Feeding of carob (Ceratonia siliqua) to sheep infected with gastrointestinal nematodes reduces faecal egg counts and worm fecundity

Abstract The present study explored the anthelmintic effects of condensed tannins (CT) in carob (Ceratonia siliqua) pods fed to sheep against gastrointestinal nematodes. Three independent in vivo trials tested whether i) carob pod (CaBP)-containing feed had an anthelmintic effect and if yes, which was the optimal concentration in the diet; ii) whether this effect could be attributed to tannins through the polyethylene glycol (PEG) test and iii) whether there were any synergistic effects when combined with another tannin-containing feed (e.g. sainfoin). In all trials 6-month old nematode-naive lambs, experimentally infected with both Haemonchus contortus and Trichostrongylus colubriformis, were used. Faecal egg counts (FEC) were performed regularly and at the end of each trial adult worm counts (AWC) and female worm fecundity were recorded. In trial 1, 35 lambs (five groups of seven lambs) were fed different CaBP concentrations ranging from 0% to 12% w/w. FEC declined up to 39.2% only in the group fed with 12% CaBP, while a declining trend (P<0.06) was demonstrated for the AWC of T. colubriformis, which was associated with the increasing concentration of CaBP in feed. Female worm fecundity was reduced in groups fed CaBP for both parasites, however this was only significant for H. contortus (P<0.001), in a dose dependent manner. In trial 2, four groups of six infected lambs each were used, which received the carob diets CaBP or CaBP+PEG, and the tannin-free diets with or without PEG (C or C+PEG). Results showed that FEC of Groups C, C+PEG, and CaBP+PEG were comparable throughout the trial, while the group receiving only CaBP showed lower FEC from DAY 25 onwards. AWC showed a reduction (67.7%) only for Η. contortus (P<0.03). Reversal of the anthelmintic effect of CaBP after PEG administration suggested that CT contributed to the anthelmintic action. However, no effect of CaBP was observed on T. colubriformis AWC and on female worm fecundity for both species. Finally, for trial 3 four groups of six lambs each received a diet based on CaBP, sainfoin (S) or a combination (CaBP+S) and were compared to a control (C) diet of lucerne. On DAY 37 FEC values in groups CaBP+S and S tended to be lower compared to the two other groups (C, CaBP), while for AWCs no significant differences were observed for both parasites. The fecundity of H. contortus and T. colubriformis demonstrated significant differences between the treated and control groups, with lower values in the animals receiving CaBP+S. Overall, the results supported the hypothesis that carob had an anthelmintic effect due to its CT, but there was no clear indication of a synergistic effect with sainfoin

Effect of α-zearalenol on development of porcine conceptuses in vitro

Zearalenone (ZEN) is a mycotoxin with estrogenic activity produced by Fusarium grain molds. After ingestion via contaminated cereals, ZEN may lead to animal fertility disturbances and other reproductive dysfunctions. It has therefore been classified to the endocrine disrupting compounds (EDCs). Zearalenone and its metabolites interact with estrogen receptors (ERs). The major in vivo metabolite of zearalenone in the pig is α-zearalenol (α-ZEN) which possesses both a greater affinity to estrogen receptors (ERs) and a greater estrogenic potency than ZEN. Thus, conversion of ZEN to α-ZEN is considered as activation of the toxin. Swine are the domestic species most affected by consumption of ZEN. Among other symptoms, ZEN can increase early embryonic mortality and cause loss of pregnancy when ingested by gilts or sows during early pregnancy. The most sensitive stage of early pregnancy in the pig is the maternal recognition of pregnancy on day 12. The signal for the maternal recognition of pregnancy is embryonic oestradiol-17β. Oestradiol-17β produced by porcine conceptuses is one of the most important hormonal signals that regulate pregnancy recognition signalling, endometrial differentiation and function, uterine-conceptus interactions and uterine receptivity for conceptus implantation. Porcine 12-day conceptuses secrete a variety of growth factors, cytokines and proteases, apart from oestradiol-17β. The urokinase-type plasminogen activator (u-PA) is considered to be an important indicator of cellular migration and differentiation of the 12-day conceptus tissues, while it also takes part in the initiation of implantation by activating other proteases that interact with embryonic and uterine extracellular matrix molecules. The aim of the present study was to investigate the in vitro effect of α-ZEN on oestradiol-17β concentration and plasminogen activator activity of the culture medium of day-12 porcine conceptuses. Fourteen gilts aged 6 to 6.5 months were oestrus synchronized using eCG and hCG and artificially inseminated on the first day of standing oestrus (day 0) and 12 hours later. Gilts were sacrificed on day 12 after first insemination and filamentous conceptuses were recovered post mortem. Each uterine horn was irrigated with 120 ml PBS and collected conceptuses were evaluated under a stereoscope and equally distributed among four groups with respect of their weight. Conceptus tissues that ranged between 93.5 and 404.5 mg were cultured individually in a controlled atmosphere chamber for 24 h. Culture medium samples were obtained every 8 h. α-ZEN was diluted in DMSO (1%) and added to each of the cultures in the α-ZEN treatment groups at the onset of incubation period. Two concentrations of α-ZEN were used (1 and 10 μg/ml), and one group served as a DMSO control. A further group of cultures free of any additive served as control. Oestradiol-17β concentration in the culture medium was determined by radioimmunoassay (RIA) and expressed in pg of oestradiol-17β per ml of culture medium. Oestradiol-17β was also expressed in pg per million cells as estimated by a DNA-assay of each culture at the end of the incubation period. Plasminogen activator activity (PAA) of the conceptus culture medium was determined spectrophotometrically and expressed in IU of urokinase type plasminogen activator per 10 μl of culture medium.Το πιο επικίνδυνο στάδιο για την πρόκληση πρώιμων εμβρυϊκών θανάτων, στο χοίρο, είναι το στάδιο της μητρικής αναγνώρισης της κυοφορίας, δηλαδή η 12η ημέρα από τη γονιμοποίηση. Ειδικά στο ζώο αυτό, το ερέθισμα για τη μητρική αναγνώριση της κυοφορίας είναι η οιστραδιόλη-17β, που εκκρίνουν τα έμβρυα. Η οιστραδιόλη-17β ασκεί τόσο παρακρινή δράση στο ενδομήτριο όσο και αυτοκρινή και παρακρινή δράση στα έμβρυα. Εξάλλου, η δράση της συμβάλλει αποφασιστικά στην ανάπτυξη των εμβρύων, στην εμβρυομητρική επαφή και στην έναρξη της εγκατάστασής τους στη μήτρα, κατά τη 13η ημέρα της κυοφορίας. Ένας από τους σημαντικότερους δείκτες της κυτταρικής διαφοροποίησης των ιστών των εμβρύων, της φυσιολογικής ρύθμισης των μηχανισμών ανάπτυξής τους, καθώς και της δυνατότητας προσκόλλησής τους στο ενδομήτριο είναι ο ενεργοποιός του πλασμινογόνου του τύπου της ουροκινάσης (u-PA). Ο u-PA, που απελευθερώνουν τα έμβρυα χοίρου, ηλικίας 12 ημερών, συνδέεται λειτουργικά με την εκκρινόμενη οιστραδιόλη-17β, μέσω πολύπλοκων αλυσιδωτών μηχανισμών. Στην παρούσα εργασία, λοιπόν, μελετήθηκε η in vitro επίδραση του πλέον τοξικού μεταβολίτη της ζεαραλενόνης, της α-ζεαραλενόλης (α-ΖΕΝ) στη συγκέντρωση της οιστραδιόλης-17β και στη δραστηριότητα των ενεργοποιών του πλασμινογόνου στο υπόστρωμα καλλιέργειας εμβρύων χοίρου, ηλικίας 12 ημερών. Για τους σκοπούς της έρευνας χρησιμοποιήθηκαν 14 νεαρές ένηβες σύες που υποβλήθηκαν σε συγχρονισμό του οίστρου [(eCG, 700 IU/ζώο, i.m., εφάπαξ, ημέρα -5) και (hCG, 700 IU/ζώο, i.m., εφάπαξ, ημέρα -2)] και τεχνητή σπερματέγχυση και θανατώθηκαν 12 ημέρες μετά την έναρξη του οίστρου. Μετά από πλύση του κάθε κέρατος της μήτρας, τα νηματοειδή έμβρυα, ηλικίας 12 ημερών, συλλέχθηκαν, εκτιμήθηκαν, υπό στεροεοσκοπική παρατήρηση, χωρίστηκαν σε τέσσερις πειραματικές ομάδες και καλλιεργήθηκαν μεμονωμένα. Στο υπόστρωμα της in vitro καλλιέργειας των εμβρύων προστέθηκε α-ΖΕΝ, σε δύο διαφορετικές συγκεντρώσεις (1 και 10 μg/ml). Σε μία επιπλέον ομάδα εμβρύων προστέθηκε μόνο ο διαλύτης της α-ΖΕΝ (DMSO, 1%), ενώ παράλληλα υπήρχε και μία ομάδα εμβρύων μαρτύρων (στο υπόστρωμα της καλλιέργειάς των οποίων δεν έγινε καμία προσθήκη). Η συνολική διάρκεια της καλλιέργειας ήταν 24 ώρες. Οι δειγματοληψίες υποστρώματος καλλιέργειας, καθώς και η ποιοτική εκτίμηση των εμβρύων επαναλαμβάνονταν ανά 8 ώρες, από την έναρξη (ώρα 0) έως και το τέλος της καλλιέργειας (24 ώρες). Η συγκέντρωση της οιστραδιόλης-17β στο υπόστρωμα καλλιέργειας των εμβρύων προσδιορίστηκε με ραδιοανοσολογική μέθοδο και εκφράστηκε σε pg οιστραδιόλης-17β ανά ml υποστρώματος καλλιέργειας εμβρύων. Ακολούθως, μετά από τον προσδιορισμό της συγκέντρωσης του DNA της κάθε καλλιέργειας, εκφράστηκε και σε pg/106 κύτταρα. Η δραστηριότητα των ενεργοποιών του πλασμινογόνου στο υπόστρωμα καλλιέργειας των εμβρύων προσδιορίστηκε με φασματοφωτομετρική μέθοδο και εκφράστηκε σε διεθνείς μονάδες (IU) ενεργοποιού του πλασμινογόνου του τύπου της ουροκινάσης (u-PA) ανά 10 μl υποστρώματος

Anthelmintic effect of carob pods and sainfoin hay when fed to lambs after experimental trickle infections with Haemonchus contortus and Trichostrongylus colubriformis

The aim of the study was to compare the in vivo anthelmintic activity of sainfoin hay (Onobrychis viciifolia) and carob pod meal (Ceratonia siliqua) against gastrointestinal nematodes. Seven days before infection, 64 naive lambs were assigned to four different groups: Group S received sainfoin hay and group CAR was fed with carob pods. The remaining lambs received lucerne hay (Medicago sativa) and were assigned to positive (non-treated, NT) and negative (treated, T) control groups (treatment with albendazole). On day 0, lambs were artificially trickle infected for 6 weeks, with a mixture of infective larvae of Haemonchus contortus and Trichostrongylus colubriformis. Parasitological and pathophysiological parameters were measured repeatedly during the 2-month study. Compared to the NT group, decreases in egg excretion were observed in the CAR and S groups with significant differences only found for sainfoin (p < 0.05). At necropsy, group S showed decreases in the total worm numbers of both nematode species with significant differences for H. contortus. In contrast, no differences were noticed for the CAR group. Compared to the NT group, lower values for fecundity of female H. contortus were found in the S and CAR groups, however differences were non-significant. No differences in body weight gains were found between groups. Consistent results were found showing significantly higher packed cell volume (PCV) values in the T and S groups compared to NT and CAR groups. Overall, these results confirm a positive effect associated with the feeding of lambs with tanniniferous resources on host resilience (PCV values) and against gastrointestinal parasitic nematodes by affecting some biological traits of worm populations (e.g. eggs per gram of faeces and worm numbers). However, the anthelmintic effects differed between the two tannin-containing resources, which might be associated with the quantity and/or quality of secondary metabolites (condensed tannins and/or other polyphenols)

Anthelmintic effect of carob pods and sainfoin hay when fed to lambs after experimental trickle infections with Haemonchus contortus and Trichostrongylus colubriformis

The aim of the study was to compare the in vivo anthelmintic activity of sainfoin hay (Onobrychis viciifolia) and carob pod meal (Ceratonia siliqua) against gastrointestinal nematodes. Seven days before infection, 64 naive lambs were assigned to four different groups: Group S received sainfoin hay and group CAR was fed with carob pods. The remaining lambs received lucerne hay (Medicago sativa) and were assigned to positive (non-treated, NT) and negative (treated, T) control groups (treatment with albendazole). On day 0, lambs were artificially trickle infected for 6 weeks, with a mixture of infective larvae of Haemonchus contortus and Trichostrongylus colubriformis. Parasitological and pathophysiological parameters were measured repeatedly during the 2-month study. Compared to the NT group, decreases in egg excretion were observed in the CAR and S groups with significant differences only found for sainfoin (p < 0.05). At necropsy, group S showed decreases in the total worm numbers of both nematode species with significant differences for H. contortus. In contrast, no differences were noticed for the CAR group. Compared to the NT group, lower values for fecundity of female H. contortus were found in the S and CAR groups, however differences were non-significant. No differences in body weight gains were found between groups. Consistent results were found showing significantly higher packed cell volume (PCV) values in the T and S groups compared to NT and CAR groups. Overall, these results confirm a positive effect associated with the feeding of lambs with tanniniferous resources on host resilience (PCV values) and against gastrointestinal parasitic nematodes by affecting some biological traits of worm populations (e. g. eggs per gram of faeces and worm numbers). However, the anthelmintic effects differed between the two tannin-containing resources, which might be associated with the quantity and/ or quality of secondary metabolites (condensed tannins and/or other polyphenols)