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Item Studies of the effects of "mimosa bark extract" containing condensed tannins on milk production by grazing dairy cows and on ruminal protein metabolism in sheep(Massey University, 1996) MashudiStudies of the effects of "Mimosa bark extract" containing condensed tannins on milk production by grazing dairy cows and on ruminal protein metabolism in sheep. M.Agr.Sc. Thesis, Massey University, Palmerston North, New Zealand. Tannins, particularly condensed tannins (CT), either added to the diet or occuring naturally in the forage are advantageous because they protect dietary protein from degradation in the rumen. The aim of this study was to measure the effect of Mimosa bark extract which contained approximately 70% CT on grazing dairy cow performance and on ruminal protein metabolism in sheep. Two experiments were carried out over the spring season (September and October 1994). In experiment I, effects of CT in Mimosa bark extract upon blood urea concentration, milk yield and milk composition, liveweight and condition score of grazing dairy cows were evaluated. Thirty Friesian cows were allocated at random to 3 treatments :(1) Control : no Mimosa bark extract (no CT); (2) Low CT : 50 g/cow daily of Mimosa bark extract (2.4 g CT/ kg DM eaten); (3) high CT : 100 g/cow daily of Mimosa bark extract (4.8 g CT/kg DM eaten). Mimosa bark extract was given twice daily as a suspension by oral drenching during each milking. In experiment II, effects of CT in Mimosa bark extract on ruminal protein metabolism in sheep were evaluated. Six mature Romney sheep fitted with permanent ruminal cannulae were randomly assigned into 2 treatments in a cross-over design. The two treatments were (1) Control : no Mimosa bark extract (no CT); (2) High CT : 6.66 g/sheep daily of Mimosa bark extract (4.8 g CT/kg DM eaten). Mimosa bark extract was given twice daily as a suspension by oral drenching just after feeding. Dry matter intake, rates of DM disappearance by the in sacco method, rumen ammonia and blood urea concentration and apparent digestibility of dry matter and and nitrogen were measured. In experiment I, liveweight and condition score as well as milk yield and composition, were not influenced by CT. Lactose concentrations were higher in the low CT group than in the high CT group in all weeks of the experiment. Cows drenched with high CT had a lower (P<0.05) blood urea concentration than cows in the control group, and, in week I they were lower (P<0.05) than cows in the low CT group. In experiment II, rumen metabolism parameters, including dry matter intake, in sacco DM disappearance parameters (A, B, C and A+B) and apparent digestibility of DM and N were not influenced by Mimosa bark extract. However sheep drenched with high CT had lower rumen ammonia and blood urea concentrations (P<0.05) than the control in the whole period. These results indicate that Mimosa bark extract had no significant effect on milk production. However it did consistently and significantly reduce blood urea concentration in both cows (high CT group) and sheep and it reduced rumen ammonia concentration in sheep. This indicates that the CT did have some biological effect in the rumen namely, a reduced protein degradation in the rumen.Item Phenolics and condensed tannins from sulla (Hedysarum Coronarium) leaves and their biological significance : this thesis was presented in partial fulfilment of the requirements for the Master of Science degree in Chemistry at Massey University, Palmerston North, New Zealand(Massey University, 2003) Tibe, OlekileThe objective of this study was to isolate and characterise condensed tannins (CT) and phenolic compounds from the leaves of the forage legume sulla (Hedysarum Coronarium) and evaluate their structure-activity relationships with in vitro parasite assays. The study was performed on samples which were collected over different seasons (spring-23/09/02, spring-05/08/02, and summer-21/12/01) from the same site. The effects of processing in different manners, for both freeze-dried and fresh frozen plant material was examined. CT extracts were purified using step and gradient Sephadex LH-20 chromatography methods. The CT fractions obtained were analysed using thiolytic degradation, electrospray ionisation mass spectrometry (ESI-MS), matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF-MS) and l3C-nuclear magnetic resonance (NMR) spectroscopy. Acid catalysed degradation of the CT polymer with benzyl mercaptan afforded catechin, epicatechin, gallocatechin and epigallocatechin in both the terminal and extender units. Epigallocatechin was the major extender unit (69%) while gallocatechin was the major terminal unit (54%) with the overall characteristic of a predominantly prodelphinidin-type CT. Sulla CT oligomers and polymers had variable chemical composition with procyanidin:prodelphinidin ratios ranging from 27:73 to 11:89 and cis:trans ratios ranging from 56:44 to 82:18. The CT oligomers of gradient LH-20 fractions obtained from fresh frozen material (spring-05/08/02) ranged between 2.9 and 6.9 mean degree of polymerisation (mDP), while CT fractions from the freeze-dried material (summer-21/12/01) varied between 3.1 and 9.1 mDP as determined by thiolysis. The CT polymer from step LH-20 fractions had CTs ranging from 12 to 26 mDP as determined by thiolysis. A medium molecular weight CT with mDP of 46 was identified. No high molecular weight CT (mDP> 50) was obtained. Screening the LH-20 fractions collected in spring (23/09/02) by HPLC-PDA indicated that there was no extractable CT. No seasonal or freeze-drying effects were observed on the chemical composition of CT. 13C NMR provided information on the stereochemistry of the heterocyclic C-ring and the existence of procyanidin and prodelphinidin units in the B-ring. The 13C NMR spectrum confirmed sulla CTs to be predominantly of the cis-stereochemistry composed of prodelphinidin units. Analysis of the CT oligomers from the 100% MeOH fractions from the gradient LH-20 with ESI-MS provided information on the molecular weight distribution and the procyanidin and prodelphinidin unit composition. Singly charged species from dimers to trimers, doubly charged species from tetramers to octamers, and triply charged species from nonamers to undecamers were detected. MALDI-TOF-MS verified the ESI-MS data and fractions were found to contain singly charged ions up to hexamers. The ions consisted of homogenous and heterogeneous CT oligomers, with the overall characteristic of a PD-type CT. This technique demonstrated that the gradient LH-20 method improved separation with fractionation of CT oligomers from polymers. Investigation of the low molecular weight phenolics (flavonoids) was performed using ESI-MS and atmospheric pressure chemical ionisation mass spectrometry (APCI-MS). Chlorogenic acid, quercetin-7-O-α-L-rhamnosyl-3-O-glucosylrhamnoside, rutin, quercetin-3-O-α-L-rhamnosyl-7-O-glucoside, kaempferol, kaempferol-3-O-β-D-glucoside-dirhamnoside, genistein-7-O-β-D-glucosyl-6"-O-malonate, formononetin-7-O-β-D-glucoside-6"-O-malonate and afrormosin were isolated for the first time from sulla. Chlorogenic acid and rutin were confirmed using authentic standards and by comparison with data from the literature. Primary metabolites were evaluated by wet chemistry methods including the available carbohydrate (g/l00g) content of 12.38 and, 14.11 and individual sugars (g/l00g) were quantified; glucose (3.68 and 5.40), fructose (0.98 ad 1.69), galactose and/or rhamnose (0.46 and 0.32), sucrose (1.63 and 5.50) in spring (23/09/02) and summer (21/12/01), respectively. Nutritional composition data (g/l00g) by near infrared (NIR) spectroscopy has shown sulla to be a nutritious forage legume with high crude protein (CP; 24.4-25.1), non-structural carbohydrates (NSC; 17.1-19.7), lower neutral detergent fibre (NDF; 12.5-16.4), acid detergent fibre (ADF; 15.5-18.3) and lipid (2.6-3.3). The butanol-HCl assay showed the extractable CT content (g/l00g) to be 7.6% and 5.3%, with 2.0% protein bound CT and 0.3% fibre bound CT and 1.3% protein bound CT and had no fibre bound CT, from summer (21/12/01) and spring (23/09/02) respectively. The effects of LH-20 fractions on egg hatching (EH) and larval development (LD) assays for the nematode, Trichostrongylus colubriformis under in vitro conditions were investigated. The fractions (freeze-dried summer (21/12/01) step method 50% MeOH eluent) containing flavonoids were effective in inhibiting EH at 500 and 1000 μg/mL, while the CT-containing fractions were not effective. All the fractions from the fresh frozen material on the step method were not effective in inhibiting EH. However, the CT-containing fractions (LH-20 70% acetone eluent) from the freeze-dried (summer-21/12/01) and fresh frozen (spring-05/08/02) material were effective in inhibiting LD (p<0.00l) with certain fractions completely inhibiting the LD process. Oligomeric and polymeric CT (gradient LH-20 100% MeOH and 70% acetone eluents) fractions were effective and inhibited the larval development process at 100 μg/mL. The anti-parasitic activity of fractions in the LD and EH assay can be attributed to both the flavonoid and CT content.Item Phenolics and condensed tannins of forage plants from Botswana and their biological significance : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Turitea campus, Palmerston North, New Zealand(Massey University, 2012) Tibe, OlekileThe main objectives of this study were to isolate and characterise phenolics and CT from Botswanan forage plants, and to investigate their potential anti-parasitic and immunostimulatory properties. Viscum rotundifolium, Viscum verrucosum, Tapinanthus oleifolius, Grewia flava and Ipomoea sinensis forage leaves and small stems were harvested over two summers (February 2009 and 2010), extracted twice with acetone:water (7:3) and subsequently defatted with dichloromethane. Each crude extract (6 g) was loaded onto a Sephadex LH-20 column and eluted with aqueous methanol (1:1) to yield four fractions, and subsequently eluted with acetone:water (7:3) to yield three fractions. Phytochemical screening of the fractions for the presence of CT and phenolics was conducted by a reverse phase high performance liquid chromatography coupled to a photodiode array (RP-HPLC-PDA) detector at 280 nm. The butanol-HCl colorimetric assay revealed that the total CT concentrations from the forage plants ranged from 0.2 to 12.7 (g/100g dry matter). These results indicated that significant amounts of CT were present in V. verrucosum, T. oleifolius and G. flava. The potential impact of each purified CT fraction was evaluated for anti-parasitic effects using a larval development assay (LDA). Three different species of gastrointestinal nematodes (Haemonchus contortus, Trichostrongylus colubriformis and Teladorsagia circumcincta) from sheep were tested with the fractions at 100 and 500 μg/mL. CT fractions from V. rotundifolium and I. sinensis samples, collected in 2009 and 2010, did not inhibit larval development (L1 and L2) to the infective L3 stage. CT isolated from V. verrucosum and T. oleifolius which were collected in 2009 completely inhibited the development of all parasite species at both concentrations. Also, complete inhibition of larval development of all tested parasite species was obtained in CT fractions from G. flava collected in 2009, with the lowest inhibitory activity at 62.5 μg/mL. These findings suggest that CT extracts have anti-parasitic effects in vitro, which may be translated into reduction of the effects of parasitism in ruminants in vivo. The potential impact of the extracts on priming of γδ T cells from the peripheral blood of lambs, calves and kids at 5 and 10 μg/mL was also evaluated in vitro. Condensed tannins (CT) from G. flava significantly primed γδ T cells in kids by up to 64.75% at 10 μg/mL, which was statistically significant relative to the negative control at 22.66% (p=0.004). CT from T. oleifolius also induced priming of γδ T cells in kids, while fractions from V. rotundifolium and V. verrucosum induced minimal priming of γδ T cells. These findings suggest that CT from a selected Botswanan forage plants can stimulate the immune system in vivo in selected ruminant species. The anti-parasitic and immunostimulatory effects could be influenced, among other things, by the chemical structure and concentration of CT in the forage sample. The 13C-NMR and thiolysis results revealed that CT from V. verrucosum, T. oleifolius and G. flava were procyanidin (PC) and cis dominant. Further purification of the thiolysis adducts of CT from these plants led to the isolation of (-)-epicatechin which was found to be the dominant compound in the extension units of the CT polymer. The final stage of the research was the re-chromatography of methanolic fractions containing low molecular weight phenolics. The low molecular weight phenolics from the methanolic fractions were successfully purified and isolated. The characterisation of flavonoids by NMR and LC-ESI-MS/MS showed that quercetin was predominant in the purified fractions with attached sugars such as rhamnose, glucose and apiose.Item Comparative studies on the implications of condensed tannins in the evaluation of Holcus lanatus and Lolium spp. swards for sheep performance : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand(Massey University, 1995) Montossi, FabioThe series of experiments which form the basis of the present study concentrated on evaluations of: (i) diet selection, grazing behaviour, herbage intake, and sheep performance between Holcus lanatus (Yorkshire fog) and Lolium spp. (perennial or annual ryegrass) swards both associated with Trifolium repens (White clover), and in one study with the presence or absence of Lotus corniculatus (Birdsfoot trefoil), and (ii) the effects of condensed tannins (CT) on the behaviour and performance of sheep grazing those swards. The effects of CT on sheep production were assessed by twice daily oral administration of polyethylene glycol (PEG; Molecular weight 4,000) to half of the lambs on each sward combination. Three grazing experiments are reported; the first two (Experiments 1 and 2) were carried out at Massey University (New Zealand) from 1992 to 1993, while the final trial was undertaken at INIA Tacuarembó Research Station (Uruguay) during 1994. In the first experiment (Chapter 3), relationships amongst sward, grazing behaviour, and animal performance variables were studied on perennial ryegrass/white clover and Yorkshire fog/white clover swards rotationally grazed by ewes at medium and high daily allowances (6% and 12% of liveweight as herbage dry matter respectively) during late autumn in 1992. The next experiment (Chapter 4) was designed to investigate the effects of low concentrations of condensed tannins (CT) on lambs grazing perennial ryegrass/white clover or Yorkshire fog/white clover swards at a constant height of approximately 6 cm from December 1992 to March 1993. The final experiment (Chapter 5) was carried out from August to early November 1994 to examine differences in behaviour and performance between annual ryegrass/white clover and Yorkshire fog/white clover swards, both with presence or absence of birdsfoot trefoil, rotationally grazed by lambs Results from Experiments 1 and 2 showed that herbage intake achieved by sheep grazing perennial ryegrass swards was 15 - 27% higher than that achieved on Holcus lanatus swards. Bite weight was 13 - 38% greater for Holcus lanatus than for ryegrass, associated with the 15 - 25% greater sward bulk density. There was a consistent advantage (1 - 5%) in the organic matter digestibility of the herbage selected in favour of ryegrass swards. Sheep on both pasture types concentrated grass rather than clover in the diet. Sheep grazing on ryegrass swards had higher liveweight gains (8 - 51%), clean wool growth (6%), carcass weight (7%), GR values (22%), and carcass dressing out percentage (2%) than sheep grazing on Yorkshire fog swards. The stocking rate maintained on ryegrass plots was 25% greater than that on Yorkshire fog plots. Similar low concentrations of CT were recorded in the diets of ryegrass and Yorkshire fog swards (≤ 0.2% on a DM basis). These results were confirmed by measurements of NH3 concentration in the rumen fluid. The low levels of CT had no significant effects on diet selection, herbage intake, grazing behaviour patterns or lamb performance. However, the lambs grazing on Yorkshire fog swards showed small and non-persistent responses to CT in terms of faecal egg counts, wool growth and liveweight gain. Experiment 3 indicated that the organic matter digestibility of the diet selected and the herbage intake of lambs grazing on Yorkshire fog swards were higher than those on annual ryegrass (5% and 24% respectively), reflecting the higher contents in the diet of grass green leaf and of legume and the lower content of dead material in favour of Yorkshire fog swards. Lambs grazing on both swards showed similar behaviour patterns. Those on Yorkshire fog swards had higher clean wool growth (15%). greater fibre diameter (48%), and longer fibre length (5%), greater liveweight gains (41%), final weight (11%), carcass weight (29%), carcass weight gains (29%), GR value (38%), and lower faecal egg count (FEC) values (20%). Slightly higher CT dietary concentrations were recorded in Yorkshire fog swards than in annual ryegrass (0.420 vs 0.365 ± 0.02% on a DM basis). These low CT levels increased clean wool growth (11%), fibre diameter (4%), although differences in carcass measurements were relatively small, and tended to reduce FEC values (15%). The effects of CT on animal performance were greater in Yorkshire fog swards than in perennial ryegrass swards. CT had no significant effects on diet selection, herbage intake, or grazing behaviour patterns. The very small effects of lotus on sward composition, sward structure and on lamb performance were explained by its very low contribution to both swards. The major conclusions of the first two experiments are as follows: (i) under high fertility conditions and intensive grazing management, perennial ryegrass/white clover swards appeared to have higher feeding value than Yorkshire fog/white clover swards for sheep production; (ii) the results of these experiments confirmed the presence of limited CT concentrations in Holcus lanatus, and provided further evidence that low CT concentrations also exist in perennial ryegrass; (iii) these low CT concentrations (≤ 0.2% on a DM basis) present in both swards did not influence sheep performance significantly. Finally, the conclusions of the last experiment were: (i) under low to moderate soil fertility conditions and lax rotational grazing management, Yorkshire fog swards had better composition and structure for lamb production than annual ryegrass, as a consequence of the early reproductive development in annual ryegrass; (ii) low CT concentrations (range 0.36 to 0.42% on a DM basis) consistently increased wool production and liveweight gains, particularly in Yorkshire fog swards. The findings of these studies are discussed (Chapter 6) in the context of the role of Holcus lanatus for grazing systems and of the potential benefits of low dietary CT concentrations in Holcus lanatus and Lolium spp. for animal production. Keywords: Lolium perenne (perennial ryegrass); Lolium multiflorum (annual ryegrass); Holcus lanatus (Yorkshire fog); Trifolium repens (White clover); Lotus comiculatus (Birdsfoot trefoil); Polyethylene glycol (PEG); Condensed tannins (CT); herbage intake; diet selection; grazing behaviour and lamb production.Item The effect of condensed tannin upon the protein nutritional value of solvent extracted cottonseed meal for ruminant and monogastric animals : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University(Massey University, 1995) Yu, Feng; Yu, FengA series of indoor experiments were conducted at Massey University and AgResearch Grasslands, Palmerston North, New Zealand, to study the effect of cottonseed condensed tannin (CT) upon the nutritional value of solvent extracted cottonseed meal (CSM) for ruminant and monogastric livestock. Ruminant nutrition experiments were conducted using samples suspended in situ in the rumen of fistulated sheep and by incubating samples with rumen fluid in vitro, to study effects upon solubility and degradability of cottonseed proteins. Monogastric nutrition experiments were done initially with laboratory rats as a model for production animals such as the pig, and then with pigs. In all cases half of the animals were supplemented with polyethylene glycol (PEG; MW 3500). PEG specifically binds and inactivates CT and can be used to deduce the effects of CT by comparing control animals (CT acting) with PEG supplemented animals (CT inactivated). 1. Experimental varieties of cottonseed and of industrial CSM were analysed for extractable and bound CT and free gossypol, crude protein, oil and fibre. CT was present in the hulls of all varieties, with higher concentrations recorded for high tannin and glandless selections (55 g kg-1 and 58 g kg-1 DM) than for the multiple host plant resistant and high gossypol selections (38 g kg-1 DM). CT was present in trace amounts in the kernels of high tannin selections, but was not detected in the kernels of all other selections. On average for the hulls of all varieties, approximately 22, 60 and 18% of total CT was present in the extractable, protein-bound and fibre-bound forms, respectively. Free gossypol was mainly found in the kernels, with negligible amounts being found in the hulls of the experimental varieties. Kernels of high gossypol selections contained higher concentrations of free gossypol (18 g kg-1 DM) than kernels of multiple host plant resistant, high tannin and commercial selections (10-12 g kg-1 DM), with free gossypol concentration being very low (0.8 g kg-1 DM) in the kernels of glandless cottonseed. A negative correlation (r = -0.50, P<0.05) between free gossypol in the kernels and total CT in the hulls was found. The mean crude protein, oil and fibre (neutral detergent fibre; NDF) contents of the kernels were 346, 348 and 228 g kg-1 DM, respectively. The hulls contained small amount of crude protein (37 g kg-1 DM) and oil (12 g kg-1 DM), and large amount of fibre (NDF; 891 g kg-1 DM). Commercially produced CSM contained 8-15 g CT kg-1 DM, due to the presence of some hulls, and 0.8 g free gossypol kg-1 DM. The mean contents of crude protein, oil and fibre (NDF) in CSM were 449, 70 and 253 g kg-1 DM, respectively. The results are discussed in relation to plant defence mechanisms against insect attack and in relation to the nutritive value of CSM for ruminant and monogastric livestock. 2. The effect of adding cottonseed hulls upon the solubility of protein in unheated solvent extracted cottonseed kernels was studied using both in vitro incubation in mineral buffer and the in situ polyester bag technique. The latter technique was also used to study effects on rumen DM digestion. Cottonseed hulls contained 51 g CT kg-1 DM, with 56 and 20% of the total CT being bound to protein and fibre, respectively; no CT was detected in kernel. In the absence of hulls, 42% of the total nitrogen (N) in cottonseed kernel was soluble in mineral buffer in vitro, whilst potential in situ N solubility and predicted rumen N solubility (corrected for rumen outflow rate) were 99 and 86% respectively. Addition of hulls linearly reduced both in vitro N solubility and potential in situ N solubility, with 100% hulls addition (i.e. 1 g kernel + 1 g hulls) reducing potential N solubility and predicted rumen N solubility to 94 and 79% respectively. PEG addition had no effect upon the protein solubility of kernels, but increased N solubility in mixtures of hulls and kernels in vitro but not in situ. Two mg PEG mg-1 total CT was shown to reverse the effect of CT in reducing in vitro protein solubility. Potential in situ DM digestion and predicted rumen DM digestion (corrected for rumen outflow rate) were substantially lower for cottonseed hulls (41 and 33%) than for kernels (99 and 88%). Increasing the addition of hulls to kernels lowered the rumen DM digestion of mixtures in a quadratic manner, with increasing rate of hulls causing progressively smaller depressions. Addition of PEG had no effect upon the digestion of kernel DM, but increased potential DM digestibility and predicted rumen DM digestion of hulls to 47 and 40% respectively. It was concluded that the high protein solubility of unheated solvent extracted cottonseed kernels can be linearly reduced by the addition of cottonseed hulls, with the magnitude of the reduction being small, and that the presence of bound CT in hulls substantially depressed fibre digestion by rumen micro-organisms. 3. A 24 h in vitro rumen incubation procedure was developed to measure the effect of adding cottonseed hulls upon degradation of the 52 and 48 kDa major seed storage proteins present in unheated solvent extracted cottonseed kernels. Proteins were fractionated using sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the protein bands quantified using imaging densitometry. A set of in vitro experiments was conducted, in which degradation rate and potential degradability were measured. In the absence of hulls, potential degradability of both kernel proteins was very high (99%), with approximately 97% of this taking place within 8 h, and the addition of PEG did not effect degradation. Increasing rates of hull addition reduced the potential degradability of both proteins in a linear manner, but did not affect degradation rate. Equal weights of hulls and kernel (i.e. 100% hulls) reduced potential degradability of the 52 and 48 kDa proteins by approximately 10% units. Addition of PEG increased degradation of both proteins in incubations involving mixtures of hulls and kernels, with 2 mg PEG mg-1 CT being required to maximise this effect. However, the increase obtained accounted for only 50% of the depression in protein degradation caused by the addition of hulls. In all experiments, the 52 and 48 kDa proteins were similarly affected by the treatments applied. It was concluded that in vitro rumen degradability of the 52 and 48 kDa storage kernel proteins was very high and close to 100%, and that this could be reduced by the addition of hulls in a linear relationship, with approximately half of the depression in potential degradability caused by hulls being due to effects of CT. 4. The effect of CT in cottonseed hulls on endogenous ileal amino acid flow in the growing rat was evaluated. Twenty-four rats were allocated to four semi-synthetic diets, which contained enzymically hydrolysed casein (EHC) as the sole source of dietary N and chromic oxide as an indigestible marker. Two of the diets contained no hulls while the remaining two contained 50 g kg-1 hulls. At each level of hull inclusion, PEG was added to one of the diets. The rats were given their respective experimental diets ad libitum for 14 days. Samples of digesta were collected at slaughter from the terminal 15 cm of ileum. The digesta samples were centrifuged and the supernate ultrafiltered. The precipitate plus retentate (MW> 10,000) fraction affords an estimate of endogenous loss. Inclusion of hulls in the EHC based diet increased ileal flow of total N (1387 vs. 1623 mg kg-1 dry matter intake; p<0.05), increased ileal flow of total amino acids (23%; p<0.01), and significantly increased ileal flow of several individual amino acids. There was no significant effect of PEG and no PEG x diet interaction, showing that the hull effects could not be explained by action of CT. The presence of some hulls in commercial CSM will contribute to lowering apparent ileal amino acid digestibility, due to its effect in increasing endogenous loss. 5. The effect of adding cottonseed hulls to casein and to cottonseed kernel based diets on the apparent and true ileal digestibility of N and amino acids, and the proportion of this effect accounted for by CT, was determined using the growing rat. Sixty rats were allocated randomly to 10 semi-synthetic diets, containing either casein (4 diets) or purified unheated solvent-extracted cottonseed kernel (6 diets) as the sole protein sources, with chromic oxide added as an indigestible marker. Two of the casein diets contained no hulls whilst the remaining two diets contained 70 g hulls kg-1. Two of the cottonseed kernel based diets contained no hulls, with two containing 23 g hulls kg-1 and the remaining two containing 46 g hulls kg-1. For each pair of diets, PEG was either included or excluded. Samples of digesta were collected at death from the terminal 15 cm of ileum. The inclusion of hulls depressed the apparent and true ileal digestibility of N and amino acids, but with the response differing between diets. With the casein based diet the mean apparent and true ileal amino acid digestibilities were significantly depressed from 0.89 and 0.96 to 0.85 and 0.92, respectively, by the inclusion of hulls in the diet, and addition of PEG then restored these to 0.89 and 0.95. All of the depression could be explained by the CT content of the hulls. However, with the cottonseed kernel based diet the response fell into three categories. The apparent and true ileal digestibilities of the essential amino acids cystine and methionine were not affected by hull addition, ileal digestibilities of leucine, isoleucine, lysine, threonine and valine were markedly depressed by hull addition with approximately 50% of the depression being explained by CT, whilst the ileal digestibilities of histidine, arginine and phenylalanine were depressed by hull addition but little or none of this effect could be explained by CT. With the cottonseed kernel based diet it seems that unknown components of the hulls other than CT also depressed the apparent and true ileal digestibility of N and amino acids. 6. The effects of heat treatment, with or without the addition of cottonseed hulls, on the chemical composition of CSM and upon reactivity of the CT were studied. Heat treatment (100°C for 2 h, in a forced draught oven) reduced the concentrations of free gossypol and fluorodinitrobenzene (FDNB) available lysine by small amounts, reduced measurable total CT content by 13%, reduced the solubility of total N, and reduced potential degradability of the 52 and 48 kDa cottonseed storage proteins by mixed rumen micro-organisms. Addition of hulls further depressed solubility of total N and ruminal degradation of the two major storage proteins in cottonseed kernel. The action of PEG in vitro indicated that only part of the depression caused by hull addition could be explained by the presence of CT in the hulls, and that the effects of CT upon N solubility and potential degradability in heated CSM were similar to that in unheated CSM. Addition of hulls also substantially reduced FDNB available lysine. Although application of heat inactivated 13% of the total CT such that it could no longer be detected with butanol/HCl, it did not seem to change the reversible reactivity of CT with kernel proteins. Commercial CSM produced from the Brisbane mill had a lower total CT content, lower N solubility and ruminal protein degradation rate than CSM produced from the Narrabri mill, but a similar level of FDNB available lysine. 7. The effect of CT from heated and unheated cottonseed on the apparent ileal digestibility of amino acids for the growing rat and pig was determined. In Experiment 1, twenty-four rats were allocated to four semi-synthetic diets, which contained cottonseed kernel/hulls as the sole protein source. Two of the diets contained unheated solvent extracted cottonseed kernel/hulls while the remaining two diets contained similar material but which had been heat-treated by autoclaving at 110°C for 120 min. In Experiment 2, twelve rats and twelve pigs were fed four semisynthetic diets containing commercial CSM as the sole protein source. Chromic oxide was added to all diets as an indigestible marker. For each pair of diets in both experiments, PEG was either included or excluded. Ileal contents from the terminal 15 and 45 cm of ileum were collected at slaughter for the rats and pigs, respectively. Apparent ileal amino acid digestibility for rats fed the cottonseed kernel/hulls diet was significantly depressed by the heat treatment, particularly for lysine and threonine. On average, apparent ileal amino acid digestibility in the diets without PEG was decreased from 0.80 to 0.70 by heat treatment. Dietary cottonseed CT depressed apparent ileal protein digestibility in the pig and in the rat. The addition of PEG to the diets significantly increased the apparent ileal digestibility of N and some amino acids for the pigs and the rats. The mean increase in apparent ileal digestibility due to PEG addition for the 14 amino acids was 2% units in both species fed the commercial CSM diets, and 2 or 4% units in rats fed the unheated or the heated cottonseed kernel/hull diets, respectively. The effect of PEG was similar in the heated and unheated cottonseed kernel/hulls for most amino acids, but responses to PEG for lysine, threonine and tyrosine were greater in heated than unheated CSM. For several of the amino acids there were significant animal species differences in apparent ileal digestibility. Studies into the effects of cottonseed CT should be carried out in the target animal species. The commercial CSM had a low apparent ileal amino acid digestibility overall, particularly for the essential amino acids lysine and threonine. It was concluded that effects of heating did not eliminate the reversible reactivity of cottonseed CT on amino acid digestion in rats and pigs but rather appeared to increase it for threonine, tyrosine and lysine in Experiment 1, causing large reductions in apparent ileal digestibility. It was concluded that CT in CSM are mainly in the protein- and fibre-bound forms (80%), with only 10-20% of CT being extractable. In the absence of heat, adding graded levels of hulls reduced both the solubility and degradability of protein by rumen microorganisms, giving a rumen 'by-pass' effect. However, the magnitude of these effects was small and they are not regarded as being nutritionally significant under practical conditions of ruminant nutrition. Adding hulls significantly depressed the digestion of amino acids in the small intestine of the monogastric animals. This effect is considered large, and is probably one of the factors responsible for the generally low levels of amino acid availability found for commercially produced CSM. Approximately half of the 'hull effect' is due to their content of CT and the cause of the other 50% is unknown. As there is no CT in cottonseed kernels, this explains the lack of response to PEG when the diet does not contain cottonseed hulls. The study has also shown that the large amount of heat applied during normal commercial CSM processing reduces the digestion of amino acids by monogastric animals. A combination of heat and CT from hulls is especially damaging for lowing ileal amino acid digestion in monogastric species, particularly for the limiting essential amino acids lysine and threonine.Item The effects of condensed tannins upon nutrient digestion and metabolism and upon animal production in sheep fed Lotus corniculatus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University(Massey University, 1995) Wang, YuxiA series of indoor metabolism and grazing experiments were conducted at AgResearch Grasslands and Massey University, Palmerston North, New Zealand, to study the effects of condensed tannins (CT) in Lotus corniculatus (birdsfoot trefoil; cv. Grasslands Goldie) upon nutrient digestion and metabolism and upon animal production using sheep as the experimental animal. Half of the animals in each experiment were supplemented with polyethylene glycol (PEG; MW 3500). PEG specifically binds and inactivates CT without affecting other nutrients and is indigestible; hence effects of CT were specifically defined by comparing control sheep (CT acting) with PEG supplemented sheep (CT inactivated). A rotational grazing system with some restriction in feed allowance was used in most of the grazing experiments. For the indoor experiments, sheep were held in metabolism crates and fed fresh L. corniculatus from overhead belt feeders at hourly intervals. 1. The effects of CT in L. corniculatus (35 g total CT/kg DM) upon nutrient digestion and upon metabolism of methionine, cystine and inorganic sulphate in plasma were determined. PEG was continuously infused into the rumen of half the sheep. Principal measurements in the two groups were plasma irreversible loss rate (IRL) and interconversions of methionine, cystine and inorganic sulphate using 35S labelling. Action of CT considerably reduced the concentration of ammonia and molar proportions of iso-butyric acid, iso-valeric acid and n-valeric acid in rumen fluid, but had no effects upon total volatile fatty acid (VFA) concentration and molar proportions of major VFAs. CT greatly increased the IRL of plasma cystine (13.1 vs 7.0 µmol/min) and reduced IRL of plasma inorganic sulphate (36.8 vs 48.1 µmol/min) but had no effect upon methionine IRL. Action of CT increased transulphuration of methionine to cystine (4.37 vs 1.24 µmol/min) and increased cystine flux to body synthetic reactions (11.89 vs 5.41 µmol/min). CT had no effect upon the proportion of methionine total flux transferred to sulphate (0.05 vs 0.06; i.e. oxidation) but markedly reduced the proportion of cystine flux transferred to sulphate (0.09 vs 0.27). CT had no effect upon the apparent digestion of cellulose and minerals, slightly depressed DM, OM and hemicellulose apparent digestion and markedly reduced the apparent digestion of nitrogen (N). Action of CT also reduced protozoa numbers in rumen fluid. 2. The productivity of lactating ewes (Exp 1) and weaned lambs (Exp 2) grazing swards of L. corniculatus, lucerne (Medicago sativa; cv. Grasslands Oranga) and a mixture of lucerne and lotus were compared in two grazing experiments in the 1991/1992 summer. Total CT content was 32-57 g/kg DM for lotus, 8-10 g/kg DM for the mixture and was negligible for lucerne (< 2 g/kg DM). In Experiment 1, ewe wool production and lamb live weight gain (LWG) did not differ between forages, but ewe LWG was greater on lotus than on lucerne (251 vs 65 g/d), with the mixture being intermediate (115 g/d). In Experiment 2, voluntary feed intake (VFI; 1.76 vs 1.65 kg OM/d), LWG (228 vs 183 g/d), wool production (2.78 vs 2.25 kg) and carcass weight (20.4 vs 17.8 kg) were greater for lambs grazing lotus than lucerne; lambs grazing the mixture had similar VFI (1.63 kg OM/d) to those grazing lucerne, but wool production (2.49 kg) was intermediate between lucerne and lotus lambs. Lotus did not affect carcass fatness (GR 13.1 vs 12.8 mm). It was concluded that L. corniculatus supported high levels of sheep productivity, with the results suggesting that part of the response may be due to increased protein supply from action of CT in the digestive system. 3. A grazing experiment was conducted for 22 weeks in the 1992/93 summer to study the effects of CT upon lamb LWG, wool growth and rumen metabolism, and to compare the productivity of lambs grazing L. corniculatus and lucerne. PEG was given orally twice daily to half of the lambs grazing each sward. Lotus contained 34 g total CT/kg DM in the diet selected, whilst there was essentially no CT in lucerne. Compared to lambs grazing lucerne, lambs grazing lotus had slightly lower VFI, and higher LWG, carcass weight gain, carcass dressing-out percent and wool growth. PEG supplementation had no effect on these measurements or upon the composition of rumen fluid in lambs grazing lucerne. However, in lambs grazing lotus, PEG supplementation reduced wool growth (10.9 v. 12.1 g/day), slightly reduced LWG (188 v. 203 g/day), increased rumen ammonia concentration, and increased the molar proportions of iso-butyric, iso-valeric and n-valeric acids and protozoa numbers in rumen fluid. PEG supplementation did not affect carcass gain, carcass fatness or the molar proportion of rumen acetic, propionic or n-butyric acids in lambs grazing lotus. 4. The effects of CT in L. corniculatus upon the lactation performance of ewes rearing twin lambs was measured in an 8 week grazing experiment in the spring/summer of 1993. Half of the ewes were supplemented orally twice daily with PEG. Lotus contained 45 g total CT/kg DM in the diet selected, with an in vitro digestibility of 73%. The results showed that action of CT slowed down the decline in milk production and secretion rates of protein and lactose after the attainment of peak lactation, resulting in more milk (21%), more milk protein (14%) and more milk lactose (12%) secretion in mid and late lactation compared to CT inactivated ewes. CT reduced milk fat percentage but not fat secretion rate. CT had no effect upon VFI, LWG and wool growth of lactating ewes rearing twin lambs. Plasma urea and glucose concentrations were reduced due to action of CT. CT had no effect upon concentrations of non-esterified fatty acids (NEFA), growth hormone and insulin in the plasma, had no effect upon molar proportions of acetic, propionic and n-butyric acids in rumen fluid, but markedly reduced concentrations of ammonia and molar proportions of iso-butyric, iso- and n-valeric acids in rumen fluid. 5. The effect of CT upon the true and apparent digestion of methionine and cysteine in the small intestine (SI) of sheep fed L. corniculatus containing 30 g total CT/kg DM were determined, using sheep prepared with rumen and abomasal cannulae. An indigestible liquid phase marker chromium ethylene diamine tetra acetic acid (Cr-EDTA) was continuously infused into the rumen of all sheep, and PEG was continuous infused into the rumen of half the sheep. The true digestibility of methionine and cysteine in the SI and their absorption sites in the SI were measured from continuous intra-abomasal infusion of plant homogenate from L.corniculatus containing 35S-labelled protein. Action of CT substantially reduced the true digestibility of methionine (0.72 v 0.88) and cysteine (0.66 v 0.81) in the SI, but increased the total amount of plant methionine and cysteine absorbed in the SI due to reduced rumen degradation. Action of CT slowed the digestion of both 35S-methionine and 35S-cysteine in the SI, and increased the flux of both amino acids in the mid and latter thirds of the SI. CT increased abomasal flux (as a proportion of eaten) of total methionine (0.88 v 0.76) and total cysteine (0.74 v 0.62), and increased absorption of total methionine (0.72 v 0.63 g/g eaten) but not total cysteine (0.49 v 0.48 g/g eaten) from the SI. Calculated endogenous loss of cysteine at the terminal ileum was greater than for methionine and both appeared to be increased by action of CT. It was concluded that action of CT in L. corniculatus increased wool growth rate in high wool producing sheep and increased milk production in lactating ewes without affecting VFI, thus improving production efficiency. It was deducted that the improved animal production was probably due to the action of CT reducing rumen protein degradation, increasing non-ammonia nitrogen (NAN) flux into the SI, increasing essential amino acid (EAA) especially methionine absorption from the SI, and increasing the flux of cystine to body synthetic reactions. Further researches are needed to study the effects of CT in the range 10-20 g/kg DM on animal production and nutrient metabolism, and to study the effects of forage CT upon milk production and composition in dairy cows.Item Comparative evaluation of diet selection, herbage intake and performance of lambs grazing Yorkshire fog (Holcus lanatus L.), perennial ryegrass (Lolium perenne L.), and tall fescue (Festuca arundinacea Schreb.) and assessment of effects of condensed tannins (CT) in the grasses on lamb performance : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Plant Science Department of Massey University, Palmerston North, New Zealand(Massey University, 1996) Liu, FuyuanA series of grazing experiments was carried out at the Pasture and Crop Research Unit, Massey University, Palmerston North, New Zealand, to compare Yorkshire fog (Holcus lanatus cv. Massey Basyn)/white clover (Trifolium repens cv. Grasslands Tahora) with perennial ryegrass (Lolium perenne cv. Grasslands Nui)/white clover cv. Grasslands Tahora and tall fescue (Festuca arundinacea cv. Grassland Roa)/white clover cv. Grasslands Tahora pastures with reference to grazing behaviour, diet selection, herbage intake and performance of lambs, and to specifically assess the effects of low concentrations of condensed tannins (CT) in grasses on body growth and wool growth of animals. Half of the lambs were drenched with polyethylene glycol (PEG) (CT inactivated) and the remainder were drenched with water as a control (CT acting) in each experiment. PEG is assumed to specifically bind and inactivate CT without influencing the digestion of other nutrients. In the first experiment, a comparative study of grazing behaviour, diet selection, herbage intake and performance of lambs grazing Yorkshire fog/white clover and ryegrass/white clover swards was made from late May to late June, 1993. Thirty-six lambs, balanced in sets of six based on initial fasted weight continuously grazed paddocks with three replicates of the two pasture treatments for 7 weeks. Yorkshire fog had greater sward bulk density and intake per bite (68 vs 51 ± 7.1 mg OM/bite) than ryegrass. Perennial ryegrass had a consistent superiority over Yorkshire fog in organic matter digestibility (OMD) (80 vs 77 ± 0.3 %, P≤0.05) and herbage OM intake (1117 vs 930 ± 31.5 g/day, P≤0.1), resulting in faster liveweight gain (174 vs 144 ± 9.7 g/day), significantly higher carcass weight (17.7 vs 16.3 ± 0.1 kg, P≤0.05) and dressing out % (49 vs 48 ± 0.2, P≤0.05). Low CT concentrations (1.7 - 2.2 g/kg DM) were found in the diets selected from both the grasses; these low CT concentrations had no effect on grazing behaviour, diet selection and herbage intake. Small responses to PEG administration were observed in initial liveweight gain, but PEG had no effects on overall liveweight gain, carcass weight, carcass weight gain and GR (depth of total soft tissue over the 12th rib at a point 11 cm from the mid carcass). The next two experiments were designed to compare Yorkshire fog cv. Massey Basyn)/white clover cv. Grassland Tahora with tall fescue cv. Grassland Roa/white clover cv. Grassland Tahora pastures in terms of grazing behaviour, diet selection, herbage intake and performance of lambs, and to further quantify the effects of low CT concentrations in the grasses on lamb performance, especially on initial liveweight gain of lambs, under rotational grazing management in late spring, summer and early autumn, 1993/1994. The comparisons between Yorkshire fog and tall fescue pastures were made under similar sward conditions. Previous grazing experience on the appropriate pasture and sex were designed as treatments as well as pasture species and PEG supplementation. Forty-eight lambs balanced for previous grazing experience and sex in sets of sixteen were used in each experiment. One group of 16 was slaughtered as the initial group at the start of each experiment to measure the carcass weight. The other two groups of lambs grazed six paddocks of each pasture treatment in a 30-day rotation. Tall fescue had higher total N than Yorkshire fog in early December (3.56 vs 3.43±0.018 % DM, P≤0.05) and late February (3.24 vs 2.91 ±0.022 % DM, P≤0.0001), and had higher OMD in early December (81 vs 78 ±0.6 %, P≤0.01) and late February (72 vs 68 ± 1.1 %, P≤0.05), but lower OMD in early February (71 vs 74 ± 1.1 %, P≤0.05). Yorkshire fog produced faster liveweight gain (99 vs 76 ± 6.7 g/day, P≤0.1), greater carcass weight (14.7 vs 13.9 ± 0.2 kg, ≤0.05) and faster carcass weight gain (32 vs 20 ± 3.1 g/day, P≤0.05) than tall fescue in late spring and summer (Experiment 2), but not in late summer and early autumn (Experiment 3). Male lambs had faster liveweight gain than female lambs in Experiment 2 (95 vs 80 ± 3.3 g/day, P≤0.05) and in Experiment 3 (80 vs 72 ± 2.3 g/day, P≤0.05). Previous grazing experience had no effects on final liveweight gain, carcass weight, carcass weight gain. There was no significant effect of interaction between previous pasture and current pasture on these parameters. The results of the experiments further confirmed the low CT concentrations (1. - 2.1 g/kg DM) present in Yorkshire fog in Experiment 1 and small responses of lambs to PEG supplementation in initial liveweight gain only in Yorkshire fog (101 vs 92 ± 4.1 g/day, P≤0.1) in Experiment 2. There were no significant effects on carcass weight, dressing out, GR and wool growth rate. Lower faecal egg counts in lambs in Yorkshire fog than in tall fescue suggested some potential of Yorkshire fog for parasite control. The low CT concentrations in the Yorkshire fog reduced to some extent rumen ammonia concentration, but were not enough to effectively promote animal performance. The relatively low CT concentrations detected in tall fescue were probably an artifact, because there was no PEG effect on rumen ammonia concentration for tall fescue. The final grazing experiment was conducted to evaluate grazing behaviour, herbage intake and performance of lambs as affected by grazing selection opportunity and low condensed tannin (CT) concentrations in Yorkshire fog/white clover pasture under rotational grazing management from late November, 1994 to early February, 1995. Twelve lambs were slaughtered as the initial group at the start of the experiment to measure the preliminary carcass weight. Forty-eight lambs were allocated to two groups in sets of twenty-four and rotationally grazed eight paddocks each of 0.1 ha, in which a "leader" group of 24 lambs grazed each paddock for four days, followed by a similar "follower" group of 24 lambs grazing for four days. The leader/follower grazing regime created the desired contrasts in herbage quality and quantity. The superiority in sward allowance, selection opportunity, diet quality and reduced possibility of infection by worm parasites resulted, as expected, in faster body growth and wool growth rate in the 'leader' lambs than in the 'follower' lambs. The results of the experiment further confirmed the findings of the influence of low CT concentrations in Yorkshire fog on rumen ammonia concentration. The effect of low CT concentrations on animal performance was not different for generous and restricted grazing. The following general conclusions can be drawn from this series of experiments: 1) The diets selected by sheep comprised more green material and less dead material than the swards offered to the animals, while diet composition was determined largely by the structure and distribution of sward components rather than by deliberate selection by the animals. 2) Herbage OM intake was influenced to a greater extent by nutritional factors than by behavioral limitations, there being substantially higher herbage OMD and OM intake on ryegrass than on Yorkshire fog in winter. 3) Perennial ryegrass/white clover pasture tended to have higher animal production than Yorkshire fog/white clover pasture under continuous grazing management in winter, and Yorkshire fog pasture produced slightly higher animal performance than tall fescue in rotational grazing management in late spring and early summer. 4) The results of the trials confirmed that low CT concentrations (0.18 - 0.32 % on a DM basis) were present in Yorkshire fog, and provided evidence that perennial ryegrass contained relatively low CT concentrations, but the low CT concentrations detected in tall fescue were probably an artifact of the current procedures of analysing CT. 5) The low CT concentrations in the grasses had no effect on grazing behaviour, diet selection or herbage intake. The low CT concentrations in Yorkshire fog to some extent reduced rumen ammonia concentration, but were not enough to effectively improve animal production.