for 14 days. Results showed that a single dose of the product caused high fecal egg count reduction (97.4%) compared to control sheep. Egg counts were significantly reduced by Day 2 (P<0.0001). Thus, the orange oil emulsion may potentially be useful in the control of ovine haemonchosis.

Comparison of diffuse reflectance fourier transform mid-infrared and near-infrared spectroscopy with grating-based near-infrared for the determination of fatty acids in forages.

Diffuse reflectance Fourier transform mid infrared (FTMIR) and near-infrared spectroscopy (FTNIR) were compared to scanning monochromator-grating-based near-infrared spectroscopy (SMNIR), for their ability to quantify fatty acids (FA) in forages. A total of 182 samples from thirteen different forage cultivars and three different harvest times were analyzed. Three calibration analyses were conducted for lauric (C12:0), myristic (C14:0), palmitic (C16:0), stearic (C18:0), palmitoleic (C16:1), oleic (C18:1), linoleic (C18:2), and alpha-linolenic (C18:3) acids. When all samples were used in a one-out partial least squares (PLS) calibration, the average R (2) were FTNIR (0.95) > SMNIR (0.94) > FTMIR (0.91). Constituents C18:2 and C16:0 had among the highest R (2) regardless of the spectroscopic method used. The FTNIR did better for C12:0, C14:0, and C18:3. The SMNIR did better for C16:0, C16:1, C18:0, C18:1, and C18:2. A second set of calibrations developed with half of the samples as the calibration set and the rest as the validation set showed that all the methods produce acceptable calibrations, with calibration R (2) above 0.9 for most constituents. However, the SMNIR had a better average calibration relative error than the FTNIR, which was slightly better than the FTMIR. A third set of calibration equations developed using 100 random PLS runs with the 182 samples split randomly also shows that the three spectral methods are satisfactory for predicting FA. It is not clear whether any of the spectral methods is distinctly better than another. Calibration R (2) and validation R (2) were higher for most FA with the SMNIR than the FTMIR and FTNIR.

Quantification of fatty acids in forages by near-infrared reflectance spectroscopy.

Near-infrared reflectance spectroscopy (NIRS) was evaluated as a possible alternative to gas chromatography (GC) for the quantitative analysis of fatty acids in forages. Herbage samples from 11 greenhouse-grown forage species (grasses, legumes, and forbs) were collected at three stages of growth. Samples were freeze-dried, ground, and analyzed by GC and NIRS techniques. Half of the 195 samples were used to develop an NIRS calibration file for each of eight fatty acids, with the remaining half used as a validation data set. Spectral data, collected over a wavelength range of 1100-2498 nm, were regressed against GC data to develop calibration equations for lauric (C12:0), myristic (C14:0), palmitic (C16:0), stearic (C18:0), palmitoleic (C16:1), oleic (C18:1), linoleic (C18:2), and alpha-linolenic (C18:3) acids. Calibration equations had high coefficients of determination for calibration (0.93-0.99) and cross-validation (0.89-0.98), and standard errors of calibration and cross-validation were < 20% of the respective means. Simple linear regressions of NIRS results against GC data for the validation data set had r2 values ranging from 0.86 to 0.97. Regression slopes for C12:0, C14:0, C16:0, C18:0, C16:1, C18:2, and C18:3 were not significantly different (P = 0.05) from 1.0. The regression slope for C18:1 was 1.1. The ratio of standard error of prediction to standard deviation was > 3.0 for all fatty acids except C12:0 (2.6) and C14:0 (2.9). Validation statistics indicate that NIRS has high prediction ability for fatty acids in forages. Calibration equations developed using data for all plant materials accurately predicted concentrations of C16:0, C18:2, and C18:3 in individual plant species. Accuracy of prediction was less, but acceptable, for fatty acids (C12:0, C14:0, C18:0, C16:1, and C18:1) that were less prevalent.

Influence of cultivation site on sesquiterpene lactone composition of forage chicory (Cichorium intybus L.).

The forage potential of chicory (Cichorium intybus L.) has not been realized in southern West Virginia (WV) because ruminants are reluctant to consume the herbage. Chicory contains bitter sesquiterpene lactones that can adversely impact palatability. This study was undertaken to determine whether sesquiterpene lactone concentrations in chicory grown in southern WV differ from those in chicory grown in central Pennsylvania (PA) where chicory is grazed readily. Herbage was collected in 1997 and 1998 from cultivars Grasslands Puna (Puna), INIA le Lacerta (Lacerta), and Forage Feast established at research sites near State College, PA, and Beckley, WV. The total concentration of sesquiterpene lactones in WV-grown cultivars was 0.58% (dry matter basis) in Puna, 0.59% in Lacerta, and 0.79% in Forage Feast in 1997 and ranged from 1.03 (Lacerta) to 1.52% (Forage Feast) in 1998. In PA-grown cultivars, sesquiterpene lactones represented 0.16 (Puna), 0.18 (Lacerta), and 0.27% (Forage Feast) of the forage dry matter in 1997 and ranged from 0.32 (Lacerta) to 0.55% (Forage Feast) in 1998. Concentrations of lactucin, lactucopicrin, and total sesquiterpene lactones in Forage Feast exceeded those in the other cultivars grown at the same site. The lowest concentrations of lactucopicrin and total sesquiterpene lactones observed among WV-grown cultivars were higher (2-fold or more) than the highest concentrations present in cultivars grown the same year in PA. Mineral analyses of soils from the two cultivation sites indicate that P availability may influence sesquiterpene lactone composition of chicory herbage. Results provide a foundation for future studies of environmental effects on sesquiterpene lactone composition and palatability of chicory herbage.

Fatty acid composition of traditional and novel forages.

Managing the fatty acid composition of grazing ruminant diets could lead to meat and milk products that have higher conjugated linoleic acid (CLA) concentrations, but forage fatty acid dynamics must be more fully understood for a range of forages before grazing systems can be specified. The fatty acid profiles of 13 different forages, including grasses, legumes, and forbs, grown under greenhouse conditions, were determined. Three separate harvests, at 3-week intervals, were made of each plant material. alpha-Linolenic [C18:3, 7.0-38.4 mg g(-1) of dry matter (DM)], linoleic (C18:2, 2.0-10.3 mg g(-1) of DM), and palmitic (C16:0, 2.6-7.5 mg g(-1) of DM) acids were the most abundant fatty acids in all species at each harvest, together representing approximately 93% of the fatty acids present. Concentrations of fatty acids declined as plants developed, but the fractional contribution of each fatty acid to total fatty acids remained relatively stable over time. Grasses had a uniform composition across species with a mean of 66% of total fatty acids provided by C18:3, 13% by C18:2, and 14% by C16:0. The fractional contribution of C18:3 to total fatty acids was lower and more variable in forbs than in grasses. Intake of fatty acid by grazing ruminants would be affected by the forage species consumed.