A.L. Molan

Effect of flavan-3-ols on in vitro egg hatching, larval development and viability of infective larvae of Trichostrongylus colubriformis.

The effects of flavan-3-ols (the monomer units of condensed tannins (CT)) and their galloyl derivatives on the viability of eggs, the development of first stage (L1) larvae, and the viability of the infective larvae of Trichostrongylus colubriformis were investigated under in vitro conditions. Each of the flavan-3-ol gallates showed some inhibition of egg hatching at 100 microg/ml, and 100% inhibition at 1000 microg/ml, with epigallocatechin gallate being the most effective in the egg hatch (EH) assay. In contrast, none of the flavan-3-ols were able to completely inhibit egg hatching. The flavan-3-ols and galloyl derivatives dose-dependently inhibited the development of infective larvae as assessed by the larval development (LD) assay. A larval migration inhibition (LMI) assay was used to assess the effect of flavan-3-ols and their galloyl derivatives on the motility of the infective third-stage (L3) larvae of T. colubriformis. In general, the flavan-3-ol gallates were more effective than the flavan-3-ols at immobilising the infective larvae as evidenced by their ability to inhibit more (P<0.05-0.01) larvae from passing through the LMI sieves. At 500 microg/ml, epigallocatechin gallate inhibited significantly more (P<0.1) larvae from passing through the sieves than did catechin gallate, epicatechin gallate, or gallocatechin gallate. Comparisons were made between the flavan-3-ols and their galloyl derivatives with the in vitro effects of CT extracts from several forage legumes, which have exhibited effects on parasites in vivo. The forage legumes tested at 200-500 microg/ml reduced the proportion of eggs that hatch, with comparable results to those obtained using the flavan-3-ols. The activities may be influenced by the prodelphinidin: procyanidin (PD:PC) ratios: CT extracts from Lotus pendunculatus and sainfoin have PD:PC ratios of 70:30 and 77:23, respectively, whereas the less active CT extract from Lotus corniculatus has a PD:PC ratio of 27:73. The active CT extracts from forage legumes have epigallocatechin as the dominant flavan-3-ol extender unit, and epigallocatechin is the most active flavan-3-ol in both the EH and LD assays.

Effect of condensed tannins on egg hatching and larval development of Trichostrongylus colubriformis in vitro

The effects of condensed tannins extracted from seven forages on the viability of the eggs and first stage (L1) larvae of the sheep nematode Trichostrongylus colubriformis were evaluated in in vitro assays. The extracts of condensed tannins were obtained from Lotus pedunculatus (LP), Lotus corniculatus (LC), sulla (Hedysarum coronarium), sainfoin (Onobrychus viciifolia), Dorycnium pentaphylum (DP), Dorycnium rectum (DR) and dock (Rumex obtusifolius). Extracts containing 200 to 500,μg/ml reduced the proportion of eggs that hatched. The larval development assay was used to evaluate the effect of the extracts on the development of either eggs or LI larvae to L3 infective larvae. Development was allowed to proceed for seven days by which time the larvae in control incubations had reached the infective L3 stage. Extracts containing 200 μg/mI from LP, DP, DR or dock prevented egg development, and only 11, 8 and 2 per cent of the eggs developed to 13 larvae with extracts from LC, sulla and sainfoin, respectively. When the concentration was 400 μg/mI no eggs developed to L3 larvae. The addition of the extracts after hatching also inhibited the development of Ll to L3 larvae; 200 μg/ml extracted from LP, LC, sulla, sainfoin, DP, DR and dock resulted in only 14, 18, 17, 15, 14, 16 and 4 per cent of Ll larvae developing to the L3 stage compared with 85 per cent for controls, and 400 μg/ml further reduced the development of Li larvae. Statistical analyses showed that when the extracts were added before hatching they were significantly (P<0.001) more effective at inhibiting the larval development than when they were added after hatching. The condensed tannins from dock had the greatest inhibitory effect on egg development followed by the tannins from DR, sainfoin, DP, LP, sulla and LC.

Effects of condensed tannins and crude sesquiterpene lactones extracted from chicory on the motility of larvae of deer lungworm and gastrointestinal nematodes

The objective of this study was to determine the effects of condensed tannins (CT) and an extract containing crude sesquiterpene lactones (CSL) from chicory (Cichorium intybus) on the motility of the first-(L1) and third-stage (L3) larvae of deer lungworm Dictyocaulus viviparus and the L3 larvae of gastrointestinal nematodes in vitro, using the larval migration inhibition (LMI) assay. The CT and CSL had a profound effect on the motility of the larvae displayed by their ability to inhibit larval passage through nylon mesh sieves. Incubation of lungworm L1 larvae in rumen fluid (collected from deer fed pasture) containing 100, 400 and 1000 microg CT/ml, inhibited 12, 28 and 41% of the larvae from passing through the sieves, respectively, while the incubation of L3 larvae with rumen fluid (pH 6.6) containing the same concentrations inhibited 26, 37 and 67% of L3 larvae from passing through the sieves, respectively. Gastrointestinal larvae seem more susceptible to CT than lungworm larvae especially at higher concentrations. CT inhibited 27, 56 and 73% of gastrointestinal larvae from passing through the sieves when used at a concentration of 100, 400 and 1000 microg/ml, respectively. CT were more effective (P<0.001) at reducing the motility of lungworm L1 and L3 larvae when added to the rumen fluid than when added to the abomasal fluid (pH 3.0). Addition of 2 microg polyethylene glycol/microg CT eliminated the inhibitory effect of CT against L1 and L3 larvae especially during incubation in rumen fluid, confirming the effect as due to CT. The CSL extract also showed similar inhibitory activity against L1 and L3 lungworm and L3 gastrointestinal larvae in both fluids, indicating that this extract was not affected by the pH of the fluid, and was more effective against L3 than L1 lungworm larvae. Condensed tannins appeared to be more effective than CSL at inactivating L1 and L3 lungworm and L3 gastrointestinal larvae in rumen fluid, but CSL were particularly effective against L3 lungworm larvae in abomasal fluid. Activity of these secondary compounds explains the reduced parasite problem of young deer grazing chicory.

Effect of condensed tannins extracted from four forages on the viability of the larvae of deer lungworms and gastrointestinal nematodes

The inhibitory activity of condensed tannins extracted from four forage legume plants were evaluated by using a larval migration inhibition assay. The first (LI) and third (L3) stages of deer lungworm (Dictyocaulus viviparus), and the third stage (L3) of deer gastrointestinal nematodes were incubated with tannins extracted from Lotus pedunculatus, Lotus corniculatus, sulla (Hedysarum coronarium) and sainfoin (Onobrychus viciifolia). The tannins extracted from all the forages had inhibitory activity as measured by their ability to paralyse the larvae and inhibit them from passing through sieves. At the highest concentration used (1200 μg/ml) the tannins extracted from sainfoin had the highest activity against ensheathed L1 lungworm larvae (58 per cent), followed by L pedunculatus (45 per cent), sulla (42 per cent) and L corniculatus (35 per cent) when the larvae were incubated at 37°C. The same trend, but with lower activities, was observed when the larvae were incubated at 22°C. Anthelmintic activity against L3 lungworm larvae was evaluated by measuring the death rate of ensheathed L3 larvae after incubation with condensed tannins for two, 24 and 48 hours at room temperature (22°C). The death rate was significantly higher (P<0.001) after 48 hours incubation than after two hours or 24 hours, and significantly higher (P<0.001) after 24 hours than after two hours incubation. Condensed tannins from sainfoin had the highest inhibitory activity followed by L pedunculatus, sulla and L corniculatus. The tannins from sainfoin also had the highest activity against L3 larvae of gastrointestinal nematodes, followed by L pedunculatus, sulla and L corniculatus. Exsheathed larvae of gastrointestinal nematodes were significantly more susceptible to the action of the tannins than ensheathed larvae.

Condensed tannins from Lotus corniculatus and Lotus pedunculatus exert different effects on the in vitro rumen degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein

Condensed tannins (CT) or proanthocyanidins (PA), which occur in a restricted range of forages, have the ability to interact with proteins and enzymes and can influence the digestion of plant protein in the rumen. We compared the effects of CT extracts from Lotus corniculatus and pedunculatus on degradation of the principal leaf protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), by rumen microorganisms. Total soluble leaf protein extracted from white clover (Trifolium repens ) was incubated with fresh rumen fluid from sheep and a range of concentrations of each CT extract. The rate of degradation of the large (LSU) and small subunit (SSU) of Rubisco was quantified by fractionating the proteins in samples taken from in vitro rumen incubations using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and imaging densitometry. To deduce the effects of the CT extracts, experiments were performed in the presence (CT inactive) and absence (CT active) of polyethylene glycol (PEG; MW 3350). The two CT extracts differed markedly in their effects on the degradation of the LSU and SSU of Rubisco. At concentrations of 0.89 and 1.79 mg CT mg −1 total soluble leaf protein, the CT extract from L pedunculatus was more effective at preventing the degradation of the LSU and SSU by rumen microorganisms than the CT extract from L corniculatus. At a concentration of 1.79 mg CT mg −1 total soluble leaf protein, the CT extracts from L corniculatus and pedunculatus prevented about 0.75 and 0.83 of the LSU and about 0.69 and 0.86 of the SSU, respectively, from being degraded. Addition of PEG removed the inhibition and almost complete degradation of these proteins occurred, as was the case in incubations without CT extracts. The results of this study suggest that the concentration of CT in the diet and the chemical structure which affects the activity of the CT needs to be considered when assessing the effects of CT on protein metabolism in ruminants. © 1999 Society of Chemical Industry

Effects of grazing undrenched weaner deer on chicory or perennial ryegrass/white clover pasture on the viability of gastrointestinal nematodes and lungworms

This study determined the in vitro effects on the viability of internal parasites of grazing undrenched weaner deer on either chicory (Cichorium intybus) or perennial ryegrass (Lolium perenne)/white clover (Trifolium repens) pasture. One experiment investigated the hatching and development of gastrointestinal nematode eggs and larvae, and the development and motility of Li lungworm (Dictyocaulus eckerti) larvae, and a second experiment used larval migration inhibition assays to test the viability of LI lungworm larvae extracted from the faeces of weaner deer grazed on either chicory or pasture when they were incubated with rumen and abomasal fluids from fistulated deer also grazing on chicory or pasture. The incubations were undertaken with and without added condensed tannins purified from chicory and with or without polyethylene glycol (PEG) to bind the tannins. Chicory had no effect on the hatching and development of gastrointestinal nematode eggs and larvae. Grazing chicory reduced the number of lungworm larvae developing to the L3 stage, and Li lungworm larvae from the faeces of chicory-grazed deer were less viable in rumen and abomasal fluid than larvae from pasture-grazed animals. Abomasal fluid was significantly (P<0.001) less inhibitory to the migration of Li lungworms than rumen fluid. When the larvae were incubated in rumen and abomasal fluids from chicory-grazed deer, their passage through sieves was significantly (P<0.001) reduced in comparison with when they were incubated in the fluids from pasture-razed deer. Adding condensed tannins to rumen fluid increased the inhibition of the migration of Li lungworm larvae but PEG removed this inhibition; this effect was not observed with abomasal fluid.