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Item Dynamics of nitrogen in three contrasting pastures grazed by sheep : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Soil Science at Massey University(Massey University, 1991) Ruz Jerez, Belarmino EmilioThe dynamics of nitrogen (N) were studied during two years (March 1989 - May 1991) in three contrasting pastures grazed by sheep. The pastures were: ryegrass-white clover, herbal ley (a legume-based pasture of interest for "organic" agriculture) and pure ryegrass receiving 400 kg fertiliser N/ha/yr. This study was undertaken on a recent alluvial soil at DSIR Grasslands. Palmerston North. Treatments were replicated as small paddocks, and periodically mob-grazed with sheep. Frequent soil measurements provided estimates for leaching and denitrification. Herbage yields and botanical composition were recorded, and symbiotic N2 fixation was measured in swards of the two treatments containing forage legumes. Soil total N and carbon were measured annually, providing estimates of the partial mass balance for N. The soil mineral N pool was dominated (especially in the systems receiving no fertiliser N) by the highly concentrated pulse of N returned in the excreta of grazing animals to a small proportion of the grazed area. In the pure grass sward the large inputs of fertiliser N had a significant effect in increasing the amount of mineral N available in the top 45 cm of soil. On average, in composite samples including urine-affected and non-affected areas, about 30 kg/ha-45 cm more mineral N was available throughout the year in the ryegrass fertilised with N than in the legume-based pastures. This consistently high level of soil mineral N in the ryegrass+N sward was responsible for the greatest annual herbage yield; however annual losses by leaching and denitrification were 5 to 6 times greater than in the legueme-based pastures. A common feature of the three pastures was the small amount of N recovered in animal products, with most of the N that circulated through the plant to the sheep being returned to the soil in urine. This concentrated input was localised in about 10% of the area, which provided the major avenues for N escape from the pastures receiving no fertiliser N. It was estimated that a little more than half the nitrate leached (total, about 6 kg NO3-N/ha/yr) arose from this restricted area, but in the grass+N pasture the contribution of animal-induced losses was proportionally smaller than in the legume-based pastures. Fertiliser N. by increasing soil mineral N, offered more site opportunities for N leaching and denitrification, in addition to that from urine. Here, only one-quarter of leached nitrate (total, 41 kg NO3-N/ha/yr) arose from urine patches. Denitrification accounted for 4-5 kg N/ha/yr from the legume-based pastures, but 20 kg N/ha/yr from swards receiving fertiliser N. Ammonia volatilisation, which was estimated using data from previous studies at this site, was enhanced by direct emission from the fertiliser N (urea) as it is hydrolysed on the soil surface. Calculation of N inputs and outputs for these three pastures indicated that the two legume-based systems were more or less in balance, but in the pasture receiving fertiliser N some 180 kg N/ha/yr was unaccounted for. This difference may reflect incorporation of N into soil organic matter, as indicated by a small increase in soil total N during the second year. Pasture production (average of two years) from the herbal ley was about 15 t DM/ha/yr, or about 90% of the yield from pasture receiving fertiliser N, and some 25-30% more than from ryegrass-clover. Symbiotic N2 fixation, estimated by the acetylene reduction assay to have been 140-150 kg N/ha/yr, was similar in both systems based on forage legumes. The herbal ley utilised soil N more efficiently than the ryegrass-clover and ryegrass+N pastures, hence achieving an outstanding yield of herbage. It is argued that this apparently better exploitation of soil N was brought about largely by stimulation of microbial biomass in the rhizosphere around chicory roots, with the additional N that was scavenged by bacteria being made available to this herb after protozoan digestion of the bacteria. A herbal ley offers the possibility of sustaining a high level of forage production, but with reduced N emissions to the environment.Item Digestion and metabolism of sulphur containing amino acids in sheep fed fresh forage diets : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand(Massey University, 1990) McNabb, Warren CharlesSulphur amino acids (SAA) are important in the sheep industry because they appear in many instances, to limit wool protein synthesis. Experiments were carried out to study two methods with the potential to increase the absorption of SAA from the small intestine of sheep fed fresh forage diets. The first experiment evaluated the effects of condensed tannins (CT) in Lotus pedunculatus upon the following; (1) Rumen-S metabolism, and (2) the digestion of methionine and cystine. (3) The metabolism of plasma SAA and inorganic sulphate, and (4) The solubility and (5) degradation of protein in the rumen. The second experiment identified proteins that contained a high proportion of SAA, and assessed their potential for expression in the leaves of forage legumes using genetic engineering techniques. Two aspects were measured; (6) Rates of rumen degradation of ovalbumin and sunflower albumin 8 (SF8) proteins in vitro, and (7) The expression of SF8 protein from sunflower seeds in the leaves of transgenic tobacco. The nutritional consequences of CT in Lotus pedunculatus were assessed by infusing polyethylene glycol (PEG), into the rumen of one group of sheep (PEG sheep; CT not operating), whilst a separate group of sheep received an infusion of water (CONTROL sheep; CT operating). PEG selectively binds CT, preventing CT from binding plant proteins in the rumen, so that CT effects on digestion could be evaluated. Polyethylene glycol was also added to in vitro incubations as required. The principal results were; (1) Condensed tannins had a major effect on rumen sulphur (S) metabolism. The irreversible loss rate (IRL) of reducible-S (total non-protein S in rumen fluid, measured as sulphate after performic acid oxidation of rumen fluid) from the rumen was lower (P<0.001) in control (0.84gS/d) than PEG (2.49gS/d) sheep. This was due in part to a higher (P<0.001) flux of methionine (2.75 and 2.09g/d) and cystine (3.33 and 2.52g/d) through the abomasum in control than PEG sheep. There was no net loss of dietary methionine or cystine from the rumen in control sheep, whilst 29% of methionine (P<0.001) and 28% of cystine (P<0.001) intake disappeared from the rumen in PEG sheep. The proportion of microbial-non-ammonia-nitrogen (NAN) in whole rumen digesta-NAN was lower (P<0.001) in control (0.44) than PEG (0.71) sheep, although it was calculated that the rumen microbial-NAN pool size was similar in control (2.9g) and PEG (3.1g) sheep. These observations suggest CT reduced the degradation of forage protein and SAA in the rumen. (2) The apparent absorption of methionine from the small intestine was higher (27%; P<0.001) in control (2.11g/d) than PEG (1.66g/d) sheep, but the apparent absorption of cystine from the small intestine was similar (4%; P>0.05) for control (1.40g/d) and PEG (l.34g/d) sheep. The apparent digestibility of methionine in the small intestine was similar (0.78) for both groups, whilst for cystine, it was lower (P<0.01) in control (0.42) than PEG (0.53) sheep. The increased absorption of methionine from the small intestine with CT was due to an increased flux from the rumen, whereas the digestibility of cystine in the small intestine may have been lower as a consequence of binding to CT complexes. (3) Condensed tannin had a major effect on plasma SAA fluxes, especially cystine. Plasma cystine concentration and IRL were higher (P<0.001) in control (41.7μmol/l and 39.8:μmol/min) than PEG (27.5μmol/l and 22.4μ.mol/min) sheep. Condensed tannins resulted in a 79% increase in the transulphuration of methionine to cystine (11.7 and 6.5μ:mol/min; P<0.05) and a decrease (P<0.05) in the oxidation of cystine (3.33 and 5.2μ.mol/min) and methionine (0.2μ.mol/min and 1.2μmol/min) to sulphate in control compared to PEG sheep. The net effect of CT was to increase (P<0.05) the flux of plasma cystine to productive processes and maintenance by 110% in control (36.5μmol/min) compared to PEG (17.4μmol/min) sheep. This represented 91% of total plasma cystine flux in control sheep, compared to only 74% of total cystine flux in PEG sheep (P<0.05). Since wool growth is generally accepted as the major productive process utilising plasma cystine, these results indicate that a major effect of CT in the diet would probably be to increase wool growth. The IRL of plasma methionine was similar in control (20.5μmol/min) and PEG (19.9μmol/min) sheep, whilst the IRL of plasma sulphate was lower (P<0.01) in control (35.9μmol/min) than PEG (50.lμmol/min) sheep. (4) The rate of protein solubilization in the rumen was studied by measuring the loss of N, corrected for microbial-NAN contamination (true), from fresh minced (FM) and freeze-dried and ground (FD) Lotus pedunculatus, incubated in polyester-fibre bags suspended in the rumen of control and PEG sheep fed Lotus pedunculatus. Freshly minced Lotus was chosen as one treatment because it more closely represented the effects of chewing on cell rupture and CT release than was likely with freeze drying. Mincing resulted in a much greater initial N loss (47%) than freeze drying (14%). The true loss of N from FD Lotus was higher in PEG than control sheep at 2, 4, 6.5, 11 and 24 hours of incubation, whilst with FM Lotus, N losses were similar. Microbial-NAN adhering to FD residues was higher in PEG than control sheep at 2, 4, 6.5 and 11 hours, but was similar at 24 hours of incubation. However, microbial-NAN adhering to FM Lotus was higher in PEG compared to control sheep, only at 6.5 and 24 hours. These observations suggest that CT reduced protein solubility and microbial colonization of FD Lotus to a much greater extent than FM Lotus. (5) The rate of protein degradation in the rumen was studied in vitro by incubating FM and FD Lotus pedunculatus in rumen fluid, with and without PEG, and using sodium-dodecyl-sulphate gel electrophoresis (SDS-PAGE). After 4 hours of incubation protein from FD and FM Lotus was clearly degraded when PEG was present, whilst after 8 hours of incubation it was essentially undetectable in both incubations. In contrast, after 8 hours, leaf protein from FM and FD Lotus was still readily detectable in incubations without PEG. Therefore, CT substantially reduced the rate at which soluble protein was degraded by rumen microorganisms but had little effect on the rate at which it was solubilized, particularly when minced. (6) The rate of degradation of SF8 protein was compared to the degradation of the LSU and small subunit (SSU) of Fraction 1 leaf, vicilin and ovalbumin proteins using in vitro incubations and SDS-PAGE. The SF8 protein had a rate of proteolysis of 0.23h-1 and a half-life of 3.0 hours, but the principal degradation product of SF8, which had a half-life of 69 hours, was extremely resistant to rumen degradation. Proteolysis of the LSU of Fraction 1 leaf protein was resolved into two components. The first product had a degradation rate of 0.06h-1 and a half-life of 11.6 hours, whilst the second component of proteolysis, which occurred from 12 hours onwards, had a degradation rate of 0.45h-1 and a half-life of 4.6 hours. The proteolysis of the SSU of Fraction 1 leaf protein had a degradation rate of 0.04h-1 and a half-life of 17.3 hours. Ovalbumin was not degraded during the initial 16 hours of incubation, but was then degraded at a rate of 0.08h-1, with a half-life of 8.7 hours. Vicilin had a rate of proteolysis of 4.3h-1 and a half-life of about 10 min. Both SF8 protein and ovalbumin were found to be more resistant to rumen proteolysis than the LSU of Fraction 1 leaf protein, but different mechanisms were involved in conveying resistance. Therefore it is worthwhile to introduce expression of genes coding for these proteins into the leaves of important agricultural legumes, using genetic engineering techniques, with a view to increasing the availability of SAA for sheep. (7) The gene for SF8 is normally expressed only in seeds. Therefore a SF8 cDNA clone was genetically engineered for expression in the leaves of tobacco plants and inserted into a gene delivery system in Agrobacterium tumefaciens and transferred to tobacco. Transcription of the SF8 synthetic (chimeric) gene occurred in the leaves of transformed tobacco, with the level of SF8 mRNA varing over a 100 fold range, but in the highest expressor, it represented 14% of the SF8 mRNA level found in sunflower seeds. However, SF8 protein was not detected in the leaves of transformed tobacco. Consequently, the level of SF8 in the leaves of transgenic tobacco must have been less than 0.03% of total leaf protein. The highest SF8 mRNA expressor contained nine times more SF8 mRNA than an ovalbumin-transformed tobacco contained ovalbumin mRNA. As the ovalbumin transformed tobacco produced 0.01% of its leaf protein as ovalbumin, there is at least sufficient SF8 mRNA to support up to nine times that level of protein expression. As this level of SF8 protein expression would be detectable using sensitive immunological procedures, it seems that either SF8 mRNA is not translatable in tobacco plants or SF8 protein is very unstable in tobacco leaves. Translatability of SF8 mRNA was tested in E. coli using an expression vector, pJLA602 without success. If SF8 protein was unstable in tobacco leaves, then SF8, which is a seed storage protein, should be stably accumulated in tobacco seeds. The expression of the SF8 chimeric gene was monitored in tobacco seeds, and again the results were negative. It would appear that the SF8 cDNA coding region was untranslatable so that DNA sequencing of the SF8 chimeric gene will be necessary to correct the DNA sequence by oligonucleotide-directed, site specific mutagenesis. (8) Both CT and proteins, resistant to degradation in the rumen and with a high proportion of SAA, have the potential to increase the absorption of SAA from the small intestine in sheep grazing fresh forages. However, further research is required to examine; (a) the effectiveness of lower dietary CT concentrations than were examined in the present studies, on increasing SAA absorption and metabolism, and (b) what level of foreign gene expression is required in transgenic legumes to stimulate wool growth.Item The effect of feeding willow upon the death of established parasites and upon parasite fecundity : a thesis presented in partial fulfilment of the requirements for the degree of Animal Science at Massey University, Palmerston North, New Zealand(Massey University, 2010) Mupeyo, BornwellTwo indoor feeding experiments were conducted at the Animal Physiology Unit (APU) of Massey University, involving young sheep, to investigate the effect of feeding forage willow upon the death of established parasites and upon parasite fecundity, using chaffed lucerne as the control diet. Experiment 1: Twenty-four (24) parasite-free weaned hoggets weighing 29.9 ±1.2 kg (SD) were individually penned and fed chaffed lucerne ad libitum during a preexperimental adaption period of 5 weeks. They were then fed either lucerne chaff or chopped willow for a further 5 weeks (n = 12/group) and intakes were adjusted such that the DMI of the two groups was similar during weeks 9 & 10. All lambs were infected with L3 larvae parasites comprising 20,650 Teladorsagia, 1,320 Trichostrongylus and 330 Cooperia through oral drenching 12 days before willow feeding started. This was done after confirmation that the sheep were free of nematodes through FEC analysis. Total faeces were collected for 3 day periods towards the end of weeks 9 & 10, to measure diet digestibility and total faecal egg excretion. The sheep were slaughtered at the end of week 10. Voluntary feed intake (VFI), FEC and liveweight were measured weekly, whilst burdens of individual parasites and carcass characteristics were measured after slaughter. Duplicate samples of each feed offered and individual animal refusals were taken daily and pooled weekly per animal for chemical analysis. Female worm fecundity was calculated by two methods. Blood samples for immunological analysis were collected on days 20, 34, 51 and 70, and analysed for components of white blood cells (WBC) and for lymphocyte subsets. Experiment 2: A 2 x 2 changeover experiment was conducted, involving two time periods (Period 1 and Period 2 each of 14 days) with the same diets as used in Experiment 1, fed to 9 individually penned parasite-free young sheep randomly allocated to experimental diets. The parameters investigated were FEC and larvae hatching. Initially, a period of 7 days was allowed for acclimatisation in which both groups were fed on half willow and half lucerne chaff. This was followed by Period 1 with 4 lambs fed lucerne and 5 fed willow, after which the diets were changed over for Period 2. Total faeces produced were collected from all animals on the last day of each period using bagged sheep. A known number of Teladorsagia eggs (500 epg) was then added to faecal samples from these sheep and faeces-egg mixtures were made from which FEC was determined, to see if egg recovery was affected by these diets. Faecal samples for Period 2 with added eggs were also incubated for 10 days to measure hatchability. The recovery of added Teladorsagia eggs in Experiment 2 was 85% in lucerne-fed lambs and 53% willow-fed lambs (P<0.001); these were used as correction factors for Experiment 1 data. Larvae that hatched per gram of wet faeces in Experiment 2 tended to be lower for sheep fed willow than lucerne chaff (71% vs 83% of eggs added; P=0.08). Willow feed offered had lower DM (P<0.001) and CP (P<0.05) content, but had a significantly higher OM content (P<0.01) than lucerne chaff. Condensed tannin content of chopped willow was 27 g/kg DM, with only traces for lucerne. Apparent digestibility for DM (62.4% vs 59.5%; P≤0.05), OM (64.8% vs 59.9%; P≤0.001), DOMD (58.1% vs 55.0%; P≤0.01) and calculated ME (9.48 MJ/kg vs 8.96 MJ/kg; P≤0.01) were higher for the willow diet. VFI was similar for both groups during the adaption period (P>0.05) but declined with the introduction of willow in week 6 (P<0.001) and then progressively increased until it was similar to lucerne-fed sheep in weeks 9 & 10 (P>0.05). Calculated DM intake per head/day during the last two weeks of Experiment 1 was similar for the two groups (P>0.05); while the willow group had higher ME (P<0.01) and CP (P<0.001) intake per animal/day. Liveweight increased for the two groups during the adaption period (P>0.05), then declined for willow-fed lambs in week 6 (P<0.001) but later increased and by week 10 was similar to that of lucerne-fed lambs. The willow-fed lambs had lower carcass GR than the lucerne-fed lambs (P<0.01) when carcass weight was used as a covariate. Adjusted total daily egg production in Experiment 1 was lower in willow-fed sheep than lucerne-fed sheep, due to reductions for Haemonchus spp. (P<0.05) and Teladorsagia spp. (P<0.05). The per capita fecundity for Haemonchus worm spp. (P<0.05) and the in utero fecundity in both abomasal Teladorsagia spp. and small intestinal Trichostrongylus spp. (P<0.001) were lower for willow-fed sheep. There was reduced production of larvae for both Haemonchus spp. and Teladorsagia spp. (P<0.05) in willow-fed sheep. Feeding willow reduced the burden of Haemonchus adult worms in the abomasum (P<0.01) but reduced female worm burden only in Teladorsagia spp. (P<0.05) and reduced Cooperia spp. in the small intestines (P<0.01). Total WBC, total lymphocytes, subsets of lymphocytes and other white-cell groups were not affected by willow feeding (P>0.1). It was concluded that feeding chopped willow to young sheep reduced nematode worm burdens in the abomasum, especially both male and female Haemonchus spp., and reduced female worm burdens of Teladorsagia spp. Female worm fecundity of both species was also reduced by willow feeding. These reductions have been associated with CT content in the willow feed and the reduced worm burdens have been attributed to the death of the established worms by CT, since there was no evidence of immune priming in willow-fed sheep. Compounds present in the faeces of willow-fed sheep have been found to mask some of the nematode eggs, making them invisible by microscopic examination while keeping their viability. It is postulated that this could be due to binding of nematode eggs to insoluble CT associated with indigestible fibre in the faeces of willow-fed sheep. Conventional methods of measuring FEC therefore underestimated nematode eggs present in the faeces of willow-fed sheep and this needs to be checked for other CT-containing forages.Item Evaluation of multipurpose fodder trees in Nepal : a thesis submitted in partial fulfilment of the requirement for the degree of Doctor of Philosophy (Ph. D.) in Forestry, Institute of Natural Resources, Massey University, Palmerston North, New Zealand(Massey University, 2007) Kshatri, Bhoj BahadurThis PhD thesis consists of nine chapters describing aspects of the subsistence farms of western Nepal in general, and a need-based evaluation of multipurpose fodder trees (MFT) as a source of dry-season forage for ruminants in particular, as a basis for mitigating the current high rate of land degradation and loss of productivity in livestock production systems in the region. Understanding the complex farming systems that provide a living for 65% of the 27.1 million people in Nepal is the key to designing effective programmes of research and development. Evaluation methods include review of past work, farmers group workshops to identify current practice in the use of MFT in Nepal, studies on biomass production of Artocarpus lakoocha and Ficus glaberrima trees older than 50 years in Nepal and the propagation of F. benjamina, comparison of the feeding preferences of sheep for alternative browse species, and study of the nutritive value of alternative forage diets for lactating buffalo. Reviews showed 2.2 million cattle and 1 million buffalo are an extra burden to steep land where productivity is declining at the rate of 1.25% per year. Indigenous knowledge identified Ficus glaberrima with its three varieties (Maghe, Chaite and Jethe), A. lakoocha, F. benjamina and Bassia butyracea as the best four MFT for renovating degraded lands. A survey study showed significantly higher dry matter (DM) production by F. glaberrima than A. lakoocha (154 vs 91 kg DM /tree/year) during dry periods at low altitude (800 - 1000m). There was no significant difference in production of fat - corrected milk (FCM ) between buffalos eating A. lakoocha, F.glaberrima or a diet of 53% straw and 47% F. glaberrima (DM basis). Metabolisable energy balance (MJME/day) was greater in Artocarpus than Ficus, with the mixed diet intermediate (+1.60, -0.34 and -12.94 MJ ME/buffalo/day respectively, relative to requirements, P=0.0318). When fed together in an indoor trial, poplar (48% = 106 g DM/sheep/day) and willow (43% = 95 g DM/sheep/day) were preferred to Ficus benjamina (8% = 18 g DM/sheep/day) by sheep, reflecting the greater maturity and structural strength of leaves of Ficus. These results are used to develop recommendations for choice of MFT species and management strategies to improve the sustainability and productivity of livestock systems incorporating fodder treesItem Effects of willow (Salix spp.) browse upon ewe reproduction and rumen microbiology under drought feeding conditions : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Animal Science at Massey University, Palmerston North, New Zealand(Massey University, 2007) Pitta, Dipti WilhelminaA series of grazing experiments were conducted in the summer/autumn of 2003 and 2004 at Massey University's Riverside dryland farm near Masterton in Wairarapa on the East Coast of NZ, to study the effects of grazing willow fodder blocks (6,000 stems/ha) upon the production and reproductive performance of ewes relative to ewes grazing drought pastures. Drought pastures were simulated in this study and included short drought pasture and long drought pasture. Pasture with a low pre-grazing mass of approximately 1500 kg OM/ha, a dead matter content of >50 % and a sward height of 5-7 cm was defined as short drought pasture typical of drought conditions. Long drought pasture was similar to pasture growing in the willow fodder blocks, with a pre-grazing pasture mass of >4000 kg OM/ha, a sward height of > 30cm and a dead matter content of 30-60 % . Willow fodder blocks were established on low-lying wet, marshy areas of the farm that had very low or zero productivity in the undeveloped state. Pasture development in the fodder blocks was noticed with the growth of unsown grasses and legumes, as the areas dried up following the planting of willow stakes, due to evapotranspiration from the trees. Forage in the willow fodder blocks included both trees and pasture that was grown under the trees. The nutritive value of short drought pasture was low with an ME of 8 MJ/kg O M ; long drought pasture ranged between 8- 1 0 MJ ME/kg DM; willow pasture contained 8 MJ M Elkg DM in 2003 and 1 0 MJ ME/kg OM in 2004. The nutritive value of edible willow tree (<5 mm diameter) was superior to drought pasture with an ME of > 10 MJ/kg OM. The concentrations of the secondary compounds such as condensed tannins (CT ; 30- 40 glkg OM) and phenolicglycosides ( PG ; 1 5-35 g/kg DM) were higher in willow trees compared to their concentrations (CT ; 2-3 g/kg DM) and (PG; 2-9 g/kg OM) in control drought pastures. Experiments involving short drought pasture, long drought pasture and willow fodder blocks as treatment groups were grazed by ewes for 10 weeks in regular breaks from mid February to early May. Ewes were mated during this period and were joined together after mating and grazed on normal pasture until weaning. Live weight (LW) change and body condition score (BCS) were recorded throughout the experiments, whilst reproductive performance of ewes was measured as the number of lambs recorded at ultrasound pregnancy scanning, lambing, docking and weaning. Measurements on wool production were also recorded at weaning. In 2003, experimental ewes grazed control drought pastures (short and long) and willow fodder blocks (restricted and full access) as treatment groups (n= 1 00 ewes/group; Chapter 2). Ewes grazing short drought pasture had an allowance of 0.8 k g DM/ewe/d whilst ewes with restricted access had an allowance of 0.8 kg DM/ewe/d from drought pasture and 0 .4 kg OM/ewe/d from willow fodder blocks. Ewes in full access treatment group had no access to pasture but were confined to willow fodder blocks at an allowance of 2.0 kg OM/ewe/d, which was the same allowance given to long drought pasture ewes. Ewes grazing short drought pasture lost weight at approximately 1 00g/d and recorded a low reproductive rate (90 lambs weaned/100 ewes mated) with a high proportion of single lamb births. Live weight loss was significantly reduced to 40 g/d in ewes grazing willow fodder blocks (full access) with a 20% units increase in reproductive rate due to more multiple births (P <0. 05). Ewes grazing long drought pasture performed intermediate to ewes with full access to fodder blocks and ewes grazing short drought pasture, whilst ewes with restricted access performed similar to ewes grazing short drought pasture. In 2004 (Chapter 3), the restricted access to willow fodder blocks treatment was eliminated from the study and the number of ewes was increased to 165 ewes per treatment group. Performance of ewes grazing short drought pasture was similar to that of ewes grazing short drought pasture in 2003 , with ewes loosing live weight (40g/d) and a low reproductive rate (90 lambs weaned/l00 ewes mated) whilst ewes grazing long drought pasture gained L W (54 g/d) and had a higher reproductive rate (P<0.05). Ewes grazing willow fodder blocks performed better than ewes grazing short drought pasture by maintaining L W and their reproductive rate was intermediate to ewes grazing short and long drought pasture. In 2005, a short grazing trial with rumen fistulated sheep was conducted to study the effect of supplementing willow to ewes grazing drought pastures upon plasma amino acid concentrations (Chapter 4) and upon the microbiology of the rumen (Chapter 5 and 6). Grazing occurred during summer/autumn for 10 weeks with two treatment groups; control (short drought pasture; n=7) at an allowance of 0.8 kg DM/ewe/d and ewes grazing short drought pasture at 0.8 kg DM/ewe/d plus a supplement of fresh willow at 1.4 kg fresh willow/ewe/d (n=7) . Blood samples for the quantification of plasma amino acids were collected at week 5 and 10, with L W and BCS measured at fortnightly intervals. Short drought pasture in this experiment had a low pasture mass (2000 k g DM/ha) and a low nutritive value (8 MJ/kg DM), whilst willow had a higher M E of 10 MJ/kg OM. Both groups of ewes lost live weight at the rate of 50 g/d. Plasma concentration of 3 methylhistidine (3-MTH; 88 vs 127μ mole/L) at week 5 and non essential amino acids (NEAA; 1082 vs 1417μ mo1e/L) at week 5 and ( 1155 vs 1324 μ mole/L) at week 10, were substantially lower (P<0 .05) in w illow supplemented ewes than control ewes. It was concluded that the increased reproductive rate from willow supplementation in ewes grazing drought pasture might be partly explained by reduced body protein catabolism, besides also increasing plasma branched chain amino acids CBCAA) and essential amino acids (EAA) concentrations. To investigate the effects of willow supplementation on rumen microbes, rumen samples were collected during the 2005 experiment with fistulated ewes over a 10 week period. The study involved the use of a molecular technique ( Chapter 5), denaturing gradient gel electrophoresis (DGGE), to compare the rumen microbial populations between the control and supplemented ewes and a cultivation technique (Chapter 6) to study the effect on rumen bacteria of ewes grazing drought pastures with and with out willow supplementation. DGGE analysis of the V3 region of 16S ribosomal RNA genes in DNA extracted from samples of rumen contents taken fortnightly over a 10 week feeding period showed a distinct difference in banding patterns between treatment groups which progressively developed over time, showing rumen microbial adaptation to willow supplementation. However, phylogenetic analysis of the DNA sequences retrieved from the DGGE bands from willow-supplemented and control ewes did not cluster by treatment group. It was deduced that willow supplementation induced a change in rumen bacterial populations through selecting sub-populations of organisms already present in the rumen. The changes in the rumen bacterial populations is attributed to the ability of these bacteria to metabolise secondary compounds in willow such as phenolicglycosides and flavanoid monomers and their ability to resist the inhibitory effects of condensed tannins. The cultivation study involved enumeration, isolation and purification of bacterial colonies on Complete Carbohydrate, Salicin, Xylan, Cellulose and Willow media followed by full characterisation of a representative set of pure bacterial cultures. Total bacterial counts on the above media at week 5 and week 10 were generally lower in willow-supplemented ewes compared to control ewes and the 16S rRNA gene sequences of the majority of iso lates characterised from both Salicin and Xylan media, were most closely related to species from the Pseudobutyrivibrio genus. Isolates from Willow medium clustered as two distinct groups. One group (mostly isolated from control ewes) was made up of mainly of organisms not usually associated with the rumen and probably represent non-resident organisms that are passing through the rumen. The other group of bacteria were mainly retrieved from willow-supplemented ewes and were most closely related to species of the Ofsenella genus. Compared to bacteria isolated on Salicin and Xylan media, isolates on Willow medium showed little ability to ferment various carbohydrates or trypticase (hydrolysed protein) but were able to utilise secondary compounds from willow. It was concluded that willow fodder blocks are useful sources of supplementary fodder for mating ewes during drought situations. Both the field and m icrobiological studies showed adaptation to the willow supplementary diet, including the detection of Olsenelfa-like bacteria for the first time in the rumen. It is suggested that the principal purpose of the rumen investigation is the degradation of secondary compounds present in willow.