The nutritional and anthelmintic effects of Calliandra calothyrsus condensed tannin in the gastrointestinal tract of merino sheep

Calliandra calothyrsus is a high protein, high condensed tannin legume with potential nutritional value and possible anthelmintic properties. The objectives of these studies were to determine the in vivo and in vitro nutritional and anthelmintic properties of condensed tannin from this tropical shrub legume. Comparisons were made between the methods of Terrill et al. (1992b) and Perez- Maldonado (1994) analysing the three fractions of condensed tannin (free, protein and fibre-bound) to determine which method is more suited for routine analysis of both plant material and digesta. Analysis of plant material for total condensed tannin was not different using either of the methods tested. However, when the three fractions of condensed tannin, free, protein-bound and fibre-bound, were analysed, then the modified method of Terrill et al. (1992b) gave a higher result for analysis of plant material. Total condensed tannin in rumen digesta when analysed with the method of Perez-Maldonado (1994) compared to the modified method of Terrill et al. (1992b) did result in a significantly higher value, however, results for the different fractions of condensed tannin were not significantly different. The in vivo experiment compared digesta from lambs fed high condensed tannin (Calliandra calothyrsus) or no condensed tannin (lucerne) diets in the presence and absence of either of the worm species Haemonchus contortus and Trichostrongylus colubriformis to determine any interactions between worms, diet, digesta pH and condensed tannin fractions. The effect of the presence of these nematodes on the concentration of condensed tannin fractions in different segments of the gastrointestinal tract was also investigated. There was a significant (P≤ 0.005) diet and worm interaction in the abomasum. Diet had a significant effect on pH values in the abomasum, and the presence of worm infections significantly affected pH values in the ileum, caecum and colon. The presence of worms had no effect on total or fractions of condensed tannin in any of the gastrointestinal segments. The presence of worms had a significant effect on the concentration of all fractions of condensed tannin and total condensed tannin for overall means (pooled for worm species and no worms) in gastrointestinal segments. Protein-bound, fibre-bound, free and total condensed tannin changed significantly along the gastrointestinal tract as influenced by segments. The relationship between pH and free condensed tannin (R² = 0.196) and also between pH and total condensed tannin (R² = 0.171) was correlated. The protein-condensed tannin complex is possibly the most important condensed tannin complex with respect to nutritional interactions in the gut and anthelmintic effects. The greatest percentage of protein-bound condensed tannin in the gastrointestinal tract was found in the rumen; therefore, condensed tannin could potentially complex with dietary proteins, rumen microbes and nematode third stage larvae. The majority of condensed tannin is in the free form in the abomasum and duodenum, which are the segments inhabited by adult nematodes. A higher portion of condensed tannin was protein-bound in the ileum and possibly the jejunum (extrapolation from pH values), suggesting that there would be significant amounts of condensed tannin bound to dietary protein, digestive enzymes or nematode eggs. It was also evident that the condensed tannin in the ingested feed could not always be detected in the digesta samples. The total condensed tannin concentration in feed and abomasum were comparable, however, the concentration detected in all other gastrointestinal segments were lower. In vitro assays were conducted to examine binding and dissociation of condensed tannin extracted from Calliandra calothyrsus, with Rubisco and cellulose, at pH 1-9 and from 0.5 to 24 h of complexing without the influence of digesta and gastrointestinal secretions. Complexing time had no significant effect on condensed tannin binding. This study confirmed that in vitro binding of condensed tannin with and dissociation from protein is pH dependant; however, this was not the case for the fibre-bound condensed tannin complexing. The pH range at which protein formed the most stable complex with condensed tannin was from 3-9. Enzyme inhibition studies demonstrated that Calliandra calothyrsus condensed tannin does inhibit the activity of the digestive enzymes, trypsin (R²= 0.965) and pancreatic α-amylase (R²= 0.903) with the relationship being well correlated. The in vitro assays demonstrated that Calliandra calothyrsus condensed tannin is effective at inhibiting egg hatching, first stage larvae feeding and infective third stage larvae exsheathment and motility in Haemonchus contortus susceptible, Haemonchus contortus macrocyclic lactone resistant and Trichostrongylus colubriformis susceptible strains. The implications are that Callinadra condensed tannin has anthelmintic potential to disrupt the lifecycle of Haemonchus contortus and Trichostrongylus colubriformis. The eggs and larvae were sensitive to low condensed tannin concentrations with the condensed tannin concentrations demonstrated to have in vitro effects well below that found in the GI segments of lambs fed a 100% Calliandra calothyrsus diet. There were differences in the effect of Calliandra calothyrsus condensed tannin to the different worm species for egg hatch and the larval exsheathment assays only, with the Haemonchus contortus susceptible strain being more sensitive than the Haemonchus contortus macrocyclic lactone resistant and Trichostrongylus colubriformis susceptible strains. Calliandra calothyrsus condensed tannin had a greater effect on the Haemonchus contortus macrocyclic lactone resistant strain for the larval exsheathment assay. The mimosa extract only had effects on egg hatching and first stage larvae feeding. The mechanism by which the condensed tannin is having in vivo anthelmintic effects by reducing egg production (Cresswell, 2007) may well be due to the effect of condensed tannin on the larvae in the rumen. Alternatively condensed tannin may also complex with the adult nematodes living in the abomasum and duodenum. The Haemonchus contortus are abomasal blood suckers and would not be ingesting condensed tannin directly, however, the duodenal grazer, Trichostrongylus colubriformis would be ingesting condensed tannin and so may be having an effect via ingestion. Although it was shown that condensed tannin was mostly found as free condensed tannin in the abomasum and duodenum, there were small percentages of protein-bound condensed tannin which, as seen in the in vitro complexing, digestive enzyme and egg and larvae assays that condensed tannin is capable of exerting large effects with small concentrations, so this possibility must not be overlooked. Calliandra calothyrsus condensed tannin is capable of disrupting different stages of the lifecycle in vitro of Haemonchus contortus susceptible, Haemonchus contortus macrocyclic lactone resistant and Trichostrongylus colubriformis susceptible strains. However, not all results from in vitro studies can be correlated with what happens in vivo due to the physiological conditions present in the gastrointestinal tract and the interactions with constituents of digesta and secretions. Egg hatching, first stage larvae feeding and infective third stage larvae exsheathment and motility were all inhibited due to the effects of Calliandra calothyrsus condensed tannin. Calliandra calothyrsus condensed tannin complexing dynamics does have nutritional and anthelmintic effects. These studies have shown conditions in the rumen and post duodenum to be conducive to condensed tannin complexing with proteins and therefore the potential to complex with dietary proteins, rumen microbes, digestive enzymes and nematode larvae. By-pass proteins formed in the rumen and proteincondensed tannin complexes post duodenum, would be contributing to increased faecal nitrogen output and decreased dry matter digestibility as reported by Cresswell (2007)