Froehlich, Kelly A.2020-09-012020-09-012020https://hdl.handle.net/10182/12594Loline is an alkaloid produced by Epichloë endophytes in pastoral grass species with potential antimicrobial properties. Loline has several known derivatives; N-formyl loline (NFL), N-acetyl loline (NAL), N-acetyl norloline (NANL), N-methyl loline (NML), and loline base. Although considered non-toxic to mammals there is little information available about its metabolism or potential antimicrobial effects. Therefore, through a series of experiments this study investigated concentration and forms of loline metabolized in rumen fermentation and digestion, its absorption through gastrointestinal epithelia, and distribution in the body (tissues, blood, etc), and lastly determine if any anti-microbial effects exist and their impact on rumen microflora and gastrointestinal nematodes. Chapter 3 used in-vitro methods to determine the fate of lolines during rumen fermentation and digestion through examining forms and concentrations, and its effects on rumen microbes through the production of volatile fatty acids and ammonia. Festuca pratensis seeds either with (LOL) or without (NIL) lolines (150-200 µg/mL) were incubated in either sterile or viable rumen fluid or in either HCL/pepsin (pH 2) or water (pH 7). At 72 hours LOL in sterile rumen fluid had 23%, 26%, and 45% more NFL (P=0.05), NANL (P=0.04), and loline base (P=0.01), respectively, with 27% more total loline present (P=0.06), and 65% more NML (P=0.09) compared with LOL in viable rumen fluid. Loline tended to alter the fermentation pattern as NIL produced 11% more ammonia (P=0.07), and 5% less propionate (P=0.06) than LOL. In HCL/pepsin there were 38%, 49%, and 39% more total lolines (P=0.05), NANL (P=0.02), and NFL (P=0.04) respectively, compared with water. Rumen and abomasal digestion appeared to have a small effect on loline’s form and concentration with only slight alteration to the rumen fermentation pattern. Presence of lolines had little evidence of an antimicrobial effect on rumen microbes. Chapter 4 was an extension of chapter 3 to explore lolines effect on rumen fermentation using an in-vitro gas production technique in two experiments. Experiment 1 treatments were Festuca pratensis seeds either with (LS) or without (NS) loline (150-200 µg/mL) as sole substrate and experiment 2 used Lolium perenne as a substrate with treatments being ryegrass only (RGS) or ryegrass with (RGL) or without (RGN) a loline seed extract incubated in buffered ruminal fluid. Cumulative gas production was measured over 24-hours and apparent dry matter digestibility and pH were collected at the end. In experiment 1, NS had a 9% greater gas yield than LS after 24-hours (P=0.0001) with no difference in apparent dry matter digestibility (P=0.11). In experiment 2 predicted potential gas production was not different (P=0.67) for ryegrass treatments using an Ørskov nonlinear model. However, the fractional rate of gas production was greater (P=0.0001), in RGN (0.112 ± 0.0002) than RGL (0.109 ± 0.0002), and RGL was greater than RGS (0.093 ± 0.0002). Apparent dry matter digestibility was greater in RGS than RGL (P=0.05) and RGN (P=0.02). Loline had little antimicrobial effect on rumen microbes as shown by potential gas production but the addition of the water-soluble fraction of seed extracts to rumen fluid increased the soluble pool readily available to microbes, changing the fermentation kinetics. Therefore, loline could be explored as a potential pharmaceutical on gut-dwelling organisms without negatively affecting rumen fermentation or function. Loline metabolism in ruminants is believed to be rapid, surviving digestion and relying on hepatic metabolism prior to fast but low urinary excretion. It is unclear however where loline is absorbed and information on its distribution throughout tissues is not available. Chapter 5 investigated passive absorption of Festuca pratensis seed extract (1034 µg/mL) and caffeine (22.1 µg/mL) suspended in a solution in either rumen, abomasal, duodenal, ileum, large intestine, or colon tissues mounted in an Ussing chamber. Loline distribution was measured in gastrointestinal (small intestine, abomasum), organs (kidney, liver), and blood (plasma, red blood cells) of lambs deliberately bottle-fed loline. Little passive absorption occurred in ruminal or abomasal tissues (<2%). Ileum tissues appeared to have the greatest absorption capacity (5%, P=0.04) at 2 hours incubation compared with abomasal tissues (0.11%). Of the loline forms, loline base and NFL were passively absorbed across all gastrointestinal tissues with NAL and NANL only crossing small intestine tissues. Recovery of loline in-vivo was low for NFL in blood plasma (46 µg/g DM), and greatest for loline base (296 µg/g DM), and loline base was the only metabolite found in liver (126 µg/g DM) and kidney (112 µg/g DM) of lambs. The low recovery of loline in organs and blood are in line with its reported fast metabolism however, the low absorption rates through gastrointestinal tissues were unexpected. Potentially indicating either the majority of loline is not passively absorbed or membrane integrity was affected as suggested by lack of caffeine absorption. Chapter 6 investigated the validation of using loline as a natural anthelmintic for the gastrointestinal nematodes in sheep of Trichostrongylus colubriformis, Teladorsagia circumcincta, and Haemonchus contortus through a series of in-vitro and in-vivo studies. In-vitro experiments revealed that larval migration of T. colubriformis was not affected by Festuca pratensis seed extract (P=0.29) nor was there an extract x concentration interaction (P=0.52). However, T. circumcincta establishment was decreased (P<0.01) by 59% in excised tissues of lambs fed loline compared with non-treated lambs. This suggested loline could have potential anthelmintic properties on larvae when in close contact with tissues or blood. In a pilot study, the numbers of L4 mucosal browsers T. colubriformis and T. circumcincta in-vivo were reduced by 23, and 75% in lambs treated with a loline seed extract via abomasum 10 days post infection. However, dosing loline 23 days post larval infection when T. colubriformis and T. circumcincta were an adult had no effect. In a follow up pilot study, adult H. contortus in-vivo was reduced 34 and 36% in lambs fed loline 23 days post larval infection either orally or via abomasum compared with controls. Further investigation examined the effect of loline seed extract given orally in-vivo and compared non-treated lambs (CON) or lambs treated with Festuca pratensis seed extract without loline (NIL) in lambs infected with a mix of L4 T. circumcincta, T. colubriformis, and adult H. contortus. Lambs were dosed H. contortus on day 0, and L3 T. circumcincta, T. colubriformis on day 14 from dosing H. contortus. Loline was dosed orally starting on day 13, and every other day until slaughter on day 28. Worm burdens of T. circumcincta (P=0.96), T. colubriformis (P=0.43), and H. contortus (P=0.15) showed no difference in lambs loline treated compared with CON or NIL. Furthermore, no treatment difference (P=0.39) was observed in faecal egg counts, weight gain (P=0.51), feed intake (P=0.18) between treatments. However, average growth efficiency (kg LWG/ kg DM intake) was 0.18 in CON treated lambs which was less (P=0.01) than LOL (0.24) or NIL (0.23) treated lambs. Overall, it was concluded there is limited evidence to support an in-vivo anti-parasitic effect of lolines despite in-vitro studies indicating potential benefits when parasites are in either a developmental stage in close contact with gastric mucous layers or adult stage consuming blood. The discrepancy between in-vivo and in-vitro results may be dependent on the length of time loline has in contact with larvae and potentially mode of ingestion or the forms of loline present. In summary, these results demonstrate that rumen and abomasal digestion have little effect on loline form and concentration. There were slight alterations of ammonia, propionate, and gas production in the presence of loline in the rumen but overall showed limited antimicrobial effect on rumen microbes and, perhaps more importantly, microbial digestion did not affect the level of loline present. Surviving digestion with little effect on rumen fermentation meant loline could be explored as a potential pharmaceutical on gut-dwelling organisms without negative effects. Unexpectedly, only small amounts of loline were passively absorbed across gastrointestinal epithelia with the most absorption occurring in ileum tissues compared with abomasal tissues, indicating the majority of loline is not a passively absorbed. In-vivo only small amounts of NFL and loline base were found in kidney, liver, and blood plasma of lambs that may reflect its suggested fast metabolism, which is supported by, loline base being the metabolite found in the greatest concentration. Although, loline metabolites are found in-vivo there was limited evidence to support an anti-parasitic effect of loline although some benefit may arise when parasites are in close contact with the gastric mucous layers or at a blood feeding stage.enlolinealkaloidmeadow fescueruminal digestionabomasal digestiongas productionUssing chambersgastrointestinal parasitesThesisANZSRC::07 Agricultural and Veterinary SciencesANZSRC::070707 Veterinary Microbiology (excl. Virology)Q112952051https://creativecommons.org/licenses/by-nc-nd/4.0/Attribution-NonCommercial-NoDerivatives 4.0 International