Molecular biology has provided new opportunities to understand better the functioning of the immune system and to exploit this information for the construction of specific antibodies against a wide variety of antigens including the pathogens of humans and animals. In spite of the economic importance of cattle, many aspects of the immunology of this animal remain uncharacterised and tools to understand better bovine infections are lacking. This project has addressed aspects of both issues. The bovine immunoglobulin (Ig) system resembles that of other domesticated mammals in some respects, but other properties (eg the length of the third antigen-binding region of the heavy chain) appear unique. The first area for investigation in this project was to characterise the bovine JH locus and to understand why Ig rearrangement apparently favours a single JH segment. PCR was used to recover JH sequence from genomic DNA, either from non-lymphoid tissues or lambda vectors isolated and studied by other investigators. A region of 3200 bp was characterised which included the DQ52 segment, 6 JH segments and the heavy chain enhancer. The bovine DQ52 sequence is longer than those of other species and differs in sequence from a common consensus. For the most part, the JH locus is homologous to that of the sheep. The sixth JH segment identified appears to undergo rearrangement and is expressed in a minority of cattle antibodies. However, none of the segments carried the sequence which is most commonly expressed in bovine Ig. To identify which segment participates in this process, sequence was recovered from the rearranged genomic DNA of isolated bovine B cells using PCR with primers against VH and JH regions. This implicated the rearrangement of the fourth JH segment in the formation of bovine Igs but as the sequence differed between germline and rearranged copies, it appears that a non-conventional process operates in cattle. It is proposed that a gene conversion event or modified rearrangement process introduces sequence to form the fourth framework region of bovine Ig which does not exist at the JH locus in the germline. The mechanism of this modification requires more investigation. The second part of this project aimed to construct a library of recombinant bovine Fab antibodies from the Ig repertoire of a calf vaccinated against Mannheimia haemolytica (previously named Pasteurella haemolyticd).

The aim of the present work was to study the protein utilization during energy undernutrition in sheep. In four different trials, the effects of varying levels of protein with submaintenance amounts of energy on the energy &'38 N balances of sheep were studied using the intragastric infusion technique and the respiration chamber. The effects of change in energy and protein supply on plasma metabolites and hormonal concentrations were also studied. When animals were given protein well in excess of their maintenance requirement with little or no non-protein energy, they attained positive N balance although they were in negative energy balance, apparently by efficient utilization of endogenous energy (presumably body fat). As body fat was used to fuel the energy needed for protein retention, fat and protein deposition were negatively correlated. However, at very high level of casein infusion, the oxidized component of the supplied protein can contribute up to 36&'37 of the total ME requirement. About 16 kJ of endogenous energy was used for each g of protein accretion. The efficiency of endogenous energy utilization ranged between 0.56 to 0.60. There was no clear evidence, that there is any minimum level of body fatness which is necessary before body fat can be utilized to support protein retention during exogeneous non-protein energy restriction. Protein utilization during exogenous energy restriction was found to be more affected by the growth potential than the adiposity of the animal. Both fasting-heat production &'38 N excretion were reduced when the glucogenic needs of animals were met. Similarly plasma glucose, -hydroxybutyrate and free fatty acids concentrations were not affected by the energy status of the animals, when glucose requirements of the animals were met.

The studies reported in this thesis were carried out to evaluate the effects of enzyme treatment on the nutritive value of three lupin species (L. luteus, L. albus and L. angustifolius) and evaluate their suitability as a source of protein for growing broilers. Some preliminary in vitro work on the effect of quinolizidine alkaloids on bacteria from poultry excreta was carried out. Furthermore the use of near infrared spectroscopy (NIRS) as an alternative technique for assessing the chemical composition and nutritive value (such as metabolizable energy and digestibilities) was investigated. Compositional analyses of the various lupins, fractions and diets were conducted. The chemical composition of the lupin seeds used in this study showed a high crude protein content and a variable content in ether extract and neutral detergent fibre. The alkaloid content of the seeds was determined and allowed the classification of the lupin into bitter (L. luteus cv Cardiga), semi-sweet (L. albus cv Estoril) and sweet (L. albus from France and L. angustifolius from Australia) seeds. Alkaloids from lupins decrease feed consumption and may affect the digestive capacity of the animal. A preliminary study on the effects of sparteine and alkaloids extracted from L. luteus seeds on bacteria from poultry excreta indicated that extracts of alkaloids from L. luteus had an inhibitory effect on coliform growth that was not, however, as great as that of sparteine. Sparteine, essentially prevented growth of coliforms at concentrations of 10 g dm-3. Lactobacilli were apparently not directly affected by the lupin extract of alkaloid or the isolated sparteine. This in vitro work suggests that it may be possible to influence the gut microflora in a beneficial manner by using the appropriate concentration of the appropriate alkaloids. A set of tube feeding experiments (by gavage) were carried out to evaluate the effect of different enzymes on the nutritive value of L. luteus (cv. Cardiga) and L. albus (cv. Estoril) seeds. The first experiment was a preliminary one which allowed the modification of the tube feeding assay in order to suit the characteristics of the test materials used in this study. The following six experiments tested the effects of pre-incubation, as a wet mash, of a polygalacturonase, five proteases, a pectinase and an [Special character omitted]-galactosidase at variable levels, on the nutritive value of L. luteus (cv Cardiga) and L. albus (cv. Estoril). The nutritive value of the lupin seeds was evaluated essentially by measuring metabolizable energy (ME) and amino acid (AA) digestibilities. Results indicated that L. albus seeds, irrespective of enzyme treatment, had a higher ME expressed as TMEn than L. luteus seeds. Carbohydrase containing enzyme preparations, caused variable improvements in the ME value of lupin seeds. Polygalacturonase caused a significant increase in the ME of L. luteus seeds, which may have been mediated by an increase in protein digestibility as shown by improvements in the AA digestibility. Increased concentrations of pectinase tended to be more effective in the improvement of ME of L. albus, while increasing concentrations of a-galactosidase were so for L. luteus. The fact that the carbohydrases acted differently upon the two lupin species was probably due to their different concentration in constituent carbohydrates. Pre-incubation with exogenous proteases decreased the ME of the lupin seeds. The reasons for the depression were not clear, however high concentrations of proteases, expressed as U kg-1 of lupin seeds, may have interfered with the gastro intestinal tract and also with the endogenous enzymes of the bird resulting in reduced utilisation of the pre-incubated material. A growth experiment was design to investigate the effects of the inclusion of L. luteus (cv. Cardiga), L. albus (from France) or L. angustifolius (from Australia) as the main or secondary source of protein in diets for growing broilers. Lupin seeds were either pre-incubated as a wet mash, or the lupin containing diets were supplemented with a carbohydrase, a protease or a mixture of both. Diets were fed to the animals over a three week period. The results obtained tended to support the hypothesis that the enzymes had little effect on the other dietary ingredients but had their main effects on the lupins in the diets.

Chapter One. A general introduction on feed resources in the coastal lowland Kenya, use of the legume tree forages, and their effect when used as supplements to low quality roughage is presented. This is followed by a review of the literature on the role of microbes in the rumen and the synthesis of microbial protein. The factors that influence the feed intake in ruminants are also reviewed. The role of the leguminous tree forages as supplements to low quality basal diet is presented with more emphasis on the effects of feeding forages from gliricidia and leucaena trees, on intake, digestion and animal productivity. The effects of deleterious compounds with particular reference to tannin is reviewed. Chapter Two. Twenty intact and five fistulated crossbred steers (Ayrshire/Brown Swiss x Sahiwal) were used in Experiment One. The materials and methods used in this experiment had much in common with those of the three following experiments. Therefore procedures similar to all experiments are described in this chapter and only briefly mentioned in the subsequent chapters. The objective of the experiment was to describe the response in production as a result of supplementing napier grass basal diet with incremental levels of gliricidia forage. Napier grass basal diet was offered ad libitum alone or supplemented with 7.5, 15, 22.5 or 30 g DM/kg W0.75/d. Intake and live weight changes were measured for 49 days, and digestibility, and microbial N supply during the last week of the experiment with the twenty steers, in a randomized design. The rumen parameters were measured in the five fistulated steers in a 5 x 5 latin square design. Total Dry matter (DM) intake tended to increase (5.2, 5.1, 5.2, 5.4 and 5.7 kg DM/d, s.e.d 0.21; P 0.05) with supplementation, but this was accompanied by a linear decrease in napier grass intake (5.2, 4.7, 4.5, 4.3 and 4.2 kg DM/d, s.e.d 0.21; P 0.05). The diet digestibility, rumen pH and in sacco DM degradation of napier grass remained unchanged, while the rumen ammonia concentrations (NH3-N) were increased linearly (P 0.05) from 130 to 215 mg/1 for control and highest level of gliricidia forage offered. When gliricidia was offered as proportionally 0.26 of the diet, animal weight gains were increased proportionally by about 0.56. The relationship between the amount of gliricidia offered and the average daily gain was such that, every 10 g DM /kg W0-75 increment of gliricidia forage resulted in 49 g/d of live weight gain. The estimated microbial N supply were lowest in the control group and supplementation tended to increase the yield, but with no significant difference across the treatments. Chapter Three. The objective of Experiment Two was to examine the effect of supplementing napier grass with incremental levels of leucaena forage on voluntary food intake, diet digestibility, rumen fermentation, and live weight gains in steers. The same animals used in Experiment One were used in this experiment. The levels of leucaena offered were 0, 7.5, 15, 22.5 or 30 gDM/kg W0.75/d, the same as those of gliricidia in Experiment One. Increasing the proportion of leucaena forage in the diet of steers offered napier grass had no significant effect on the intake of the napier grass (5.2, 5.3, 5.3, 5.3 and 5.0 kg DM/d, s.e.d. 0.21). The total DM intake increased linearly (5.2, 5.8,6.2,6.6 and 6.7 kg DM/d, s.e.d 0.31; P 0.001). The response was such that for every increment of 10 g DM/kg W0.75 of leucaena, the total intake was increased by 0.52 kg DM/d. Diet digestibility tended to increase, while the rumen pH and in sacco DM degradation characteristics remained unchanged with supplementation. Rumen NH3-N was significantly increased by approximately 11 mg/1 for every 10 g DM/kg W0.75/d of leucaena forage offered. The growth of the animals was improved with supplementation, and the relationship between the amount of leucaena in the diet and the gain was such that for every 10 g DM/kg W0-75/d increment of leucaena forage intake, the gains were increased by 96 g/d. It could be predicted that 1 kg of leucaena offered would result in 171 g/d of gain. At a highest level of leucaena offered ie. proportionally 0.27 of the diet, the weight gains were increased by 0.57 compared with the control group. Chapter Four. The objective of Experiment Three was to examine the effect of incremental levels of gliricidia forage on voluntary food intake, digestion, microbial N supply and live weight gains. Twenty crossbred steers (Ayrshire/Brown Swiss x Sahiwal) and five fistulated steers of the same breed were used for the trial. Intake, diet digestibility, microbial N supply and live- weight changes were measured using the twenty steers and the rumen parameters using the five fistulated steers. Maize stover was offered ad libitum plus 1 kg of maize bran alone, or supplemented with 7.5, 15, 22.5 or 30 g DM/kg W0.75/d of gliricidia forage. Supplementation with gliricidia forage significantly increased the total DM intake (3.0, 3.0, 3.2, 3.5, 3.5 kg DM/d, s.e.d. 0.10; P 0.001) but depressed the intake of maize stover. The response in total DMI was an additional of 0.21 kg DM/d for an increment of 10 g DM/kg W0.75 of the legume. Diet digestibility was not significantly changed, nor was the rumen pH. The degradation parameters was slightly higher in the supplemented group, however, there were no difference in the degradation characteristics of the feeds incubated in steers offered different level of legume forage. Rumen NH3-N and live weight gains were significantly increased with supplementation. The relationship between the proportion of the supplement in the diet and the rumen ammonia or daily gains were such that, for every increment of 10 g DM/kg W0 75 of the legume offered the NH3-N and daily gain were increased by 16 mg/1 and 69 g/d respectively. The microbial N supply tended to be higher in the supplemented groups than the control (30.8, 37.2, 32.9, 34.9, 32.3, s.e.d. 1.06; P 0.05). Chapter Five. Experiment Four compared the type (gliricidia or leucaena) and levels (15 or 30 g DM/kg W0.75) of legume forages given as supplements to maize stover. The effects on intake, diet digestibility, rumen parameters, microbial N supply and live weight gains are presented and discussed. The same steers used in Experiment Three were used in this experiment (Twenty for the growth trial, and the five fistulated animals for the measurements of rumen parameters. The steers were offered maize stover ad libitum plus 1 kg maize bran alone, (2 kg for the fistulated animals), or supplemented with either 15 or 30 g DM/kg W0.75/d of gliricidia or leucaena (Control, G15, G30, L15 or L30). The intake of the basal diet was significantly (P 0.05) increased from 2.3 to 2.5 and 2.3 to 2.7 kg DM/d when of either gliricidia or leucaena respectively were offered as 0.17 of the diet. At higher level of supplementation (to about 0.33 of diet), the maize stover intake tended to decline. The total DM intake were increased (P 0.001) at both levels of both supplements offered (3.2,4.1,4.6,4.3 and 4.6 DM/kg, s.e.d 0.05, being the intakes for control, G15, G30, L15 and L30 respectively). Supplementation at the lower level significantly increased the diet digestibility. The rumen pH remained unchanged, the rumen NH3-N increased (31, 80, 101, 95, 111 mg/1 s.e.d 20.4; P 0.001), and the in sacco DM characteristics of the feeds tended to increase with supplementation. Both the DM and nitrogen (N) in leucaena forage were degraded at a slower rate than in gliricidia. The control group had the lowest (P 0.001) gains, compared with the supplemented groups. The leucaena supplemented group tended to have higher live weight gains than the gliricidia group, but the differences were not significant. Gains were 81, 355, 695, 396, 753 g/d s.e.d 44.5 for control, G15, G30, L15 and L30 groups respectively. With all parameters measured, there were no significant difference between supplementation with gliricidia or leucaena forages, at either level.

The objective of the present work was to elucidate some of the possible choice determinants involved in diet selection by sheep and goats. The first experiment investigated the ingestive and digestive responses of these two species to a range of forage chemical composition attributes (i.e. nitrogen and fibre content). Forage DM intake was positively associated with their nutritive quality. Goats had higher voluntary DM intakes (P<0.05) and lower apparent digestibility coefficients (P<0.001) for any given forage than sheep, but the intake of digestible DM was similar for the two species. When forages were offered in pairs in a preference trial, the two species ranked forages similarly by favouring those of higher nutritive quality. The second experiment investigated the effects of different staple lengths of a single hay on the ingestive and digestive responses of sheep and goats. Goats had higher voluntary DM intakes (P<0.05) and lower apparent digestibility coefficients (P<0.001) for any given length than sheep, but the intake of digestible DM was similar for the two species. Goats allocated less time (P<0.001) than sheep to chewing the feed consumed and the mean retention time of the undigested residues in the animals' digestive tract was significantly shorter (P<0.01) for goats than sheep. When staple lengths of the grass hay were offered in pairs none of them was favoured and the choices were not significantly different between goats and sheep. In a third experiment a new method of feed presentation was developed and compared with conventional feed-bins. Goats had higher voluntary intakes than sheep (P<0.05), whereas there was no effect of the feed presentation method on DM intake.

A problem with the captive breeding of grouse in this and other studies has been that cocks may attack hens in the confined space of an enclosure. One approach used to avoid severe injury to the hens has been to limit the amount of time each cock spends with his hen. However, separating pairs by hand is labour intensive and disruptive to the grouse. During this study a pen was developed which was separated into two parts by a 60cm tall barrier: this kept the wing-clipped cock on one side but allowed the unclipped hen to flutter to either side and so escape from the cock when she wanted to. The hatchability of eggs produced by captive grouse was unaffected by the type of hatcher used. Both still-air and forced-draught hatchers produced a hatchability of 52-57%. In 1989 and 1990 a total of 107 grouse aged 6-9 weeks was released into a wild grouse population. Data from 22 grouse fitted with radio transmitters showed a similar pattern in survival in both years. Over half died within three weeks of the release date, and most carcasses were discovered in a thin and emaciated condition. After three weeks the main cause of death was predation and only 2 of the 107 released grouse survived to the following breeding season. Initially, released grouse were reluctant to fly, but with time they did take to the wing more readily. In 1989 the release pens were located at the edge of the moor and many released grouse were found on the surrounding farmland. In 1990 the release pens were relocated in the middle of the moor and most grouse were subsequently found on the moor. In 1989 none of the released grouse were shot. In 1990 the total number of grouse shot on the release area was 14 and of this 6 were released grouse. The breeding density of the wild population on the release area declined during the period of the release experiments. However, on an adjacent control area where grouse were not released the breeding density increased. The causes of these patterns were not established.

The objective of this study was to examine the effects of rearing temperature on the meat yield of broilers. Four experiments were conducted. Experiment 1 examined meat yield at different temperatures and on different diets when birds were grown to a fixed age. Experiments 2 and 3 were aimed at the effects of temperature and of the interaction between temperature and stocking density respectively on the meat yield of broilers when killed at a specified liveweight (i.e. allowing the rearing period to vary). Experiment 4 was designed to assess whether depression of growth rate at high temperature affected the meat yield in broilers.

Foetal piglets do not obtain immunoglobulins during pregnancy and rely on colostrum immunoglobulins until active immunity develops. The amount of immunoglobulin that piglets obtain depends upon: colostrum immunoglobulin concentration; quantities of colostrum ingested and when closure occurs. Closure describes the change that takes place between 12-36h of life, when the piglet gut can no longer absorb immunoglobulins. A series of studies were undertaken to examine colostrum IgG concentrations and IgG acquisition by piglets. The first two trials demonstrated that IgG concentration between sows and piglet IgG intake was very variable. Piglet plasma IgG concentrations were remarkably consistent however, suggested that piglets were regulating IgG uptake. The third trial reduced piglet IgG intakes by delaying suckling. Results showed that piglets with reduced IgG intakes had reduced plasma IgG concentrations and could not compensate for lowered IgG intake. The results suggested that piglets regulated IgG uptake when IgG was in excess, but had only limited ability to regulate IgG uptake when IgG supply was limited. The fourth trial set out to examine the effects of maternal diet and vaccination on piglet IgG acquisition. A 2x2 factorial design, sows were fed diets either with vitamins A, C and E at recommended amounts or 3-5 times recommended amounts and either vaccinated or not. Results showed that maternal vaccination or diet did not affect maternal plasma IgG concentration or colostrum IgG concentration. Supplementing maternal diet with vitamins significantly increased piglet plasma IgG concentrations, as did maternal vaccination. Of ingested IgG, approximately 0.6-0.7 appeared in piglet plasma. It was possible to influence piglet plasma IgG concentrations by manipulating maternal diet and immune status. With increasing pressure on the use of antibiotics in agriculture and increasing animal welfare/health demands, the effects of other macro and micronutrients on piglet IgG status need to be investigated.

Three experiments were carried out to assess the energy and protein requirments of single and twin-bearing beef cows during the last three months of pregnancy and also to observe the effects of pregnancy nutrition on the subsequent lactational performance of cows and calves. A wide range of energy level was applied to single and twin-bearing cows. As assessed from body weight change, calf birthweight, milk yield, calf growth rate and blood parameters, it was found that there was no benefit in giving high levels of dietary energy to cows during pregnancy. It was concluded that feeding 50 and 70 MJ ME/day to single and twin-bearing cows respectively together with a high proportion of UDP may be acceptable levels during the latter part of gestation, provided that the cows are in good body condition at the start of the last trimester of pregnancy and they are given adequate nutrition during early lactation. Another three experiments were conducted with pregnant ewes, wethers, pregnant heifers and steers to measure the digestibility and to determine the metabolisable energy value of two mixed diets which differed in rumen degradability of protein. Such diets were used in experiments with pregnant cows. A comparison was made between the digestive capacity in different species and sex of animals. Digestibility co-efficients of two diets, two sexes and two species did not differ significantly. Therefore, it was concluded that metabolisable energy value determined for mixed diets using sheep can be used for cattle. ME value of mixed diets was less than that calculated in these experiments but the extent of the decrease may be acceptable under farm conditions.

Theileria annulata is a protozoan parasite of cattle, that causes the disease tropical theileriosis throughout sub-tropical regions of the Old World. Theileria parasites have the ability to immortalise the host leukocyte they infect causing clonal expansion and dissemination of infected leukocytes throughout the host. This property has allowed the development of an in vitro system for the culture of bovine cells infected by the macroschizont stage of the parasite. In addition, differentiation of the parasite towards the next life cycle stage, the merozoite, can be induced in culture. The signals that cause the macroschizont to differentiate into merozoites are not fully understood, although it is known that this event is associated with a major elevation in merozoite gene expression (Shiels et al., 1994). Recently a small family of parasite genes that are negatively regulated early during differentiation to the merozoite were identified. One member, known as TashAT2 contained predicted AT hook DNA binding motifs and was shown to be localised to the host cell nucleus. It has been postulated that the TashAT2 polypeptide may play a role in the regulation of macroschizont or modulation of host cell gene expression (Swan et al., 1999). The focus of this project was to characterise TashAT1, a second member of the TashAT gene family. To this end, the TashAT1 gene was sub-cloned and sequenced and mapped to a region of the genome containing TashAT2 and a third Task AT gene, TashAT3. The 1.4kb open reading frame of TashAT1 was virtually identical to the five prime end of TashAT3, indicating that TashAT1 or TashAT3 (TashAT1/3) were derived from a recent duplication event. The predicted amino acid sequence of TashAT1/3 contained four AT hook motifs, a nuclear localisation signal and a signal sequence. Northern blot analysis revealed that TashAT1, TashAT2 and TashAT3 mRNA were down regulated early, during differentiation to the merozoite in vitro. However, no down regulation was observed for any of the TashAT transcripts in a cell line that was severely attenuated with respect to parasite differentiation. Sequence analysis of the upstream regions of TashAT1/3 identified a motif element (TashUM) located 43bp upstream of the putative transcription start site of TashAT1/3 that was highly related to a sequence upstream of TashAT1 and another, unrelated macroschizont gene, Tash1. Preliminary electromobility band shift analysis of TashUM revealed that it bound to a factor found in host and parasite enriched nuclear extract, which appeared to decrease in abundance as the parasite differentiated towards merogony. Antisera generated against a region of TashAT1 failed to recognise a TashAT1 polypeptide by Western blot analysis. However, a 180kDa polypeptide that was down regulated with respect to merogony and co-localised to the host nucleus was specifically recognised. The detected polypeptide was identified as TashAT3 on the basis of size, sequence identity and predicted expression profile. Immunofluorescence analysis showed that the anti-TashAT1 antisera reacted against both the host nucleus and parasite. This reactivity was lost as the parasite differentiated to the merozoite. The host reactivity was probably due to recognition of TashAT3, while it could not be concluded that the parasite reactivity was directed against TashAT1. Taken together, the results indicated that TashAT3 and possibly TashAT1 are additional candidates for parasite encoded factors that are translocated to the host nucleus, bind to DNA and alter host cell gene expression. This modulation of gene expression could directly or indirectly alter the phenotype of the host cell and be involved in parasite dependent regulation of leukocyte cell division.